Stapf, a Medicinal Herb from the Trans-Himalayan Region of Nepal

Carbohydrate Research 341 (2006) 2161–2165

Note New ﬂavonoid glycosides from Aconitum naviculare (Bru¨hl) Stapf, a medicinal herb from the trans-Himalayan region of Nepal Bharat Babu Shrestha,a Stefano Dall’Acqua,b Mohan Bikram Gewali,c Pramod Kumar Jhaa and Gabbriella Innocentib,* aCentral Department of Botany, Tribhuvan University, Kirtipur, Kathmandu, Nepal bDepartment of Pharmaceutical Sciences, University of Padova, Italy cResearch Center for Applied Science and Technology, Tribhuvan University, Kirtipur, Kathmandu, Nepal Received 27 March 2006; received in revised form 7 May 2006; accepted 16 May 2006 Available online 9 June 2006

Abstract—Three new ﬂavonoid glycosides, 3-O-[b-D-glucopyranosyl-(1!3)-(4-O-trans-p-coumaroyl)-a-L-rhamnopyranosyl-(1!6)- b-D-glucopyranosyl]-7-O-[b-D-glucopyranosyl-(1!3)-a-L-rhamnopyranosyl]kaempferol, 3-O-[b-D-glucopyranosyl-(1!3)-(4-O- trans-p-coumaroyl)-a-L-rhamnopyranosyl-(1!6)-b-D-glucopyranosyl]-7-O-[b-D-glucopyranosyl-(1!3)-a-L-rhamnopyranosyl]quercetin and 7-O-[b-D-glucopyranosyl-(1!3)-a-L-rhamnopyranosyl]quercetin were isolated from the aqueous extract of the aerial parts of Aconitum naviculare. Their structures were elucidated by spectral analysis (HRAPI-TOF MS, 1H, 13C NMR, HMQC, HMBC, DFQ-COSY, ROESY and TOCSY). 2006 Elsevier Ltd. All rights reserved.

Keywords: Aconitum naviculare; Ranunculaceae; Manang; Flavonoid glycosides

Aconitum naviculare (Bru¨hl) Stapf (Ranunculaceae), to its high effectiveness in traditional medicine, it may commonly called Ponkar, is a biennial medicinal herb be a potential source of income for mountain people of alpine grassland [>4000 m above sea level (asl)] found and a source of important natural compounds for phar- in the trans-Himalayan region of Nepal (Manang, Mus- maceutical companies. Although the chemical constitu- tang and Dolpa districts). The whole plant is used by ents of plants of the genus Aconitum have been mountain people against colds, fevers and headaches extensively studied,2,3 there is only one paper regarding (Shrestha, personal observation), and in Tibetan folk alkaloids1 available, and there are no phytochemical medicine, it is also used as a sedative, analgesic and feb- studies reported on A. naviculare. The present study rifuge.1 According to the Manangi people’s tradition, describes the isolation and characterization of three A. naviculare is the most effective of the various medici- new flavonoid glycosides from the aerial parts of nal plants used. Although the whole plant is medicinally A. naviculare. important, the Manangis collects only the aerial parts Three new flavonoid glycosides were identified from (stem, leaves and flowers) during the flowering season the aqueous extract obtained by partition with organic (September–October). About 1 g of air-dried plant solvents of crude methanol extract (Fig. 1). material is boiled with two glasses of water for about Compound 1. The HRAPI-TOF MS spectrum 30 min, and the bitter concoction is drunk twice a day. showed a negative ion [MH] at m/z 1209.3555, indi- 1 Although the Manangis living in and outside Manang cating a molecular formula of C54H66O31. The H use A. naviculare extensively, it is not marketed. Due NMR spectrum showed the typical signal pattern for a kaempferol derivative (Table 1). A pair of doublets (d 7.61, 6.31) with a coupling constant of 15.95 Hz revealed * Corresponding author. Tel.: +39 049 8275370; fax: +39 049 the presence of a trans olefinic double bond. Two more 8275366; e-mail: [email protected] ortho-coupled doublets at d 7.54 and 6.89 (J 8.0 Hz),

OH O O H3C O O O O H C R' 3 O OH O H C HO R = OH 2 OH O R'' O HO O HO O OH HO HO O HO OH O HO OH HO OH OH

1: R' = H, R'' = R 2:' R' = OH, R'' = R 3: R' = R'' = OH

Figure 1. Structure of flavonoid derivatives. each integrating for two proton signals, showed a further Compound 2 was isolated as a yellow powder. The o, p-disubstituted aromatic ring, indicating the presence HRAPI-TOF MS spectrum showed a negative ion of a coumaric acid residue. Complete structure assign- [MH] at m/z 1225.3515 corresponding to the mole- 1 ments were achieved by HMQC, HMBC, DFQ-COSY, cular formula of C54H66O32. The H NMR spectrum TOCSY and ROESY experiments and by comparison showed the characteristic pattern of a glycosylated quer- of spectral data previously published for similar com- cetin derivative. A pair of doublets (d 7.59, 6.29) with a pounds.4–6 The anomeric signals of five sugar units were coupling constant of 15.5 Hz revealed a trans olefinic observed at d 4.24 (J 8.0 Hz), 4.63 (J 0.8 Hz), 4.69 (J double bond. Two more ortho-coupled doublets at d 7.8 Hz), 5.15 (J 7.5 Hz) and 5.59 (J 0.9 Hz). The HMQC, 7.50 (J 8.0 Hz) and 6.84 (J 8.9 Hz), each integrating DQF-COSY and TOCSY data showed that sugar for two proton signals, indicated a second o, p-disubsti- appeared as three b-D-glucopyranose units and two a-L- tuted aromatic ring, suggesting a coumaric acid residue, rhamnopyranose units. ROESY correlations were also as in 1. Five sugar units were revealed by five signals of observed between the anomeric proton signal of RhaI (d anomeric protons at d 4.21 (J 7.6 Hz), 4.61 (J 0.9 Hz), 5.59) and the doublets at d 6.64 (H-8) and 6.50 (H-6). In 4.64 (J 0.8 Hz), 5.24 (J 8.0 Hz) and 5.51 (J 10.0 Hz), addition, HMBC correlations were observed between respectively. Exhaustive analysis of DQF-COSY, H-1 of RhaI and carbon resonance at d 163.2 (C-7). These TOCSY and HMQC data determined sugar units as I data revealed the linkage between Rha and the C-7 of the two b-D-glucopyranose and three a-L-rhamnopyranose kaempferol unit. HMBC correlation was also observed units. The ROESY and HMBC data completed struc- between H-1 of GlcI (d 4.69) and the carbon resonance ture assignments, showing that compound 2 differed C-3 of RhaI at d 82.7, indicating that the sugar portion, from 1 only due to the OH group in position 30. This evi- which is linked to position 7 of the kaempferol moiety, dence led us to conclude that compound 2 was 3-O-[b-D- was a b-D-glucopyranosyl-(1!3)-a-L-rhamnopyranosyl glucopyranosyl-(1!3)-(4-O-trans-p-coumaroyl)-a-L-rham- residue. The fact that a long-range correlation was nopyranosyl-(1!6)-b-D-glucopyranosyl]-7-O-[b-D-gluco- II observed between the proton signal at d 5.15 (H-1 of Glc ) pyranosyl-(1!3)-a-L-rhamnopyranosyl]quercetin. and the carbon resonance at d 135.3 (C-3), as well as the Compound 3 was isolated as a yellow powder. The ROESY correlation between this signal at d 5.15 and pro- HRAPI-TOF MS spectrum yielded a negative ion tons H-20–60 (d 8.20), suggested that the second position [MH] at m/z 609.1485, indicating a molecular for- 1 of glycosidation is C-3 of the kaempferol unit. HMBC mula of C27H30O16. The H NMR spectrum was consis- correlations between the H-1 of RhaII (d 4.63) and the tent with the presence of a glycosylated quercetin C-6 of GlcII (d 68.8) and between the H-1 of GlcIII (d derivative. Two proton signals were ascribable to ano- 4.24) and the C-3 of RhaII (d 79.1) established that the res- meric protons at d 5.58 (J 0.9 Hz) and 4.63 (J 8.0 Hz), idue was linked to position C-3 as a b-D-glucopyranosyl- respectively. Data obtained from DQF-COSY, NOESY (1!3)-a-L-rhamnopyranosyl-(1!6)-b-D-glucopyranosyl and HMQC spectra established the sugars as one b-D- II unit. The HMBC correlation between the H-4 of Rha glucopyranose and one a-L-rhamnopyranose units. (d 5.05) and the carbon resonance at d 168.9 (C-900) also The glycosidation position was revealed both by the revealed esterification of the coumaric acid at position 4 HMBC correlation between H-1 Rha (d 5.58) and of this second rhamnose unit. Thus, 1 was identified as carbon resonance at d 164.0 (C-7), and by the NOESY 3-O-[b-D-glucopyranosyl-(1!3)-(4-O-trans-p-coumaroyl)- correlations between H-1 Rha and the doublet signals a-L-rhamnopyranosyl-(1!6)-b-D-glucopyranosyl]-7-O- of H-6 (d 6.46) and H-8 (d 6.75) of the quercetin skele- [b-D-glucopyranosyl-(1!3)-a-L-rhamnopyranosyl]kaemp- ton. Thus, compound 3 was identified as 7-O-[b-D- ferol. glucopyranosyl-(1!3)-a-L-rhamnopyranosyl]quercetin. B. B. Shrestha et al. / Carbohydrate Research 341 (2006) 2161–2165 2163

Table 1. 1H and 13C NMR data of compounds 1–3 in MeODa Position 123

dH (J in Hz) dC dH (J in Hz) dC dH (J in Hz) dC 1 — —— —— — 2 — 158.9 — 158.0 — 149.9 3 — 135.3 — 135.2 — 135.0 4 — 178.8 — 180.0 — 176.2 5 — 163.0 — 161.7 — 163.3 6 6.50 d (1.8) 100.8 6.43 d (1.5) 99.6 6.46 d (1.7) 100.3 7 — 163.2 — 162.4 — 164.0 8 6.64 d (1.8) 95.7 6.60 d (1.5) 94.2 6.75 d (1.7) 95.6 9 — 157.4 — 156.5 — 158.0 10 — 107.4 — 106.9 — 107.0 10 — 121.8 — 133.8 — 124.6 20 8.20 d (8.5) 132.7 7.89 d (1.6) 116.7 7.77 d (1.5) 116.2 30 6.96 d (8.5) 116.3 — 144.6 — 148.0 40 — 161.6 — 149.0 — 147.4 50 6.96 d (8.5) 116.3 6.90 d (8.0) 115.7 6.89 d (7.8) 116.4 60 8.20 d (8.5) 132.7 7.67 d (8.0; 1.6) 122.0 7.67 dd (7.8; 1.5) 122.4 RhaI (7) 1 5.59 d (0.9) 99.7 5.51 d (1.0) 98.2 5.58 d (0.9) 99.9 2 4.37 m 71.0 4.30 m 69.6 4.31 m 71.2 3 3.99 m 82.7 3.93 m 80.9 3.95 m 83.3 4 3.67 m 71.0 3.60 m 71.0 3.66 m 71.8 5 3.71 m 71.3 3.65 m 69.0 3.70 m 71.5 6 1.31 d (6.0) 18.3 1.25 d (7.0) 18.1 1.28 d (6.0) 18.5 GlcI 1 4.69 d (7.8) 105.7 4.64 d (8.0) 105.0 4.63 d (8.0) 106.4 2 3.41 m 75.5 3.36 m 77.1 3.28 m 75.2 3 3.50 t (9.0) 77.6 3.40 m 71.0 3.37 m 77.8 4 3.37 m 70.9 3.36 m 71.0 3.64 m 71.9 5 3.38 m 77.6 3.48 m 78.0 3.47 m 78.2 6 3.99–3.65 m 62.3 3.93 dd (12.0; 2.0)–3.72 dd (12.0; 5.0) 62.0 3.72–3.86 m 62.7 GlcII (3) 1 5.15 d (7.5) 105.6 5.14 d (7.8) 105.8 2 3.88 m 72.7 3.81 m 75.2 3 3.69 m 75.0 3.69 m 73.7 4 3.30 m 77.0 3.34 m 77.0 5 3.20 m 77.5 3.61 m 71.0 6 3.80–3.57 m 68.8 3.87 m–3.76 m 68.5 RhaII 1 4.63 d (0.8) 102.3 4.61 d (0.9) 101.9 2 3.95 dd (8.8; 3.3) 71.7 3.95 m 71.8 3 3.85 m 79.1 3.86 m 79.0 4 5.05 dd (8.0; 8.2) 73.6 5.00 dd 72.6 5 3.85 m 67.9 3.80 m 68.2 6 0.98 d (6.5) 17.9 0.95 d (6.5) 18.0 GlcIII 1 4.24 d (8.0) 105.7 4.21 d (7.6) 105.2 2 3.16 brt (9.0) 74.7 3.14 m 73.7 3 3.31 m 77.5 3.26 m 77.2 4 3.35 m 75.5 3.28 m 76.0 5 3.26 m 71.0 3.30 m 71.0 6 3.60 dd (11.2; 5.0)–3.80 dd (11.8; 2.0) 62.2 3.70–3.80 m 62.1 Coumaric 1 — 127.4 — 126.8 2–6 7.54 d (8.0) 131.4 7.50 d (8.0) 129.8 3–5 6.89 d (8.0) 117.1 6.84 d (8.9) 115.7 4 — 161.3 — 161.1 7 7.61 d (15.95) 147.0 7.59 d (15.5) 149.6 8 6.31 d (15.95) 115.5 6.29 d (15.5) 114.1 9 — 168.9 — 167.4 a Assignments were conﬁrmed by DFQ-COSY, HMQC and HMBC. 2164 B. B. Shrestha et al. / Carbohydrate Research 341 (2006) 2161–2165

To our knowledge, 1–3 are reported here for the first 1.3. Extraction and isolation time, as new, naturally occurring compounds from A. naviculare. Fresh plant material was spread in a shaded, windy environment for 2 days, and was then packed in thin cotton bags and hung in the shade until it was dry. Dry mate- 1. Experimental rial was then ground into pieces. Air-dried powdered aerial parts (100 g) were exhaus- 1.1. General methods tively extracted in a Soxhlet apparatus with MeOH. The solvent was evaporated under reduced pressure Sephadex LH-20 was used for column chromatography. (230–250 mbar) and a semisolid MeOH extract was Silica gel plates were used for analytical TLC (E. Merck obtained (8 g). The MeOH extract (about 5.0 g) was cat. 5715). Semipreparative HPLC was performed on a suspended in a mixture of 9:1 H2O–MeOH (200 mL) Gilson series 305 liquid chromatograph using a LiChro- and the pH was adjusted to pH 2 using 5% aq HCl. spher 100 RP-18 column (particle size 10 lm, The solution was then partitioned first with CHCl3 250 · 10 mm ID, E. Merck). The mobile phase was sol- (5 · 50 mL) and then with EtOAc (5 · 50 mL). Solvents vent A, 0.1% aq HCO H, and solvent B, AcCN. The 2 were removed under vacuum obtaining the CHCl3 flow rate was 6 mL/min. The separation gradient was fraction CL-I (850 mg) and EtOAc fraction EA-I from 75% to 45% of A in 25 min. (330 mg). The pH of the residual aqueous layer was then Analytical HPLC was performed on an Agilent adjusted to pH 8 using diluted NH3. The aq layer was 1100 series liquid chromatograph with a diode array partitioned with CHCl3 (5 · 50 mL) and EtOAc UV–vis detector. A LiChrospher RP-18 column (parti- (5 · 50 mL). Solvents were removed under vacuum cle size 5 lm, 250 · 4.6 mm ID) equipped with guard yielding the CHCl3 fraction CL-II (250 mg) and EtOAc column was used. Chromatographic conditions were fraction EA-II (133 mg). The pH of the aqueous layer as follows: solvent A, 0.1% aq HCO2H, solvent B, was then adjusted to pH 7.0, and the solvent was MeOH and solvent C, AcCN, in the following gradi- removed by freeze-drying, giving fraction AQ (3.3 g). ent elution. Fraction AQ was applied to a Sephadex LH-20 column (300 mL) and eluted with MeOH. The eluted frac- Min %A %B %C tions were analyzed by TLC and pooled on the basis of 0801010their chromatographic behaviour into eight groups (1– 10 50 25 25 8). Further chromatographic steps on TLC (10:5:1 30 50 50 0 CHCl3–MeOH–H2O) and semipreparative HPLC 35 50 50 0 yielded new compounds 1 (6.2 mg), 2 (7.8 mg) and 3 (4.8 mg). The purity of the isolated compounds was checked by NMR spectra in CD3OD were obtained on a Bruker HPLC and found to be 97%. Retention times (tR) were AMX-300 spectrometer operating at 300.13 MHz for as follows: 1 15.3 min, 2 14.0 min, 3 17.5 min. 1H NMR and 75.03 MHz for 13C NMR. 2D experiments, 1H–1H DQF-COSY, ROESY, TOCSY and inverse-detected 1H–13C HMQC and HMBC spectra 1.4. 3-O-[b-D-Glucopyranosyl-(1!3)-(4-O-trans-p-cou- were performed using UXNMR software. Exact masses maroyl)-a-L-rhamnopyranosyl-(1!6)-b-D-glucopyran- were measured on an API-TOF spectrometer (Mariner osyl]-7-O-[b-D-glucopyranosyl-(1!3)-a-L-rhamnopyran- Biosystems). Samples were diluted in a mixture of 1:1 osyl]kaempferol (1) H O–AcCN with 0.1% NH and directly injected at a 2 3 m z flow rate of 10 lL/min. Yellow amorphous powder; HRAPI-TOF MS: / 1209.3555 (calcd m/z 1209.3510 for C54H66O31 H). 1 13 1.2. Plant material H and C NMR data, see Table 1.

Aerial parts including stem, leaves, flowers and imma- 1.5. 3-O-[b-D-Glucopyranosyl-(1!3)-(4-O-trans-p-cou- ture fruits were collected from Ladtar (4100 m asl, upper maroyl)-a-L-rhamnopyranosyl-(1!6)-b-D-glucopyran- Manang) during the last week of September 2004. A osyl]-7-O-[b-D-glucopyranosyl-(1!3)-a-L-rhamnopyran- voucher specimen was collected during a field survey osyl]quercetin (2) and deposited at Tribhuvan University Central Herba- rium (TUCH); identification was confirmed by Profes- Yellow amorphous powder; HRAPI-TOF MS: m/z sor Ram Prasad Chaudhary of the Central Department 1225.3515 (calcd m/z 1225.3459 for C54H66O32 H). of Botany, Tribhuvan University, Kathmandu. 1H and 13C NMR data, see Table 1. B. B. Shrestha et al. / Carbohydrate Research 341 (2006) 2161–2165 2165

1.6. 7-O-[b-D-Glucopyranosyl-(1!3)-a-L-rhamnopyran- Foundation (Germany) Project in Nepal for a Ph.D. osyl]quercetin (3) fellowship.

Yellow amorphous powder; HRAPI-TOF MS: m/z 1 References 609.1485 (calcd m/z 609.1456 for C27H30O16 H). H and 13C NMR data, see Table 1. 1. Gao, L.; Wei, X.; Yang, L. J. Chem. Res. 2004, 307–308. 2. Pelletier, S. W.; Mody, N. V. In The Alkaloids; Manske, R. H. F., Rodorigo, R. G. A., Eds.; Academic Press: New York, 1981; Vol. 17, pp 1–103. Acknowledgements 3. Pelletier, S. W.; Mody, N. V.; Joshi, B. S.; Schramm, L. C. In Alkaloids, Chemical and Biological Perspectives; Pelletier, We are grateful to Dr. Meena Rajbhandary for her S. W., Ed.; Wiley: New York, 1983; Vol. 2, pp 206–462. help during the extraction process. One of the authors, 4. Agrawal, P. K.; Bansal, M. C. Carbon-13 NMR of B.B.S., thanks the University of Padova (Italy) and Flavonoids; Elsevier: New York, 1989; pp 283–363. 5. Fico, G.; Braca, A.; De Tommasi, N.; Tome`, F.; Morelli, I. Tribhuvan University (Kathmandu, Nepal) for Phytochemistry 2001, 57, 543–546. providing the opportunity to visit the University of 6. Diaz, J. G.; Ruiz, J. G.; Dias, B. R.; Gavin Sazatornil, J. A.; Padova as a Research Scholar; and the Volkswagen Herz, W. Biochem. Syst. Ecol. 2005, 33, 201–205. Local Effects of Global Changes in the Himalayas: Manang, Nepal, 2007, pp 171-181. Eds.: Ram P. Chaudhary, Tor H. Aase, Ole R. Vetaas, Bhim P. Subedi Publishers: Tribhuvan University, Nepal and University of Bergen, Norway

Chapter 12

Ethnomedicinal Use and Distribution of Aconitum naviculare (Brühl) Stapf. in Upper Manang, Nepal

Bharat B. Shrestha1, Pramod K. Jha1 and Mohan B. Gewali2

Abstract

Ethnomedicinal use of Aconitum naviculare was documented and its’ population was sampled in Upper Manang (Nyeshang) during September 2005. Aconitum naviculare has been used against fever, headache, jaundice, high blood pressure and cold, and was considered as the most effective among the medicinal plants used by Manangis. There was no commercial collection of this species in Manang. Aconitum naviculare is found only on dry, south facing slope between 4000 and 4700 m a.s.l. In Ice Lake area, Manang hill and upper Khangsar small population of Aconitum naviculare was restricted to a specific pocket habitat whereas in Ledar the population of this plant was relatively large. Aconitum naviculare mostly grows along with thicket forming alpine scrubs such as species of Juniperus, Berberis, Caragana, Astragalus, Spiraea, etc which protect Aconitum naviculare from animal damage. Maximum population density in different sites ranged from 4.07 - 8.66 plants/m2 in their natural habitat. Population density of this plant appears to be declining mainly due to collection of plant before seed dispersal. Distribution and population status of Aconitum naviculare is reported in this paper and it can serve as a reference for population dynamics study in the future and for similar kind of work in other regions.

12.1 Introduction Himalayan mountains are rich in medicinal plants and mountain people have long tradition of using these medicinal plants for their health care (Watanabe et al. 2000, Thomas et al. 2005). In subalpine and alpine regions of Nepal, collection of medicinal plants from wild for sale in market is an integrative part of mountain peoples’ livelihood and has significant share in local household as well as national economy (Olsen and Larsen 2003). Due to socio-economic transformation, urban people rely mostly on modern allopathic medicine but mountain people still use selected medicinal plants

1 Central Department of Botany, Tribhuvan University, Kirtipur, Kathmandu, Nepal 2 Research Center for Applied Science and Technology (RECAST), Tribhuvan University, Kathmandu, Nepal 172 Bharat B. Shrestha et al. and formulations prepared from them. When compounded and prescribed appropriately the safety of traditional herbal medication is high due to moderate bioreactivity of natural plant products (Elvin-Lewis 2001). There is a long tradition of transferring cultural knowledge of using medicinal plants from generation to generation. In the last few decades these ethnomedicinal knowledges have been exploited for preparing modern medicine, especially in search for novel bioactive natural products which can be used for treatment of serious diseases. Ethnomedicinal knowledge can provide a lead for this venture (Hamilton 2003). Species of Aconitum are well known for the presence of, either poisonous or medicinally important bioactive compounds (Pelletier and Mody 1981, Pelletier et al. 1983). Out of thirty four species of Aconitum recorded from Nepal (Press et al. 2000), ethnomedicinal uses of only five species have been described in detail by Manandhar (2002), and eleven species by Baral and Kurmi (2006). However sixteen species of Aconitum have been listed in Medicinal and Aromatic Plant Database of Nepal (MAPDON) which also included Aconitum naviculare (Shrestha et al. 2000). Aconitum naviculare, a Himalayan endemic (Stainton 1997), is an ecologically and phytochemically less studied medicinal plant growing in alpine grassland of trans-Himalayan regions. Most of the medicinally important Aconitum species have been categorized as critically endangered in their natural habitat (Anonymous 1998) but detailed study on distribution and population status has not yet been made. Ecological studies of a few medicinal plants in Lower Manang (Gyasumdo valley) were undertaken (e.g. Ghimire et al. 1999, Gahire 2003) but such study in Upper Manang (Nyeshang), where Aconitum naviculare is found, has been lacking though it is rich both in floral diversity (Pokharel 2004) and cultural/indigenous knowledge of their use (Bhattarai et al. 2006). Hence, in this paper the focus is on ethnomedicinal use and distribution of Aconitum naviculare in Upper Manang.

12.2 Study area and Methods 12.2.1 Study area. The study area (28° 41”- 28° 46” N, 83° 57”- 84° 04” E, elevation 4150 - 4650 m a.s.l.) lies in upper part of Manang district, central Nepal. The upper Manang (Nyeshang) has U-shaped, glacially formed valley (3000-3400 m a.s.l.) traversed by the river Marsyangdi and surrounded by high mountains (>6000 m a.s.l.). It lies on rain shadow area of trans- Annapurna region with an annual rainfall of 255 mm (DHM 2000, based on nearest climate station in Jomsom which has similar weather condition to Upper Manang). North facing slope is moist with three types of subalpine forests (Pinus wallichiana forest, Abies spectabilis forest and Betula utilis forest) while the south facing slope is dry and forest is mostly absent except Pinus wallichiana forest near the valley floor and Betula utilis, the availability of plants is limited to moist pockets. South facing slope is mainly Ethnomedicinal Use and Distribution of Aconitum naviculare (Brühl) Stapf. 173 dominated by dry alpine scrub with dwarf and prostate junipers (Juniperus indica, J. squamata), Rhododendron lepidotum, Rosa species, Berberis species, Caragana species, Ephedra gerardiana, etc. Among the herbs Cremanthodium species, Swertia species. Gentiana species, Aconitum naviculare, etc are common. From reconnaissance study the following sites were selected for the present study: Ice Lake area (Braga Village Development Committee (VDC) area), Manang Hill (Manang VDC area), upper Khangsar (Khangsar VDC area), Ledar (Tanki Manang VDC area) and near Thorong Phedi (Tanki Manang VDC area). First three sites were near human settlement (about two hours walk) whereas in Ledar and Thorong Phedi there was no permanent human settlement except a few hotels for tourists (Figure 12.1). Livestock grazing was common at all sites but grazing pressure appears to be high in Ice Lake area, Manang hill and upper Khangsar. 12.2.2 Species characters. Aconitum naviculare (Brühl) Stapf. (Local name: ponkar/bongkar (Gurung), Fam. Ranunculaceae, (Figure 12.2) is a biennial herb with tuberous white root; leaves mostly basal, palmately divided into 3- 5 segments, upper leaves sessile; flowers stalked; sepals whitish or violet, flushed blue purple with prominent darker veins, uppermost sepal boat shaped; flowering in September-October (Stainton 1997). It is endemic to Himalayas and found from west Nepal to Bhutan. Aconitum naviculare has been widely used in Tibetan medicine (Gao et al. 2004) as well as for traditional health care in Manang. 12.2.3 Field data collection and analysis. Ethnomedicinal Uses. Ten local residents including shepherds (four), farmers (four), herbal medicine practitioner (one) and staff of tourist information center (one) were interviewed individually. They were asked about medicinal use of Aconitum naviculare, parts used, time and mode of collection, storage, dose, relative effectiveness and local distribution. Distribution and Population. We sampled a total of 240 sub-plots (1 m x 1 m) lying within thirty large plots (10 m x 10 m) during September 2005. Each large plot was divided into four quarters (5 m x 5 m) and two sub-plots (1 m x 1 m) were studied in each quarter. In each sub-plot individuals of Aconitum naviculare of all age groups were counted to determine density (pl/m2). Major associated species were recorded. Due to patchy distribution of Aconitum naviculare sampling plots were manually located at those spots where the species was more common and covered an area >100 m2. Thus calculated density should be considered as the maximum density in its habitat and the data should not be extrapolated to the entire study area. The sampling plots were also examined for trampling damage and browsing. Due to unequal population size the number of plots studied was not equal; three large plots were sampled at Ice Lake, five at each of Manang and Khangsar, 174 Bharat B. Shrestha et al.

Figure 12.1 Ledar of Tanki Manang village development committee area. It has the largest population of Aconitum naviculare in upper Manang. Inset - a flowering Aconitum naviculare [Photograph taken in late September].

Figure 12.2 Decoction of air-dried aerial parts of Aconitum naviculare prepared for immediate use by a shepherd at Ledar, Tanki Manang. Ethnomedicinal Use and Distribution of Aconitum naviculare (Brühl) Stapf. 175 and seventeen at Ledar. Thorong Phedi area was not sampled because there had been heavy collection of this plant before we reached there. Analysis of variance (ANOVA, SPSS 2002, Version 11.5) was done to test the significance of the difference in density among the sites.

12.3 Results and Discussion 12.3.1 Ethnomedicinal use. Aconitum naviculare is an important medicinal plant which has been used for health care of mountain people in trans- Himalayan region. It has been used against fever, headache, jaundice, high blood pressure and cold. Although the whole plant including the tuberous root is medicinally important, Manangis collect only aerial parts (stem, leaves and flowers) during peak flowering season (September – October) (Figure 12.2). In absence of flowers Aconitum naviculare is often confused with associated species of Geranium and Delphinium. Therefore, people prefer to collect it when it is flowering. Though tuberous root is equally, perhaps more important than the aerial parts, people generally did not collect it. Most of the collectors were aware of the negative impact of collecting underground tuber to regeneration of plant in the following years. The collected plant material is air dried in shade by hanging inside the house. In some households, the dried plant material is crushed and stored in airtight bottles. During administration, about one gram of dried plant material is boiled with two glasses of water for about half an hour and the black, bitter decoction (Figure 12.2) is drunk twice a day. According to the respondents the bitter decoction makes the patient weak, probably due to presence of antibiotics in the plant material. To restore vigor the decoction is often taken along with ghee (butter). Shepherds were the main collectors of this plant. They brought the plant down to the village and shared with relatives and privileged people in the society. In Ledar, Buddhist monks also collected Aconitum naviculare to use as medicine in Gumba (Bhuddhist shrine). The collection pressure depended on the demand in the villages of Manang and among Manangis in Kathmandu. A shepherd in Thorong Phedi collected about 3.5 kg (air dry) Aconitum naviculare during September 2006 and sent to Manang village. Most of the households in Manang generally used 200- 500 g/household (air dry mass) in a year. Although Manangis living in and outside Manang widely use Aconitum naviculare, it has not been commercially marketed from Manang. Generally Manangis used Aconitum naviculare, Neopicrorhiza scrophulariiflora and Swertia species separately for cold, fever and headache. According to Manangis Aconitum naviculare is the most effective of these three medicinal plants. Although it is widely used in Tibetan folk medicine (Gao et al. 2004), ethnomedicinal use of Aconitum naviculare in Nepal has not been mentioned in either ‘Useful Plants of Manang District’ (Pohle 1990) or in ‘Plants and People of Nepal’ (Manandhar 2002); the latter being considered most inclusive contribution to 176 Bharat B. Shrestha et al. ethnobotany of Nepal. However, this species has been listed in Medicinal and Aromatic Plant Database of Nepal (MAPDON) (Shrestha et al. 2000) and A Compendium of Medicinal Plants in Nepal (Baral and Kurmi 2006), but the details of its use have not been mentioned. Recently, Bhattarai et al. (2006) reported ethnomedicinal use of Aconitum naviculare along with 91 other plant species from Manang. In Dolpa, which has similar climatic condition as Manang, this plant has been used against poisoning and fever (Lama et al. 2001). 12.3.2 Distribution and Population density. Aconitum naviculare is mainly found on dry, south-west facing slope (average aspect 228° SW) of the valley in Braga (Ice Lake area), Manang (Manang hill), Khangsar (upper Khangsar) and Tanki Manang (Gunsang, Yak Kharka, Ledar and Thorong Phedi) Village Development Committee (VDC) area between 4000 and 4700 m a.s.l. Thorong Phedi lies on south-east facing slope (average aspect: 105o ES) where a small population of Aconitum naviculare was found between 4350 and 4450 m a.s.l. Total area of Aconitum naviculare habitat in upper Manang was estimated to be 40 ha. Aconitum naviculare has also been reported from Dolpa district (Dho-Tarap, 4200 m a.s.l.) where it is found on open rocky dry slopes (Shrestha 2004). Aconitum naviculare mostly grows along with thicket forming alpine scrubs such as species of Juniperus, Caragana, Astragalus, Berberis, Spiraea, Ephedra, etc. These thicket forming plants are thorny and protect Aconitum naviculare from grazing and trampling damage by animals and also collection by humans. It may also form a suitable micro-habitat with high soil moisture content. Population density of Aconitum naviculare was not uniform and there was significant difference (ANOVA, F3, 236 = 10.6, p <0.001) among the sites. In Ice Lake area, Manang hill and upper Khangsar Aconitum naviculare became a scarce species. In Ice Lake area a small population was confined to a small patch (ca 2.5 ha) but population density was relatively high (Table 12.1). Here the plant has been growing along with spiny Caragana species which has protected the plant from animal trampling and grazing. In Manang hill and upper Khangsar Aconitum naviculare was growing in a relatively large area but it was quite sparse with low population density. The density was lowest in Manang which was near two villages (Manang and Tanki Manang) with comparatively high human population density and high collection pressure. Ledar had the largest population of Aconitum naviculare in the valley and distributed in a large area (ca 55 ha). Both population density and above ground biomass were highest in Ledar (Table 12.1).

Ethnomedicinal Use and Distribution of Aconitum naviculare (Brühl) Stapf. 177

Table. 12.1 Physical location of study sites and mean population density of Aconitum navicualare. Sites Elevation range Average slope Average Aspect Density + SD (m a.s.l.) (plants/m2) Ice Lake 4400-4550 32° 193° SW 8.66 + 3.37 Manang 4150-4450 30° 237° SW 4.07 + 2.34 Khangsar 4400-4650 29° 208° SW 6.42 + 6.35 Ledar 4200-4500 30° 236° SW 8.34 + 4.47 Thorong Phedi 4350-4450 38° 105° NE - Mean - - - 7.34 + 4.78

Population density of Aconitum naviculare (average 7.34 plant/m2), reported in this study is higher than the density reported for other Aconitum species such as A. orochryseum from Gyasumdo valley of Manang (4 plants/m2, Ghimire et al. 1999), and A. balfourii and A. heterophyllum from Garhwal Himalayas, India (1.87 and 1.48 plants/m2 respectively, Nautiyal et al. 2002). Low population density (0.33 to 3.3 plants/m2) of four Aconitum species (A. balfourii, A. heterophyllum, A. rotundifolium and A. violaceum) was also reported from protected areas of three northwestern states of Indian Himalayan region (Kala 2005). However, these differences in population density of different Aconitum species may be due to differences in sampling, habitat, human pressure, growth strategy and conditions. Above mentioned studies had employed random sampling but in the present study the sampling plots were laid manually where density of the plant was seemingly high. Thus the density reported in the present study should be considered as the maximum value for Aconitum naviculare in Manang. Using similar sampling method Kala (2000) reported that the population density of two rare species of Aconitum (A. rotundifolium and A violaceum) in Indian trans-Himalaya was found to range from 2.0 to 7.7 plants/m2. It is thus apparent that most species of Aconitum have restricted distribution to specific pocket habitat and their population density is low in their natural habitat. Previous data on distribution and population density of Aconitum naviculare is not available. Lack of population details including harvesting trend and impact assessments are the major research gaps of most Himalayan medicinal plants, except a few high profile taxa (Dhar et al. 2000). According to local collectors Aconitum naviculare was a common herb in the past in and around their pasture lands but these days its population has been declining. Rare occurrence with restricted distribution was also reported from Dolpa (Lama et al. 2001). Small and declining population in Ice Lake area, Manang hill and upper Khangsar appears to be related with factors like grazing and early collection of aerial plant parts for medicinal use. These areas are close to human settlements and have several pasturelands (Kharka). Aconitum naviculare is not a preferred species of livestock though it is browsed incidentally by goat, sheep and blue sheep. 178 Bharat B. Shrestha et al.

Heavy trampling and to some extent browsing, damage the plant in its’ vegetative stage which could suppress flowering and fruit production. Cattle grazing have been reported as highly destructive activity in the natural habitats of high altitude medicinal plants such as species of Aconitum, Nardostachys and Dactylorhiza in Darjeeling Himalayas (Chhetri et al. 2005). Trampling may also reduce seed germination and seedling survival as also reported for wild population of Meconopsis species, an alpine medicinal herb growing in Chinese Himalayan region (Xie et al. 2005). Collection of Aconitum naviculare at flowering stage prevents seed production and negatively affects regeneration. Collection of whole plant or parts of medicinal plant before seed set has been reported as a major constraint for regeneration of high altitude medicinal plants including Aconitum species in Gyasumdo valley of Manang (Ghimire et al. 1999) and in other parts of Himalayas such as in Sikkim (Rai et al. 2000). This mode of collection might have reduced population size and population density of Aconitum naviculare in Upper Manang. Rarity of medicinal plants even in absence of commercial exploitation was also reported from Spiti of Indian trans- Himalayan region (Kala 2000). Therefore collection of aerial parts before seed set and damage due to trampling by livestock appear to be the main causes of population decline of Aconitum naviculare in Upper Manang. Although Aconitum navicvulare was widely used in Tibetan medicine and by local communities in Manang, this is not a trading item from Nepal, probably due to low yield. Above ground biomass (oven dry mass: 5 g/m2; BB Shrestha, unpublished data) was relatively low. Although chemical constituents of plants of the genus Aconitum have been extensively studied (Pelletier and Mody 1981, Pelletier et al. 1983) only a few references are available regarding the phytochemistry of Aconitum naviculare (e.g. Gao et al. 2004, Shrestha et al. 2006). Since most of the research and conservation efforts have been focused on commercially exploited medicinal plants, medicinally effective species such as Aconitum naviculare has got no priority in this regard. This species is not currently in trade but this can be a potential source of bioactive natural products and necessary measures need to be taken to conserve the species.

12.4 Conclusion Aconitum naviculare is considered to be one of the most effective medicinal plants used by Manangis. The species is found mostly on dry, south facing slope between 4000 m and 4700 m a.s.l. Although Aconitum naviculare has not been collected for commercial purpose, population density of this plant appears to be declining mainly due to collection of plant before seed set and damage by livestock trampling. Conservation management should focus not only on the species in trade but also the other species such as Aconitum naviculare which are not currently in trade but are Ethnomedicinal Use and Distribution of Aconitum naviculare (Brühl) Stapf. 179 considered highly effective in ethnomedicine and can be a potential source of natural product for modern medicine. Distribution and population status of Aconitum naviculare as reported in this paper can serve as reference for population dynamics study in the future and for similar kind of work in other habitats such as in Mustang and Dolpa.

Acknowledgements Financial support for this research from VW Foundation (Germany) is thankfully acknowledged. We are thankful to Annapurna Conservation Area Project for permission to work in Manang. We thankfully acknowledge the help from Prof. RP Chaudhary, Prof. HD Lekhak and Prof. VNP Gupta, Central Department of Botany, Tribhuvan University, Shandesh Bhattarai (Ph.D. scholar) and RK Sharma during field study. Karma Gurung, Pemba Sherpa and Aite Pariyar of Manang provided information about distribution of the plant and accompanied BBS during field works. BBS is thankful to Karma and Chhiring dai of Tilicho Hotel at Manang village and all the respondents for their valuable information during the field work. We are also thankful to Dr. SK Ghimire (CDB, TU) for valuable comments on the manuscript.

References Anonymous. 1998. Executive Summary Report of BCPP CAMP on Selected Medicinal Plants of Northern, North-eastern and Central India. Forest Department of Uttar Pradesh, Lucknow, India. Baral, S.R. and Kurmi, P.P. 2006. A Compendium of Medicinal Plants in Nepal. R. Sharma, Kathmandu. Bhattarai, S., Chaudhary, R.P. and Taylor, R.S.L. 2006. Ethnomedicinal plants used by the people of Manang district, central Nepal. Journal of Ethnobiology and Ethnomedicine, 2:41, doi: 10.1186/1746-4269-2-41. Chhetri, D.R., Basnet, D., Chiu, P.F., Kalikotay, S., Chhetri, G. and Parajuli, S. 2005. Current status of ethnomedicinal plants in the Darjeeling Himalaya. Current Science, 89: 264-268. Dhar, U., Rawal, R.S. and Upreti, J. 2000. Setting priorities for conservation of medicinal plants: A case study in the Indian Himalaya. Biological Conservation, 95: 57-65. DHM. 2000. Preliminary Monthly Weather Summary of Nepal. Department of Hydrology and Meteorology (DHM), Kathmandu. Elvin-Lewis, M. 2001. Should we be concerned about herbal remedies. Journal of Ethnopharmacology, 75: 141 164. Gahire, S. 2003. Ecology, distribution and trade of kutki [Neopicrorhiza schrophulariiflora (Pennell) Hong.] in Manang district, Nepal: A case study of Chame and Tachi Bagarchhap Village Development Committees (VDCs). Central Department of Botany, Tribhuvan University, Kathmandu (M. Sc. Thesis). Gao, L., Wei, X. and Yang, L. 2004. A new diterpenoid alkaloid from a Tibetan medicinal herb Aconitum naviculare Stapf. Journal of Chemical Research, Number 4, pp. 307-308. 180 Bharat B. Shrestha et al.

Ghimire, S.K., Sah, J.P., Shrestha, K.K. and Bajracharya, D. 1999. Ecological study of some high altitude medicinal and aromatic plants in the Gyasumdo valley, Manang, Nepal. Ecoprint, 6: 17-25. Hamilton, A. 2003. Medicinal Plants and Conservation: Issues and Approaches. International Plant Conservation Unit, WWF-UK, Surrey, UK. Kala, C.P. 2000. Status and conservation of rare and endangered medicinal plants in the Indian trans-Himalaya. Biological Conservation, 93: 371-379. Kala, C.P. 2005. Indigenous uses, population density and conservation of threatened medicinal plants in protected areas of Indian Himalayas. Conservation Biology, 19: 368-378. Lama, Y.C., Ghimire, S.K. and Aumeeruddy-Thomas, Y. 2001. Medicinal Plants of Dolpo: Amchis' Knowledge and Conservation. WWF Nepal Program, Kathmandu. Manandhar, N.P. 2002. Plants and People of Nepal. Timber Press Inc. Portland, Oregon, USA. Nautiyal, B.P., Prakash, V., Bahuguna, R., Maithani, U., Bisht, H. and Nautiyal, M.C. 2002. Population study for monitoring the status of rarity of three Aconite species in Garhwal Himalayas. Tropical Ecology, 43: 297-303. Olsen, C.S. and Larsen, H.O. 2003. Alpine medicinal plant trade and Himalayan mountain livelihood strategies. The Geographical Journal, 169: 243-254. Pelletier, S.W. and Mody, N.V. 1981. Diterpenoid alkaloids. In: The Alkaloids (Ed.: R.H.F. Manske), vol. 17, pp. 1-103. Academic Press, New York. Pelletier, S.W., Mody, N.V., Joshi, B.S. and Schramm, L.C. 1983. 13C and proton NMR shift assignments and physical constants of C19- diterpenoid alkaloids. In: Alkaloids: Chemical and Biological Perspectives (Ed.: S.W. Pelletier), Vol. 2, pp. 206-462. John Wiley Sons, New York. Pohle, P. 1990. Useful Plants of Manang Districts: A Contribution to the Ethnobotany of the Nepal Himalayas. Nepal Research Center Publication No 16, Franz Steiner Verlag Wiesbaden GmbH, Stuttgart, Germany. Pokharel, P. 2004. Contribution to the Flora of Nyeshang, Upper Manang, Central Nepal (Gymnospermae and Dicotyledonae). Central Department of Botany, Tribhuvan University, Kathmandu (M. Sc. Thesis). Press, J.R., Shrestha, K.K. and Sutton, D.A. 2000. Annotated Checklist of the Flowering Plants of Nepal. The Natural History Museum, London. Rai, L.K., Prasad, P. and Sharma, E. 2000. Conservation threat to some important medicinal plants of the Sikkim Himalaya. Biological Conservation, 93: 27-33. Shrestha, M.R. 2004. Trans-Himalayan Dicot Flora of northwest Nepal: Dolpa and its surroundings. Central Department of Botany, Tribhuvan University. Katmandu (M. Sc. Thesis). Shrestha, B.B., Dall’Acqua, S., Gewali, M.B., Jha, P.K. and Innocenti, G. 2006. New flavonoid glycosides from Aconitum naviculare, a medicinal herb from the trans-Himalayan region of Nepal. Carbohydrate Research, 341: 2161-2165. Shrestha, K.K., Tiwari, N.N. and Ghimire, S.K. 2000. MAPDON - Medicinal and Aromatic Plant Database of Nepal. In: The Himalayan Plants, Can they save us? (Eds:. T. Watanabe, A. Takano, M.S. Bista, and H.K. Saiju), pp. 53-74. Proceedings of Nepal – Japan joint symposium on conservation and utilization of Himalayan medicinal resources. November 6 – 11, 2000, Kathmandu, Nepal. Ethnomedicinal Use and Distribution of Aconitum naviculare (Brühl) Stapf. 181

Stainton, A. 1997. Flowers of the Himalayas: A supplement. Oxford University Press, Delhi. Thomas, Y., Karki, M., Gurung, K. and Parajuli, D. (Eds.). 2005. Himalayan Medicinal and Aromatic Plants, Balancing Use and Conservation. Proceedings of the Regional Workshop on Wise Practice and Experiential Learning in Conservation and Management of Himalayan Medicinal Plants, December 15- 20, 2002, Kathmandu, Nepal, 533p. Watanabe, T., Takano, A., Bista, M.S. and Saiju, H.K. (Eds.). 2000. The Himalayan Plants, Can They Save Us? Proceedings of Nepal – Japan joint Symposium on Conservation and Utilization of Himalayan Medicinal Resources, November 6 – 11, 2000, Kathmandu, Nepal, 358p. Xie, H., Fang, J. and Zhendong. 2005. Conservation biology of Himalayan poppies (Meconopsis species) in North-west Yunnan, China: in situ and ex situ cultivation. In: Himalayan Medicinal and Aromatic Plants, Balancing Use and Conservation (Eds.: Y. Thomas, M. Karki, K. Gurung, and D. Parajuli), pp. 328- 350. Proceedings of the Regional Workshop on Wise Practice and Experimental Learning in Conservation and Management of Himalayan Medicinal Plants, December 15-20, 2002, Kathmandu, Nepal.

This Issue is Dedicated to Professor Franco F. Vincieri on the Occasion of his 70th Birthday

Volume 3. Issue 12. Pages 1941-2166. 2008 ISSN 1934-578X (printed); ISSN 1555-9475 (online) www.naturalproduct.us NPC Natural Product Communications

EDITOR-IN-CHIEF

DR. PAWAN K AGRAWAL ADVISORY BOARD Natural Product Inc. 7963, Anderson Park Lane, Prof. Viqar Uddin Ahmad Prof. Francisco Macias Westerville, Ohio 43081, USA Karachi, Pakistan Cadiz, Spain [email protected] Prof. Øyvind M. Andersen Prof. Anita Marsaioli Bergen, Norway Campinas, Brazil EDITORS Prof. Giovanni Appendino Prof. Imre Mathe PROFESSOR GERALD BLUNDEN Novara, Italy Szeged, Hungary The School of Pharmacy & Biomedical Sciences, University of Portsmouth, Prof. Yoshinori Asakawa Prof. Joseph Michael Portsmouth, PO1 2DT U.K. Tokushima, Japan Johannesburg, South Africa [email protected] Prof. Maurizio Bruno Prof. Ermino Murano

PROFESSOR ALESSANDRA BRACA Palermo, Italy Trieste, Italy

Dipartimento di Chimica Bioorganicae Biofarmacia, Prof. Carlos Cerda-Garcia-Rojas Prof. Virinder Parmar Universita di Pisa, Mexico city, Mexico Delhi, India via Bonanno 33, 56126 Pisa, Italy [email protected] Prof. Josep Coll Prof. Luc Pieters Barcelona, Spain Antwerp, Belgium PROFESSOR DEAN GUO State Key Laboratory of Natural and Biomimetic Drugs, Prof. Geoffrey Cordell Prof. Om Prakash Chicago, IL, USA Manhattan, KS, USA School of Pharmaceutical Sciences, Peking University, Prof. Samuel Danishefsky Prof. Peter Proksch Beijing 100083, China New York, NY, USA Düsseldorf, Germany [email protected] Dr. Biswanath Das Prof. William Reynolds

Hyderabad, India Toronto, Canada PROFESSOR J. ALBERTO MARCO Departamento de Quimica Organica, Prof. A.A. Leslie Gunatilaka Prof. Raffaele Riccio Universidade de Valencia, Tucson, AZ, USA Salerno, Italy E-46100 Burjassot, Valencia, Spain Prof. Stephen Hanessian [email protected] Prof. Ricardo Riguera Montreal, Canada Santiago de Compostela, Spain

PROFESSOR YOSHIHIRO MIMAKI Prof. Michael Heinrich School of Pharmacy, Prof. Satyajit Sarker London, UK Coleraine, UK Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan Prof. Kurt Hostettmann Prof. Monique Simmonds [email protected] Lausanne, Switzerland Richmond, UK

PROFESSOR STEPHEN G. PYNE Prof. Martin A. Iglesias Arteaga Prof. Valentin Stonik Department of Chemistry Mexico, D. F, Mexico Vladivostok, Russia University of Wollongong Prof. Jerzy Jaroszewski Prof. Hermann Stuppner Wollongong, New South Wales, 2522, Australia Copenhagen, Denmark Innsbruck, Austria [email protected] Prof. Teodoro Kaufman Prof. Apichart Suksamrarn PROFESSOR MANFRED G. REINECKE Rosario, Argentina Bangkock, Thailand Department of Chemistry, Texas Christian University, Prof. Norbert De Kimpe Prof. Hiromitsu Takayama Forts Worth, TX 76129, USA Gent, Belgium Chiba, Japan [email protected] Prof. Hartmut Laatsch Prof. Karin Valant-Vetschera PROFESSOR WILLIAM N. SETZER Gottingen, Germany Vienna, Austria

Department of Chemistry Prof. Marie Lacaille-Dubois Prof. Peter G. Waterman The University of Alabama in Huntsville Dijon, France Lismore, Australia Huntsville, AL 35809, USA [email protected] Prof. Shoei-Sheng Lee Prof. Paul Wender Taipei, Taiwan Stanford, USA

PROFESSOR YASUHIRO TEZUKA Institute of Natural Medicine Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan [email protected]

INFORMATION FOR AUTHORS

Full details of how to submit a manuscript for publication in Natural Product Communications are given in Information for Authors on our Web site http://www.naturalproduct.us.

Authors may reproduce/republish portions of their published contribution without seeking permission from NPC, provided that any such republication is accompanied by an acknowledgment (original citation)-Reproduced by permission of Natural Product Communications. Any unauthorized reproduction, transmission or storage may result in either civil or criminal liability.

The publication of each of the articles contained herein is protected by copyright. Except as allowed under national “fair use” laws, copying is not permitted by any means or for any purpose, such as for distribution to any third party (whether by sale, loan, gift, or otherwise); as agent (express or implied) of any third party; for purposes of advertising or promotion; or to create collective or derivative works. Such permission requests, or other inquiries, should be addressed to the Natural Product Inc. (NPI). A photocopy license is available from the NPI for institutional subscribers that need to make multiple copies of single articles for internal study or research purposes.

To Subscribe: Natural Product Communications is a journal published monthly. 2008 subscription price: US$1,395 (Print, ISSN# 1934-578X); US$1,095 (Web edition, ISSN# 1555-9475); US$1,795 (Print + single site online). Orders should be addressed to Subscription Department, Natural Product Communications, Natural Product Inc., 7963 Anderson Park Lane, Westerville, Ohio 43081, USA. Subscriptions are renewed on an annual basis. Claims for nonreceipt of issues will be honored if made within three months of publication of the issue. All issues are dispatched by airmail throughout the world, excluding the USA and Canada. 2008 NPC Natural Product Communications Vol. 3

No. 12 Diterpenoid Alkaloids and Phenol Glycosides from 1985 - 1989 Aconitum naviculare (Brühl) Stapf.

Stefano Dall’Acquaa*, Bharat B. Shresthab , Mohan Bikram Gewalic, Pramod Kumar Jhab , Maria Carrarad and Gabbriella Innocenti a aDepartment of Pharmaceutical Sciences, University of Padova, Padova, Italy bCentral Department of Botany, Tribhuvan University, Kirtipur, Kathmandu, Nepal cCentral Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal dDepartment of Pharmacology and Anesthesiology, University of Padova, Padova, Italy [email protected]

Received: June 20th, 2008; Accepted: October 28th, 2008

Phytochemical investigation of the aerial parts of Aconitum naviculare, a medicinal plant used in traditional Nepalese medicine, led to the isolation and characterization of two new diterpenoid alkaloids, navirine B (1), and navirine C (2), along with (+) chellespontine (3), kaempferol-7-O-β-D-glucopyranosyl(1→3)α-L-rhamnopyranoside (4), kaempferol-7-O α-L- rhamnopyranoside,3-O-β-D-glucopyranoside (5), p-coumaric-4-O-β-D-glucopyranoside acid (6), and ferulic-4-O-β-D- glucopyranoside acid (7). The structures of the isolated compounds were elucidated on the basis of extensive analyses of 1D and 2D NMR spectra (HMQC, HMBC, COSY, ROESY) and HR-MS data. The antiproliferative activity of alkaloids 1-3 against human tumor cell lines (LoVo and 2008) was also evaluated.

Keywords: Aconitum naviculare, diterpenoid alkaloids, antiproliferative activity, traditional medicine.

Aconitum species are well-known for their contents potential source of income for mountain people. of C19 and C20 diterpenoid alkaloids. Some of these Little information is available regarding the (e.g., aconitine) are highly toxic and some exhibit phytochemical composition of A. naviculare. Gao et powerful biological activities, for example al. [4] reported the isolation of a new diterpenoid antiarrhythmic [1,2], analgesic [2], antiinflammatory alkaloid and some atisine-like alkaloids. We recently [2], antiepileptic [2] and antiproliferative [3], making reported three novel glycosylated flavonoids from them potential new pharmaceutical entities. Aconitum this plant [6]. Here we report the isolation and naviculare (Brühl) Stapf (Ranunculaceae) is a characterization of two new diterpenoid alkaloids, biennial medicinal herb of Alpine grassland (>4000 navirine B (1) and navirine C (2), and five known m a.s.l.) found in the trans-Himalayan region of compounds (3-7) from A. naviculare. Nepal, i.e., the Manang, Mustang and Dolpa districts. The aerial parts are used in Nepalese and Tibetan folk The isolated diterpenoid alkaloids 1-3 were also medicine against cold, fever and headache, as well as studied for their ability to affect tumor cell for sedative and analgesic remedies [4,5]. In the proliferation. In particular, antiproliferative activity Manang region, the aerial parts are collected during against ovarian (2008 cells) and colon (LoVo cells) flowering. The local inhabitants usually dry the adenocarcinoma was tested. whole plant and prepare a bitter decoction, which is used for various medicinal purposes. Although Compound 1 had a molecular formula of C30H40N2O3 Manangis living in and outside Manang commonly on the basis of the protonated molecular ion [M+H]+ use A. naviculare, it has not yet become an item of displayed at m/z 477.3122 in the HRAPITOFMS. The trade. Due to its supposed high effectiveness in IR spectrum showed absorption bands supporting the traditional healthcare, A. naviculare may become a presence of an imino group (1670 cm-1). 1986 Natural Product Communications Vol. 3 (12) 2008 Dall’Acqua et al.

13 13 13 12 17 12 17 12 17 11 20 11 11 O O 16 16 16 1 14 OH 1' 14 1' 2' 9 2' 9 9 14 15 15 2 15 1 6' 10 8 OH 1 6' 2 10 8 22 N H 2 10 8 H 3' H 5 H 3' 3 5 3 4 7 3 5 4 7 5' N 4 7 5' 4' N 4' 6 6 O H OH 6 1 H 2 7' 19 18 3 19 7' 19 18 18 ' 8' 8 N 9' N 9' 9 9' '

Figure 1: Structure of isolated compounds 1-3.

1 The H NMR spectrum showed signals ascribable to with the carbon at δC 52.5 (C-8’) suggested the one methyl group (δ 1.05 s, 3H), an N,N-dimethyl presence of one hordenine moiety in the molecule. group (δ 2.40 s, 6H), and one olefinic proton (δ 5.67 brs, 1H). Two ortho coupled doublets (J = 8.0) in the NOESY data and comparisons with the spectral aromatic region (δ 6.85 and 7.12, 2H each) also data of Gao et al. [4] for navirine evidenced the indicated the presence of an o-p disubstituted relative stereochemistry of the molecule, supporting aromatic ring. The structure of compound 1, a an α orientation for groups at positions 10, 8 and 12. diterpenoid alkaloid, was obtained by exhaustive NOESY cross-peaks were also obtained between the analysis of COSY, HMQC and HMBC data. Various exchangeable proton signal at δ 3.48 (OH-5) and H-9 spin systems were detected when COSY and HMQC (δ 1.64), which were assigned as β on the basis of data were compared, establishing the connectivity previous references [4,7], suggesting a β orientation between CH2 in positions 1,2,3 and 6,7 and between for the hydroxy group. With all this evidence, positions 9,11,12,13. Further connectivity between compound 1 was established as a new alkaloid, called the highly deshielded CH-19 (δH 7.43 s, 1H; δC navirine B (5β-hydroxy navirine). 169.9) and CH-20 (δH 3.57 brs, 1H; δC 80.6) and also between the olefinic CH-15 (δH 5.67 brs, 1H; δC The HRAPITOFMS of compound 2 displayed a + 130.8) and CH2-17 (δH 4.55 brs, 1H; δC 68.8) were protonated molecular ion [M+H] at m/z 461.3532, seen in the COSY spectrum. Diagnostic long-range corresponding to a molecular formula of C31H45N2O. (HMBC) correlations were observed from the The 1H NMR spectrum of compound 2 was quite methyl group 18 and carbon resonances at δ 30.9 similar to that of compound 1, but there were some (C-3), 44.9 (C-4), 72.6 (C-5) and 169.9 (C-19). differences. It lacked the signal for the imino proton, HMBC correlations, observed from the proton signal and the proton signal of H-20 was shifted downfield of CH-20 (δ 3.57) with carbon resonances C-19 and to δ 2.53 (δ 3.57 for 1). In addition, a singlet signal C-5, supported the presence of a six-member ring integrating for nine protons was observed at δ 2.50, containing an imino group. Diagnostic long-range supporting the presence of three nitrogen linked correlations were observed from the same proton methyl groups. Extensive analyses of COSY, HMQC signal (H-20) with C-1 (δ 27.9), C-9 (47.1), C-5 and HMBC data revealed a similar diterpenoid (72.6) and C-8 (43.8). HMBC correlations of skeleton, as well as the same hordenine moiety as the proton at δ 5.67 (H-15) with C-9 (δ 47.1), C-12 found in compound 1. In the HMBC spectrum, (δ 31.7) and C-17 (68.8) were also seen. These long-range correlations were observed between the observations, as well as analysis of COSY and methyl group at δ 1.00 (CH3-18) and carbon HMQC data, suggested two more hexa-atomic rings resonances at δ 34.1 (C-3), 41.7 (C-4), 53.5 (C-5) and in the compound. Long-range correlations observed 58.0 (C-19), which supported the presence of amino- from proton signals at δ 1.91-1.56 (CH2-13) with linked CH2 at position 19 (δH 2.30; δC 58.0), as well carbon resonances at δ 146.8 (C-16) and 80.6 (C-20) as a CH group at position 5 (δH 1.26; δC 53.5). The β supported the linkage between positions 20 and 14. configuration for H-5 was established on the basis of Long-range correlations observed from the aromatic the NOESY correlation observed from the methyl doublet δH 7.12 (H-3’ 5’) with the carbon at δC 33.3 group at δ 1.00 and the proton signal at δ 1.26 (H-5). (C-7’) and from the methyl group at δH 2.59 (CH3-9’) Complete analyses of 1D and 2D NMR data afforded a novel structure, called navirine C.

Diterpenoid alkaloids and glycosides from Aconitum naviculare Natural Product Communications Vol. 3 (12) 2008 1987

Compound 3 showed a protonated molecular ion whereas, in 2008 cells, only compound 1 induced a [M+H]+ at m/z 344.2590, establishing its molecular marked inhibition of cell growth, as shown in Table 1 1 formula as C22H33NO2. The H NMR spectrum of 3 as IC50 values. displayed one aldehydic proton signal (8.71 s, 1H), one exocyclic methylene (δ 5.02 d, 5.12 d, 1H each; Our study on the phytochemical composition of A. J = 1.0 Hz), one methyne signal linked to an naviculare may be useful in improving scientific electronegative atom (δ 3.70 m, 1H), and one methyl knowledge of this Nepalese medicinal plant. group (δ 1.06 s, 3H). Its COSY, HMQC and HMBC data revealed a diterpenoid skeleton. Compound 3 Experimental possessed a hydroxyl group at position 15, as Optical rotations were measured on a Jasco P2000 supported by the HMBC correlation between H-17 digital polarimeter, IR spectra on a Perkin Elmer (δ 5.05 and 5.12) and the carbon resonance at δ 74.7 1600 FT-IR spectrometer, and NMR spectra, in (C-15). The β orientation of the OH group at position CDCl or CD OD, on a Bruker AMX-300 15 was deduced on the basis of NOESY cross-peaks 3 3 spectrometer, operating at 300.13 MHz for 1H NMR observed between H-15 and H-13 and H-14. The and 75.03 MHz for 13C NMR. 2D experiments, 1H-1H proton and carbon resonances at position 15 were DQF-COSY, and inverse-detected 1H-13C HMQC at δ 3.70 and δ 74.7 respectively, due to the β-OH H C and HMBC spectra were performed with UXNMR group. The H 9 configuration in compound 3 was software. established by observing the NOESY correlations from the methyl group 18 (δ 1.06) and H-5 (δ 1.38), HRMS were obtained on an API-TOF spectrometer and from this latter and the H-9 signal (δ 2.19). On (Mariner Biosystems). Samples were diluted in a the basis of the spectral data, compound 3 was mixture of H O-AcCN (1:1), with 0.1% formic acid established as chellespontine [8]. 2 for positive ion mode, and directly injected at a flow

rate of 10 µL/min. Sephadex LH 20 and silica gel 60 To our knowledge alkaloid 3, flavonol glycosides 4 were used for column chromatography. Silica gel and 5, and phenylpropanoid glycosides 6 and 7 have plates were used for preparative and analytical TLC been isolated and characterized from A. naviculare (Merck cat. 5717 and 5715). Compounds on TLC for the first time. were detected with a UV lamp (254 nm) and by

treating plates with Dragendorff’s reagent. Recently some Aconitum alkaloids have been evaluated for their antiproliferative activity against Semi-preparative HPLC was performed on a Gilson A172 malignant cells [3]. For this reason, we decided series 305 liquid chromatograph equipped with a to study this activity for the isolated diterpenoid LiChrosphere 100 RP-18 column (particle size 10 alkaloids 1-3. μm, 250 x 10 mm ID, E. Merck). Table 1: Antiproliferative activity of compounds 1-3. LoVo cellsa 2008 cellsa Plant material: Aerial parts, including stems, leaves, Compound IC50 (confidence limits) μM IC50 (confidence limits) μM flowers and immature fruits of Aconitum naviculare 1 22 (19-25) 33 (30-37) were collected from Ladtar (4100 m a.s.l., upper 2 nd nd Manang) during the last week of September 2004. A 3 38 (33-41) nd voucher specimen was collected during a field survey Cisplatinb 33.3 (28.7-36.1) 13.8 (11.5-!6.5) and deposited at Tribhuvan University Central aLoVo: colon cell line; 2008: ovarian cell line. Herbarium (TUCH; n° ANV904); identification was bCisplatin was used as a reference compound. confirmed by Prof. Ram Prasad Chaudhary of the nd: IC50 is not determined. Central Department of Botany, Tribhuvan University, Kathmandu. Compounds 1 and 3 showed a significant antiproliferative activity, whereas compound 2 was Extraction and isolation: Air-dried powdered aerial inactive. The capacity of the compounds to affect parts (100 g) were exhaustively extracted in a Soxhlet tumor cell growth revealed a dose-dependent effect. apparatus with MeOH. The solvent was evaporated In particular, colon cell line LoVo was more sensitive under reduced pressure (230–250 mbar) and a than ovarian cells 2008. In fact, compounds 1 and 3 semisolid MeOH extract was obtained (8 g). About were able to decrease cell proliferation in LoVo cells, 1988 Natural Product Communications Vol. 3 (12) 2008 Dall’Acqua et al.

5.0 g of this extract was suspended in a mixture of (H-20), 6.85 d (8.0) (H-2’/6’), 7.12 d (8.0) (H-3’/5’), 9:1 H2O–MeOH (200 mL), and the pH was adjusted 2.99 brt (H-7’), 3.02 brt (H-8’), 2.40 s N-(CH3)2-9’. 13 to 2 with 5% aq HCl. The solution was then C NMR (75.03 MHz, CDCl3): 27.9 (C-1), 30.1 partitioned first with CHCl3 (5 x 50 mL) and then (C-2), 30.9 (C-3), 44.9 (C-4), 72.6 (C-5), 30.9 (C-6), with EtOAc (5 x 50 mL). Solvents were removed 20.9 (C-7), 43.8 (C-8), 47.1 (C-9), 41.0 (C-10), 43.2 under vacuum, yielding the CHCl3 fraction CL-I (850 (C-11), 31.7 (C-12), 43.5 (C-13), 69.8 (C-14), 130.8 mg) and EtOAc fraction EA-I (330 mg). The pH of (C-15), 146.8 (C-16), 68.8 (C-17), 19.1 (C-18), 169.9 the residual aqueous layer was then adjusted to 8 with (C-19), 80.6 (C-20), 158.2 (C-1’), 114.9 (C-2’/6’), diluted NH3. It was then partitioned with CHCl3 (5 x 129.2 (C-3’/5’), 125.0 (C-4’), 33.3 (C-7’), 52.5 50 mL) and EtOAc (5 x 50 mL). Solvents were (C-8’), 34.3 (N-(CH3)2-9’). + removed under vacuum, yielding the CHCl3 fraction HRAPITOFMS: m/z [M + H ] calcd for C30H40N2O3: CL-II (250 mg) and EtOAc fraction EA-II (133 mg). 477.3117; found: 477.3122. The pH of the aqueous layer was then adjusted to 7.0, and the solvent was removed by freeze-drying, giving Navirine C (2) fraction AQ (3.3 g). amorphous solid.

[α] : +7.5 (c 0.070, CH OH). TLCs of the fractions CL-II and EA-II in several D 3 IR (KBr): 3350, 3030, 3018, 1650, 1605, 1508, solvents (toluene/diethylamine/EtOAc 80:5:20; 820 cm-1. v/v CHCl /MeOH/NH 85:15:0.5; v/v) showed 3 3 1H NMR (300 MHz, CDCl ): 1.74-2.08 m (H-1), 1.62 spots that gave a positive reaction to Dragendorf 3 m (H-2), 1.25-1.78 m (H-3), 1.26 m (H-5), 1.83-2.14 reagent. Fraction CL-II was repeatedly subjected m (H-6), 1.80 m (H-7), 1.34-1.68 m (H-11), 2.87 m to preparative thin layer chromatography (PTLC) (H-12), 1.33 m (H-13), 5.63 brs (H-15), 4.52 brs with (toluene/diethylamine/EtOAc 80:5:20 v/v, (H-17), 1.00 s (H-18), 2.30 m (H-19), 2.53 m (H-20), CHCl /MeOH/NH 85:15:0.5 v/v). as eluents, 3 3 6.84 d (8.0) (H-2’/6’), 7.10 d (8.0) (H-3’/5’), 2.89 m yielding compounds 1 (10.1 mg) and 2 (6.5 mg). (H-7’), 2.89 m (H-8’), 2.55 s (N-(CH3)2-9’). Fraction EA-II was subjected to silica plate 13 chromatography using chloroform/methanol 4:1 as C NMR (75.03 MHz, CDCl3): 21.8 (C-1), 28.5 eluent. Further purification was achieved by (C-2), 34.1 (C-3), 41.7 (C-4), 53.5 (C-5), 33.9 (C-6), semi-preparative HPLC with aqueous 0.1% HCOOH 34.1 (C-7), 44.4 (C-8), 44.5 (C-9), 44.0 (C-10), 28.1 (A) and AcCN (B) as eluents. Gradient elution was (C-11), 31.1 (C-12), 44.1 (C-13), 45.0 (C-14), 132.4 used from 90% (A) to 85% (A) in 4 min, and then to (C-15), 145.8 (C-16), 68.7 (C-17), 27.8 (C-18), 58.0 40% (A) in 22 min, yielding compound 3 (5.2 mg). (C-19), 76.7 (C-20), 158.8 (C-1’), 115.1 (C-2’/6’), 129.7 (C-3’/5’), 128.0 (C-4’), 33.2 (C-7’), 60.5 (C-8’), 44.3 (N-(CH3)2-9’). The AQ fraction was subjected to several semi- + preparative HPLC steps (Spherisorb C-18, 1 x 300 HRAPITOFMS: m/z [M + H ] calcd for C31H44N2O: 461.3532; found: 461.3512. mm, 10 μm) with aqueous 0.1% HCOOH (A) and methanol (B) as eluents. Gradient elution used for the Chellespontine (3) isolation of compounds 4-7 was as follows: from 90% (A) to 85% (A) in 10 min, and then to 70% (A) amorphous solid. 20 in 15 min, yielding compounds 4 (11.0 mg) and 5 [α] D: +5.5 (c 0.052, CHCl3). -1 (13.0 mg); from 75% (A) to 45 % A in 25 min for the IR (KBr) νmax: 2915, 1730, 990, 892 cm . 1 isolation of compounds 6 (8.0 mg) and 7 (6.5 mg). H NMR (300 MHz, CDCl3): 1.68 m (H-1), 1.31 m (H-2), 1.48-1.74 m (H-3), 1.38 m (H-5), 1.70 m Navirine B (1) (H-6), 1.61-2.05 (H-7), 2.19 m (H-9), 1.16-1.93 m amorphous solid. (H-11), 2.40 m (H-12), 1.80-1.97 m (H-13), 1.30- 1.93 m (H-14), 3.70 m (H-15), 5.05-5.12 d (J = 1.0) [α]D: +12.0 (c 0.083, CH3OH). IR (KBr): 3350, 3030, 3020, 1670, 1650, 1610, 1508, (H-17), 1.06 s (H-18), 3.77 m (H-19), 4.00 m (H-20), -1 3.75 m (H-21), 8.71 s (H-22). 820 cm . 13 1 C NMR (75.03 MHz, CDCl3): 25.7 (C-1), 19.6 ( C- H NMR (300 MHz, CDCl3): 1.65-1.78 m (H-1), 1.30 m (H-2), 1.75-1.98 m (H-3), 1.87-2.02 m (H-6), 1.26- 2), 40.5 (C-3), 33.5 (C-4), 45.6 (C-5), 19.7 (C-6), 1.58 m (H-7), 1.64 m (H-9), 1.42-1.88 m (H-11), 2.54 34.2 (C-7), 37.2 (C-8), 39.8 (C-9), 46.0 (C-10), 27.9 m (H-12), 1.56-1.91 m (H-13), 5.67 brs (H-15), 4.55 (C-11), 36.1 (C-12), 25.3 (C-13), 27.9 (C-14), 74.7 brs (H-17), 1.05 s (H-18), 7.43 brs (H-19), 3.57 brs Diterpenoid alkaloids and glycosides from Aconitum naviculare Natural Product Communications Vol. 3 (12) 2008 1989

(C-15), 154.8 (C-16), 109.7 (C-17), 24.0 (C-18), 59.5 Cell growth was determined by the MTT reduction (C-19), 56.5 (C-20), 65.0 (C-21), 183.3 (C-22). assay [12] after 72 h of incubation. Briefly, 20 μL of + HRAPITOFMS: m/z [M + H ] calcd. for C22H33NO2: MTT solution (5 mg/mL in PBS) was added to each 344.2590; found: 344.2550. well and plates were incubated for 4 h at 37°C. DMSO (150 μL) was added to all wells and mixed Compounds 4-7 were characterized on the basis of thoroughly to dissolve the dark blue crystals. reported data [9-11]. Absorbance was measured on a micro-culture plate reader (Titertek Multiscan) at a test wavelength of Antiproliferative activity: Human ovarian carcinoma 570 nm and a reference wavelength of 630 nm. (2008) and human intestinal adenocarcinoma(LoVo) Experiments were performed at least in triplicate, and cell lines were used. The 2008 cells were maintained results were statistically evaluated using Student's in RPMI 1640, and LoVo cells in Ham’s F 12, in t-test [13]. IC50 95% confidence limits were estimated both cases supplemented with 10% heat-inactivated with the Litchfield and Wilcoxon method. FCS, 1% antibiotics and 1% 200 mM L-glutamine (all products of Biochrom KG Seromed). Cells were Acknowledgment - The authors are grateful to seeded on 96-well tissue plates (Falcon) at 5 x 104 MIUR for financial support. cells/mL, and treated 24 h later with various concentrations of the compounds 1-3.

References

[1] Amiya T, Bando H. (1988) Aconitum alkaloid, in The Alkaloids: Chemistry and Biology, Brossi A. (Ed) Academic Press Inc, San Diego, CA, Vol. 34, Chapter 3, 95-177. [2] Ameri A. (1998) The effects of Aconitum alkaloids on the central nervous system. Progress in Neurobiology, 56, 211-235. [3] Wada K, Hazawa M, Takahashi K, Mori T, Kawahara N, Kashiwakura I. (2007) Inhibitory effects of diterpenoid alkaloids on the growth of A172 human malignant cells. Journal of Natural Products, 70, 1854-1858. [4] Gao L, Wei X, Yang L. (2004) A new diterpenoid alkaloid from a Tibetan medicinal herb Aconitum naviculare Stapf. Journal of Chemical Research, 307-308. [5] Shrestha BB, Jha PK, Gewali MB. (2007) Ethnomedicinal use and distribution of Aconitum naviculare (Bruhl) Stapf in upper Manang, central Nepal. In: Local Effects of Global Changes in the Himalayas: Manang, Nepal. Chaudhary RP, Aase TH, Vetaas OR, Subedi BP. (Eds.) Tribhuvan University, Nepal, and University of Bergen, Norway, 171-181. [6] Shrestha BB, Dall’Acqua S, Gewali MB, Jha PK, Innocenti G. (2006) New flavonoid glycosides from Aconitum naviculare (Bruhl) Stapf, a medicinal herb from the trans-Himalayan region of Nepal. Carbohydrate Research, 341, 2161-2165. [7] Pelletier SW, Keith LH. (1970) In The Alkaloids: Chemistry and Physiology, Manske RHF. (Ed.) Academic Press, New York, Vol. 12, Chapter 2, 143-155. [8] Desai HK, Joshi BS, Pelletier SW, Sener B, Bingol F, Baykal T. (1993) New alkaloids from Consolida hellespontica. Heterocycles, 36, 1081-1089. [9] Du M, Xie J. (1995) Flavonol glycosides from Rhodiola crenulata, Phytochemistry, 38, 809-810. [10] Pauli GF. (2000) Higher order and substituent chemical shift effects in the proton NMR of glycosides. Journal of Natural Products, 63, 834-838. [11] Galland S, Mora N, Abert-Vian M, Rakotomanomana N, Dangles O. (2007) Chemical synthesis of hydroxycinnamic acid glucosides and evaluation of their ability to stabilize natural colors via anthocyanin copigmentation, Journal of Agricultural and Food Chemistry, 55, 7573-7579. [12] Mosmann T. (1983) Rapid colorimetric assay for cellular growth survival: application to proliferation and cytotoxic assay. Journal of Immunoogical Methods, 65, 55-63. [13] Tallarida RJ, Murray RB. (1987) Manual of Pharmacological Calculations with Computer Programs. Springer-Verlag.

Impurities in Herbal Substances, Herbal Preparations and Herbal Medicinal Products, IV. Heavy (toxic) Metals SFSTP Commission, Didier Guédon, Michèle Brum, Jean-Marc Seigneuret, Danièle Bizet, Serge Bizot, Edmond Bourny, Pierre-Albert Compagnon, Hélène Kergosien, Luis Georges Quintelas, Jerôme Respaud, Olivier Saperas, Khalil Taoubi and Pascale Urizzi 2107

A Fresh Insight into the Interaction of Natural Products with Pregnane X Receptor Salvador Máñez 2123

Natural Products as Gastroprotective and Antiulcer Agents: Recent Developments Rosa Tundis, Monica R Loizzo, Marco Bonesi, Federica Menichini, FilomenaConforti, Giancarlo Statti and Francesco Menichini 2129

Phytochemistry and Pharmacology of Boronia pinnata Sm. MassimoCurini, Salvatore Genovese, Luigi Menghini, Maria Carla Marcotullio and Francesco Epifano 2145

Therapeutic Potential of Kalanchoe Species: Flavonoids and other Secondary Metabolites Sônia S. Costa, Michelle F. Muzitano, Luiza M. M. Camargo and Marcela A. S. Coutinho 2151

In vitro Apoptotic Bioactivity of Flavonoids from Astragalus verrucosus Moris Joseph A. Buhagiar, Alessandra Bertoli, Marie Therese Camilleri-Podesta and Luisa Pistelli 2007

Qualitative Profile and Quantitative Determination of Flavonoids from Crocus sativus L. Petals by LC-MS/MS Paola Montoro, Carlo I. G. Tuberoso, Mariateresa Maldini, Paolo Cabras and Cosimo Pizza 2013

HPLC/DAD/ESI-MS Analysis of Non-volatile Constituents of Three Brazilian Chemotypes of Lippia alba (Mill.) N. E. Brown Patrícia Timóteo, Anastasia Karioti, Suzana G. Leitão, Franco Francesco Vincieri and Anna Rita Bilia 2017

Optimization and Validation of an HPLC–Method for Quality Control of Pueraria lobata Root Lidiya Bebrevska, Mart Theunis, Arnold Vlietinck, Luc Pieters and Sandra Apers 2021

Pharmacokinetics of Luteolin and Metabolites in Rats Sasiporn Sarawek, Hartmut Derendorf and Veronika Butterweck 2029

Complete Characterization of Extracts of Onopordum illyricum L. (Asteraceae) by HPLC/PDA/ESIMS and NMR Luisella Verotta, Laura Belvisi, Vittorio Bertacche and Maria Cecilia Loi 2037

Phenolic Profiles of Four Processed Tropical Green Leafy Vegetables Commonly Used as Food Sule Ola Salawu, Marzia Innocenti, Catia Giaccherini, Afolabi Akintunde Akindahunsi and Nadia Mulinacci 2043

(Bio)Sensor Approach in the Evaluation of Polyphenols in Vegetal Matrices M. Camilla Bergonzi, Maria Minunni and Anna Rita Bilia 2049

In vitro Radical Scavenging and Anti-Yeast Activity of Extracts from Leaves of Aloe Species Growing in Congo Annalisa Romani, Pamela Vignolini, Laura Isolani, Sara Tombelli, Daniela Heimler, Benedetta Turchetti and Pietro Buzzini 2061

Antioxidant Principles and Volatile Constituents from the North-western Iberian mint “erva-peixeira”, Mentha cervina Matteo Politi, César L Rodrigues, Maria S Gião, Manuela E Pintado and Paula ML Castro 2065

Chemical Composition of Thymus serrulatus Hochst. ex Benth. Essential Oils from Ethiopia: a Statistical Approach Bruno Tirillini, Roberto Maria Pellegrino, Mario Chessa and Giorgio Pintore 2069

GC MS Analysis of the Volatile Constituents of Essential Oil and Aromatic Waters of Artemisia annua L. at Different Developmental Stages Anna Rita Bilia, Guido Flamini, Fabrizio Morgenni, Benedetta Isacchi and Franco FrancescoVincieri 2075

Do Non-Aromatic Labiatae Produce Essential Oil? The Case Study of Prasium majus L. Claudia Giuliani, Roberto Maria Pellegrino, Bruno Tirillini and Laura Maleci Bini 2079

Olive-oil Phenolics and Health: Potential Biological Properties Francesco Visioli, Francesca Ieri, Nadia Mulinacci, Franco F. Vincieri and Annalisa Romani 2085

Traceability of Secondary Metabolites in Eucalyptus and Fagus Wood derived Pulp and Fiber Aline Lamien-Meda, Karin Zitterl-Eglseer, Heidrun Fuchs and Chlodwig Franz 2089

Potential Anticancer Activity Against Human Epithelial Cancer Cells of Peumus boldus Leaf Extract Juan Garbarino, Nicolas Troncoso, Giuseppina Frasca, Venera Cardile and Alessandra Russo 2095

Antihyperalgesic Effect of Eschscholzia californica in Rat Models of Neuropathic Pain Elisa Vivoli, Anna Maidecchi, Anna Rita Bilia, Nicoletta Galeotti, Monica Norcini and Carla Ghelardini 2099

Problems in Evaluating Herbal Medicinal Products Jozef Corthout 2103

Natural Product Communications 2008 Volume 3, Number 12

Contents

Page

1968-2008: 40 Years of Franco F. Vincieri’s Natural Products Research Anna Rita Bilia 1941

Effects of Terpenoids from Salvia willeana in Delayed-type Hypersensitivity, Human Lymphocyte Proliferation and Cytokine Production Anna Vonaparti, Anastasia Karioti, María C. Recio, Salvador Máñez, José L. Ríos, Eleani Skaltsa and Rosa M. Giner 1953

Characterization of By-products of Saffron (Crocus sativus L.) Production Pamela Vignolini, Daniela Heimler, Patrizia Pinelli, Francesca Ieri, Arturo Sciullo and Annalisa Romani 1959

Antitrypanosomal and Antileishmanial Activities of Organic and Aqueous Extracts of Artemisia annua Anna Rita Bilia, Marcel Kaiser, Franco Francesco Vincieri and Deniz Tasdemir 1963

Secondary Metabolites from the Roots of Salvia palaestina Bentham Antonio Vassallo, Ammar Bader, Alessandra Braca, Angela Bisio, Luca Rastrelli, Francesco De Simone and Nunziatina De Tommasi 1967

Cancer Chemopreventive Potential of Humulones and Isohumulones (Hops α- and Iso-α-acids): Induction of NAD(P)H:Quinone Reductase as a Novel Mechanism Gregor Bohr, Karin Klimo, Josef Zapp, Hans Becker and Clarissa Gerhäuser 1971

A Polar Cannabinoid from Cannabis sativa var. Carma Giovanni Appendino, Anna Giana, Simon Gibbons, Massimo Maffei, Giorgio Gnavi, Gianpaolo Grassi and Olov Sterner 1977

HPLC-DAD-MS Fingerprint of Andrographis paniculata (Burn. f.) Nees (Acanthaceae) Sabrina Arpini, Nicola Fuzzati, Andrea Giori, Emanuela Martino, Giacomo Mombelli, Luca Pagni and Giuseppe Ramaschi 1981

Diterpenoid Alkaloids and Phenol Glycosides from Aconitum naviculare (Brühl) Stapf. Stefano Dall’Acqua, Bharat B. Shrestha, Mohan Bikram Gewali, Pramod Kumar Jha , Maria Carrara and Gabbriella Innocenti 1985

Inhibition of PGHS-1 and PGHS-2 by Triterpenoid Acids from Chaenomelis fructus Eveline Reininger and Rudolf Bauer 1991

Preparative Isolation of Antimycobacterial Shoreic Acid from Cabralea canjerana by High Speed Countercurrent Chromatography Gilda G. Leitão, Lisandra F. Abreu, Fernanda N. Costa, Thiago B. Brum, Daniela Fernandes Ramos, Pedro Eduardo A. Silva, Maria Cristina S. Lourenço and Suzana G. Leitão 1995

Antiplasmodial Effects of a few Selected Natural Flavonoids and their Modulation of Artemisinin Activity Anna Rita Bilia, Anna Rosa Sannella, Franco Francesco Vincieri, Luigi Messori, Angela Casini, Chiara Gabbiani , Carlo Severini and Giancarlo Majori 1999

Comparative Analysis of Antimalarial Principles in Artemisia annua L. Herbal Drugs from East Africa Silvia Lapenna, Maria Camilla Bergonzi, Franco Francesco Vincieri and Anna Rita Bilia 2003

(Continued inside backcover)

J. Mt. Sci. (2009) 6: 66–77 DOI 10.1007/s11629-009-0209-1

Habitat Range of two Alpine Medicinal Plants in a Trans- Himalayan Dry Valley, Central Nepal

Bharat Babu SHRESTHA* and Pramod Kumar JHA

Central Department of Botany, Tribhuvan University, Kathmandu, Nepal

* corresponding author, email: [email protected]

Abstract: Understanding of the habitat range of damage by livestock, the population of A. naviculare threatened Himalayan medicinal plants which are was found absent in open areas in five of the six declining in their abundance due to high sampling sites and it was confined only within the anthropogenic disturbances is essential for bushes of alpine scrubs. For N. scrophulariiflora, developing conservation strategies and agro- high probability of complete receding of small technologies for cultivation. In this communication, glaciers may be a new threat in future to its habitat. we have discussed the habitat range of two alpine The information about habitat conditions, together medicinal plants, Aconitum naviculare (Brühl) Stapf with the information from other areas, can be useful and Neopicrorhiza scrophulariiflora (Pennel) Hong to identify potential habitats and plan for cultivation in a trans-Himalayan dry valley of central Nepal, or domestication of the two medicinal plants. Manang district. They are the most prioritized medicinal plants of the study area in terms of Key words: Aconitum naviculare; Neopicrorhiza ethnomedicinal uses. A. naviculare occurs on warm scrophulariiflora; habitat degradation; nitrogen; and dry south facing slopes between 4090~4650 m organic carbon; radiation; Manang; Nepal asl along with sclerophyllous and thorny alpine scrubs, while N. scrophulariiflora is exclusively found on cool and moist north facing slope between Introduction 4000 and 4400 m asl where adequate water is available from snow melt to create a suitable habitat for this wetland dependent species. The soil in Himalayan mountains are rich in medicinal rooting zone of the two plants differs significantly in plants and the mountain people have rich organic carbon (OC), organic matter (OM), total knowledge to use these plants for their primary nitrogen (N) and carbon to nitrogen (C/N) ratio. Due health care (e.g. Pohle 1990, Lama et al. 2001, to cool and moist condition of N. scrophulariiflora Manandhar 2002). In addition to local uses, habitat, accumulation of soil OC is higher, but soil N collection and trade of medicinal plants are content is lower probably due to slow release from litter, higher leaching loss and greater retention in important parts of livelihood of mountain people perennial live biomass of the plant. The C/N ratio of (Olsen and Larson 2003). In absence of alternative soil is more suitable in A. navuculare habitat than modern medicine and belief on the traditional that of N scrophulariiflora for N supply. Warm and medicine, plants will continue to be a major source sunny site with N rich soil can be suitable for of medicine in near future for the people living in cultivation of A. naviculare, while moist and cool site high Himalayas. with organic soil for N. scrophulariiflora. The Collection of high altitude medicinal plants for populations of both the plants are fragmented and use by local herbal healers (e.g. amchis: herbal small. Due to collection by human and trampling healers trained in Tibetan medicine) appears to be sustainable and the collectors have good Received: 18 July 2008 knowledge of habitat, life history traits and Accepted: 1 September 2008

66 J. Mt. Sci. (2009) 6: 66–77

regeneration of medicinal plants they collect Chandra 2004, Chauhan and Nautiyal 2005). A (Ghimire et al. 2005). However, untrained and study on morphological parameters and energy ‘alien’ commercial collectors employed destructive exchange characteristics of central Himalayan harvest methods to supply as much as large alpine medicinal plants grown at lower elevations volume of medicinal plants to lowland markets, (550 and 2100 m asl) showed the possibility of which is more responsible for declining the their cultivation at lower elevation with better populations of high altitude medicinal plants than growth (Chandra 2004). the collection for traditional uses. Because most of Aconitum naviculare and Neopicrorhiza the medicinal plants in global market have been scrophulariiflora are among the most prioritized supplied by wild collection and demand of high- medicinal plants in Manang in terms of value medicinal plants has increased in global ethnomedicinal uses (Bhattarai et al. 2007). A. market (Grunwald and Buttel 1996), harvest naviculare shows narrow distribution range in the pressure on medicinal plants in wild is likely to Himalayas, and it has been reported only from increase in future. Market driven harvest of trans-Himalayan regions, such as Tibet (Gao et al. medicinal plants from wild habitats has already 2004) and some parts of Nepal including Dolpo threatened some of the species and these are now (Lama et al. 2001), Mustang (Bista and Bista 2005, legally banned for collection and trade (MFSC Chetri et al. 2006, Ohba et al. 2008) and Manang 2002). However, livelihood of mountain people (Bhattarai et al. 2007, Shrestha et al. 2007a). N. has heavily depended on trade of medicinal plants scrophulariiflora widely distributes in moist (Olsen and Larson 2003), and legal ban can not be alpine regions on southern declivity of the enforced adequately in remote mountains. Himalayas. However, a small population of this Protection of wild populations and cultivation species is also found on northern declivity of the for commercial purpose are two important Himalayas in the upper Manang Valley of central strategies, which can prevent the species from Nepal. Since A. naviculare and N. being extinct. In the context of the declining scrophulariiflora are high-value medicinal plants populations of Himalayan medicinal plants, the in traditional medicine of Manangis, changes in need of domestication and cultivation are widely status of these two plant species can influence local felt (Chhetri et al. 2005) which decrease harvest health care system. The populations of the two pressure on wild populations. However, there are species are fragmented and population sizes some major problems in cultivation such as logistic appear to be declining though there is no problems in cultivation of plants at high altitude commercial collection at present. Thus, cultivation and decline in medicinal value of plant materials can be the immediate step to conserve their wild from cultivation (Hamilton 2003, Schippmann et populations. This paper discusses the habitat al.. 2002). Among the native Himalayan medicinal ranges, adaptation and threat to the natural plants, only 20% are under cultivation which habitats of the two medicinal plants in the upper reflects the severe deficiency of research and Manang Valley, which will be helpful for selecting development (R&D) efforts to domesticate farmlands for their cultivation and planning for Himalayan native medicinal plants (Dhar et al. conservation management of the species in their 2000). A successful cultivation without any decay natural populations. in medicinal value of the plant may need replication of wild habitat condition in the farm land, which is virtually impossible but can be 1 Materials and Methods maximized if we have detail information on distribution range and habitat requirements in the 1.1 Study species wild condition. However, the objective information on distribution of medicinal plants is an important gap in the research of Himalayan medicinal plants Aconitum naviculare (Brühl) Stapf with its (Dhar et al. 2000). Attempts have been made to local name as ponkar/bongkar (Gurung) and cultivate Himalayan alpine medicinal plants at family name as Ranunculaceae (Figure 1) is a lower elevation than their natural distribution (e.g. perennial alpine medicinal herb with biennial

67 J. Mt. Sci. (2009) 6: 66–77

Aconitum Neopicrorhiza naviculare scrophulariiflora North facing slope North facing South facing slope South facing

BB Shrestha

Figure 1 Upper Manang valley with the Marsyangdi River on the valley floor. Aconitum naviculre is found on the south facing slope and Neopicrorhiza scrophulariiflora on the north facing slope. There is cultivation of wheat and buckwheat at the base of the south facing slope.

white tuberous root and whitish violet flower. The Bhattarai et al. 2006, Shrestha et al. 2007a). morphology of this plant has been described by Neopicrorhiza scrophulariiflora (Pennell) Stainton (1997) and Ohba et al. (2008). It is Hong (Syn. Picrorhiza scrophulariiflora Pennell), endemic to the Himalayas and found from west with its local name as kutki and family name as Nepal to Bhutan. A. naviculare has been used in Scrophulariaceae (Figure 1) is a threatened the Nepalese and Tibetan folk medicines against perennial medicinal herb of alpine region, growing cold, fever, headache and bile disorder as well as on moist rocky slopes. The morphology, growth sedative and analgesic purposes (Lama et al. 2001, and regeneration of this species have been Gao et al. 2004, Bista T. and Bista G. 2005, described by Polunin and Stainton (2000) and

68 J. Mt. Sci. (2009) 6: 66–77

Ghimire et al. (2005). In Nepal, the creeping floor. The valley floor predominantly has blue pine rhizomes of N. scrophulariiflora have been used (Pinus wallichiana) forest, while the upper belt on for treating cold, fever, headache, anemia, high north facing slope has firs (Abies spectabilis) and blood pressure, sore throat, gastritis, intestinal birch (Betula utilis) forests up to tree line (4200 m pains and conjunctivitis in traditional medicine asl) (Shrestha et al. 2007b). The moist alpine scrub (Pohle 1990, Lama et al. 2001, Manandhar 2002, above tree line on the north facing slope was Bhattarai et al. 2006). In regional market, the dominated by Rhododendron anthopogon, rhizomes of N. scrophulariiflora, together with Juniperus indica, Caragana species, etc. On the those of Picrorhiza kurrooa Royle (which is listed dry southern slope the alpine scrub has dwarf and in CITES Appendix II) have been sold under a prostrate junipers (Juniperus indica, J. recurva, J. common trade name kutki (Mulliken 2000). Nepal squamata), Rhododendron lepidotum, Rosa spp., is the main supplier of kutki to international Berberis spp., Ephedra gerardiana, etc. market, followed by India and Bhutan (Olsen 2005). In fiscal year 2003/04, the government of 1.3 Field sampling Nepal collected royalty of 5200 kg rhizome of N. scrophulariiflora (Department of Forest 2004). Informal discussions were held with local A. naviculare and N. scrophulariiflora are people (herdsman, shepherds, farmers and local among the most prioritized medicinal plants in herbal healers) in order to locate the specific Manang in terms of use and efficacy (Bhattarai et localities where the target plant species could be al. 2007) but during the study period there was no found. With one of the local informants we visited commercial collection of the two medicinal plants all the potential areas and marked some of them from the upper Manang Valley. A. naviculare has for detail sampling. Six sites for A. naviculare (Ice been collected in a considerable amount for lake area, Khangsar, Manang hill, Yak Kharka, traditional uses by Manangis living in and outside Ledtar and Thorong Phedi) and three for N. Manang (Shrestha et al. 2007a). People collect N. scrophulariiflora (Humde, Millereppa and scrophularriflora in small amount for their Chonkar) were identified for sampling. According domestic uses. to locals, N. scrophulariiflora was also found on northern slope opposite to Khangsar village, but 1.2 Study area the site could not be sampled due to physical inaccessibility. All sampling sites of A. naviculare The study area (28°37'~46' N, 83° 57'~84°06' have been used for grazing. The sampling site of N. E and alt. 4000~4700 m asl) lies on the upper scrophulariiflora at Humde was a post fire site Manang Valley of Manang district, central Nepal. (Betula utilis forest was damaged completely by The glacially formed U-shaped valley (3000~3400 fire about forty years ago), and also it was a debris m asl), also called Nyishang, is traversed by the deposited site at Millereppa and scree at Chonkar. Marsyangdi River and surrounded by high These two species have different growth forms and mountains (>6000 m asl). The peaks are formed of distribution patterns. Thus, we used different paragneiss and schists encroached by quartzites sampling strategies for them. For A. naviculare 42 with layers of haematite, slates and limestone with plots (10 m × 10 m) were identified for sampling. clays and marl (Hagen 1969). The valley lies on a Each plot was divided into four quarters and single rain shadow area of the trans-Annapurna soil sample was collected from each quarter, thus mountain range (elevation >8000 m) with a mean totaling 168 samples. During soil collection, a best annual precipitation of 444 mm (at 3420 m asl) looking individual of A. naviculare was completely and temperature 6.2° C (Miehe et al. 2001). Crop uprooted and the soil of rooting zone (5~10 cm) growing season (late April to early October) mean was collected. For N. scrophulariiflora, ten best temperature on valley floor is 5~12°C (Aase and looking and least disturbed patches were identified Vetaas 2007). The area is covered by snow during within a large 50 m × 50 m plot in each site. Three winter for about five months (November to March). soil samples (250 g) were collected randomly in Forest vegetation is mostly confined on the moist each patch from rooting depth (5~10 cm), totaling north-facing slope of Annapurna, and the valley 90 soil samples. Soil samples were air dried in

69 J. Mt. Sci. (2009) 6: 66–77

shade for 7~8 days and stored in zip lock plastic statistical analyses. bags until laboratory analysis. The physical locations (elevation, aspect, slope, latitude and longitude) of the sampling plots were recorded 2 Results using clinometer and geographic positioning system (GPS). The physical locations were also A. naviculare was often found with recorded at points of occurrence of the species sclerophyllous and thorny species, such as outside the sampling plots. Important plant Juniperus squamata, Ephedra gerardiana, species found in the habitats of these two species Cotoneaster microphyllus, Caragana gerardiana were also collected and identified with the help of and Berberis spp. (Table 1). In Ice Lake, Manang references (Stainton 1997, Pollunin and Stainton hill, Khangsar, Yak Kharka and Thorong phedi, 2000). where its population size was small, A. naviculare was confined within the scrub of these 1.4 Laboratory analysis sclerophyllous and thorny species. The individual plants growing in such habitats had long petioles Soil organic carbon (OC, %), organic matter and internodes so that their leaves and flowers (OM, %) and total nitrogen (N, %) contents were could reach above the height of scrubs. In Ledtar, determined in air dried soil samples after passing this species was also found in open places outside through fine sieve (mesh size 0.5 mm). The OC and the bushes. N. scrophulariiflora was often found OM contents were determined by Walkley and in the form of patch due to clonal propagation. In Black Method, and total N by micro-Kjeldahl the post fire site, the plants mostly formed method as described by Gupta (2000). We compact mat on the exposed soil surface, and other analyzed 165 soil samples (three were damaged species were poorly represented within the mat. during transportation) from A. naviculare habitat But at debris deposited site and scree, the patches and 90 from N. scrophulariiflora habitat. of N. scrophulariiflora were comparatively loose and other herbaceous (Bistorta macrophylla, Primula spp., Aster spp. and Geranium spp.) and 1.5 Numerical Analysis spreading shrubs (Salix calyculata, Bistorta affinis From the values of aspect (Ω), slope (β) and and Potentilla frutocosa) well represented (Table 1). Rhododendron anthopogon was common latitude (Φ), relative radiation index (RRI) of habitats of A. naviculare and N. scrophulariiflora dominant shrub of the plant community where N. was calculated following Ôke (1987) [RRI = scrophulariiflora was found, while Epilobium angustifolium was found only at the post fire site. {Cos(180 - Ω).Sinβ. SinΦ} + Cosβ. CosΦ]. The RRI gives a relative value (+1 to −1) of how much solar A. naviculare was found within the elevation radiation a particular spot receives at noon at range from 4090 to 4650 m asl (Table 2). When individual sites are considered it was found within equinox. For each soil sample carbon to nitrogen ratio (C/N ratio) was calculated. a narrow elevation band spanning 100 to 300 m. N. The mean and standard deviation (SD) of RRI, scrophulariiflora was found between 4000 and 4400 m asl which is also relatively narrow (Table soil OC, OM, N and C/N ratio were determined for each sampling site. The values of these parameters 3). The average slopes of the habitats of these two were pooled together separately for the two species. species are nearly equal. A. naviculare was found exclusively on south facing slope and its habitat The differences in environmental parameters among the habitats of the two species were received high light intensity for longer period compared using non-parametric Mann-Whitney (Figure 1). However, N. scrophulariiflora was found only on north facing slope and the habitat U–test. Spearman’s correlations were determined between soil OC and N. A scattered diagram was received low light. A. naviculare received more developed between soil OC and N to show their light (p = 0.05) than N. scrophulariiflora in their habitats. The soil of A. naviculare habitat was plot-wise variation. Statistical Package for Social Sciences (SPSS version 11.5, 2002) was used for all found at average with 7.79% OC, 13.57% OM and

70 J. Mt. Sci. (2009) 6: 66–77

Table 1 Plant species commonly associated with Aconitum naviculare and Neopicrorhiza scropulariiflor

Plant species associated with A. naviculare Plant species associated with N. scrophulariiflora

Caragana gerardiana Royle* Rhododendron anthopogon D. Don

Ephedra gerardiana Wall. ex Stapf* Epilobium ungustifolium L.

Rhododendron lepidotum Wall ex D.Don Salix calyculata Hook f. ex Andersson

Cotoneaster microphyllus Wallich ex Lindley* Bistorta affinis (D. Don) Greene

Cremanthodium arnicoides (DC. ex Royle) R. Good Bistorta macrophylla (D. Don) Soják

Spiraea arcuata Hook. f.* Potentilla frutocosa L.

Juniperus squamata Buch. – Ham. ex D. Don* Geranium species

Berberis sp. or spp.* Primula species

Swertia species Aster species

Tanacetum species

Potentilla species

Thallictrum species

Rosa species*

* Thorny and sclerophyllous plant species

Table 2 Mean values of physical environmental factors and soil attributes of Aconitum naviculare habitats in upper Manang. The highest and lowest mean among the sites are shown in bold face.

Elevation Slope Aspect Soil OC Soil OM Soil N C/N Sites RRI (m asl) (°) (°) (%)* (%)* (%)* ratio* 193±20 0.980 4.96± 8.55± 0.40± 12.22± Ice Lake 4400~4550 32±3 SW ±0.015 1.66a 2.87a 0.11a 1.79a 237±37 0.859± 6.59± 11.37± 0.56± 11.70± Manang 4150~4450 30±8 SW 0.107 2.36ab 4.07ab 0.19ab 1.38a 208±50 0.908± 7.13± 12.30± 0.55± 12.87± Khangsar 4400~4650 29±7 SW 0.149 3.23ab 5.57ab 0.24ab 1.41a Yak 239±21 0.847± 8.99± 15.5± 0.67± 13.45± 4090~4270 36±1 Kharka SW 0.077 3.89b 6.7b 0.27ab 0.72a 240±24 0.873± 8.61± 14.85± 0.81± 11.39± Ledtar 4200~4500 28±5 SW 0.093 3.35b 5.77b 0.41b 2.70a Thorong 108±13 0.778± 6.35± 10.95± 0.55± 11.80± 4350~4450 38±7 Phedi SE 0.073 1.52ab 2.61ab 0.16ab 1.33a Mean/ 227±42 0.870± 7.87± 13.57± 0.70± 11.78± 4090~4650 30±6 Range SW 0.098 3.29 5.67 0.37 2.35

* In these columns values with different letters differed significantly at p = 0.05. The data were analyzed by Duncan’s Homogeneity test of one-way analysis of variance (ANOVA). The same below

71 J. Mt. Sci. (2009) 6: 66–77

Table 3 Mean values of physical environmental factors and soil attributes of Neopicrorhiza scrophulariiflora habitats in upper Manang. The highest and lowest mean among the sites are shown in bold face.

Sites Altitudinal Slope Aspect Soil OC Soil OM Soil N C/N range RRI ( ) ( ) (%)* (%)* (%)* ratio* Land uses Name (m asl) ° ° Post fire 344 11.94± 20.59± 0.53± 22.1± Humde 4000~4200 38 0.41 site NW 5.5b 9.44b 0.2a 3.5b Debris 8.60± 14.83± 0.60± 16.2± deposited Milereppa 4100~4400 18 36 NE 0.72 3.8a 6.48a 0.36a 5.8a site 9.25± 15.94± 0.65± 14.8± Scree Chonkar 4250~4350 40 350 N 0.37 3.3a 5.71a 0.25a 4.0a 0.50± 9.93± 17.12± 0.59± 17.7± Mean/range 4000~4400 32 - 0.19 4.50 7.72 0.28 5.2

Table 4 Comparison of soil attributes and relative radiation index between habitats of Aconitum naviculare and Neopicrorhiza scrophulariiflora

RRI Soil OC Soil OM Soil N C/N ratio A. naviculare habitat 0.87±0.098 7.87±3.29% 13.57±5.67% 0.70±0.37% 11.78±2.35 N. scrophulariiflora 0.50±0.19 9.93±4.50% 17.12±7.72% 0.59±0.28% 17.7±5.2 habitat Mann-Whitney U 60 5438 5438 6316 2086 Significance level (p) 0.000 0.000 0.000 0.049 0.000 Total sample size 105 255 255 255 255

Note: total sample size is the total number of samples of each of the parameters analyzed

0.70% total N with C/N ratio of 11.78 (Table 2). soil OC and N was stronger in the soil of A. Site specific variation in soil OC, OM and N was naviculare habitat (r = 0.90, p = 0.01 and N = 165) significant (p<0.05) but C/N ratio did not vary than that of N. scrophulariiflora habitat (r = 0.83, significantly with sites. The soil of N. p = 0.01 and N = 90). For a given value of soil OC, scrophulariiflora habitat had 9.93% OC, 17.12% the N content was higher in the soil of A. OM and 0.59% N with C/N ratio of 17.7 (Table 3). naviculare habitat (Figure 2). For the combined The soil of the post fire site had higher soil OC and data, the lowest soil N content was measured in N. OM than the other two sites; but soil N and C/N scrophulariiflora habitat and the highest in A. ratio did not vary significantly among the sites. naviculare habitat. Since the RRI of A. naviculare habitat was higher than that of N. scrophulariiflora habitat, the former received more light than the latter. 3 Discussion There were also significant differences in soil OC, OM, N and C/N ratio of the two species’ habitats. 3.1 Habitat range and adaptation The soil OC, OM and C/N ratio were higher in N. scrophulariiflora habitat, while the soil N was A. naviculare was exclusively found on dry higher in A. naviculare habitat. However, the south-facing slope of the valley. The south-facing difference in soil N content between the two was slope warms up more than north-facing slope, due only marginal (Table 4). The correlation between to high solar radiation (Larcher 1995). In the study

72 J. Mt. Sci. (2009) 6: 66–77

area only a few nights had temperature below zero low temperature. Density of A. naviculare is higher on the south-facing slope during growing season at the plots with higher RRI (Shrestha et al. (Aase and Vetaas 2007). Alpine meadows, such as unpublished data). Therefore, this species is the habitat of A. naviculare, have high ground completely absent from the moist north facing surface temperature than the subalpine forests at slope where several nights are cold with low elevation (Körner 2007). Permanent snow is temperature below zero during growing season absent on most of the mountain tops and south- (Aase and Vetaas 2007). facing slope. Thus, soil moisture recharge from N. scrophulariiflora was exclusively found on snow melt water during growing season could be the moist north facing slope which receives low low. Since annual precipitation is very low (444 light but abundant water. Due to the presence of mm; Miehe et al. 2001) in the valley, snow melt permanent snow cover on the mountain tops, the water is the major source of moisture for the north facing slope receives ample water which is vegetation. High evapotranspiration is coupled adequate even to support the growth of subalpine with low soil moisture recharge on the south- forests (Shrestha et al. 2007b), though annual facing slope, and the plants growing in this aspect precipitation is very low (444 mm; Miehe et al. are most likely subjected to drought stress during 2001). Snow melt water moves down along the growing season. Thus, A. naviculare is adapted to thin surface soil over the steep slope where the high solar radiation, relatively warm temperature infiltration of water is prevented by underlying and mild water deficit, but it may not tolerate very rock. During growing seasons, temperature

2.5

2.0

1.5

1.0

Soil nitrogen (%) SITE

N. scrophulariiflora .5 Rsq = 0.5030

A. naviculare 0.0 Rsq = 0.6242 0 10 20 30

Soil organic carbon (%)

Figure 2 Relationship between soil nitrogen and organic carbon content in sampling plots. Each mark indicates values of soil nitrogen and organic carbon in a soil sample. The fitted lines are obtained from linear regression (n = 165 for Aconitum naviculare and 90 for Neopicrorhiza scrophulariiflora). The relation was significant at p=0.01.

73 J. Mt. Sci. (2009) 6: 66–77

dropped below zero in many nights (Aase and mountain slopes with higher mean annual Vetaas 2007) but the prostrating growth form, precipitation (Yimer et al. 2006). Low radiation such as of N. scrophulariiflora, creates favorable and high water supply can lower the soil warmer microclimate during the day (Körner temperature, thereby limiting the activity of 2003). Since N. scrophulariiflora is a wetland decomposers. Due to abundant supply of water dependent species (IUCN Nepal 2004), abundant on the north facing slope, biomass production supply of water makes the north facing slope a (and hence litter production) of alpine scrub is suitable habitat for this plant. In lower part of higher than that on the south facing slopes, Manang district (Gyasumdo valley) too, this which could also increase organic matter pool of plant is found in moist sites such as the banks of the soil. High OM content helps retain a streams and gullies, as well as on cliffy substantial amount of snow melt water, which is mountains (Ghimire et al. 1999). Another closely otherwise lost by gravity flow or seepage. Since N. related species Picrorhiza kurrooa is also found scrophulariiflora is a wetland dependent species mainly on moist (Kala 2000) and rocky slopes (IUCN Nepal 2004), OM rich soil creates a (Uniyal et al. 2006). The south facing slope is dry suitable niche for its growth by holding abundant and it is not suitable for the growth of this plant water. The soil of debris deposited site and scree resulting in its complete absence on the south had lower soil OM (Table 3), and appears to be facing slope. dry due to accumulation of coarse stone on the Temperature and moisture exert a dominant surface. But fine soil accumulates in the inner influence on the amounts of OM and N in soil matrix of the coarse stones and retains plentiful (Brady 1995). The average soil OC and N in the moisture (Pérez 1998, Körner 2003), sufficient to habitats of these two species (Tables 2 and 3) are support the growth of the wetland dependent N. close to mid values of the global range for scrophulariiflora. subtropical and tropical alpine vegetations (OC Soil N content of A. naviculare habitat was 1.2~19%, N 0.1~1.6%; Körner 2003). A. higher than that of N. scrophulariiflora, a trend naviculare and N. scrophulariiflora are found opposite to that observed in soil OC. Correlation within similar elevation range (Table 2 and 3), between soil N and OC was stronger for A. but there are significant differences in soil OC naviculare habitat (r = 0.90) than for N. and N of their habitats (Table 4). This could be scrophulariiflora (r = 0.83), and for a given possible because soil OC and N pool are mainly value of OC the N content of soil was higher for A. determined by temperature and moisture which naviculare habitat (Figure 2). Lower soil N vary with topographic (e.g. radiation, slope, content in N. scrophulariiflora habitat could be aspect) and geochemical (e.g. age, parent rocks) due to slow release of nutrients from litter, factors in addition to elevation in the mountains higher leaching loss and greater retention in (Körner 2003, Yimer et al. 2006). The soil OC perennial biomass of the plant. Nitrogen supply and OM were higher in N. scrophulariiflora to alpine soil predominantly depends on habitat which lies on the moist north facing slope. biological activities (dinitrogen fixation and These values were also higher than those of the recycling) (Körner 2003), which could be higher subalpine birch forests lying just below the on the warmer south facing slope. The two plants present sampling sites (Shrestha et al. 2007b). are growing in the same elevation range but they Higher OM content in the habitats of N. are subjected to large thermal contrast due to scrophulariiflora could be attributed to the different exposures (topographic aspects). direct negative effects of low temperature on During growing season the area is generally snow organic matter recycling, and the negative free but temperature dropped below freezing feedbacks of soil OM accumulation on the soil point in several nights on the north facing slope, milieu via acidification (Körner 2003), the latter but only in few nights on south facing slope (Aase being more pronounced in cool and moist and Vetaas 2007). High temperature on the environments. This is also obvious from the south facing slope enhances litter decomposition, present results that soil OM was the lowest and thus increases the nutrient release to soil. among the habitats of the both species at the The extent of formation of needle ice (In moist sites where RRI (hence soil temperature) was the alpine area soil moisture change into needle ice highest (Table 2 and 3). In the Bale Mountains during night and it melts during day. This cycle (Ethiopia) soil OC stock was higher in the makes the soil loose and prone to loss of fine soil)

74 J. Mt. Sci. (2009) 6: 66–77

and subsequent heaving could be higher in N. habitat (> 4000 m asl). In 2006, the plants scrophulariiflora habitat. Diurnal freeze-thaw flowered and produced fruits. However, cycle, when combined with moisture load from assessment of yield and economic viability has snow melt water may lead to loss of substantial not been done. amount of fine soil and available nutrients (Körner 2003). Due to higher availability and 3.2 Habitat condition flux of water through soil on the north facing slope leaching of elements was higher than on Grazing pressure was high on the sunny the south facing slope where water availability south facing slope which is the habitat of A. and flux through soil is very low (Egli et al. 2006). naviculare. Though the shrub vegetation is Since there is negative correlation between sparser on the south facing slope than on the annual biomass production and total soil N northern, livestock prefers south facing slope for concentration (Körner 2003), soil N grazing due to warm temperature. Since most of concentration could be high on the south facing the old human settlements are on the southern slope with sparse vegetation cover. At least 51 % slope, it is easier to bring their livestock to the of peak season leaf N content of N. pastures on the southern slope. A. naviculare is scrophulariiflora is retranslocated before leaf not a preferred species for livestock, but it is senescence (Shrestha et al. 2007c). Relative frequently browsed by goat and sheep, because proportion of perennial live biomass of N. latter two animals are non-selective in foraging scrophulariiflora (rhizome and root) is higher (Chandrashekhar et al. 2007). Trampling damage than that of A. naviculare (tuberous root). Thus, is, however, more important than browsing. a large fraction of N pool could be stored in Fortunately, in most sites, A. naviculare was biomass. found associated with thorny and sclerophyllous The average C/N ratio in the soil of A. species (Table 1) which reduces the chances of naviculare habitat (Table 2) is similar to the damages by livestock and collection by human. value (11.5~12.78) reported for the Bale These thorny species are generally excluded by Mountains of Ethiopia (Yimer et al. 2006) and it large animals such as cow, yak and dzomos with lies within the global range (6~15) for alpine wide mouth (Chandrashekhar et al. 2007). In the region of tropical and subtropical mountains sites of high grazing pressure such as Ice lake (Körner 2003), but the C/N ratio of the soil of N. area, Manang hill, Khangsar, Yak Kharka and scrophulariiflora was above the range (Table 3). Thorong Phedi, A. naviculare is virtually absent The A. naviculare habitats are more arid and in open areas and is exclusively confined in the warmer than N. scrophulariflora habitats; both bushes of the thorny and sclerophyllous species. of these conditions lower the C/N ratio (Brady It appears that the plant disappeared from the 1995). The average C/N ratio of the soil of A. open areas due to collection and trampling naviculare habitat is very close to upper limit of damage. Thus, protection of these areas from C/N ratio for fertile soil with stable organic over grazing is vital for the conservation of A. matter (10~12, Biswas and Mukherjee 1999). naviculare. Thus, in term of C/N ratio, the soil of A. N. scrophulariiflora habitat is subjected to naviculare habitat had more favorable N supply relatively low anthropogenic disturbances. to plants than that of N. scrophulariiflora habitat. Grazing pressure is relatively low and the two Thus, the two alpine medicinal plants have sites (post fire site and scree) are not easily different habitat preferences. A. naviculare accessible due to steep slope. There is no prefers warm and sunny sites with nitrogen rich commercial collection at present, but according soil, whereas N, scrophulariiflora prefers moist to local people, there was large scale collection in and cool sites with organic soil. While selecting the past (per. comm. Abu Karki, age 65, Oct farmlands for cultivation, considering these 2005). There are no historical data on the conditions can maximize yield and benefit. distribution of N. scrophulariiflora in the upper Domestication of A. naviculare has not been Manang Valley, but the populations of this plant observed in the study area but a commercial in the sampling sites could be the relicts of herbal farm located on the valley floor (alt. 3300 previously large populations. Kala (2000) m asl) has successfully transplanted N. encountered a very small patch (1 m2) of A. scrophulariiflora in small scale from its natural violaceum in one of the six sampling areas in

75 J. Mt. Sci. (2009) 6: 66–77

Indian trans-Himalaya, despite the common wetland dependent species, is found exclusively occurrence of habitats suitable for this plant. It on cool and moist north facing slope. Collection appears that most of the threatened medicinal by human and trampling damage by livestock are plants of the Himalayas have localized the major causes of population decline and distribution (Kala 2005) and present small fragmentation of A. naviculare. High probability localized population may represent the relict of of complete receding of small glaciers may be a large population in the past. new threat to the habitat of N. scrophulariiflora. Ongoing glacier retreat may be a potential Based on the range of natural habitats of the two new threat to N. scrophulariiflora. In the upper species in the study area, it can be concluded that Manang glaciers are mostly confined on the warm and sunny sites with nitrogen rich soil north facing slopes. The glaciers with small (e.g. could be suitable for cultivation of A. naviculare. Braga glacier) and large (e.g. Gangapurna glacier) while moist and cool sites with organic soil for N. reservoir are retreating rapidly but the glaciers scrophulariiflora. with smaller reservoirs are likely to disappear in the nearest future (Vetaas 2007). Complete disappearance of these small glaciers may lead to Acknowledgments acute shortage of water to wetland dependent species such as N. scrophulariiflora in and We are thankful to Volks Wagen Foundation around the water courses. Two of the three (Germany) for funding the research project and habitats (debris deposited site and scree) to Annapurna Conservation Area Project sampled in the present study lie on the glacier (Manang) for the permission to work in the melt water courses. The two populations will be protected areas. We acknowledge the helps of subjected to water shortage when the glaciers D.P. Rijal, R.K. Sharma, K. Gurung and A. above them disappear. Pariyar during field sampling. We thank K. Gurung, C. Gurung, A. Karki, M. Gurung and P. Sherpa of Manang for providing information 4 Conclusions about potential areas of distribution of A. naviculare and N.scrophulariiflora in the upper A. naviculare and N. scrophulariiflora have Manang. We are also thankful to Dr. S.K. non-overlapping distribution in the study area. A. Ghimire for helping in statistical analysis and naviculare distributes in warm and dry south making critical comments on the first draft of the facing slope, while N. scrophulariiflora, a manuscripts.

References

Aase T.H. and Vetaas O.R. 2007. Risk Management by Brady N.C. 1995. The Nature and Properties of Soils. 10th Communal Decision in Trans-Himalayan Farming: edition. New Delhi, India: Prentice-Hall of India Pvt. Ltd. Manang Valley in Central Nepal. Human Ecology 35: 453- Pp. 621 . 460. Chandra S. 2004. Effect of Altitude on Energy Exchange Bhattarai S., Chaudhary R.P. and Taylor R.S.L. 2006. Characteristics of some Alpine Medicinal Crops from Ethnomedicinal Plants Used by the People of Manang Central Himalayas. Journal of Agronomy and Crop District, Central Nepal. Journal of Ethnobiology and Sciences 190: 13-20. Ethnomedicine 2: 41 doi: 10.1186/1746-4269-2-41. Chandrashekhar K., Rao K.S., Maikhuri R.K. and Saxena K.G. http://www.ethnobio med .com/ content /2/1/41 2007. Ecological Implications of Traditional Livestock Bhattarai S., Chaudhary R.P. and Taylor R.S.L. 2007. Husbandry and Associated Land Use Practices: A Case Prioritization and Trade of Ethnomedicinal Plants by the Study from the Trans-Himalaya, India. Journal of Arid People of Manang District, Central Nepal. In: RP Environments 69: 299-314. Chaudhary, TH Aase, OR Vetaas and BP Subedi (eds.) Chauhan R.S. and Nautiyal M.C. 2005. Commercial viability Local Effects of Global Changes in the Himalayas: Manang, of cultivation of an endangered medicinal herb Nepal. Kathmandu, Nepal: Tribhuvan University (Nepal) Nardostachys jatamansii at three different agroclimatic and University of Bergen (Norway). Pp. 151-169. zones. Current Science 89: 1481-1488. Bista T. and Bista G. 2005. Himalayan Doctors and Healing Chetri M., Chapagain N.R. and Neupane B.D. 2006. Flowers Herbs: The Amchi Tradition and Medicinal Plants of of Mustang: A Pictorial Guide Book. Kathmandu, Nepal: Mustang. Mustang, Nepal: Lo-Kunphen Mensikhang. Pp. National Trust for Nature Conservation, Annapurna 96 . Conservation Area Project, Upper Mustang Biodiversity Biswas T.D. and Mukherjee S.K. 1999. Text Book of Soil Conservation Project. Pp. 196. Science. 2nd ed. New Delhi, India: Tata McGraw-Hill Chhetri D.R., Basnet D., Chiu P.F., Kalikotay S., Chhetri G. Publishing Company Limited. Pp. 433 . and Parajuli S. 2005. Current Status of Ethnomedicinal

76 J. Mt. Sci. (2009) 6: 66–77

Plants in the Darjeeling Himalaya. Current Science 89: Ohba H., Iokawa Y. and Sharma L.R. (eds). 2008. Flora of 264-268. Mustang, Nepal. Tokyo, Japan: Kodansha Scientific Ltd., Department of Forest. 2004. Our Forest. Kathmandu, Nepal: Shinjuku-ku. Pp. 506. Department of Forest, Ministry of Forest and Soil Ôke T.R. 1987. Boundary Layer Climate. New York, USA: Conservation. Pp. 211. (In Nepali) Metheuen and Co. Pp. 435. Dhar U., Rawal R.S. and Upreti J. 2000. Setting Priorities for Olsen C.S. 2005. Trade and Conservation of Himalayan Conservation of Medicinal Plants – a Case Study in the Medicinal Plants: Nardostachys grandiflora DC. and Indian Himalaya. Biological Conservation 95: 57-65. Neopicrorhiza scrophulariiflora (Pennell) Hong. Biological Egli M., Mirabella A., Sartari G., Zanelli R. and Bischof S. Conservation 125: 505-514. 2006. Effect of North and South Exposure on Weathering Olsen C.S. and Larsen H.O. 2003. Alpine Medicinal Plant Rate and Clay Mineral Formation in Alpine Soil. Catena 67: Trade and Himalayan Mountain Livelihood Strategies. The 155-174. Geographical Journal 169: 243-254. Gao L., Wei X. and Yang L. 2004. A New Diterpenoid Pérez F.L. 1998. Conservation of Soil Moisture by Different Alkaloid from a Tibetan Medicinal Herb Aconitum Stone Covers on Alpine Talus Slopes (Lassen, California). naviculare Stapf. Journal of Chemical Research No. 4: 307- Catena 33: 155-177. 308. Pohle P. 1990. Useful Plants of Manang Districts: A Ghimire S.K., Sah J.P., Shrestha K.K. and Bajracharya D. Contribution to the Ethnobotany of the Nepal Himalayas. 1999. Ecological Study of some High Altitude Medicinal Stuttgart, Germany: Nepal Research Center Publication No and Aromatic Plants in the Gyasumdo valley, Manang, 16, Franz Steiner Verlag Wiesbaden GmbH. Pp. 65. Nepal. Ecoprint 6: 17-25. Polunin O. and Stainton A. 2000. Flowers of the Himalayas. Ghimire S.K., McKey D. and Aumeeruddy-Thomas Y. 2005. 4th Impression. New Delhi, India: Oxford University Press. Conservation of Himalayan Medicinal Plants: Harvesting 518 p + 128 plates. Patterns and Ecology of two Threatened Species, Schippmann U., Leaman D.J. and Cunningham A.B. 2002. Nardostachys grandiflora DC. and Neopicrorhiza Impact of Cultivation and Gathering of Medicinal Plants on scrophulariiflora (Pennell) Hong. Biological Conservation Biodiversity: Global Trends and Issues. In: FAO (ed.) 124: 463-475. Biodiversity and the Ecosystem Approach in Agriculture, Grunwald J. and Buttel K. 1996. The European Forestry and Fisheries. Satellite Events on the Occasion of Phytotherapeuticc Market. Drug Made in Germany 39:6-11. the Ninth Regular Session on the Commission on Genetic Gupta PK. 2000. Methods in Environmental Analysis: Water, Resources for Food and Agriculture, Interdepartmental Soil and Air. New Delhi, India: Agrobios (India). Pp. 409. Working Group on Biological Diversity. October 12-13, Hagen T. 1969. Report on the Geological Survey of Nepal. Vol 2002 Rome. Pp. 21. 1: Preliminary Reconnaissance. Zurich, Switzerland: Shrestha B.B., Jha P.K. and Gewali M.B. 2007a. Denkschrift der Schweizerischen Naturforschenden Ethnomedicinal Use and Distribution of Aconitum Gesellschaft 86/1. Orell Füssli. 185 p. + 5 plates + 136 naviculare (Bruhl) Stapf. in upper Manang, Nepal. In: RP figures. Chaudhary, TH Aase, OR Vetaas and BP Subedi (eds.) Hamilton A. 2003. Medicinal Plants and Conservation: Issues Local Effects of Global Changes in the Himalayas: Manang, and Approaches. Surrey, UK: International Plant Nepal. Kathmandu: Tribhuvan University (Nepal) and Conservation Unit, WWF-UK. Pp. 51. University of Bergen (Norway). Pp. 171-181. IUCN Nepal. 2004. A Review of the Status and Threats to Shrestha B.B., Ghimire B.K., Lekhak H.D. and Jha P.K. 2007b. Wetlands of Nepal. Kathmandu: IUCN Nepal. Pp. 80. Regeneration of Treeline Birch (Betula utilis D.Don) Forest Kala C. 2000. Status and Conservation of Rare and in a Trans-Himalayan Dry Valley in Central Nepal. Endangered Medicinal Plants in the Indian Trans- Mountain Research and Development 27: 259-267. Himalaya. Biological Conservation 93: 371-379. doi:10.1659/ mrdd.0784. Kala C. 2005. Indigenous Uses, Population Density and Shrestha B.B., Gewali M.B. and Jha P.K. 2007c. Ecology of Conservation of Threatened Medicinal Plants in Protected Neopicrorhiza scrophulariiflora (Pennell) Hong. Growing Areas of the Indian Himalayas. Conservation Biology 19: under Different Land Uses in a Trans-Himalayan Dry 368-376. Valley of Central Nepal. International Journal of Ecology Körner Ch. 2003. Alpine Plant Life: Functional Plant Ecology and Environmental Sciences 33: 233-241. of High Mountain Ecosystems. 2nd edition. Berlin SPSS 2002. Statistical Package for Social Sciences (SPSS), Heidelberg: Springer-Verlag. Pp. 344. version 11.5, USA: SPSS Inc. Körner Ch. 2007. Climate Treelines: Conventions, Global Stainton A. 1997. Flowers of the Himalayas: A Supplement. Patterns, Causes. Erdkunde 61: 316-324. Delhi, India: Oxford University Press. 86 p+128 plates Lama Y.C., Ghimire S.K. and Aumeeruddy-Thomas Y. 2001. Uniyal S.K., Kumar A., Lal B. and Singh R.D. 2006. Medicinal Plants of Dolpo: Amchis' Knowledge and Quantitative Assessment and Traditional Uses of High Conservation. Kathmandu: WWF Nepal Program. Pp. 150. Value Medicinal Plants in Chhota Bhangal Area of Larcher W. 1995. Physiological Plant Ecology, 3rd ed. Berlin Himanchal Pradesh, Western Himalaya. Current Science (Germany): Springer Verlaag. Pp. 493. 91: 1238-1242. Manandhar N.P. 2002. Plants and People of Nepal. Oregon, Vetaas O.R. 2007. Global Change and its Effect on Glaciers USA: Timber Press Inc. Pp. 599 . and Cultural Landscape: Historical and Future MFSC. 2002. Nepal Biodiversity Strategy. Kathmandu, Nepal: Considerations. In: RP Chaudhary, TH Aase, OR Vetaas Ministry of Forest and Soil Conservation (MFSC), His and BP Subedi (eds.) Local Effects of Global Changes in the Majesty’s Government of Nepal. Pp. 170. Himalayas: Manang, Nepal. Kathmandu: Tribhuvan Miehe G., Winiger M., Böhner J. and Zhang Y. 2001. The University (Nepal) and University of Bergen (Norway). Pp. climate diagram map of High Asia: purpose and concepts. 23-39. Erdkunde 55: 94-95. Yimer F., Ledin S. and Abdelkadir A. 2006. Soil organic Mulliken T. 2000. Implementing CITES for Himalayan Carbon and Total Nitrogen Stocks as Affected by Medicinal Plants Nardostachys grandiflora and Picrorhiza Topographic Aspect and Vegetation in the Bale Mountains, kurrooa. TRAFFIC Bulletin 18: 63-72. Ethiopia. Geoderma 135: 335-344.

77 Fitoterapia 80 (2009) 279–282

Contents lists available at ScienceDirect

Fitoterapia

journal homepage: www.elsevier.com/locate/fitote

Two phenolic glycosides from Curculigo orchioides Gaertn

Stefano Dall'Acqua a,⁎, Bharat Babu Shrestha b, Stefano Comai a, Gabbriella Innocenti a, Mohan Bikram Gewali c, Pramod Kumar Jha b a Department of Pharmaceutical Sciences, University of Padova, Via Marzolo 5, 35131 Padova, Italy b Central Department of Botany, Tribhuvan University, Kirtipur, Kathmandu, Nepal c Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal article info abstract

Article history: One new glycoside derivative from syringic acid and one new phenol glycoside, curculigoside E Received 23 December 2008 (1) and orchioside D (2), were isolated and characterized from the rootstock of Curculigo Received in revised form 23 February 2009 orchioides collected in the Nawalparasi District (Nepal). The structures of the new isolated Accepted 3 March 2009 compounds were elucidated by means of spectroscopic methods such as 1D, 2D NMR and MS. Available online 11 March 2009 © 2009 Elsevier B.V. All rights reserved.

Keywords: Curculigo orchioides Nepal Ethnomedicine Isolation

1. Introduction Nepal, we undertook the phytochemical work on the ground parts of C. orchiodes collected in the Nawalparasi District, Curculigo orchioides is a medicinal herb distributed central Nepal. In this paper, we describe the isolation and throughout Nepal from tropical to subtropical regions [1–3]. structural elucidation of one new glycoside derivative from It is also diffused in Japan, China, Malaysia, India and syringic acid and one new phenol glycoside, curculigoside E Australia. Dried rhizomes are used as tonic in the traditional (1) and orchioside D (2), from the rootstocks of C. orchioides. Chinese medicine [2]. In Nepal, the rootstocks of C. orchioides have several ethnomedicinal uses such as aphrodisiac and 2. Experimental tonic as well as against asthma and jaundice [1,2]. Secondary metabolites so far isolated and characterized from ground 2.1. General parts mainly belong to three classes: glycosylated phenolics, triterpenic saponins and aliphatic ketones. Several phenolic Optical rotation was performed on a Jasco P2000 digital glycosides of syringic acid, 2,6-dimethoxybenzoic acid and polarimeter and UV spectra on a Perkin Elmer lambda 25 orcinol (5-methyl resorcinol) derivatives have been pre- spectrophotometer. NMR spectra were obtained with a Bruker viously reported [1–4], and dichloro and methoxy derivatives AMX 300 spectrometer in CD3OD. HR-MS spectra were of the orcinol were also isolated [5]. Moreover orchioside A, recorded on a Mariner Biosystem API-TOF spectrometer. HPLC curculigosides A–D [5–9], cycloartane type derivatives [10] separations were carried out with an Agilent 1100 series liquid and other saponins [5,11–13] have also been obtained from C. chromatograph equipped with a Diode array detector. Semi- orchiodes. Phytochemical studies seem to be lacking in the preparative HPLC was obtained on a Gilson 305–306 series Nepali specimen of this medicinal plant. As a part of our equipped with a Perkin Elmer spectrophotometer using a ongoing collaborative work on the medicinal plants from Merck Lichrocart C-18 10 μm (10×250 mm, ID). GC-MS analysis were performed on a Varian Saturn 2000 GC equipped with ion ⁎ Corresponding author. trap MS detector, using a fused-silica-capillary column (DB-5, E-mail address: [email protected] (S. Dall'Acqua). 30 m×0.25 mm×0.25 μm ﬁlm thickness, J&W Scientiﬁc,

1 13 Folsom, CA, USA). Temperature of the injector was set to 220 °C, 789.2453); HNMR(CD3OD, 300 MHz) and CNMR(CD3OD, temperature gradient was as follows: 120 °C for 2 min, then to 75 MHz) see Table 1. 25 180 °C at 5 °C/min, kept at 180 °C for 10 min and then to 300 °C Orchioside D (2) (yield 0.002%): White powder; [α] D- at 10 °C/min. 14.4 (c 1.04, EtOH); HRAPITOF m/z 459.1307 [M–H]−, 1 (calculated for C23H24O10–H, 459.1291); H NMR (CD3OD, 13 2.2. Plant material 300 MHz) and C NMR (CD3OD, 75 MHz) see Table 1.

Rootstocks of C. orchioides Gaerten (Hypoxidaceae) were 3. Results and discussion collected in Nawalparasi District (500 m asl), central Nepal, in

August, 2006, and were authenticated by one of the authors Compound 1 had a molecular formula of C34H46O21 as (BB Shrestha). The herbarium specimen of the plant has been deduced from the HRAPIMS. The UV spectrum showed deposited at Tribhuvan University Central Herbarium (TUCH), maximum at 265 nm and a shoulder at 300 nm consistent Kathmandu. with the presence of phenol derivative. The 1H NMR spectrum revealed the presence of a singlet at δ 7.34 integrating for two 2.3. Extraction and isolation protons. Diagnostic HMBC correlations were observed from the singlet at δ 7.34 with carbon resonances at δ 172.6 (C-9), Ground dried parts of C. orchioides (400 g) were extracted 140.0 (C-4) and 154.3 (C-3 and C-5) supporting the structure with methanol at room temperature. The yield of extraction of a 4-hydroxy-3,5-dimethoxybenzoic acid moiety. In addi- was 10% on the basis of dried plant material. The solvent was tion, two doublets at δ 6.41 (J=8.0) and 6.28 (J=1.8) and removed under vacuum and the residue was used for the one doublet of doublets at δ 6.37 integrating one proton each phytochemical analysis. Methanol extract (15 g) was dis- and one singlet at δ 2.13 integrating for three protons were solved in 2000 mL of a mixture methanol/water (1/9) and observed in the 1H NMR. Long-range HMBC correlations were partitioned with petroleum ether (3 times×500 mL each) observed from both the signals at δ 6.28 and δ 6.37 with and chloroform (3 times×500 mL each). The solvents were removed under vacuum. The volume of the aqueous layer was Table 1 reduced by evaporation under vacuum followed by freeze- 1 13 H NMR and C NMR data for compounds 1 and 2 (300 and 75 MHz, CD3OD, drying yielding a solid residue of 6 g. δ in ppm , number in parenthesis are J in Hz). The residue (2 g) was applied on a silica gel column (250 mL) and eluted with a mixture of chloroform/metha- Compound 1 Compound 2 nol/water (10/5/1). Eluted fractions were collected on the C δH δCCδH δC basis of their chromatographic behaviour in four groups. 1 – 128.5 1 4.64 d (10.2) 76.1 Group 3 (450 mg) was subjected to semipreparative HPLC 2 7.34 s 108.9 2 4.00 m 82.3 – yielding in the isolation of compounds 1 (6.0 mg) and 2 3 154.3 3 2.28 dd (16.9; 5.46) 22.7 4 – 140.0 2.58 dd (16.9; 4.48) (7.9 mg). Mobile phases were aqueous formic acid (0.1%) (A) 5 – 154.3 4 – 85.6 and acetonitrile (B), and were eluted with the following 6 7.34 s 108.9 5 – 84.6 gradient: starting with 90% A, 10 min isocratic, and then to OCH3 3.88 s 57.8 1′– 133.7 ′ 80% A in 65 min. The ﬂow rate was 2.5 mL/min and was OCH3 3.88 s 57.8 2 6.88 d (1.80) 116.0 – ′– monitored at the wavelength of 230 nm. Purity of isolated 9 172.6 3 147.7 1′– 160.9 4′– 147.2 N compounds was checked by HPLC and was 98% on the basis 2′– 159.7 5′ 6.68 d (8.11) 116.9 of relative integration. For the determination of sugar residue, 3′ 6.28 d (1.8) 110.0 6′ 6.72 m 120.4 the compounds were hydrolyzed (0.5 mL of 3M TFA, 100 °C, 4′– 140.3 1″– 134.0 5′ 6.37 dd (1.8;8.0) 111.7 2″ 6.79 d (1.90) 120.3 2 h) and then evaporated to dryness under N2. The residue 6′ 6.41 d (8.0) 110.8 3″– 147.8 was dissolved in methanol and treated with NaBH4 (5 mg). 7′ 2.13 s 22.2 4″– 146.4 Acetic acid was added (0.5 mL) and the solvents were dried GlcI:1′ 5.04 d (7.5) 105.0 5″ 6.64 d (9.0) 116.3 ′ ″ under N2. The residue was treated with Ac2O (0.5 mL, 100 °C, 2 3.52 dd (8.4;7.5) 74.9 6 6.74 m 125.3 1 h). The mixture was cooled in an ice bath, diluted with 3′ 3.39 m 77.1 1‴ 4.58 d (7.43) 102.8 ′ ‴ water and extracted with ethyl ether. The ether phase was 4 3.74 m 71.2 2 3.31 m 74.1 5′ 3.51 m 76.7 3‴ 3.40 m 77.5 used for GC analysis. The acetylated sugars were compared 6′ 4.13 dd (11.8; 2.0) 69.8 4‴ 3.28 m 71.8 with authentic sample of glucose. For the determination of 3.86 dd (11.8; 5.1) 5‴ 3.34 m 77.8 the absolute conﬁguration of the glucose, a sample of each GlcII:1″ 4.46 d (7.5) 104.1 6‴ 3.86 dd (11.25;1.50) 62.7 ″ hydrolyzed compound was treated with R-2-butanol and AcCl 2 3.21 dd (9.00; 7.5) 74.9 3.64 dd (11.25; 4.05) 3″ 3.40 m 77.4 (0.1 mL) at room temperature for 24 h. The solvent was 4″ 3.19 m 72.1 evaporated and the sample was acetylated as previously 5″ 3.30 m 78.0 described. The retention time of the R-2-butanol ester 6″ 3.77 dd (11.2; 1.59) 62.1 acetylated derivative was compared with the one of standard 3.77 dd (11.2; 4.4) GlcIII:1‴ 4.80 d (7.7) 110.5 D-glucose derivative [14]. ″ β → β 2 3.51 dd (10.5;7.7) 74.6 3,5-Dimethoxy-4-O-[ -D-glucopyranosyl-(1 6)- -D- 3″ 3.61 m 77.1 glucopyranoside]-benzoic acid 5-methyl-2-O-β-D-glucopyr- 4″ 3.40 m 72.7 anoside-phenyl ester (curculigoside E) (1) (yield 0.015%). 5″ 3.25 m 77.7 25 6″ 3.72 dd (11.1; 2.0) 62.2 Colorless amorphous solid; [α] D-27.3 (c 1.10, EtOH); − 3.66 dd (11.1; 5.5) HRAPITOF m/z 789.2445 [M–H] ,(calculatedforC34H46O21–H, S. Dall'Acqua et al. / Fitoterapia 80 (2009) 279–282 281

carbon resonance at δ 22.2, supporting the presence of a Compound 2 had a molecular formula of C23H24O10 as methyl group linked to the aromatic ring. Further long-range determined from the HRAPIMS. The UV spectrum showed correlations were observed from the signal at δ 6.28 with the two maxima at 290 and 250 nm consistent with the structure carbon at δ 160.9 and 159.7 and from the one at δ 6.37 with δ of a phenol derivative. The 1H NMR showed two meta coupled 160.9 supporting the presence of two oxygenated positions. doublets at δ 6.79 (J=1.90) and 6.88 (J=1.80), two ortho Thus the second aromatic ring was deduced to be a 1,2 dioxy- coupled doublet at δ 6.64 (J=9.00) and δ 6.68 (J=8.11) and 4-methylbenzene. Furthermore, the signals of the anomeric two overlapping signals at δ 6.72 and 6.74 this latter coupling protons at δ 5.04 (J=7.5), 4.80 (J=7.7) and 4.46 (J=7.5) with the one at δ 6.64 in COSY spectrum. These data along supported the presence of three sugar moieties. Complete with pertinent HMQC and HMBC correlations revealed the sugar assignments were obtained by a combination of COSY, presence of two 1,3,4 trisubstituted aromatic ring. Further- TOCSY and HMQC data (See Table 1). These data were more, long-range (HMBC) correlations from H-2′ (δ 6.88) and consistent with the presence of three β-glucose moieties. In H-5′ (δ 6.68) with carbon resonances at δ 147.2 and 147.7 and addition the identity of sugar residues were confirmed by from H-2″(δ 6.79) and H-5″(δ 6.74) with carbon resonances at hydrolysis followed by GC analysis. One of the glucose unit δ 146.4 and 147.8 allowed us to establish the presence of was found to have glycosidic linkage at position 6 based on hydroxyl groups in the positions 3 and 4 of both aromatic the chemical shift of C-6′ (δ 69.8). Other glycosidation rings. From the COSY spectrum correlations were found positions were determined by 2D NMR experiments. Diag- between proton signals at δ 4.64 with δ 4.00 and 2.28–2.58. nostic HMBC correlation was observed from the signal at δ HMBC correlations were found between H-2′ and H-6′ with 5.04 and carbon resonance at δ 140.0, as well as NOESY C-1 revealing a first linkage between the aromatic ring and correlation was observed from the same proton signal (H-1′ the aliphatic chain of the compound. In addition, long-range glc-I) and the methoxy group (δ 3.88) supporting the linkage correlations were observed from the H-3 (δ 2.28–2.58) with between glc-I and C-4. Further long-range correlation was quaternary carbon resonances at δ 85.6 and 84.6 and from the observed from the proton signal at δ 4.46 (H-1″ glc-II) with H-2″ and H-6″ (δ 6.72) with C-5 (δ 84.6) supporting the carbon resonance at δ 69.8 (C-6′ glc-I) supporting the 1→6 presence of the triple bond in the positions 4–5 and the linkage between glc-II and glc-I. Furthermore, NOESY correla- linkage of the penta atomic chain with the second aromatic tion was observed between the H-1‴ glc-III (δ 4.80) and ring. The COSY, NOESY and HMQC data, as well as hydrolysis proton at δ 6.41 (H-6′). HMBC correlation between the experiments, supported the presence of a D-glucose unit. In same anomeric proton and carbon at δ 160.9 was also found addition, the coupling constant of the anomeric proton signal thus establishing all the glycosidation positions in the (J =7.43) is indicative of β configuration. Furthermore molecule. With all these evidences, compound 1 was diagnostic HMBC correlations between H-1 (δ 4.64) and C- identified as 3,5-Dimethoxy-4-O-[β-D-glucopyranosyl- 2‴ (δ 74.1) and between H-2 and C-1‴ (δ 102.8) revealed the (1→6)-β-D-glucopyranoside]-benzoic acid 5-methyl-2-O-β- glycosidation positions. NOESY correlations (see Fig. 1) were D-glucopyranoside-phenyl ester. The isolated compound was observed between H-2 and H-1‴ supporting the same named curculigoside E. orientation for this two protons. Furthermore, NOESY cross

Fig. 1. Structures of isolated compounds. Diagnostic NOESY correlations for compounds 1 and 2 are indicated by arrows. 282 S. Dall'Acqua et al. / Fitoterapia 80 (2009) 279–282 peaks were observed from H-1 and H-3 indicating the trans References orientation for H-1 and H-2. On the basis of the obtained data and the literature for similar compounds [7–9,13] the relative [1] Kubo M, Namba K, Nagamoto N, Nagao T, Nakanishi J, Nishimura H. fi Planta Med 1983;47:52. con guration of H-1 and H-2 was established as trans. Our [2] Shrestha BB, Dall'Acqua S, Gewali MB, Jha PK, Innocenti G. Biology and spectral data are very similar to those previously published by phytochemistry of Curculigo orchioides Gaertn. In: Jha PK, Karmacharya Li et al. about crassifoside B [15]. Significant differences were SB, Chettri MK, Thapa CB, Shrestha BB, editors. Medicinal plants in δ Nepal: an anthology of contemporary research. Kathmandu, Nepal: observed for the chemical shifts of H-1 and H-2 ( 4.81 and Ecological Society (ECOS); 2008. p. 50–67. 4.56 in crassifoside B and δ 4.64 and 4.00 in our compound) as [3] Wu Q, Fu DX, Hou AJ, Lei GQ, Liu ZJ, Chen JK, et al. Chem Pharm Bull well as in the chemical shift of C-2 (δ 75.4 in crassifolide B and 2005;53:1065. δ 82.3 in our compound) and in the H-1‴ (δ 4.83 vs 4.58) [15]. [4] Valls J, Richard T, Larronde F, Leblais V, Muller B, Delaunay JC, et al. Fitoterapia 2006;77:416. Compounds with similar structure were reported from C. [5] Garg SN, Misra LN, Agarwal SK. Phytochemistry 1989;28:1771. orchioides [6] as well as from its other species such as C. [6] Chen C, Ni W, Mei W. Acta Bot Yunnanica 1999;21:521. [7] Gupta M, Achari B, Pal BC. Phytochemistry 2005;66:659. recurvata [8], C. pilosa [9] and C. crassifolia [15]. To the best of fi fi [8] Chifundera K, Messana I, Galef C, De Vincente Y. Tetrahedron our knowledge, this is the rst report of compound 2,an 1991;47:4369. isomer of crassifoside B [15], from the natural source. We have [9] Palazzino G, Galeffi C, Federici E, Delle Monache F, Cometa MF, Palmery named compound 2 as orchioside D. M. Phytochemistry 2000;55:411. [10] Misra TM, Singh RS, Tripathi DM. Phytochemistry 1984;23:2369. [11] Xu J, Xu R, Li X. Planta Med 1991;58:208. Acknowledgments [12] Xu J, Xu R. Phytochemistry 1992;31:2455. [13] Xu J, Xu R, Li X. Phytochemistry 1992;31:233. [14] Li N, Chen J, Zhou J. Helv Chim Acta 2004;87:845. This work has been supported by M.I.U.R. Field work by BB [15] Vinogradov E, Nossova L, Korenevskt A, Beveridge TJ. Carbohydr Res Shrestha was supported by Volkswagen Foundation (Germany) 2005;340:1750. project in Nepal. Life History and Population Status of the Endemic Himalayan Aconitum naviculare Author(s): Bharat Babu Shrestha and Pramod Kumar Jha Source: Mountain Research and Development, 30(4):353-364. 2010. Published By: International Mountain Society DOI: 10.1659/MRD-JOURNAL-D-10-00003.1 URL: http://www.bioone.org/doi/full/10.1659/MRD-JOURNAL-D-10-00003.1

BioOne (www.bioone.org) is an electronic aggregator of bioscience research content, and the online home to over 160 journals and books published by not-for-profit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.

BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Mountain Research and Development (MRD) MountainResearch An international, peer-reviewed open access journal Systems knowledge published by the International Mountain Society (IMS) www.mrd-journal.org

Life History and Population Status of the Endemic Himalayan Aconitum naviculare

Bharat Babu Shrestha* and Pramod Kumar Jha * Corresponding author: [email protected] Central Department of Botany, Tribhuvan University, Kirtipur, PO Box 5275, Kathmandu, Nepal

Open access article: please credit the authors and the full source.

Sustainable management species. Plant height and petiole length were higher in of rare medicinal plants is individuals growing within juniper scrub, whereas tuber mass, becoming a major flowers/plant, and seed/follicle were higher in open areas. conservation issue in the Reproductive outputs were determined by the growth vigor of Himalaya, and the need to individual plants and associated species, and not by population consider population size. Stem mass and above-ground biomass declined with status and life history elevation, whereas density increased with relative radiation strategies for sustainable index. Soil attributes could not explain the variation in life management of these history traits and abundance. Associated shrubs reduced the plants has been pressure of human collection and destructive effects of animal expressed. We sampled Aconitum naviculare, an endemic grazing. In conclusion, a plant’s life history and responses to Himalayan medicinal plant, to study life history strategies and different natural environments can explain the variation in abundance across 6 sampling sites in Manang Valley, central abundance of rare species such as A. naviculare. Nepal. The relationship among environmental variables, life history traits, and abundance was analyzed by using regressions. Seed germination, growth characters, Keywords: Alpine; medicinal plants; conservation; reproductive output, and population density varied environmental variables; facilitation; germination; growth significantly across the sites; most of these were lowest at variation; Nepal. Khangsar, a site located at the highest elevation. Growth characters were largely governed by life forms of associated Peer-reviewed: March 2010 Accepted: August 2010

Introduction spp have been categorized as critically endangered in their natural habitats (CAMP 1998), but availability and Given their minimal access to modern health care systems, life history strategies of this taxon have not been studied people in the high mountains of Nepal have traditionally in Nepal Himalaya. Aconitum naviculare, endemic to used .50% of locally available plant species for health care Himalaya (Stainton 1997), is found from Bhutan to Nepal, (Kunwar and Bussmann 2008). In addition, trade in including southern Tibet (Grierson and Long 1984; medicinal plants collected from the wild has been an Liangqian and Kodata 2001; Ohba et al 2008). This plant integral part of the livelihood of mountain people (Olsen species has been used in Tibetan medicine against colds, and Larsen 2003), whereas market-driven collection of fever, and headache (Lama et al 2001; Bhattarai et al 2006; medicinal plants from the wild is largely unsustainable Shrestha et al 2007b); it is the most preferred species in (Ghimire et al 2005). Although some medicinal plants in the ethnomedicine in Manang, a trans-Himalayan dry valley Himalaya have been threatened because of unsustainable in central Nepal (Shrestha et al 2007b). Any decline in the harvest from the wild for trade (Kala 2000; Ghimire et al abundance of this species could have adverse effects on 2005), others are naturally rare, and populations have been the local health care system in Manang, where people decliningevenwhentheyhavebeenusedonlyfor have little access to modern medicine. traditional health care (Kala 2005). Understanding life In this article, we discuss germination, growth history and demography is essential for developing a characters, reproductive output, and the plant’s scientific management plan (Murray et al 2002). However, adaptation to alpine environments to explain the except for a few high-profile taxa, population details are distribution and abundance of this species in Manang largely lacking for most Himalayan medicinal plants (Dhar Valley. The specific objectives of the study were (1) to et al 2000), and life history strategies have been studied only understand variation in life history characters of A. for a few species of Nepal Himalaya (Ghimire et al 2005). naviculare among the populations, (2) to understand Aconitum spp are among the 10 most traded medicinal adaptation of this species to alpine environments, and (3) plants from the mountain regions of Nepal (Olsen and to explain the abundance of the species based on the life Larsen 2003). Most of the medicinally important Aconitum history characters and anthropozoogenic pressure.

Mountain Research and Development Vol 30 No 4 Nov 2010: 353–364353 http://dx.doi.org/10.1659/MRD-JOURNAL-D-10-00003.1 ß 2010 by the authors MountainResearch

FIGURE 1 Map of Manang district showing the sampling sites. (Map by Bharat Babu Shrestha and Prem Sagar Chapagain)

Material and methods pressure from livestock grazing than the other 3 sites. There were no permanent settlement near Yak Kharka, Study site Ledtar, and Phedi, but a few hotels for tourists were The study area (28u410–28u460N, 83u570–84u040E, present. elevation 4050–4650 m) lies in the upper part of Manang district, central Nepal, which has a glacially formed U- Species studied shaped valley (3000–3400 m), traversed by the Marsyangdi A. naviculare (Bru¨ hl) Stapf. (vernacular name: ponkar/ River, and surrounded by high mountains (.6000 m) bongkar; Fam. Ranunculaceae; Figure 2A–E) is a perennial (Figure 1). The valley lies in a rain shadow area of the herb with tuberous white roots; stems ascending, 10– trans-Annapurna region, with a mean annual 30 cm; leaves mostly basal, palmately divided into 2–5 precipitation of 444 mm and temperature of 6.2uC segments; flowers in loose raceme; sepals tinged dull (measured at 3420 m, Miehe et al 2001). The area is reddish purple-violet blue, with dark purple veinlets, covered by snow from November to March. Forest is lower surface pubescent, persistent in fruits, helmet mostly confined to the moist north-facing slope of navicular; petals clawed; fruit of 5 follicles, sparsely Annapurna and the valley floor. The lower belt of the pubescent (Ohba et al 2008). north-facing slope (3000–3500 m) has Pinus wallichiana and Juniperus indica forests, whereas the upper belt (3500– Sampling procedure 4200 m) has Abies spectabilis and Betula utilis forests (4200 m) We sampled 240 quadrats (1 m 3 1 m) lying within 30 (Shrestha et al 2007a). On the southern slope, there is large plots (10 m 3 10 m) during September 2005. Each Juniperus indica forest up to 3800 m, whereas the alpine large plot was divided into 4 quarters (5 m 3 5 m), and 2 scrub has junipers (Juniperus indica, Juniperus recurva, quadrats were studied in each quarter. In each quadrat, Juniperus squamata), Rhododendron lepidotum, Rosa spp, individuals of A. naviculare were counted to determine Berberis spp, Caragana gerardiana, Ephedra gerardiana, density (pl/m2). Major associated shrub species were Cotoneaster microphyllus, etc. recorded. Because of patchy distribution of A. naviculare, The sampling sites represented at least three fourths sampling plots were subjectively located at localities of the total area where A. naviculare occurred in the study where the species was relatively common and covered an area (Table 1). There were a few sites that were not easily area .100 m2. Thus, calculated density should be accessible. The Ice Lake area, Khangsar, and Manang hill considered as the maximum density in its habitat. To are nearer to the settlements and experience greater determine above-ground biomass, all the individuals of A.

Mountain Research and Development 354 http://dx.doi.org/10.1659/MRD-JOURNAL-D-10-00003.1 MountainResearch

TABLE 1 Some features of the sampling sites.a)

Elevation Associated shrub Sites WD (m) Slope (u) Aspect (u) RRI species

Ice Lake 3 4400–4550 32 6 3 193 6 20 SW 0.980 6 0.015 Caragana gerardiana Royle

Manang 2 4150–4450 30 6 8 237 6 37 SW 0.859 6 0.107 Spiraea arcuata Hook. f., Rosa spp, Berberis spp, Ephedra gerardiana Wall. ex. Stapf

Khangsar 2 4400–4650 29 6 7 208 6 50 SW 0.908 6 0.149 Juniperus squamata Buch. – Ham. ex. D.Don, Cotoneaster microphyllus Wallich ex. Lindley

Yak 4 4090–4270 36 6 1 239 6 21 SW 0.847 6 0.077 Spiraea arcuata Kharka Hook. f., Berberis spp, Cotoneaster microphyllus Wallich ex. Lindley

Ledtar 5 4200–4500 28 6 5 240 6 24 SW 0.873 6 0.093 Juniperus squamata Buch. – Ham. ex. D.Don, Spiraea arcuata Hook. f., Berberis spp

Thorong 6 4350–4450 38 6 7 108 6 13 SE 0.778 6 0.073 Spiraea arcuata Phedi Hook. f., Berberis spp

Mean*/ 4090–4650** 30 6 6* 227 6 42 SW* 0.870 6 0.098* – range** a) WD, walking distance in hours from traditional settlements; RRI, relative radiation index.

naviculare (excluding seedlings and saplings) were height, petiole length) and reproductive outputs (number collected from the single most densely populated quadrat of flowers borne by each individual plant (flowers/plant) within the large plot. Of 30 large plots, we did not sample as well as number of seeds in each follicle (seeds/follicle). 5 plots (2 at Manang hill and 3 at Khangsar) for above- Mature seeds were collected from each sampling site to ground biomass because of rarity of the plant at the determine seed mass and germination behavior. sampling sites. The single most vigorous individual was completely Laboratory analysis uprooted from each quarter to measure tuber and stem Air-dried plant materials were oven dried (60uC for mass. About 100 g of soil was collected from the rooting 72 hours), and the biomass was measured to 0.001 g. Soil depth (5–10 cm) of the collected plant. The plant and soil samples were air-dried in shade under laboratory samples were air-dried under shade. At each site, the area conditions, and the dried soil was passed through a fine of occupancy of A. naviculare was visually estimated. sieve (0.5 mm). Soil organic carbon (OC; Walkley and Because of unequal population size, the number of plots Black method) and total nitrogen (N; micro-Kjeldahl studied was not equal; 3 plots were sampled in the Ice method) were determined by following the methods Lake area, 5 each at Manang and Khangsar hills, and 17 at described by Gupta (2000). Ledtar. We did not sample the Yak Kharka and Thorong Phedi areas for density and above-ground biomass during Germination experiment: The seeds were stored at 4uC the first visit because there had been heavy collection of before germination. Germination experiments were this plant before we reached the area. During the second carried out 3 times: after 10, 15, and 20 weeks of seed visit in October 2006, we sampled individual plants again collection. Each time, 100 seeds were selected from each to measure growth characters (tuber and stem mass, plant sampling site and divided randomly into 5 groups of 20

Mountain Research and Development 355 http://dx.doi.org/10.1659/MRD-JOURNAL-D-10-00003.1 MountainResearch

FIGURE 2 A. naviculare. (A) Flowering individual; (B) underground tuberous root of previous and current years; (C) flower above the canopy of juniper; (D) fruits covered by persistent sepals; (E) seeds. (Photos by Bharat Babu Shrestha) and the pairs of sites were compared by multiple comparison by using the Dunnett C-test. Log and square root transformations did not improve normality, and homogeneity of variance in growth and reproductive characters; for these data nonparametric tests (Kruskal– Wallis and Mann–Whitney U-tests) were used for comparison of means across the sites. Similarly, morphological characters and reproductive output of individual plants occurring in juniper, other shrubs, and open areas were also compared by Kruskal–Wallis and Mann–Whitney U-tests. Regression analyses were done to understand the relationship among environmental variables (elevation, RRI, soil OC, and soil N), abundance of A. naviculare (density and biomass), growth (plant height, petiole length, stem mass, and tuber mass), and reproductive characters (flowers/plant, seeds/follicle, and mass per 100 seeds). The data were checked for normality and were log transformed if necessary. As suggested by Sokal and Rohlf (1995), during log transformation, if the original values were ,1, then each of these values was multiplied by 100 to avoid negative values after transformation. All statistical analyses were done by using the Statistical Package for Social Sciences (SPSS 2002, version 11.5). seeds each. Because of the large population, 200 seeds were taken from Ledtar for germination. After the mass of each Results group was taken, the seeds were placed in petri dishes (diameter 10 cm) with double-layered blotting papers Life history saturated with distilled water. Seeds were examined every 4 days over 32 days to analyze germination. The internal A. naviculare is a perennial alpine herb with biennial environment of petri dishes was kept saturated with tuberous roots. A. naviculare growing in open areas were moisture by frequently adding distilled water. The much more branched than the individuals growing with experiment was done under laboratory conditions with junipers and other shrubs. Aerial parts of the plant died natural photoperiod (11–12 hours of light/12–13 hours of at the end of every growing season but it perennated as a dark), mid-day temperature from 16 to 22uC, and relative tuberous root with a fully developed apical bud buried in humidity of 36 to 50%. Because most of the alpine plants the soil at a depth of 2–3 cm (Figure 2B). Thus, A. need light for seed germination (Bliss 1958), the Petri naviculare is a cryptophyte, according to Raunkiaer’s dishes were placed near a window; thus, they received classification of life forms. The mother plant produced a direct sunlight for about 4 hours a day. fusiform, tuberous white root with apical bud in the first year. After winter perennation, aerial parts developed Numerical analysis from the apical bud early in the growing season after snow melt (May–June). The plant flowered from late From the values of aspect (V), slope (b), and latitude (W), September to early October. By the time fruit was mature, the relative radiation index (RRI; Oˆ ke 1987) of each large plot was calculated by using the following formula: the development of the new tuber was completed and the old one dried up. Thus, the plant was perennial with RRI~fgCosðÞ 180{V Sinb SinW zCosb CosW biennial tuberous root and annual aerial parts. In general, a single unbranched tuber is produced by each individual Population size was estimated as the product of estimated plant. There is the presence of meristematic tissue joining area of occupancy and mean density for each sampling tuber and stem; hence, these 2 parts can easily be site. detached from each other. When aerial parts were pulled Before comparison of mean of population density, during collection, the tuberous root remained in the soil. growth characters, and reproductive output among A single plant produced 1–17 flowers (Table 2). With sampling sites, the data were tested for normality the development of fruits, the sepals dried up, becoming (Kolmogorov–Smirnov test) and homogeneity of variance papery but remained persistent and covered the (Levene test). The density was normalized when square root developing fruits until dehiscence. Seeds/follicle varied transformed, but variance remained unequal. Therefore, the from 4 to 16 (mean, 11). A single plant produced an average mean across the sites was compared by analysis of variance, of 165 seeds (3 3 5 3 11 5 165). Seeds were small (mass, 47

Mountain Research and Development 356 http://dx.doi.org/10.1659/MRD-JOURNAL-D-10-00003.1 MountainResearch

TABLE 2 Mean values of various morphological characters of A. naviculare for entire range of sampling sites.a)

Morphological traits Sample sizeb) Range Mean ± SD CVc)

Plant height (cm) 373 4–45 19 6 842

Petiole length (cm) 358 2.5–21.0 7.12 6 3.2 50

Stem mass (g/plant) 297 0.015–1.528 0.164 6 0.188 115

Tuber mass (g/tuber) 297 0.031–1.221 0.27 6 0.17 63

No. flowers/plant 395 1–17 3 6 2.5 83

No. seeds/follicle 262 4–16 11 6 2.1 19

Seed size (mg/100 seeds) 52 27–75 47 6 11 23 a) SD, standard deviation; CV, coefficient of variance. b) Sample size refers to the total number of plants or plant parts measured for the individual trait. c) CV is the SD expressed as the percentage of mean.

6 11 mg/100 seeds) and had no active mechanism of naviculare were the highest within juniper patches, whereas dispersal. At the time of seed dispersal, temperature and tuber mass/plant, flowers/plant, and seeds/follicle were the soil moisture were low, which prevented immediate highest in individuals growing in open areas. germination. Germination occurred in the next growing Among the growth and reproductive characters, season when the area became snow free in June. petiole length and seed mass increased linearly with plant height, but the tuber mass and flowers/plant showed Variation in growth and reproduction nearly unimodal relations (Figure 4). The relationship was Growth and reproductive characters differed significantly positive in 6 other pairs of characters (Figure 5); it was (P , 0.001) among the sampling sites (Figure 3). Most of the linear in 3 pairs but, in 3 other pairs, it deviated slightly characters measured were lowest at Khangsar site. The from linear relationships. greatest difference among the sites was observed in stem Stem mass/plant decreased with an increase in mass, for which the largest value (Manang hill) was 5 times elevation (linear regression, P 5 0.001, R2 5 0.37). Plant larger than the smallest one (Khangsar and Yak Kharka). A. height, petiole length, tuber mass, flowers/plant, and naviculare was generally found in association with thorny seeds/follicle did not vary significantly with elevation. We shrub species (Table 1). Quantitative measurement at were not able to find any significant relation between Ledtar showed that life forms of associated species (such as growth and reproductive characters of A. naviculare, and junipers, other shrubs, and herbs between patches of soil OC and N. shrubs) had a significant effect on the height of A. naviculare, petiole length, and flowers/plant, and a marginal Seed germination effect on tuber mass and seeds/follicle (Table 3). Average Germination after 10 and 15 weeks of seed collection was stem mass/plant was not affected by life forms of the 49% (6 35 SD, n 5 35) and 43% (6 38SD, n 5 35), associated species. Plant height and petiole length of A. respectively. Most of the seeds germinated between 8and

TABLE 3 Growth characters and reproductive output of A. naviculare in microhabitats at Ledtar.a)

Test statisticsb)

Attributes Juniper Other shrubs Open NPvalue x2

Plant height (cm) 29.5c (9.25) 19.5b (9.75) 18a (9.5) 186 ,0.001 42.7

Petiole length (cm) 13.5b (3.25) 7.0a (3.5) 6.5a (2.0) 188 ,0.001 38.3

Stem mass/plant (g) 0.090a (0.06) 0.096a (0.112) 0.116a (0.142) 187 0.312 2.3

Tuber mass/plant (g) 0.194a (0.168) 0.225b (0.176) 0.249b (0.254) 156 0.021 7.7

No. flowers/plant 2.0a (1.0) 3.0b (3.0) 4.0c (5.0) 191 ,0.001 18.1

No. seeds/follicle 10.25a (4.33) 11.14ab (2.58) 11.45b (2.05) 152 0.039 6.5 a) Main entries are medians, whereas the values in parentheses are interquartile range; values in each row with the same letter in the superscript are not significantly different at P , 0.05. b) Based on Kruskal–Wallis test; N, sample size (total number of plants or plant parts measured for each trait).

Mountain Research and Development 357 http://dx.doi.org/10.1659/MRD-JOURNAL-D-10-00003.1 MountainResearch

FIGURE 3 (A–F) Morphological characters and reproductive output of A. naviculare (1, Ice lake area; 2, Manang hill; 3, Khangsar; 4, Yak Kharka; 5, Ledtar; 6, Phedi). Main entries above bars are medians and values inside parentheses are interquartile range; values with same letter are not different significantly at P , 0.05. N 5 total sample size.

16 days after sowing, and germination of new seeds fourths of the total area of occupancy of this plant in continued until 24 days (Figure 6A). Seed germination Manang Valley, the estimated total area of occupancy could varied across the sampling sites from 17 to 97% after be about 50 ha. In Manang hill, Khangsar, and Phedi, it was 10 weeks and 6 to 99% after 15 weeks of collection found in small and fragmented patches, whereas at the Ice (Figure 6B). After 20 weeks, average seed germination Lake area, it was found only in a single patch. In Ledtar, A. declined to 7% with no germination (Khangsar site) to naviculare was found in a large area where the population 24% (Yak Kharka). Germination was epigeal (sensu Baskin density, estimated population size, and above-ground and Baskin 1998). biomass were also the highest. The density of A. naviculare was the highest at the Ice Lake area and the lowest at Population status and conservation Manang hill, the latter being at the nearest distance from The total area of occupancy of A. naviculare at sampling sites the densely populated Manang village (Table 4). Average in Manang Valley was estimated to be 40 ha (Table 4). density increased with increasing RRI (Figure 7A). No Because the present sampling sites represented nearly three relationship was detected between population density and

Mountain Research and Development 358 http://dx.doi.org/10.1659/MRD-JOURNAL-D-10-00003.1 MountainResearch

FIGURE 4 (A–D) Variation of growth characters and reproductive output of A. naviculare with height of the plant. The fitted lines, equations, and other test statistics were obtained by linear or quadratic regression analyses.

any of the soil variables measured. Above-ground biomass Seeds/follicle was the highest at Manang hill (Figure 3) of A. naviculare declined with elevation (Figure 7B). where density of A. naviculare was the lowest (Table 4). Bosch and Waser (1999) reported a positive correlation Discussion between density and seed set in A. columbianum and Delphinium nuttallianum, and the decline in seed set in Variation in growth and reproductive characters across sparse populations was attributed to receipt of inbreed the sites pollens or harsh environmental conditions. But at Growth and reproductive characters varied significantly Manang hill, despite low population density, growth vigor across the sites, which could be attributed to large was high as evident from high biomass (Figure 3, Table 4). microhabitat differences in alpine regions (Bliss 1956). The This indicates favorable physical environmental lowest value of most of the characters measured (Figure 3), conditions, and the low density could be attributed to together with the lowest seed mass (Figure 6B) and the high anthropogenic pressure. Thus, it appears that seed smallest population size (Table 4) at Khangsar, could be an set was determined by growth vigor of individual plants, indication of harsh environmental conditions, including and population density was not low enough to prevent low temperature (because of upper elevation limit of A. effective pollination, fertilization, and seed set at Manang naviculare in Manang) and high anthropogenic pressure hill. (grazing and collection). Plant height and petiole length were determined mainly by height and canopy structure of Variation in growth and reproductive output with life forms of associated species. Lower height and petiole length at associated species Khangsar, Ice Lake area, and Yak Kharka were caused by Variation in growth characters and reproductive outputs the dominance of dwarf shrub species, such as C. among the individuals of A. naviculare growing in juniper, microphyllus and C. gerardiana as associated species (Table 1). other shrubs, and open areas (Table 3) represent growth Highest flowers/plant at Ledtar (Figure 3) were caused by responses to resource availability and can be considered dominance of individuals in open areas in the population as phenotypic plasticity. The variation in plant height and of A. naviculare because individuals growing in open areas petiole length was an adaptive response to light. Plasticity produced more flowers/plant than individuals growing of shoot helps to place the leaves in areas of high light (de within patches of shrubs (Table 3). Kroon and Hutchings 1995). Herbaceous dicot plants

Mountain Research and Development 359 http://dx.doi.org/10.1659/MRD-JOURNAL-D-10-00003.1 MountainResearch

FIGURE 5 (A–F) Relationship among growth characters and reproductive output of A. naviculare. The fitted lines, equations, and other test statistics were obtained by linear or quadratic regression analyses.

growing in open habitats often respond to shading by 2003) rather than adaptive response. To position the elongating internodes and petiole length (Huber 1996), leaves and flower in the upper layer of canopy, relative and improved access to light (Falster and Westoby 2003). allocation of biomass to stem increased in shade Because of the long stem and petiole of A. naviculare conditions (Huber 1996), which could have reduced tuber growing with shrubs, the leaf lamina were exposed to full biomass of A. naviuclare growing in juniper scrubs. sunlight and flowers to pollinators. This variation allowed Relatively low stem and tuber biomass of A. naviculare A. naviculare to grow and reproduce in open areas as well growing in juniper scrub was also indicative of a stressful as with dense shrubs. environment, which was expressed as the lowest flowers/ Stem mass of A. naviculare did not vary significantly plant of A. naviculare growing in juniper scrub (Table 3). among the 3 habitats (Table 3). There appeared to be a trade-off between height and thickness of stem. In shaded Adaptation to the alpine environment conditions, the stems are thinner and longer than in full A. naviculare showed a number of adaptive features to sunlight (Huber 1996). The lowest tuber biomass of this alpine environments with strong wind. The flower did not plant growing within juniper scrubs could be the result of open completely and was globular, with a small opening the ‘‘inevitable effect of resource limits’’ (sensu Sultan on the lateral side. A globular shape has been suggested as

Mountain Research and Development 360 http://dx.doi.org/10.1659/MRD-JOURNAL-D-10-00003.1 MountainResearch

FIGURE 6 Seed germination pattern of A. naviculare. (A) Mean seed germination after 10 and 15 weeks of collection; (B) seed mass and germination. Seed mass is the mean value of 3 experiments. plants to develop seeds and fruits completely (Molau 1993). Persistent sepals could help to maintain relatively high temperature inside for rapid development of fruits and protect developing fruits from the desiccating effects of strong wind and solar radiation. Burial of perennating bud and meristem below the soil surface may prevent damage from low temperature (Ko¨ rner 2003). A. naviculare also used food reserves from the previous year for early growth during the growing season. All these characters have been considered important adaptive features of plants to alpine environments (Bliss 1962; Ko¨ rner 2003).

Seed size and germination Seed size of A. naviculare (mean, 47 mg/100 seeds 5 470 mg/ seed) was very close to mean seed size (467 mg/seed) of alpine forbs from various latitudes compiled by Ko¨ rner (2003). There was wide variation in germination of seed across the sampling sites. However, we could not detect any consistent pattern in the relationship between population size and seed germinability of A. naviculare. Some earlier studies also failed to detect the effect of population size on germination (eg Oostermeijer et al 1994; Morgan 1999). Seeds collected from Khangsar had the smallest size and lost germination after 20 weeks of storage (Figure 6B), which could be an indication of harsh environmental conditions at that site. Low seed germination at the Khangsar site, at the upper elevation limit, could be attributed to low temperature. In late flowering alpine plants like A. naviculare, low temperature may result in underdeveloped seeds with low seed mass, poor germination, and short viability (Molau 1993). an adaptation to minimize mechanical damage by strong Relatively high temperature at the Ice Lake area because wind in alpine environments (Garg and Husain 2003). The of the highest RRI among the sampling sites (Table 1) temperature inside the blue flower is higher than in allowed complete seed and fruit development in a short flowers of other colors (Bliss 1962). This could, to some period before the plant died. The habitat of A. naviculare extent, compensate the problem of the short time span at Yak Kharka represented the lower elevation limit with favorable air temperature, enabling late flowering (4090–4270 m) of distribution in upper Manang valley,

TABLE 4 Estimated area of occurrence, population density, estimated population size (number of individuals), and aboveground biomass of A. naviculare at the 6 sampling sites.a)

Area of Population density Estimated Above-ground Sites occupancy (ha) (pl/m2)b) population size biomass (g/m2)

Ice lake 2.0 8.67c 6 3.37 173,200 2.67 6 0.24

Manang Hill 5.5 4.08a 6 2.45 223,850 5.02 6 1.32

Khangsar 1.3 6.43ab 6 6.35 80,250 3.06 6 1.4

Yak Kharka 7.3 na na na

Ledtar 21.0 8.35bc 6 4.47 1,751,400 5.73 6 2.71

Phedi 3.0 na na na

Total*/mean** 40.1* 7.35 6 4.75** 2,228,700* 4.99 6 2.58** a) na, data not available. b) Values were square root transformed before performing ANOVA; values with same letter in superscript are not significantly different at P , 0.05.

Mountain Research and Development 361 http://dx.doi.org/10.1659/MRD-JOURNAL-D-10-00003.1 MountainResearch

FIGURE 7 (A, B) Relationship between mean density of A. naviculare and relative radiation index (A), and biomass and elevation (B). Fitted line, equation, and other test statistics were based on linear regression analysis. to 7.7 pl/m2 for A. violaceum. A. naviculare was found only in small areas in 3 of the 6 sampling sites (Table 4), and the population was either much fragmented (Yak Kharka) or confined to a single patch (Ice Lake area). Population density of A. naviculre increased with increasing RRI (Figure 7A); it was highest at Ice Lake area (Table 4) where RRI was the highest (Table 1). The highest density might also be because of the high seed germination at Ice Lake area (Figure 6B). Population density was the lowest in Manang hill (Table 4), which is close to the densely populated villages of Manang and Tanki Manang. The lowest density at Manang hill could be attributed to the high pressure of human collection and animal grazing. At Ledtar, A. naviculare was more commonly found in open areas than within patches of shrubs, which may indicate low animal damage. This was the least disturbed site because of its location far from traditional settlements. The great abundance (high density, above-ground biomass, and the largest population) of A. naviculare at Ledtar might be attributed to the lowest pressure of human collection and animal damage. The destructive effect of cattle grazing, and collection of aerial parts by local people before seed set, appeared to be responsible for declining abundance of A. naviculare and the very low density at some sites. The natural habitats of A. naviculare are near to human settlements, with high pressure of livestock grazing (Shrestha and Jha 2009). Large animals with wide mouths often avoid thorny shrub species (Chandrashekhar et al 2007), which are the most common associates of A. naviculare (Table 1). The population density of this species was the highest at Ice Lake area (Table 4), but which could be relatively warm. Seed development could it was only found in a single patch among a thorny shrub C. be complete there too, leading to the highest seed gerardiana, which could protect A. naviculare from damage by germination (approximately 98%) (Figure 6B). livestock and collection by humans. The protective role of shrubs is often important for the persistence of rare species Population status, harvesting pattern, and (Facelli and Temby 2002). Because A. naviculare was found conservation prospects only within alpine scrubs at the localities with high grazing Population density of Aconitum spp found in the Himalaya pressure (Ice Lake area, Manang hill, Khangsar, Yak Kharka, 2 was relatively low (,1to8pl/m)(Ghimireetal1999;Kala and Phedi), the importance of protective roles, as 2000, 2005; Nautiyal et al 2002). The mean density (7.3 pl/ hypothesized by Bertness and Callaway (1994), was high at 2 m )ofA. naviculare in the present study area was these localities. The protective role of associated scrubs 2 comparable with the mean density (6.7 pl/m )ofA. violaceum against animal damage and human collection is a kind of in the Garhwal Himalaya, India (Nautiyal et al 2002). facilitation, as proposed by Callaway (2007), and it is Among 3 Aconitum spp (Aconitum balfourii, Aconitum important for the persistence of this species in the study heterophyllum,andAconitum violaceum) found in Garhwal area. Himalaya, the highest density was recorded for A. violaceum, For sustainable management of alpine medicinal which was attributed to habitat inaccessibility and less plants in their natural habitats, the harvesting pattern and exploitation by humans (Nautiyal et al 2002). The smaller life history characters should be considered together size of Aconitum spp found in the alpine zone could also have (Ghimire et al 2005). The entire plant is used in the case of contributed to their higher density than other Aconitum spp alpine species of Aconitum (eg A. violaceum, A. naviculare, found at lower elevation. The mean density of A. naviculare Aconitum gammiei) (Baral and Kurmi 2006). Although whole in Manang was relatively high in comparison to reported plant of A. naviculare is medicinally important, people in values for other Aconitum spp. However, the present data the study area have been collecting only aerial parts at the represented the maximum density of this species because flowering stage (Shrestha et al 2007b). This method is less the sampling plots were selected subjectively. By using a destructive in terms of parts removed, but collection of similar sampling strategy, Kala (2000) reported density up the plant at flowering stage did not allow regeneration by

Mountain Research and Development 362 http://dx.doi.org/10.1659/MRD-JOURNAL-D-10-00003.1 MountainResearch

seeds. This might have a negative impact on increasing or associated species. Reproductive outputs were determined maintaining population size, as has been also reported by by growth vigor of individual plants and associated species. Ghimire et al (1999). The negative impact could be more Seed germination varied across the sites. Temperature and critical for areas with a small populations and/or low anthropozoogenic pressure appeared to be the most density, such as Ice Lake area, Manang hill, and Khangsar. important environmental factors that determine distribution and abundance of A. naviculare. The presence Conclusions of tuberous root with well-developed winter perennating bud, shoot, and leaf meristem buried inside soil, globular The growth characters and reproductive outputs of A. blue flower, and persistent sepals, and rapid germination of naviculare varied significantly across the sites; most of them seed after moistening are likely adaptive features of this were lowest at Khangsar, a site at the highest elevation, plant in alpine environments. Variation in plant height and which indicates harsh environmental conditions for this petiole length enabled this plant to grow from open areas to plant at Manang. Variation in plant height, petiole length, dense juniper scrubs. Alpine scrub seems to have protected and flowers/plant were largely governed by life forms of this plant from livestock damage and human collection.

ACKNOWLEDGMENTS

Financial support from the Volkswagen Foundation (Germany) is gratefully University, during the field study. K. Gurung, P. Sherpa, A. Pariyar, C. Gurung, acknowledged. We are thankful to Annapurna Conservation Area Project for and M. Gurung of Manang helped at various times during field sampling. We permission to work in the protected area. We acknowledge the help of Prof RP thank P. S. Chapagain of the Central Department of Geography, Tribhuvan Chaudhary and RK Sharma of the Central Department of Botany, Tribhuvan University, for preparing the map of the study area.

REFERENCES Ghimire SK, Sah JP, Shrestha KK, Bajracharya D. 1999. Ecological study of some high altitude medicinal and aromatic plants in the Gyasumdo valley, Manang, Nepal. Ecoprint 6:17–25. Baral SR, Kurmi PP. 2006. A Compendium of Medicinal Plants in Nepal. Grierson AJC, Long DG. 1984. Flora of Bhutan. Vol 1, Part 2. Edinburgh, United Kathmandu, Nepal: R. Sharma. Kingdom: Royal Botanical Garden. Baskin CC, Baskin JM. 1998. Seeds. San Diego, CA: Academic Press. Gupta PK. 2000. Methods in Environmental Analysis: Water, Soil and Air. New Bertness MD, Callaway RM. 1994. Positive interactions in communities. Delhi, India: Agrobios. Trends in Ecology and Evolution 9:191–193. Huber H. 1996. Plasticity of internodes and petioles in erect and prostrate Bhattarai S, Chaudhary RP, Taylor RSL. 2006. Ethnomedicinal plants used by Potentilla species. Functional Ecology 10:401–409. the people of Manang district, central Nepal. Journal of Ethnobiology and Kala CP. 2000. Status and conservation of rare and endangered medicinal Ethnomedicine 2:41. http://dx.doi:10.1186/1746-4269-2-41. plants in the Indian trans-Himalaya. Biological Conservation 93:371–379. Bliss LC. 1956. A comparison of plant development in microenvironment of Kala CP. 2005. Indigenous uses, population density and conservation of arctic and alpine tundras. Ecological Monograph 26:303–337. threatened medicinal plants in protected areas of Indian Himalayas. Bliss LC. 1958. Seed germination in arctic and alpine species. Arctic 11:180–188. Conservation Biology 19:368–378. Bliss LC. 1962. Adaptation of arctic and alpine plants to environmental Ko¨rner C. 2003. Alpine Plant Life. 2nd edition. Berlin, Germany: Springer-Verlag. conditions. Arctic 15:117–144. Kunwar R, Bussmann RW. 2008. Ethnobotany in the Nepal Himalaya. Journal of Bosch M, Waser NM. 1999. Effect of local density on pollination and Ethnobiology and Ethnomedicine 4:24. http://dx.doi:10.1186/1746-4269-4-24. reproduction in Delphinium nuttallianum and Aconitum columbianum Lama YC, Ghimire SK, Aumeeruddy-Thomas Y. 2001. Medicinal Plants of (Ranunculaceae). American Journal of Botany 86:871–879. Dolpo: Amchis’ Knowledge and Conservation. Kathmandu, Nepal: WWF [World Callaway RM. 2007. Positive Interactions and Interdependence in Plant Wide Fund for Nature] Nepal Program. Communities. Dordrecht, The Netherlands: Springer. Liangqian L, Kodata Y. 2001. Aconitum L. In: Zhengyi W, Raven PH, Deyuan H, CAMP. 1998. Selected Medicinal Plants of Northern, Northeastern and Central editors. Flora of China. Vol 6. Caryophyllaceae through Lardizabalaceae. Beijing, India: Conservation Assessment and Management Plans (CAMP). Lucknow, China: Science Press, pp 149–222. India: Forest Department of Uttar Pradesh. Miehe G, Winiger M, Bo¨hner J, Zhang Y. 2001. The climate diagram map of Chandrashekhar K, Rao KS, Maikhuri RK, Saxena KG. 2007. Ecological High Asia. Purpose and concepts. Erdkunde 55:94–95. implications of traditional livestock husbandry and associated land use Molau U. 1993. Relationship between flowering phenology and life history practices: A case study from the trans-Himalaya, India. Journal of Arid strategies in tundra plants. Arctic and Alpine Research 25:391–402. Environments 69:299–314. Morgan JW. 1999. Effects of population size on seed production and de Kroon H, Hutchings MJ. 1995. Morphological plasticity in clonal plants: The germinability in an endangered, fragmented grassland plant. Conservation foraging concept reconsidered. Journal of Ecology 83:143–152. Biology 13:266–273. Dhar U, Rawal RS, Upreti J. 2000. Setting priorities for conservation of Murray BR, Thrall PH, Gill AM, Nicotra AB. 2002. How plant life history and medicinal plants: A case study in the Indian Himalaya. Biological Conservation ecological traits relate to species rarity and commonness at varying spatial 95:57–65. scales. Austral Ecology 27:291–310. Facelli JM, Temby AM. 2002. Multiple effects of shrubs on annual plant Nautiyal BP, Prakash V, Bahuguna R, Maithani U, Bisht H, Nautiyal MC. 2002. communities in arid lands of South Australia. Austral Ecology 27:422– Population study for monitoring the status of rarity of three aconite species in 432. Garhwal Himalaya. Tropical Ecology 43:297–303. Falster DS, Westoby M. 2003. Plant height and evolutionary game. Trends in Ohba H, Iokawa Y, Sharma LR, editors. 2008. Flora of Mustang, Nepal. Tokyo, Ecology and Evolution 18:337–343. Japan: Kodansha Scientific Ltd. Garg A, Husain T. 2003. Strategies adopted by the alpine genus Pedicularis L. Oˆke TR. 1987. Boundary Layer Climates. New York, NY: Methuen and Co. (Scrophulariaceae) to overcome environmental stress. Current Science 85: Olsen CS, Larsen HO. 2003. Alpine medicinal plant trade and Himalayan 1413–1414. mountain livelihood strategies. The Geographical Journal 169:243–254. Ghimire SK, McKey D, Aumeeruddy-Thomas Y. 2005. Conservation of Oostermeijer JGB, van Eijck MW, den Nijs JCM. 1994. Offspring fitness Himalayan medicinal plants: Harvesting patterns and ecology of two threatened in relation to population size and genetic variation in the rare perennial species, Nardostachys grandiflora DC. and Neopicrorhiza scrophulariiflora plant species Gentiana pneumonanthe (Gentianaceae). Oecologia 97:289– (Pennell) Hong. Biological Conservation 124:463–475. 296.

Mountain Research and Development 363 http://dx.doi.org/10.1659/MRD-JOURNAL-D-10-00003.1 MountainResearch

Shrestha BB, Ghimire BK, Lekhak HD, Jha PK. 2007a. Regeneration of treeline TH, Vetaas OR, Subedi BP, editors. Local Effects of Global Changes in the birch (Betula utilis D.Don) forest in a trans-Himalayan dry valley in central Nepal. Himalayas: Manang, Nepal. Kathmandu, Nepal: Tribhuvan University, pp 171–181. Mountain Research and Development 27:259–267. Sokal RR, Rohlf FJ. 1995. Biometry. New York, NY: WH Freeman. Shrestha BB, Jha PK. 2009. Habitat range of two alpine medicinal plants in a Stainton A. 1997. Flowers of the Himalayas: A supplement. New Delhi, India: trans-Himalayan dry valley, central Nepal. Journal of Mountain Science 6:66–77. Oxford University Press. Shrestha BB, Jha PK, Gewali MB. 2007b. Ethnomedicinal use and distribution of Sultan SE. 2003. Phenotypic plasticity in plants: A case study in ecological Aconitum naviculare (Bru¨hl) Stapf. in upper Manang, Nepal. In: Chaudhary RP, Aase development. Evolution and Development 5:25–33.

Mountain Research and Development 364 http://dx.doi.org/10.1659/MRD-JOURNAL-D-10-00003.1 Tropical Ecology 52(1): 91-101, 2011 ISSN 0564-3295 © International Society for Tropical Ecology www.tropecol.com

Reproductive ecology and conservation prospects of a threatened medicinal plant Curculigo orchioides Gaertn. in Nepal

* ** BHARAT BABU SHRESTHA , PRAMOD KUMAR JHA & DHAN RAJ KANDEL

Central Department of Botany, Tribhuvan University, Kirtipur, GPO Box 5275, Kathmandu, Nepal

Abstract: Lack of biological information is a major constraint to the development of the medicinal plant sector in Nepal. We monitored phenology, population structure, and regeneration strategies of Curculigo orchioides, a threatened medicinal herb of tropical to subtropical Asia, for 1 year at five sites in the inner Terai, Central Nepal. Only 20 - 26 % of mature individuals were in the reproductive phase during the phenologically most active months (June - July), and about 55 % of flowering individuals developed fruits. Soil moisture, stored reserves, and biotic pressure appeared to govern phenological patterns. Fruiting frequency was high under conditions of a partially open canopy and a thin litter layer. Seeds showed physiological dormancy and germinated 10 - 12 months after dispersal in natural habitats. Clonal propagation from leaves was induced by mild mechanical damage, high soil moisture, and humidity. Low regenerative potential through sexual reproduction and high vulnerability to habitat disturbance appear to be the major constraints to maintaining natural populations of C. orchioides.

Resumen: La carencia de información biológica es una limitación fuerte para el desarrollo del sector de plantas medicinales en Nepal. Nosotros monitoreamos la fenología, la estructura poblacional y las estrategias de regeneración de Curculigo orchioides, una hierba medicinal amenazada de Asia tropical y subtropical, durante un año en cinco sitios en el Terai interior, centro de Nepal. Sólo 20-26 % de los individuos maduros estuvieron en fase reproductiva durante los meses más activos fenológicamente (junio-julio), y alrededor de 55 % de los individuos que florecieron produjeron frutos. La humedad del suelo, las reservas acumuladas y la presión biótica parecieron controlar los patrones fenológicos. La frecuencia de fructificación fue alta en condiciones de dosel abierto parcialmente y una capa delgada de mantillo. Las semillas mostraron latencia fisiológica y germinaron 10-12 meses después de la dispersión en los hábitats naturales. La propagación clonal a partir de hojas fue inducida por un daño mecánico ligero, y por una humedad y un contenido de agua altos en el suelo. El potencial regenerativo bajo por medio de reproducción sexual y la alta vulnerabilidad a las perturbaciones del hábitat parecen ser las limitantes principales para el mantenimiento de las poblaciones naturales de C. orchioides.

Resumo: A falta de informação biológica é um dos principais entraves para o desenvolvimento do sector das plantas medicinaisno Nepal. Assim, durante um ano e em cinco sítios no interior de Terai, no centro do Nepal, monitorizamos a fenologia, a estrutura populacional, e as estratégias de revitalização da Curculigo orchioides, uma erva medicinal ameaçada da Ásia tropical e subtropical. Apenas 20-26 % dos indivíduos adultos estavam em fase reprodutiva durante os mesesfenologicamente mais activos (Junho- Julho), e cerca de 55 %

* Corresponding Author; e-mail: [email protected] ** Present Address: Orchid Science College, Chitwan, Nepal 92 ECOLOGY OF CURCULIGO ORCHIOIDES

dos indivíduos em floração desenvolveram frutos. A humidade do solo, as reservas armazenadas, e a pressão biótica pareceram governar os padrões fenológicos. A frequência dafrutificação foi alta em condições de coberto parcialmente aberto e de uma camada fina de folhada. As sementes apresentaram dormência fisiológica e germinaram 10-12 meses após a sua dispersão em habitats naturais. A propagação clonal das folhas foi induzidaatravés dano mecânico leve, alta humidade do solo e humidade. O baixo potencial de regeneração por propagação sexuada e uma elevada vulnerabilidade à perturbação do habitat parecem ser os principais obstáculos para a manutenção de populações naturais de C. orchioides.

Key words: Clonal propagation, phenology, population decline, reproductive success, seed set.

Introduction attributed to anthropogenic factors (over exploitation, over grazing, forest floor denudation) and to In the context of increasing global interest in the plant’s own biology, including poor seed set medicinal plants as potential sources of new and low germination (Gupta & Chadha 1995; bioactive molecules (Cordell 2000; Dahanukar et Jasrai & Wala 2001). However, empirical data to al. 2000; Ji et al. 2009), conservation of medicinal support or reject these assumptions has been plants as well as of the knowledge of their uses is lacking (Shrestha et al. 2008). We report some vital for the future of human health care. Though aspects of the reproductive ecology of C. orchioides. the volume of literature on Nepalese medicinal We also report temporal and spatial patterns in plants has been increasing rapidly, most (> 50 %) phenology, regenerative strategies and population of this is related to inventory, and only a small abundance and propose explanations for patterns proportion (< 10 %) has focused on biology (Ghimire observed. The major questions we asked were: (1) 2008). Biological information such as reproductive What is the pattern of phenological activity of C. ecology, life history strategies, distribution, habitat orchioides? (2) What are the major environmental preference, and plants' responses to environmental factors that have shaped phenology and determine perturbations are essential for conservation and regeneration and population size? and, (3) Can sustainable use of ethnomedicinal plants, especially reproductive biology help us understand the causes when they are collected in large volumes from wild of population decline of this species? populations. However, this information has been lacking for most of the ethnomedicinal plants Materials and methods native to Nepal. Curculigo orchioides Gaertn. (Hypoxidaceae) is Study area a medicinal herb of tropical to subtropical regions The study area (27° 37-42´ N, 84° 13-26´ E) is a in Asia, and occurs naturally on grassy slopes and dun valley (Chitwan valley), lying in the tropical forest understories. Due to its wide range of uses region of Central Nepal. Average temperature in ethnomedicine, both in the Ayurvedic and ranges from 15 °C in January to 28.4 °C in May Chinese systems of traditional health care, a large (Fig. 1). The area receives monsoon rain from June number of phytochemical and pharmacological in- to September, which accounts for about 70 % of the vestigations have been undertaken (Shrestha et al. total annual rainfall (2427 mm). The rest of the 2008). However, biological characters of this plant year is dry with a few showers of winter rain. The have not been investigated in detail. Wild popu- area has deciduous to semi-deciduous tropical for- lation of C. orchioides are reported to be declining ests with sal (Shorea robusta Gaertn.) forest as the rapidly, and the plant has been assigned to various dominant type, and riverine Acacia-Dalbergia for- threat categories, from vulnerable (Bhattarai et al. est as a minor component (Stainton 1972). 2001) and threatened (Manandhar 2002) to endan- Five sampling sites, which were relatively less gered (Jasrai & Wala 2001; Prajapati et al. 2003; disturbed and had large populations of Curculigo Suri et al. 1999). orchioides, were chosen subjectively. Each site lies Population decline of this plant has been within a different community-managed forest, na- SHRESTHA, JHA & KANDEL 93

top of the rootstock, which generally lies near the soil surface. Inflorescence is a spike and appears almost at ground level. Flowers are yellow; the first 1-2 flowers are bisexual and the remainders are male. Fruits are fleshy and hidden in the leaf bases. Seeds are shiny black with fine striations on the surface and a hook-like projection at the proximal end. Fruits contain 1-15 seeds (average: 7 seeds/fruit, n = 263) with a seed size of 5 mg seed–1 (average of 3 measurements with 100 seeds in each). Average seed output per flowering plant per year is estimated to be 12.

Fig. 1. Average monthly rainfall and temperature of the study area from 2003-2007 recorded at Bharatpur weather station. This station lies about 15 km east of the sampling site in Nawalparasi, and about 5 km west of the sampling site at Barandabhar. (Source: Department of Hydrology and Meteorology, Govern- ment of Nepal). mely Dhuseri, Chautari, Sundari, Sansari and Barandabhar (Table 1). Barandabhar is on the lowland plain of Chitwan district, near Beeshazar lake, and is about 20 km east of the other sites.

The Barandabhar forest is a part of the buffer zone area to the north-east of Chitwan National Park and is frequently visited by wild animals from the park (e.g., rhino, deer, and wild boar). The soil of this forest is alluvial and moist, but not flooded, due to the shallow water table and the adjoining lake. The other four forests lie in the foothills of

Mahabharat range within a distance of 5 km of each other, in Nawalparasi district, where the soil is relatively dry due to the raised topography. Wild boar and peacocks are common wildlife in these Fig. 2. Curculigo orchioides. (A) mature individual forests. with flowers and fruit, (B) flowering individual tag- Study species ged for monitoring phenology, (C) rootstock with cluster of fleshy roots at the base, (D) fruits, (E) seeds, Curculigo orchioides Gaertn. (family: Hypoxi- and (F) seedling. daceae, vernacular name: Kalo musali, Fig. 2) is a perennial herb, with a vertical black rootstock at Rootstock of this plant has been used in the base of which is a bundle of white fleshy roots. traditional medicine as an aphrodisiac, a tonic to The rootstock is the perennating organ while the restore vigor, and against asthma, bronchitis, leaves and other above ground parts senesce jaundice, dysentery, diarrhoea, leucorrhoea and annually. Leaves are narrow and crowded at the male sterility (Shrestha et al. 2008). The rootstock 94 ECOLOGY OF CURCULIGO ORCHIOIDES

Table 1. Characteristics of the sampling sites.

Sampling Elevation Latitude/ Slope Soil Litter Livestock Tree Shrub sites (m asl) Longitude (°) characters cover grazing canopy canopy (%) cover cover (%) (%) Dhuseri 450 27° 41.6´ N 0-10 Brown to 55 Banned 65 45 84° 13.7´ E reddish brown; coarse to fine Chautari 660 27° 42.5´ N 10-20 Brown; 50 Goat 35 30 84° 14.2´ E abundant grazing pebbles on the surface Sundari 450 27° 42.2´ N 10-20 Reddish brown; 64 Banned 65 55 84° 16.0´ E fine Sansari 425 27° 42.3´ N 20-30 Brown; coarse 27 Goat 30 65 84° 16.7´ E to fine grazing Barandabhar 410 27° 37.4´ N 0 Dark brown; 10 Banned† 60 50 84° 26.8´ E sandy loam

† Although wild animals like rhino, deer, wild boar, etc. come to this forest in significant number from adjoining Chitwan National Park. contains glycosilated phenolics, triterpenic sapo- when the plants began to senesce. In September, nins and aliphatic ketones, and some of these due to harsh weather conditions (continuous heavy secondary metabolites show immunostimulatory rainfall) we could not make observations. At each (Bafna & Mishra 2006; Lakshmi et al. 2003), monthly observation the phenophases of tagged antioxidative (Tang et al. 2004; Wu et al. 2005), individuals were recorded as vegetative, flowering estrogenic (Vijayanarayanan et al. 2007) and anti- or fruiting. osteoporotic activities (Cao et al. 2008). Environmental variables Phenology observations In October, litter cover (%) on the ground was At each sampling site, four plots (10 m × 10 m) estimated, and a 200 g soil sample was collected in were subjectively chosen and marked permanently. each quadrat from 10 - 15 cm depth to determine The plots were relatively undisturbed and had high organic matter and total nitrogen. There were density of Curculigo orchioides. Altogether, 200 altogether 200 soil samples, with 10 samples from quadrats lying within 20 plots were sampled. In each plot. each plot, 10 quadrats (1 m × 1 m) were located Soil samples were air dried in the shade for a randomly. The number of individuals in each week and passed through a fine sieve (mesh size phenophase - seedling, vegetative (mature but 0.5 mm). Organic matter (Walkley-Black method) and total nitrogen (mirco-Kjeldahl methods) were without flower and fruit), flowering and fruiting - determined in each soil sample following the were recorded in each quadrat to determine popu- methods described in Gupta (2000). Mean values of lation-level phenology. Observations were repeated soil organic matter and total nitrogen were every month, starting in June 2007. Tree canopy calculated for each plot. and ground vegetation (shrub layer) cover were estimated visually. Germination experiment The 20 best looking flowering individuals in each plot were tagged (Fig. 2B). Altogether, 400 In preliminary experiments, seeds of Curculigo individuals lying within 20 plots were tagged for orchioides did not germinate when placed between monitoring phenological changes. Plants were tag- moist blotting paper for 3 months, indicating ged in June 2007 and the phenological stage of dormancy. When these seeds were tested using 0.1 % each plant was recorded; monthly observation at solution of 2,3,5 triphenyl-tetrazolium chloride, the marked plots was continued until October 2007 they were found viable. SHRESTHA, JHA & KANDEL 95

Fig. 3. Phenological patterns of Curculigo orchioides. (A) phenological events in one year starting from May; vertical lines in flowering and fruiting indicate peak frequency of the respective phase. (B) Change in percentage of individuals in various phenophases during growing season across the entire sampling sites.

Then seeds were tested for permeability of MS medium were surface sterilized by 70 % etha- seed coat to water using the method followed by nol (1 min) and then 1 % sodium hypochlorite (5 Hidayati et al. (2001) with some modifications. min) before inoculation. Seeds soaked in distilled Three replicates of 20 seeds each (total seeds = 60) water for 24 h were considered as control. Seeds were weighed to the nearest 1 mg after soaking for were left to germinate and were observed for 6 about 2 minutes and subsequent surface-drying by weeks. blotting paper. This initial measurement was considered as the dry mass (M0). The seeds were Data analysis then placed between three layers of blotting paper placed in Petri dishes and saturated with distilled From the quadrat-level data, percentage of water. After 1 hour, the seeds were surface-dried individuals in various phenophases was calculated by blotting paper, and reweighed to get wet mass for each sampling month for each sampling site as (Mt). The process was repeated every hour for the well as from the combined data. Tagged indivi- first 10 hours and every 24 hours for the next duals data were also used to calculate percentage week. For each measurement time, the percentage of individuals in vegetative, flowering and fruiting increase in mass (ΔM) was calculated as: ΔM = (Mt stages for each sampling month and site. – M0) / M0 × 100. Relationship between seedling density of Cur- For germination, seeds were soaked in various culigo orchioides and litter cover on the ground concentrations of gibberellic acid (GA3) solution was analyzed by linear regression. Since most of (100, 250 and 500 ppm) for 24 h. Half of the seeds the phenological activities (e.g., germination, flo- soaked in each concentration of GA3 were placed in wering, and fruiting) occurred in July, percentage Petri dishes (five seeds in each of two Petri dishes) of individuals in each phenophase was calculated lined with a double layer of moist blotting paper. for each plot from this month’s data of quadrat Another half of the seeds were placed in sampling and tagging. The phenological data from Murashige-Skoog (MS) medium. Seeds placed in July was used to understand the variation of per- 96 ECOLOGY OF CURCULIGO ORCHIOIDES

Table 2. Percentage of tagged individuals of Curculigo orchioides producing various numbers of bouts of inflorescences across the sampling sites.

Bouts of Sampling sites inflorescences Dhuseri Chautari Sundari Sansari Barandabhar 1-bout 27 63 53 59 54 2-bouts 33 36 35 36 42 3-bouts 32 1 12 5 4 4-bouts 8 0 0 0 0 centage of individuals in various phenophases (e.g. time a plant would have only one inflorescence. flowering, fruiting) with environmental variables There was asynchrony in flowering within an such as litter cover, soil OM, total N and canopy inflorescence. The first flower produced was al- using regression analysis. Statistical Package for ways bisexual and it opened 3-5 days before sub- Social Sciences (SPSS, version 11.5, 2002) was sequent flowers did; a few individuals produced > 1 used for statistical analysis. bisexual flowers per inflorescence. Therefore, the average number of bisexual flowers/inflorescence Results was 1.13 (n = 31). The bisexual flowers mostly withered before anthesis of male flowers. Phenological patterns Table 3. Percentage of tagged individuals of Cur- The emergence of new leaves from the apical culigo orchioides in various phenophases. Individuals bud of underground rootstocks was initiated in in flowering stage were tagged in June. Values inside May (Fig. 3A) when temperature was the highest the parenthesis represent total number of indivi- and soil was moistened by a few showers of rain duals. (Fig. 1). Plants occurring at moist but sunny sites produced inflorescence together with new leaves. Months of observations June-July was phenologically the most active Phenophases period when most flowering, fruiting and seed June July August October germination occurred. During that phenologically Vegetative 0 24 61 92 active period, 27 % of individuals in June and 18 % (87) (190) (272) in July were in reproductive stage (flowering and Flowering 100 21 13 4 fruiting) while 73 % and 66 %, in June and July, (410) (76) (40) (12) respectively, were in the vegetative stage (Fig. 3B); Fruiting 0 55 26 4 in July, the remaining 16 % of the population was made up of seedlings. When only mature indivi- (204) (80) (12) duals were used in analysis, excluding seedlings, Total 100 100 100 100 74 % in June and 80 % in July were in the (410) (367) (310) (296) vegetative stage. There was a single flowering peak in June at Fruiting occurred from June to October across the beginning of the monsoon (Fig. 3). However, the sites, with a single fruiting peak during July flowering was not synchronized and it continued (Fig. 3). At the lowland moist site, Barandabhar, until October at some sites. During a growing fruiting occurred earlier than at other sites. About season some individuals produced up to four bouts 55 % of flowering individuals in June bore fruits in of inflorescences (Table 2). Average number of July (Table 3). However, the percentage of indi- inflorescence/plant was 1.63 (n = 579). If plants viduals fruiting varied across the sites; it was 89 % produced > 1 inflorescence/plant during a growing at Barandabhar while at other sites it varied season, they did this in two ways. They either between 40 and 55 %. produced inflorescences in sequence, such that a Seed maturation took nearly a month. plant would have > 1 inflorescence at a time but at Immature seeds were white and fleshy while the different developmental stages (Fig. 2A). Alterna- mature ones were dark black and hard (Fig. 2E). tively, they produced inflorescences at intervals of Mature seeds emerged from the fruit (berry) after a few weeks to a month, such that at any given gelatinization of the fleshy wall. Seeds were dis- SHRESTHA, JHA & KANDEL 97

Table 4. Results of linear regression analysis bet- tree canopy, tree × shrub canopy and soil N con- ween some reproductive (response) and environ- tent. Flowering increased with organic matter mental (predictor) variables. Degrees of freedom (d.f.) though the relation was only marginal. Fruiting for all analyses is 18. increased with increasing tree canopy, but the relation changed when shrub cover was used as a R- F- component of canopy cover. There was a unimodal Responses Predictors Significance square value relationship between fruiting and tree × shrub Flowering in Tree 0.480 16.61 0.001 canopy with highest fruiting at an intermediate June (%) canopy level of canopy cover (Fig. 4). Similar pattern of unimodal variation, though not significant, was Tree × 0.366 10.41 0.005 observed between tree × shrub canopy and the shrub frequency of flowering individuals that bore fruits canopy in July (P = 0.114). Percentage of individuals in Soil N 0.248 5.93 0.025 vegetative stage increased with increasing tree Flowering in Soil OM 0.207 4.7 0.044 canopy. The canopy and soil variables (OM and N) July (%) had no effect on frequency of juvenile individuals, Vegetative in Tree 0.198 4.43 0.05 and flowering individuals that bore fruits. July (%) canopy Seed germination persed by ants as well as by surface runoff during In natural habitats seeds of Curculigo orchio- the rainy season. Small ants used the seed content ides germinated 10 - 12 months after dispersal. as food. Seeds remained dormant until June of the Freshly collected seeds did not germinate when following year and germinated when the rainy placed between moist filter papers for more than season began in July (Fig. 3). Litter accumulation 90 days, indicating dormancy, although these seeds on the ground had a negative effect on the seedling remained viable (embryos were stained pink by population. Seedling density declined with increa- tetrazolium solution). When placed between moist 2 sing litter cover (linear regression, P < 0.001, R = filter papers, there was an increase in seed mass 0.50). by 30.5 % over air dry mass. Seeds treated with GA3 also did not germinate either on moist filter Environmental factors and phenological paper or with an MS medium. In short, we could patterns at population level not identify the environmental condition to break Flowering was influenced by canopy cover and seed dormancy of C. orchioides from the experi- soil characters (Table 4, Fig. 4). The percentage of ments that we performed. flowering individuals increased with increasing Vegetative propagation Underground rootstock was mainly a means of winter perennation. When upper part of the rootstock was damaged by human collection or animal foraging, there was often development of more than one shoot (Fig. 5A). These adventitious shoots detached from each other and became independent individuals. Clonal propagation from leaves (Fig. 5B-C) was observed during the second half of the growing season (September-October). When midribs of mature leaves were mildly damaged and the injured part came in direct contact with moist surface soil a few whitish swellings were formed along the midrib, which resembled tiny calluses. Calluses were also observed at the proximal end of Fig. 4. Impact of tree × shrub canopy cover on frui- completely detached leaves. Wild animals, especi- ting frequency. The fitted line is based on quadratic ally wild boar, were the main biotic factors regression model. of mechanical damage to the leaves or whole plant. 98 ECOLOGY OF CURCULIGO ORCHIOIDES

nally dry tropical forests, such as of the present study area, where flowering is induced by rainfall and is often concentrated in the transition from the late dry season to the early wet season (Murali & Sukumar 1994; Rathcke & Lacey 1985; van Schaik et al. 1993). Resource availability influences the timing of phenological events (Rathcke & Lacey 1985). Flowering during pre- to early monsoon in C. orchioides, together with leaf flushing, could be possible due to presence of adequate assimilate in the perennial rootstock. Moisture, temperature and the presence of adequate stored reserves could have imposed bottom-up selective pressure on the timing of flowering (Elzinga et al. 2007). An extended flowering in C. orchioides from May to October could be attributed to the production of inflorescences in one to four bouts at intervals of a few weeks to a month (Table 2).

Curculigo orchioides had short (nearly one

month) flower to fruit duration which could possi-

bly be due to utilization of reserved food for fruit

development (Murali & Sukumar 1994). Fruiting

during the early wet season is a common tendency

of plants in seasonal dry tropical forest, which

minimize seedling mortality in the next dry season Fig. 5. Clonal propagation in Curculigo orchioides. (van Schaik et al. 1993). However, early fruiting in A) Branching in rootstock after removal of uppermost C. orchioides could have no effect on seedling part, B-C) Clonal propagation from leaves. mortality in the same year. Seed dormancy postponed germination until early in the next wet During September-October, wild boar selectively season (July), which ensured establishment of foraged on the rootstock of this plant. These callus- seedlings before the next dry season began. Seed like structures developed into globular bulbils, dormancy in this plant appears to be an adaptive which produced leaves and roots. A complete mechanism to time germination so as to avoid plantlet was formed while still attached to the leaf unfavourable weather conditions for subsequent midrib. We observed this phenomenon at the three establishment (Finch-Savage & Leubner-Metzger sampling sites (Dhuseri, Sundari and Baran- 2006). During the late growing season the plant dhabhar) that were relatively moister than the was foraged extensively by wild animals for the other sites. Among them, frequency of leaves with rootstock, which is nutrient rich (with a calorific such plantlets was higher at Barandhabhar, the value of 1630 KJ/100 g dry mass; Suresh 2008) and wettest site. an aphrodisiac (Ramawat et al. 1998). Therefore, early flowering and fruiting ensured successful Discussion completion of seed production before the plant was damaged by wild animals. Any phenotype that Phenological patterns would flower and produce fruits late in the grow- Phenological patterns are the result of a ing season could most likely be damaged before the compromise between a variety of selective pres- fruits matured. sures, such as seasonal climatic changes, resource Seeds had no active mechanism of dispersal availability, and the presence of pollinators, preda- and were dispersed by ants and surface runoff tors, and seed dispersers (Fenner 1998). In Curcu- during the rainy season. It is not clear whether ligo orchioides, flowering began with the pre- there are chemical attractants in seeds to attract monsoon showers, reaching a peak during early ants, which has been considered an adaptive monsoon (Fig. 3). This pattern is common in seaso- modification in ant dispersed seeds (Bennett & SHRESTHA, JHA & KANDEL 99

Krebs 1987; Howe & Smallwood 1982). But we with increasing tree canopy (unpublished data, BB observed that ants foraged seeds of C. orchioides Shrestha & PK Jha). Reproductive success was for their nutritious cotyledons. Unlike the post- high in plots with partially open canopy and accu- monsoon fruiting peak of most understorey herbs mulation of a thin layer of litter. of moist tropical forests (Bhat & Murali 2001), the Freshly collected seeds showed dormancy. An mid-monsoon fruiting peak of C. orchioides, to- increase in seed mass by 30.5 % over dry mass gether with its unwettable seed coat could poten- when placed between moist filter paper is typical tially help seeds to float on the surface of runoff for permeable seed coat (Carol C. Baskin, Univer- water in the rainy season (Howe & Smallwood sity of Kentucky, USA, per. com. Nov. 20, 2009). 1982). It appears that fruiting phenology in C. This excluded the possibility of physical dormancy. orchioides could have been shaped by its seed Since the seeds did not germinate when treated dispersal mode and the potential damage by with GA3, it appears that seeds of C. orchioides animals during the late growing season. have deep physiological dormancy (Baskin & Curculigo orchioides showed spatial variation Baskin 1998; Carol C. Baskin, per. com. Nov 20, in timing of phenological events, with earlier unset 2009). of flowering and fruiting at the moister site. This Clonal propagation in Curculigo orchioides pattern of difference between moist and dry sites is from leaves was the combined effect of mechanical a common feature in tropical forests at the commu- damage, probably, high humidity and availability nity as well as the species level (Newstrom et al. of adequate moisture in soil. High humidity slows 1994). At the community level, flowering peaked down the drying rate of detached or damaged during the late dry season at moist sites but leaves, and provides sufficient time for the deve- during the early wet season at dry sites in dry tro- lopment of plantlets. The indiscriminate damage pical forests (Murali & Sukumar 1994). Individual to adult plants by animals might have induced species of tropical regions also followed the same regeneration capacity from leaf midrib. To our pattern (Newstrom et al. 1994). knowledge, this is the first report of natural regeneration of C. orchioides from leaves. This Reproductive success observation is supported by the earlier reports that in vitro direct regeneration of C. orchioides About one-fourth of the mature individuals of plantlet was the best from mid rib region of leaf Curculigo orchioides entered the reproductive (Prajapati et al. 2003; Suri et al. 1999). Identi- phase, and only about half of the flowering indi- fication of the specific environmental conditions viduals developed fruits (Fig. 3B, Table 3). This essential for natural regeneration of C. orchioides indicates a relatively low regenerative potential of from leaves can help to improve the techniques of C. orchioides through sexual reproduction. Repro- in vitro micro-propagation at a large scale. ductive success of this plant appeared to be There was temporal separation between sexual determined by canopy and litter cover (Table 4, reproduction and clonal propagation. Seasonality Fig. 4). A similar pattern in reproductive success in clonal propagation in a tropical understorey has been reported for the herbaceous monocots in herb (Aechmea magdalenae) was attributed to Amazonian tropical forests (Costa et al. 2002). variation in abiotic factors such as light and Effect of tree canopy on regeneration is modified moisture (Villegas 2001). However, in C. by litter accumulation (Dupuy & Chazdon 2008). orchioides, seasonality in clonal propagation also Among the sampling sites, highest percentage (89 %) appeared to be due to variation in activity of wild of tagged (in June) flowering individuals bore animals that damaged the plant. fruits in July at Barandabhar, where litter accumulation was the lowest (Table 1). Litter accumu- Implications for conservation lation had significantly negative impact on the seedlings density of C. orchioides (P = 0.001), The two regenerative strategies in Curculigo which is a common effect of litter accumulation on orchioides, sexual and vegetative, appear to be herbaceous species (Berendse 1999). Negative ef- separated spatially as well as temporally. The fect of litter accumulation has been also reported relative proportion of sexually and vegetatively for seedlings of small-seeded trees (Dupuy & produced offspring in the population is determined Chazdon 2008). The combined result of these two by the complex interplay between genetic and effects was the decline in total density of this plant environmental factors (Ceplitis 2001). The role of 100 ECOLOGY OF CURCULIGO ORCHIOIDES genetic variation in selecting the mode of repro- Krishna Pant, Kul Bahadur Gurung, Sarita Yadav duction in C. orchioides is not known at present, and Gurudatta Sharma helped variously during but some of the environmental factors such as high field sampling. humidity and soil moisture appeared to favor References bulbil formation from damaged leaves. There appeared to be a trade-off in regenerative strategies Bafna, A. R. & S. H. Mishra. 2006. Immunostimulatory between moist/shady and dry/open habitats. Sex- effects of methanol extract of Curculigo orchioides ual reproductive success was low at sites with high on immunosuppressed mice. Journal of Ethnophar- canopy cover (high shade and thereby high macology 104: 1-4. moisture) where frequency of clonal propagation Baskin C. C. & J. M. Baskin. 1998. Seed: Ecology, Biogeo- from leaves was high. Clonal propagation from graphy, and Evolution of Dormancy and Germin- leaves was virtually absent in the dry sites with ation. Academic Press, California. low canopy cover, where sexual reproductive suc- Bennett, A. & J. Krebs. 1987. Seed dispersal by ants. cess was high. This could be the reason for the TREE 2: 291-292. species having a wide range of distribution in the Berendse, F. 1999. Implications of increased litter pro- landscape. However, given the high anthropogenic duction for plant biodiversity. TREE 14: 4-5. disturbance and habitat degradation, this plant Bhat, D. M. & K. S. Murali. 2001. Phenology of under- appears to be vulnerable due to its limited disper- storey species of tropical moist forest of Western sal range of seeds and propagules. Plants with ant Ghats region of Uttara Kannada district in South or gravity dispersed seeds are the most vulnerable India. Current Science 81: 799-805. to habitat degradation and fail to re-colonize Bhattarai, N., V. Tandon & D. K. Ved. 2001. Highlights during restoration (McLachlan & Bazely 2001). Re- and outcomes of the Conservation Assessment and colonization of such species would be possible only Management Planning (CAMP) workshop, Pokhara, if they are actively reintroduced. Thus habitat Nepal. pp. 46-53. In: N. Bhattarai & M. Karki (eds.) degradation could be an important reason for Sharing Local and National Experiences in declining natural population size of C. orchioides Conservation of Medicinal and Aromatic Plants in in India as suggested by Jasrai & Wala (2001). South Asia. Proceedings of Regional Workshop at The low reproductive success of this plant Pokhara, Nepal. HMG/N, IDRC and MAPPA. observed in the present study area corroborates Cao, D. P., Y. N. Zheng, L. P. Qin, T. Han, H. Zhang, K. earlier reports, that natural populations of C. Rahman & Q. Y. Zhang. 2008. Curculigo orchioides, orchioides lack adequate regeneration (Gupta & a traditional Chinese medicinal plant, prevents Chadha 1995). Low regenerative potential through bone loss in ovariectomized rats. Maturitas 59: 373- sexual reproduction and high vulnerability to habi- 380. tat disturbance appear to be the major constraints Ceplitis, A. 2001. Genetic and environmental factors for maintaining natural population of C. orchi- affecting reproductive variation in Allium vineale. oides. Although the plant has evolved additional Journal of Evolutionary Biology 14: 721-730. strategies for regeneration through leaves and Cordell, G. A. 2000. Biodiversity and drug discovery- a rootstock, they are not adequate to maintain natu- symbiotic relationship. Phytochemistry 55: 463-480. ral populations in the context of increasing an- Costa, F. R. C., C. Senna & E. M. Nakkazono. 2002. Effe- thropogenic disturbances. Therefore, conservation cts of selective logging on populations of two tropical management of this plant in the forest understorey understorey herbs in an Amazonian forest. Bio- should focus on maintaining partial open canopy tropica 34: 289-296. and thin litter accumulation to maximize regene- Dahanukar, S. A., R. A. Kulkarni & N. N. Rege. 2000. rative potential through sexual reproduction. Pharmacology of medicinal plants and natural products. Indian Journal of Pharmacology 32: S81- Acknowledgements S118. Dupuy, J. M. & R. L. Chazdon. 2008. Interacting effects Financial support from Volks-Wagen Founda- of canopy gap, understorey vegetation and leaf litter tion (Germany) is thankfully acknowledged. We on tree seedling recruitment and composition in thank Community Forest Users’ Groups for per- tropical secondary forests. Forest Ecology and mission to work in their forests. Aanjana Devkota Management 255: 3716-3725. (Central Department of Botany, Tribhuvan Uni- Elzinga, J. A., A. Atlan, A. Biere, L. Gigord, A. E. Weis versity), Lila Nath Sharma, Sandesh Bhattarai, & G. Bernasconi. 2007. Time after time: flowering SHRESTHA, JHA & KANDEL 101

phenology and biotic interactions. Trends in Ecology Prajapati, H. A., D. H. Patel, S. R. Mehta & R. B. Subra- and Evolution 22: 432-439. manian. 2003. Direct in vitro regeneration of Curcu- Fenner, M. 1998. The phenology of growth and repro- ligo orchioides Gaertn., an endangered anti-carci- duction in plants. Perspectives in Plant Ecology, nogenic herb. Current Science 84: 747-749. Evolution and Systematics 1: 78-91. Ramawat, K. G., S. Jain, S. Suri & D. K. Arora. 1998. Finch-Savage, W. E. & G. Leubner-Metzger. 2006. Seed Aphrodisiac plants of Aravalli hills with special dormancy and the control of germination. New Phy- reference to safed musali. pp. 210-223. In: I. A. Khan tologist 171: 501-523. & A. Khanum (eds.) Role of Biotechnology in Ghimire, S. K. 2008. Medicinal plants in Nepal Himalaya: Aromatic and Medicinal Plants. Vol. 1. Ukaaz current issues, sustainable harvesting, knowledge Publications, Hyderabad, India. gaps and research priorities. pp. 25-42. In: P. K. Jha, Rathcke, B. & E. P. Lacey. 1985. Phenological patterns S. B. Karmacharya, M. K. Chettri, C. B. Thapa & B. of terrestrial plants. Annual Review of Ecology and B. Shrestha (eds.) Medicinal Plants of Nepal: An Systematics 16:179-214. Anthology of Contemporary Research. Ecological So- Shrestha, B. B., S. Dall’Acqua, M. B. Gewali, P. K. Jha ciety (ECOS), Kathmandu, Nepal. & G. Innocenti. 2008. Biology and phytochemistry of Gupta, P. K. 2000. Methods in Environmental Analysis: Curculigo orchioides Gaerten. pp. 50-67. In: P.K. Water, Soil and Air. Agrobios, New Delhi. Jha, S. B. Karmacharya, M. K. Chettri, C. B. Thapa Gupta, R. & K. L. Chadha. 1995. Medicinal and aro- & B. B. Shrestha (eds.) Medicinal Plants of Nepal: matic plant research in India. pp. 1-43. In: K. L. An Anthology of Contemporary Research. Ecological Chadha & R. Gupta (eds.) Advances in Horticulture, Society (ECOS), Kathmandu, Nepal. Medicinal and Aromatic Plants. Vol. 11. Malhotra Stainton, J. D. A. 1972. Forests of Nepal. Hafner Publi- House, New Delhi, India. shing Company, New York, USA. Hidayati, S. N., J. M. Baskin & C. C. Baskin. 2001. Suresh, S. 2008. Studies on the Wild Edible Plants of Dormancy breaking and germination requirements Paliyans and Pulayans in Palani Hills, Tamil for seeds of Symphoricarpos orbiculatus (Caprifoli- Nadu. Ph. D. Dissertation. Manonmaniam Sundara- aceae). American Journal of Botany 88:1444-1451. nar University, Tirunelveli, India. Howe, H. F. & J. Smallwood. 1982. Ecology of seed dis- Suri, S. S., S. Jain & K. G. Ramawat. 1999. Plantlet persal. Annual Review of Ecology and Systematics regeneration and bulbil formation in vitro from leaf 13: 201-228. and stem ex-plant of Curculigo orchioides, an en- Jasrai. Y, T. & B. Wala. 2001. Curculigo orchioides dangered medicinal plant. Scientia Horticulturae Gaertn. (Kali musli): an endangered medicinal herb. 79: 127-134. pp. 89-95. In: I. A. Khan & A. Khanum (eds.) Role of Tang, S. Y., M. Whiteman, J. F. Peng, A. Jenner, E. L. Biotechnology in Medicinal and Aromatic Plants. Yong & B. Halliwell. 2004. Characterization of anti- Vol. IV. Ukaaz Publication, Hyderabad, India. oxidant and antiglycation properties and isolation of Ji, H. F., X. L. Li & H. U. Xang. 2009. Natural products active ingredients from traditional Chinese medi- and drug discovery. EMBO Reports 10: 194-200. cines. Free Radical Biology and Medicine 36: 1575- Lakshmi, V., K. Pandey, A. Puri, R. P. Saxena & K. C. 1587. Saxena. 2003. Immunostimulant principles from van Schaik, C. P., J. W. Terborgh & S. J. Wright. 1993. Curculigo orchioides. Journal of Ethnopharmaco- The phenology of tropical forests: adaptive signi- logy 89:181-184. ficances and consequence for primary consumers. Manandhar, N. P. 2002. Plants and People of Nepal. Annual Review of Ecology and Systematics 24: 353- Timber Press Inc. Portland, Oregon, USA. 377. McLachlan, S. M. & D. Bazely. 2001. Recovery patterns Vijayanarayanan, K., R. S. Rodrigues, K. S. Chandra- of understorey herbs and their use as indicators of shekhar & E. V. S. Subrahmanyam. 2007. Evaluation deciduous forest regeneration. Conservation Biology of estrogenic activity of alcoholic extract of rhizome 15: 98-110. of Curculigo orchioides. Journal of Ethnopharma- Murali, K. S. & R. Sukumar. 1994. Reproductive pheno- cology 114: 241-245. logy of a tropical dry forest in Mudumalai, southern Villegas, A. C. 2001. Spatial and temporal variability in India. Journal of Ecology 82: 759-767. clonal reproduction Aechmea magdalenae, a tropical Newstrom, L. E., G. W. Frankie & H. G. Baker. 1994. A understory herb. Biotropica 33: 48-59. new classification for plant phenology based on Wu, Q., D. X. Fu, A. J. Hou, G. Q. Lei, Z. J. Liu, J. K. flowering patterns in lowland tropical rain forest Chen & T.S . Zhou. 2005. Antioxidative phenols and trees at La Selva, Costa Rica. Biotropica 26: 141- phenolic glycosides from Curculigo orchioides. 159. Chemical Pharmaceutical Bulletin 53: 1065-1067. PhD dissertation by Bharat Babu Shrestha

Ecology and Phytochemistry of Selected Medicinal Plants in Central Nepal

A Dissertation Submitted to the Central Department of Botany Institute of Science and Technology, Tribhuvan University, Nepal for the award of Doctor of Philosophy (Ph. D.) in Botany

By Bharat Babu Shrestha Central Department of Botany Tribhuvan University, Kathmandu

2010 AD (2067 BS)

PhD dissertation by Bharat Babu Shrestha

RECOMMENDATION

This is to certify that Mr. Bharat Babu Shrestha (Lecturer, Central Department of Botany, Tribhuvan University, Kathmandu) has completed dissertation entitled “Ecology and Phytochemistry of Selected Medicinal Plants in Central Nepal” for the award of Doctor of Philosophy in Botany under our supervision. The dissertation is based on the original research work and has not been submitted for any other degree.

………………………………………… ………………………………………...

Research Supervisor Research Co-supervisor

Pramod Kumar Jha, Ph.D. Mohan Bikram Gewali, Ph.D. Professor, Central Department of Botany Professor, Central Department of Chemistry Tribhuvan University, Kathmandu Tribhuvan University, Kathmandu

Date: July 4, 2010

PhD dissertation by Bharat Babu Shrestha

CERTIFICATE OF APPROVAL

On the recommendation of Prof. Dr. Pramod Kumar Jha (Central Department of Botany, Tribhuvan University, Kathmandu) and Prof. Dr. Mohan Bikram Gewali (Central Department of Chemistry, Tribhuvan University, Kathmandu), this dissertation work of Mr. Bharat Babu Shrestha is forwarded for the examination and is submitted to the Institute of Science and Technology (IOST), Tribhuvan University for the award of Doctor of Philosophy in Botany.

……………………………………

Prof. Dr. Krishna Kumar Shrestha Head, Central Department of Botany Tribhuvan University, Kathamandu, Nepal

Date: July 13, 2010

PhD dissertation by Bharat Babu Shrestha

DECLARATION

I hereby declare that the work presented in this dissertation has been carried out and put together by myself. I have not submitted any part of this work for any form of degree. All information cited in this work has been specifically acknowledged and credited to the respective authors or institutions as references.

………………………………….

Bharat Babu Shrestha

Central Department of Botany

Tribhuvan University, Kathmandu

July 4, 2010

PhD dissertation by Bharat Babu Shrestha

This work has been dedicated to my parent

Som Bahadur Shrestha and Santa Kumari

Shrestha

and

my wife Bimala Shrestha

PhD dissertation by Bharat Babu Shrestha

ACKNOWLEDGEMENTS

I am greatly indebted to my research supervisors Dr. Pramod Kumar Jha (Professor, Central Department of Botany, Tribhuvan University, Kathmandu) and Dr. Mohan Bikram Gewali (Professor, Central Department of Chemistry, Tribhuvan University, Kathmandu) for their continuous guidance, encouragement and support throughout my work. A research project “Bioprospecting of ethnomedicinal plants of Nepal for the conservation of biological and cultural resources” funded by Volkswagen Foundation, Germany, provided financial support to field and laboratory works. Tribhuvan University provided study leave for one year during preparation of this dissertation.

Collaboration between Tribhuvan University, Nepal and Padova University, Italy, provided me an opportunity to perform phytochemical analysis at the Department of Pharmaceutical Sciences, Padova University, Italy. I am indebted to Prof. Gabriella Innocenti and Dr. Stephano Dall‟Acqua of the Department of Pharmaceutical Sciences, Padova University, for their hospitality, helps and cooperation in phytochemical analyses. I am thankful to Dr. Suresh Kumar Ghimire (Central Department of Botany, TU) for critical comments and suggestion on the first draft of this dissertation. Dr. Ghimire served as internal examiner of this dissertation.

I am thankful to Dr. Krishna Kumar Shrestha, Professor and Head, Central Department of Botany, TU, for his encouragement and administrative supports. Thanks are also due to Prof. Dr. Ram Prasad Chaudhary, Prof. Dr. Hari Datta Lekhak and Prof. Dr. Vimal Narayan Gupta of the Department for their suggestions and helps during selection of plants and field works. I am grateful to my colleagues Anjana Devkota and Susan Phoboo for their valuable helps during field sampling and laboratory works. Dr. Prem Sagar Chapagain (Central Department of Geography, TU) helped in preparation of maps. Dr. Chandra Bahadur Thapa (Prithvi Narayan Campus, TU, Pokhara) kindly helped during my field visit to Syanja. Sandesh Bhattarai, Dhan Raj Kandel, Ravi Kumar Sharma, Bishnu Prasad Baral, Lila Nath Sharma and Achyut Aryal accompanied me in field samplings. Mr. Balkrishna Ghimire (Kangwon National University, Korea) helped to get micro-photographs of internal structure of seed of Curculigo orchioides. Krishna Pant helped in seed germination experiments. My friends Kumar Prasad Mainali, Shishir Poudel and Uttam Babu Shrestha, who are studying in USA, sent several references which were not accessible from Nepal. I

PhD dissertation by Bharat Babu Shrestha

am also thankful to administrative staffs and gardeners of Central Department of Botany, TU, for their cooperation and various helps.

I am thankful to Research Center for Applied Science and Technology (RECAST), TU, for allowing me to use rotavapor. National Herbarium at Godawari (KATH) and Tribhuvan University Central Herbarium (TUCH) kindly allowed me to consult the herbarium specimens deposited at respective herbaria. I am thankful to authorities of Annapurna Conservation Area Project and all Community Forests (Barandabhar, Dhuseri, Chautari, Sundari, Sansari, Jagreni and Patapati-Lulpani Community Forests) for their permission to work in their protected areas.

Following local people in Manang helped variously during field sampling: Karma Gurung, Chhiring Gurung, Abu Karki, Maya Gurung, Pemba Sherpa, Raju Gurung and Aite Pariyar. I am very much thankful to Mr. Mani Ram Regmi (Putalibazaar – 10, Satupasal, Syanja) and his family for their hospitality and helps during sampling in Syanja. Two I. Sc. students of Prithvi Narayan Campus, Pokhara, accompanied me during sampling in Syanja. Kul Bahadur Gurung, Sarita Yadav and Gurudatta Sharma helped during sampling in Nawalparasi.

Helps from my wife Bimala Shrestha have been invaluable. She took all responsibilities of family and saved time for my works. I acknowledge the helps from Anu and Madan Shrestha to my family while I was busy in laboratory and long field works.

Finally I am always indebted to my parent, other family members and relatives for their continuous encouragements and supports.

Bharat Babu Shrestha

PhD dissertation by Bharat Babu Shrestha

ABBREVIATIONS

ACAP: Annapurna Conservation Area Project AMF: Arbuscular Mycorrhizal Fungi AQ: Aqueous BA: Butyric Acid C/N: Carbon to Nitrogen Ratio CAMP: Conservation Assessment and Management Plan EN: Endangered Category of IUCN GA3: Gibberellic Acid HPLC: High Performance Liquid Chromatography INGOs: International Non Governmental Organizations IR: Infra Red IUCN: The World Conservation Union KATH: National Herbarium at Kathmandu m asl: Meter Above Sea Level MPs: Medicinal Plants MS medium: Murashige and Skoog Medium MS: Mass Spectroscopy N: Nitrogen NAA: Napthalene Acetic Acid NGOs: Non Governmental Organizations NMR: Nuclear Magnetic Resonance NT: Near Threatened Category of IUCN OC: Organic Carbon OM: Organic Matter ppm: Parts per Millions PTLC: Preparative Thin Layer Chromatography R&D: Research and Development RRI: Relative Radiation Index TCM: Traditional Chinese Medicine TLC: Thin Layer Chromatography TUCH: Tribhuvan University Central Herbarium UV: Ultraviolet

PhD dissertation by Bharat Babu Shrestha

ABSTRACT

Sustainable management of medicinal plants has become a major conservation issue all over the world. Lack of good quality information on distribution, abundance and biology of medicinal plants has hampered the development of medicinal plant sector in the Himalaya. Distribution, life history, plant-environment interaction, and phyto-constituents of two Nepalese medicinal plants Aconitum naviculare and Curculigo orchioides were studied.

Aconitum naviculare, an alpine perennial medicinal herb, is found from west Nepal to Bhutan including southern Tibet. The plant was found on south facing slope, often in association with alpine scrubs which had protective role against livestock damage and collection of this plant by human. The population density was 7.35 pl/m2. Mean plant height was 19 cm, tuber dry mass 0.27 g/plant, three flowers/plant, 11 seedlings/follicle, and seed size 0.47 mg/seed. Seed germination declined from 50% after 10 weeks of storage (4 C) to 7% after 20 weeks. Most of the growth and reproductive traits varied significantly with sampling sites and life forms of associated species. Mean density of the plant increased with relative radiation index while the biomass declined with elevation. High leaf nitrogen content (2.44 %) in A. naviculare can be considered as an adaptive response to short growing season in alpine region. A. naviculare has evolved a number of adaptational features to alpine environment and biotic disturbance which includes bluish globular flower, persistent sepal, and presence of intercalary meristem between stem and tuber.

Curculigo orchioides has a wide range of habitats from tropical riparian Shorea robusta forest to grassy slopes in subtropical region between 200 and 2100 m asl. S. robusta forest with partially open canopy of foothills was the most suitable habitat of C. orchioides. Density, rootstock biomass and yields varied significantly with types of habitats, with mean values of 7 pl/m2, 1.32 g/plant and 49 kg/ha, respectively. Flowering phenology was intermediate between synchrony and steady-state-flowering. Extended flowering was partly due to production of up to four bouts of inflorescences by some individuals and partly due to different timing of flowering among the individuals in the population. Fruiting frequency among individuals in forest habitats showed unimodal relationship with treeshrub canopy. A fruit contained seven seeds in average. The seeds are small (5 mg/seed) and the seed coat was permeable to water. They showed deep morphophysiological dormancy. Seeds were dispersed by ants and surface runoff water, and had no active mechanism of dispersal. Germination of seeds was postponed until early monsoon of the next growing season due to

PhD dissertation by Bharat Babu Shrestha

dormancy. In natural habitats seeds germinated in July which ensured seedling establishment during the same rainy season. Regenerative potential of C. orchioides through sexual reproduction was relatively low in forest habitats because less than one-fourth of the mature individuals entered into reproductive phase and only one-half of the flowering individuals developed fruits. It was partly compensated by clonal propagation from the mid rib region of leaves which was induced by mild mechanical damage to the mature leaves.

In phytochemical analysis of aerial parts of Aconitum naviculare, two new diterpenoid alkaloids (navirine B and C) and three flavonoid glycosides together with previously known one alkaloid and four phenol glycosides were isolated and characterized. Two new phenolic glycosides (curculigoside D and orchioside E) were isolated from rootstock of Curculigo orchioides.

Some of the life history traits explained distribution pattern of both plant species. Narrow range of distribution of A. naviculare can be attributed to early loss of seed viability and lack of long-range seed dispersal while the plasticity in plant height and petiole length enabled this plant to colonize from thick alpine scrubs to open areas. Spatial and temporal variation in relative importance of different regenerative strategies, along with seed dormancy, could be responsible for wide range of distribution of C. orchioides. Low regenerative potential through sexual reproduction can be attributed to rarity of this plant reported from elsewhere. These findings revealed that the life history traits are helpful in understanding the abundance and distribution patterns of plants. Phytochemical analysis of < 5% of medicinal plants of Nepal, exciting trend of isolating new secondary metabolites, and identification of seven new secondary metabolites from two medicinal plants in present study revealed that medicinal plants in Nepal can be important source of novel plant-based natural products.

Key words: Conservation, distribution, life history, secondary metabolites.

PhD dissertation by Bharat Babu Shrestha

TABLE OF CONTENTS

Page RECOMMENDATION i CERTIFICATE OF APPROVAL ii DECLARATION iii ACKNOWLEDGEMENTS v ACRONYMS vii ABSTRACT viii 1. INTRODUCTION 14 1.1 Background 1 1.2 Rationale and Implications 2 1.3 Hypothesis 4 1.4 Objectives 4 1.5 Limitations 4 2. REVIEW OF LITERATURE 513 2.1 Importance of Medicinal Plants 5 2.2 Threats to Medicinal Plants 7 2.3 Research in Medicinal Plants 8 2.4 Aconitum naviculare 11 2.5 Curculigo orchioides 12 3. ECOLOGY OF ACONITUM NAVICULARE 1450 3.1 Introduction 14 3.2 Materials and Methods 14 3.2.1 Study Area 14 3.2.2 Study Species 17 3.2.3 Field Sampling 20 3.2.4 Laboratory Analysis 22 3.2.5 Numerical Analysis 23 3.3 Results 24 3.3.1 Distribution 24 3.3.2 Abundance 24 3.3.3 Variation of Abundance with Environmental Factors 25 3.3.4 Life History 28 3.3.5 Phenology 29 3.3.6 Variation of Growth Characters and Reproductive Output 30

PhD dissertation by Bharat Babu Shrestha

3.3.7 Seed Germination 36 3.3.8 Leaf Nitrogen Content 39 3.4 Discussion 40 3.4.1 Distribution 40 3.4.2 Abundance 42 3.4.3 Variation of Growth and Reproductive Traits across the Sites 44 3.4.4 Variation in Growth and Reproductive Traits with Life Forms of 45 Associated Species 3.4.5 Seed Size and Germination 46 3.4.6 Leaf Nitrogen Content 48 3.4.7 Plant Response to Natural Environment 49 3.4.8 Life History Traits and Distribution 50 4. ECOLOGY OF CURCULIGO ORCHIOIDES 5189 4.1 Introduction 51 4.2 Materials and Methods 52 4.2.1Study Area 52 4.2.2 Study Species 56 4.2.3 Field Sampling 56 4.2.4 Laboratory Analysis 58 4.2.5 Numerical Analysis 60 4.3 Results 61 4.3.1Distribution 61 4.3.2 Abundance 61 4.3.3 Environmental Variable and Abundance 65 4.3.4 Life History 68 4.3.5 Phenological Patterns 71 4.3.6 Environmental Factors and Phenological Patterns at Population 77 Level 4.3.7 Seed Germination 77 4.3.8 Clonal Propagation 80 4.4 Discussion 82 4.4.1 Distribution 82 4.4.2 Abundance 82 4.4.3 Phenological Patterns 84

PhD dissertation by Bharat Babu Shrestha

4.4.4 Sexual Reproduction and Environmental Factors 85 4.4.5 Seed Germination 86 4.4.6 Clonal Propagation 87 4.4.7 Plant History Traits vs. Distribution 88 5. PHYTOCHEMISTRY 90103 5.1 Introduction 90 5.2 Materials and Methods 91 5.2.1 Survey of Literatures 91 5.2.2 Study Species 91 5.2.3 Collection of Plant Materials 92 5.2.4 Laboratory Analysis 92 5.3 Results 94 5.3.1 Phytochemical Research of Nepalese Medicinal Plants 94 5.3.2 Isolation and Characterization of Secondary Metabolites 94 5.4 Discussion 102 6. CONSERVATION 104112 6.1 Introduction 104 6.2 Materials and Methods 105 6.3 Results 106 6.3.1 Aconitun naviculare 106 6.3.2 Curculigo orchioides 107 6.4 Discussion 108 6.4.1 Aconitum naviculare 108 6.4.2 Curculigo orchioides 110 7. CONCLUSIONS AND RECOMMENDATIONS 113116 7.1 Conclusions 113 7.2 Recommendations 116 REFERENCES 117134 APPENDICES Appendix 1. Laboratory Protocol for Soil Analysis 135 Appendix 2. Laboratory Protocol for Estimation of Leaf Nitrogen Content 136 Appendix 3. List of Publications 137

PhD dissertation by Bharat Babu Shrestha

1. INTRODUCTION

1.1 Background

Plant derived materials have been a major ingredient in traditional medicinal formulations, and the relative importance of plant based-natural products is likely to increase in modern medicine for a foreseeable future (Craker 2007). Globally the number of plants with known medicinal values was estimated to be about 70,000 (Shippmann et al. 2006), and its number has been increasing steadily. In this modern era too, about 80% of people worldwide depend on traditional medicine for primary healthcare (Bannerman 1982), which largely depends on prescription to plant-based formulations. Using these traditional ethnomedicinal knowledge as starting point, scientists were able to isolate bioactive natural products from plants and used them successfully as drugs (Cordell 2000; Dahanukar et al. 2000; Ji et al. 2009), saving lives of millions of people every year (Roberson 2008). Due to their commercial prospects and high chances of finding new drugs against serious diseases, pharmaceutical companies and research institutions, respectively, have developed renewed interest to biomedical researches of medicinal plants. This has been combined with increasing popularity of herbal-based products in western society, leading to steady increase in volume of medicinal plants being collected from wild for trade (Koul and Wahab 2004). Himalayan ethnomedicinal plants have significant share in the international trade. Additional ethnomedicinal plants of the Himalaya are also expected to enter in to trade in the future.

Market driven extraction of medicinal plants from wild, without considering their life history and the amount that can be extracted sustainably has threatened some of them even to the level of local extinction. While some medicinal plants of the Himalayas have been threatened due to unsustainable harvest from wild population for trade (Ghimire et al. 1999, 2005; Kala 2000, 2005), others are naturally rare and populations have been declining even if they have been used only for traditional healthcare (Kala 2005). One-fifth of the plants with known medicinal uses has been estimated to be threatened (Shippmann et al. 2006), but most of the information related to status of these medicinal plants have been based on experts‟ perception rather than empirical data (Dhar et al. 2000; Mulliken and Crofton 2008). Though the number of references on medicinal plants of Nepal has been increasing rapidly, most of them were related to inventory, and only a small proportion (<10%) has focused on biology (Ghimire 2008). This data gap appears to be one of the major constraints for the development of medicinal plant sector in Himalayas including Nepal.

PhD dissertation by Bharat Babu Shrestha

In present work, ecology of two medicinal plants Aconitum naviculare and Curculigo orchioides was studied in their natural habitat ranges, and medicinally important parts of these two plants were analyzed for phytochemical composition. A. naviculare has been highly prized as antipyretic, analgesic and sedative agent in indigenous healthcare system (Lama et al. 2001; Gao et al. 2004; Bista and Bista 2005; Bhattarai et al. 2006). C. orchioides, a native to tropical to subtropical Asia, has been used in traditional medicine as aphrodisiac, tonic to restore vigor, and against asthma, bronchitis, jaundice, dysentery, diarrhea, leucorrhoea and male sterility (Shrestha et al. 2008), and the secondary metabolites present in this plant are known to have immunostimulatory, antioxidative, estrogenic, spermatogenic and antiosteoporotic activities (Lakshmi et al. 2003; Tang et al. 2004; Wu et al. 2005; Bafna and Mishra 2006; Chauhan et al. 2007; Vijayanarayanan et al. 2007; Cao DP et al. 2008; Chauhan and Dixit 2008; Jiao et al. 2009). Distribution, abundance, life history, plant-environment interaction, leaf nitrogen content and soil nutrient status (nitrogen and organic carbon) of A. naviculare and C. orchioides were studied. Patterns of distribution and abundance were discussed based on life history and plant-environment interactions. This study was accompanied by phytochemical analyses of medicinally important parts of these two plants, and a review of phytochemical studies of Nepalese medicinal plants. Conservation status of these two plants was assessed against IUCN threat categories (IUCN 2001), and the prospects for conservation management including protection of wild populations and cultivation have also been discussed.

1.2 Rationale and Implications

Origin of new diseases and drug resistance developed by several of them constitutes major challenges for medical scientists and pharmaceutical companies. They are searching new natural (plant) products, which would be more effective, safe and supplied in sustainable manner. Since plant derived natural products have been perceived as the most viable mean to ensure sustainable health service to future generation (Cordell 2002), pressure on sustainability of medicinal plants is likely to increase rapidly in a foreseeable future. A large number of medicinal plants (globally 21% of the total medicinal plants, Shippmann et al. 2006) have already been categorized as threatened. One has to keep in mind that the assessment of medicinal plants for conservation management has been largely relied on experts‟ perception rather than on actual field data related to abundance, habitat range, life history, etc. While the Himalayan region has been praised for being rich in medicinal plants and the indigenous knowledge of their uses, life history information including plant-

PhD dissertation by Bharat Babu Shrestha

environment interaction at species level is available only for a handful of medicinal plants native to this region. A successful conservation management cannot be expected unless these information are available for all medicinal plants which are being traded and/or used in various indigenous healthcare systems. Similarly, most of the ethnomedicinal plants of Nepal are waiting for detailed phytochemical analysis and scientific validation of their indigenous uses. Therefore, future development of the sector of medicinal plants of the Himalaya in general and Nepal in specific depends on the generation of empirical data on distribution, abundance, life history strategies, plant-environment interactions and phytochemical composition of medicinal plant species, and use of these data while formulating the conservation management plan. Conservation Assessment and Management Plan (CAMP) workshop had also recommended for „life history studies‟ of more than 50% of the medicinal plants assessed by the expert participants (Molur and Walker 1998). As an attempt to proceed in the direction mentioned above, distribution, abundance, life history traits, plant-environment interactions and phytochemical composition of two ethnomedicinal plants of Nepal, Aconitum naviculare and Curculigo orchioides, were studied. These two medicinal plants are from high mountains and tropical ecosystems, respectively, which are the major harvesting areas of the leading medicinal plants in diverse healthcare systems worldwide (Cunningham et al. 2008). A. naviculare is native to alpine zone in trans-Himalayan region while C. orchioides is native to tropical – subtropical region of Asia. Both of these medicinal plants are highly potential but have not got adequate attention from policy makers, researchers and traders. A. naviculare has been highly prized as antipyretic, analgesic and sedative agent in indigenous healthcare system in the study area (Manang), and any decline in abundance of this plant in nature will have adverse effect to local healthcare system. C. orchioides has been known to have secondary metabolites with immunostimulatory, antioxidative, estrogenic, spermatogenic and antiosteoporotic activities. However, ecological information together with natural range of distribution and abundance were not available for both of these medicinal plants. Furthermore, specimens of these plants collected from Nepal had not been used for any phytochemical investigation. Along with isolation of a few new secondary metabolites from these two medicinal plants, the present work has generated empirical data on distribution, abundance, life history traits and plant-environment interaction, and discussed in the context of conservation prospects. These information will be valuable for sustainable wild harvest and cultivation of these two medicinal plants.

PhD dissertation by Bharat Babu Shrestha

1.3 Hypothesis

Present work has been based on following two general hypotheses:

 Understanding reproductive biology and other life history traits can help understand the distribution and abundance of plants;  Medicinal plants in Nepal can be important source of novel plant-based natural products.

1.4 Objectives

A general objective of the present work was to generate ecological and phytochemical information of two important medicinal plants Aconitum naviculare and Curculigo orchioides. The specific objectives are:

 To document distribution and conservation status of Aconitum naviculare and Curculigo orchioides in Nepal  To understand relationship among environmental variables, life history and abundance of these two medicinal plants in their natural habitats  To perform phytochemical analysis of ethnomedicinally used parts of these two plants

1.5 Limitations

The major limitations of the present work are:

 Due to varied patterns of distribution and topography of the habitats, sampling strategies for the two plant species were also different.  Limited time and resources did not allow covering entire range (vertical and horizontal) of distribution of these plants in Nepal for sampling.  Aconitum naviculare could not be transplanted for close observation of its life cycle as it is an alpine plant and could not be grown in environmental conditions of Kathmandu.  Due to taxonomically unrelated taxa and ecologically distinct habitats, there is apparent lack of integration of the research findings of the two species in the dissertation.

PhD dissertation by Bharat Babu Shrestha

2. REVIEW OF LITERATURE

In this section, a general overview of importance of medicinal plants, existing threats to them and contemporary research frontiers have been discussed with focus on information gap and research priorities for the medicinal plant sector of Nepal. At the end, all information available related to study species (Aconitum naviculare and Curculigo orchioides) have been summarized.

2.1 Importance of Medicinal Plants

Use of plants as sources of remedies precedes recorded human history probably by thousands of years. More than 60,000 years ago, the Neanderthals, the ancestors of modern man, were also aware of medicinal properties of plants (Ji et al. 2009). Along with biological and socio-cultural evolution of human beings, the uses of plants and other biological organisms for medicinal purposes have also evolved and enriched knowledge what we call today the „traditional medicine‟. These traditional medicines such as Ayurveda and Traditional Chinese Medicine (TCM) are still the indispensible part of human health care though the modern medicine appears to dominate the health sector. More than 80% of people worldwide mostly rely on traditional remedies for their healthcare (Bannerman 1982), and the uses of medicinal and aromatic plants in culinary, medicinal and other anthropogenic applications are likely to increase in foreseeable future (Craker 2007).

Since the first isolation of natural product in pure form (morphine from opium by Friedrich Wilhelm Sertürner) in 1805 and subsequent commercialization in 1826 (Ji et al. 2009), hundreds of biological organisms have been screened, and thousands of natural products been isolated and tested for potential medicinal uses. Over 50% of prescription drugs worldwide are derived from chemicals first identified in plants (Leaman 2009). About 1 billion people worldwide depend on drugs derived from forest plants for their health care (World Bank 2004). Out of top 150 prescription drugs in USA, 118 are based on natural sources, and plant-derived anti-cancer drugs save at least 30,000 lives every year in USA (Roberson 2008). Though the isolation and identification of novel natural products to be used in drugs is costly and time consuming, they are safe and their sustainable supply can be ensured (Cordell 2002). Putting aside some uncertainties and controversies on herbal formulations (e.g. Elvin-Lewis 2001; Marks 2004; Willcox and Bodeker 2004; Ferner and Beard 2005; Guo et al. 2007), the natural products are better sources of potential drugs than

PhD dissertation by Bharat Babu Shrestha

the combinatorial chemistry and high-throughput screening (Ji et al. 2009). In practice the rate of discovery of new drugs from plant has been disappointing; however the chances of success can be increased following the leads provided by traditional medical knowledge (Hamilton 2003). In fact, about 25% of modern medicines have been derived from plants first used traditionally (Anonymous 2009). A few of the notable drugs derived from plants are: taxol (known by its trade name Paclitaxel) from Taxus spp. for treating cancer; diosgenin from Dioscorea deltoides for preparation of oral contraceptive; reserpine from Rauvolfia serpentina for treatment of hypertension; aspirin from Salix spp. for pain relief, promotion of heart-health and blood thinning; colchicine from Colchicum autumnale for cancer and gout treatment; digitalis from Digitalis purpurea for heart treatment.

There are about 70,000 plant species worldwide, out of total 422,000, that have medicinal values; and 3000 species in international trade (Shippmann et al. 2006). Global trade in medicinal plant is estimated to be worth US$ 60 billion per year (World Bank 2004) and it is increasing at the rate of 7% per year (Koul and Wahab 2004). In India, the „gold-mine of herbal medicine‟, medicinal use of more than 3000 plant species have been officially recognized and it has been estimated that over 6000 plant species have been used in traditional remedies with about 7000 manufacturing units (Dubey et al. 2004).

In absence of complete flora, it is not wise to make estimation of total number of medicinal plants in Nepal. Larger figures have appeared every time when new publications appeared on medicinal plants of Nepal. In a more recent publication, Baral and Kurmi (2006) have compiled the medicinal uses of 1764 species of Nepalese vascular plants while Ghimire (2008) has estimated it to be 1906 with 1614 native, 192 introduced/cultivated and 100 naturalized taxa. Due to poor system of data management and illegal trade of a large amount of medicinal plants, the figures published by the Forest Department and other NGOs/INGOs including independent researchers did not reflect the actual number and the volume of medicinal plants under trade in Nepal. Department of Forest in Nepal has fixed revenue for 163 botanical items (including fungi) for trade, and collected NRs. 28,780,934 (equivalent to US$ 359,761 with US$ 1 = NRs. 80) as revenue against trade of 3,380,657 kg in fiscal year 2007/2008 (Department of Forest 2009). Export of unprocessed medicinal plants is the country‟s fifth most important export commodity of Nepal (Olsen and Helles 2009). At least 143 medicinal and aromatic plant species has been considered commercially important in terms of traded volume, market demand and revenue collection (Bhattarai and Ghimire 2006). There are over 29 herbal processing centers in Nepal which have been using over 66

PhD dissertation by Bharat Babu Shrestha

medicinal plants to prepare herbal medicine in the form of extracts and mixture of wet/dry powder (Phoboo et al. 2008).

2.2 Threats to Medicinal Plants

The major threats to medicinal plants at global scale are: habitat destruction, biopiracy and over harvesting of known medicinal plants (Roberson 2008). In Europe, over-exploitation, destructive harvesting techniques, habitat change and decrease in genetic diversity have been identified as major threats to MPs (Lange 1998). At regional and national levels, lack of clear policy on common property rights, illegal trade, destructive harvest trends, high dependence on exclusive wild form, and unavailability of quality information on abundance, distribution, trade, level of sustainable harvest from wild and biological information (regenerative strategies, viable population size) are additional threats to MPs (Dhar et al. 2000; Larsen and Olsen 2007; Mulliken and Crofton 2008). Out of estimated 70,000 medicinal plants worldwide, about 15,000 species (21.4% of total) are under threat (Shippmann et al. 2006). In Darjeeling, a north-eastern part of India, 14 % (40 out of 281 species) of the ethnomedicinal plants have been assigned various categories of threats (Chhetri et al. 2005). There has been no threat assessment of most of the Nepalese MPs (but see Bhattarai et al. 2001). Out of 34 Nepalese plant species included in IUCN Red List of threatened species (IUCN 2006, cited in Bhuju et al. 2007), 16 species have medicinal values (based on Baral and Kurmi 2006). Similarly, out of 109 plant species of Nepal included in CITES list (UNEP-WCMC 2009), ethnomedicinal uses of 23 species have been described by Baral and Kurmi (2006).

About 70-80% of plant species in international trade have been collected entirely from wild, mostly on unsustainable ways (Hawkins 2008). Among 1200-1300 of the medicinal aromatic plants traded in and native to Europe, at least 90 % came from wild collection, and about 150 species out of them are threatened in at least one of the several European countries due to over-harvest from the wild (Lange 1998). In Nepal medicinal plants for export mainly came from wild harvest (Singh et al. 1979) and the situation has not been changed significantly over the last few decades, though the volume and value of medicinal plants exported has increased dramatically over the same period (Olsen and Helles 2009). There is no reliable data available on the number of Nepalese medicinal plants in trade which have been collected from wild, but a conservative estimate from various sources (Bhattarai and Ghimire 2006; Department of Forest 2009) indicates it to be more than 150

PhD dissertation by Bharat Babu Shrestha

species. Sixteen species of plants which are native to Nepal have been sources of essential oil traded from Nepal (Gurung 2009). This commercial exploitation has led to continuous declining in population size (Mulliken and Crofton 2008) and may lead to unavailability of traditional medicines to indigenous people who have relied on them for centuries (Roberson 2008).

In recent time, impact of global change (particularly global warming and land use changes) has been also viewed as a potential threat to medicinal plants. Since the Himalayan alpine ecosystems including Tibetan arid regions are the most vulnerable geographic regions of the world outside of the polar region to climate change, medicinal plants, including others, native to these geographic regions (e.g. Aconitum naviculare) are subjected to habitat shrinkage and high risk of extinction (Cavaliere 2009). This negative impact of climate change pushes Himalayan alpine medicinal plants to greater risk of extinction whose population has already been dwindling due to habitat destruction and unsustainable commercial harvesting (Mulliken and Crofton 2008). Extinction of medicinal plants not only affects livelihood of poor and marginalized herbal collectors (World Bank 2004), but also threatens the process of drug discovery (Bower 2008). Extinction of medicinal plants leads to loss of at least one potential major drug every two years (Roberson 2008).

2.3 Research in Medicinal Plants

Research in medicinal plants is a multidisciplinary approach which needs integration of several disciplines such as from molecular biology to landscape ecology. Historically, research on medicinal plants began with documenting verbal knowledge into the written texts. Ethnomedicinal survey of various indigenous communities worldwide began during the first half of 20th century and it still continued today with renewed interests. Though natural product from plant was first isolated in pure form early in 1800s (Ji et al. 2009), intensive phytochemical and biomedical researches on medicinal plant began only in second half of the 20th century after various technological innovations (Dahanukar et al. 2000; Hostettman and Marston 2002; Phillipson 2007). In recent time, a large number of institutions from botanical gardens to pharmaceutical companies have been screening ethnomedicinal plants of south America, Africa and Asia in search of sources of potential new drugs (e.g. Taylor et al. 1995; 1996; Haque et al. 2000; Balunas and Kinghorn 2005; Rajbhandari et al. 2007; Sharma et al. 2008). However, biological studies of medicinal plants such as demography, reproductive biology, life history, ecology (with particular

PhD dissertation by Bharat Babu Shrestha

emphasis to plant responses to environmental changes) and agro-technology are lagging behind in comparison to phytochemical and biomedical researches.

While the oldest medicinal text comes from ancient Mesopotamia written around 2600 BC describing the uses of 1000 plants and plant derived substances, it was Charaka Samhita (written around 900 BC) the first treaties devoted to Ayurveda, and Wu Shi Er Bing Fang (prescription for fifty two diseases) the first medical text on Traditional Chinese Medicine (TCM) written around 350 BC (Ji et al. 2009). “Shausruta Nighantu” was the first pharmacopeia of Nepal which was hand written during 7th Century AD (Subedi and Tiwari 2000). These and other historical herbal texts can provide important clues for the discovery of new potential drugs as exemplified by Buenz et al. (2006).

Documentation of ethnomedicinal knowledge is still dominating the researches on MPs of the developing countries. Majority (53%) of the references on researches on medicinal plants of Nepal have focused on ethnomedicine and related issues (Ghimire 2008). As of December 2003, out of total 590 references on ethnobotany 232 (40%) dealt with MPs (Shrestha et al. 2004). However, a large number of medicinal plants and ethnic groups are awaiting documentation of their traditional uses. In a study of herbal drugs used by Raute- tribe in far-western region of Nepal, Manandhar (1998) reported that the uses of 32% (15 out of 47 species) of the medicinal plants they documented were not reported earlier. From a survey of a small village in Dolakha district, Shrestha and Dhillion (2003) claimed that about 50 % (56 out of total 113) of the ethnomedical remedies they reported were new to ethnobotanical knowledge of Nepal. Similarly, ethnomedicinal uses of about 16% (18 out of 115 plant species) ethnomedicinal plants being used by Chepang communities of Shaktikhor village (Chitwan district) were first reported recently by Rijal (2008). Out of 1792 plant species (including lichens and fungi) described in „A Compendium of Medicinal Plants in Nepal‟ (Baral and Kurmi 2006), ethnomedicinal information of 323 (18.02%) species were completely derived from the documentations from outside Nepal. Despite major effort on ethnomedicinal documentations, aforementioned examples have shown that probability of finding new medicinal plants and traditional remedies is still high in Nepal.

About 1100 of the worlds‟ known plant species have been examined in detail for potential medicinal properties and one out of 125 plant species studied has produced a major drug (Dobson 1995). Himalayan region has been praised for high diversity in MPs, but pharmaceutical potential of native medicinal plants of this region has not been adequately

PhD dissertation by Bharat Babu Shrestha

exploited (Dhar et al. 2000). Some attempts have been made for phytochemical analysis of Nepalese medicinal plants (review in Watanabe et al. 2005), most of these phytochemical researches were done in laboratories outside Nepal though some initiatives were taken by then „Royal Drug Research Laboratory‟ early in 1970s (Singh et al. 1979). Only 17% of the references on researches on medicinal plants in Nepal have focused on phytochemical and pharmacological screening (Ghimire 2008).

Conservation of medicinal plants has been hampered by lack of good quality information on population density in wild, current commercial demand and future projection, methods of collection and threats to these plants (Hawkins 2008). Several assumptions made on the conservation issues of Nepalese medicinal plants are based on inadequate and poor empirical data without scientific validation, leading to misguided conservation efforts (Larsen and Olsen 2007). Status of medicinal plants throughout their range is generally limited and information on decline and rarity of any species largely depends on expert opinions (Mulliken and Crofton 2008), without objective assessment of threats (Dhar et al. 2000). For example, while assessing status of Curculigo orchioides, a medicinal plant of tropical to subtropical region, during Conservation Assessment and Management Plan (CAMP) workshop in India, expert participants used data based on „general field studies‟ and „informal field sighting‟ (Molur and Walker 1998). Such approach has given confused scenarios of species in question such as C. orchioides. The species has been assigned threat categories from vulnerable (Bhattarai et al. 2001), threatened (Manandhar 2002) to endangered (Suri et al. 1999; Jasrai and Wala 2001; Prajapati et al. 2003; Dennis and Alphonsa 2004). Therefore, generation of species-specific baseline data on population density in wild is a research priority for development of medicinal plant sector (Hawkins 2008) and prioritization for conservation (Dhar et al. 2000).

Cultivation of medicinal plants has been considered as a strategy to reduce harvest pressure on wild (Canter et al. 2005), however, this is still not a common practice in Nepal. Some researches on domestication of wild medicinal plants in Nepal had started early in 1970s (Singh et al. 1979), but the outcome and progress of this aspect of research on medicinal plants over the last three decades is not noteworthy. While the Government of Nepal has established infrastructures for cultivation and processing of medicinal and aromatic plants, the focus has been more on exotic species than on wild harvested and traded species of Nepal (Larsen and Olsen 2007). Survey of available literature (e.g. Shrestha and Shrestha 2004; Kunwar 2006) showed that about 40 species of medicinal and aromatic plants native

PhD dissertation by Bharat Babu Shrestha

to Nepal are under domestication, mostly at the level of field trial. Dabur Nepal Pvt. has prioritized 19 medicinal plants for cultivation (Pimprikar and Haque 2002), of which 13 were native to Nepal (based on Press et al. 2000). There has been inadequate research and development (R&D) effort to bring wild medicinal plants of Himalaya to domestication (Dhar et al. 2000). Lack of knowledge about the specific requirements for pollination, seed germination and growth may limit the success of domestication of wild medicinal plants (Canter et al. 2005). These biological information, among others, are essential not only for domestication of wild medicinal plants but also to develop a scientific management plan including strategies for sustainable harvests (Bevill and Louda 1999; Murray et al. 2002). If we consider sustainable harvest of the medicinal plants from the wild as a viable strategy to conserve cultural and biological diversity (Hamilton 2003), it is very essential to understand the life history strategies and post harvest regeneration of medicinal plants in question to develop plans for sustainable harvest. A recent simulation experiment has also indicated that knowledge on species-specific traits is essential to understand the impact of warming on medicinal (and forage) plants (Klein et al. 2008). However this part of research is lacking for medicinal plants native to Nepal in specific and the Himalaya in general. Only eight percent of the references on medicinal plants of Nepal have focused on ecology, which were mostly further limited to the level of inventory (Ghimire 2008). Some biological information (e.g. seed germination, reproductive output, population dynamics) is available only for a handful of high value medicinal plants from Nepal, such as Nardostachys grandiflora, Neopicrorhiza scrophulariiflora and Swertia chirayita (Barakoti 2002; Bhattarai and Basnet 2002; Ghimire et al. 2005; Larsen 2005; Shrestha et al. 2007a; Ghimire SK et al. 2008b). Biological information such as reproductive biology, population dynamics, response to environmental perturbations, etc. are not available for most of the wild harvested medicinal plants which has potentially high trade-value and dwindling natural populations. For example Curculigo orchioides has been emerging as a potential source of new drug (Kapoor 2008) with more than 25 peer reviewed references on phytochemistry (18) and bioassay (7), there was not a single peer reviewed reference on its population status in wild though the plant has been often quoted as endangered (e.g. Suri et al. 1999; Prajapati et al. 2003; Dennis and Alphonsa 2004; Francis et al. 2007).

2.4 Aconitum naviculare

Aconitum naviculare (Brühl) Stapf. (Ranunculaceae) is found in southern Tibet (Liangqian and Kodata 2001), northern Bhutan (Grierson and Long 1984), Sikkim (Shrivastava 1998)

PhD dissertation by Bharat Babu Shrestha

and trans-Himalayan regions of Nepal (Stainton 1997, Ohba et al. 2008). In Nepal, A. naviculare has been reported from Manang (Bhattarai et al. 2006), Mustang (Chetri et al. 2006; Ohba et al. 2008) and Dolpo (Lama et al. 2001). This alpine medicinal herb has been widely used in Tibetan system of medicine against cold, fever and headache (Lama et al. 2001; Gao et al. 2004; Bista and Bista 2005; Bhattarai et al. 2006), and it is the most prioritized species in ethnomedicine of Manang – the present study area (Bhattarai et al. 2007). In phytochemical analysis, Gao et al. (2004) reported a new diterpenoid alkaloid (navirine) along with five previously known alkaloids (isoatisine, hordenine, atisine, hetisinone and delfissinol). Recently Cao J-X et al. (2008) characterized two new diterpenoid alkaloids – naviculine A and naviculine B. There has been no recorded trade of this plant in Nepal. Population status of Aconitum naviculare was not known though it has been reported as rare in Dolpo, western Nepal (Lama et al. 2001) and Bhutan (Nawang 1996).

2.5 Curculigo orchioides

Curculigo orchioides Gaertn. (Hypoxidaceae) is found in tropical to subtropical regions of Asia and Australia. Ethnomedicinal uses, phytochemistry, bioactivity, trade and cultivation of this plant have been reviewed during present research and published (see Shrestha et al. 2008 in appendix). Therefore only a brief account of these aspects has been presented here. Rootstock of this plant has been used in traditional medicine as aphrodisiac, tonic to restore vigor, and against asthma, bronchitis, jaundice, dysentery, diarrhea, leucorrhoea and male sterility. The rootstock contains glycosilated phenolics, triterpenic saponins and aliphatic ketones. Altogether thirteen derivatives of glycosilated phenolics, sixteen triterpenic ketones and four aliphatic ketones have been isolated and characterized. Glycosilated phenolics include syringic acid, 2-6 dimethylbenzoic acid, orcinol aglycone (four derivatives), curculigine A, orchioiside A-B (two derivatives), curculigoside A-C (three derivatives) and myricetin derivatives (Tiwari and Misra 1976; Kubo et al. 1983; Garg et al. 1989; Chen et al. 1999; Wu et al. 2005; Gupta et al. 2005; Valls et al. 2006). Recently, four new phenolic glycosides (orcinosides D, E, F and G) from the rootstock of C. orchioides have been reported by Zuo et al. (2010). The triterpenic saponin includes curculigosaponins (fifteen derivatives) and curculigol (Misra et al. 1990; Xu et al. 1992a, b; Xu and Xu 1992; Chen et al. 1999). The aliphatic ketones are 27-hydroxytriacontan-6-one, 23-hydroxytriacontan-2- one, 21-hydroxytetracontan-10-one and 4-methylheptadecanoic acid (Misra et al. 1984a, b). Secondary metabolites present in rootstock of Curculigo orchioides have shown

PhD dissertation by Bharat Babu Shrestha

immunostimulatory (Lakshmi et al. 2003; Bafna and Mishra 2006), antioxidative (Tang et al. 2004; Wu et al. 2005), estrogenic (Vijayanarayanan et al. 2007), spermatogenic (Chauhan et al. 2007; Chauhan and Dixit 2008), antiasthmatic (Pandit et al. 2008) and antiosteoporotic (Cao DP et al. 2008; Jiao et al. 2009) and antihistaminic activities (Venkatesh et al. 2009).

Empirical data on the population status of C. orchioides is not available but this plant has been assigned to various threat categories from vulnerable (Bhattarai et al. 2001), threatened (Manandhar 2002) to endangered (Suri et al. 1999; Jasrai and Wala 2001; Prajapati et al. 2003; Dennis and Alphonsa 2004; Francis et al. 2007). To help commercial cultivation and subsequent ex situ conservation, various protocols have been developed for in vitro propagation of this plant (e.g. Augustine and D‟Souza 1997; Suri et al. 1999; Prajapati et al. 2003; Wala and Jasrai 2003; Dennis and Alphonsa 2004; Francis et al. 2007; Nema et al. 2008). Most of these works have shown that leaf midrib was a better source of explants for in vitro propagation, and there could be direct regeneration of plantlets from explants without intervening callus in basal medium without any growth regulators. Wala and Jasrai (2003) induced plantlet regeneration from shoot meristem in basal medium supplemented with BA and NAA. Joy et al. (2004, 2005) attempted to optimize farm conditions for commercial cultivation and found that partial shade (25%) and poultry manure were found to maximize rootstock yield of this plant. Sharma et al. (2009) evaluated the effect of native arbuscular mycorrhizal fungal (AMF) community to growth response in Curculigo orchioides and found that high AMF species richness exhibited improved growth in comparison to low AMF diversity.

In conclusions, both the species Aconitum naviculare and Curculigo orchioides are important for primary healthcare in traditional medicine of the Himalaya. A. naviculare has been reported as rare and C. orchioides as endangered, but these assignments were based on experts‟ opinions rather than empirical data. Quantitative data on distribution, abundance and life history traits were lacking for both the species. Furthermore, only a few secondary metabolites have been isolated and characterized from A. naviculare collected from Tibet. There are more than thirty secondary metabolites known in C. orchioides. However, there was not a single work published on phytochemistry of both these plants collected from Nepal.

PhD dissertation by Bharat Babu Shrestha

3. ECOLOGY OF ACONITUM NAVICULARE

3.1 Introduction

Aconitum naviculare (Brühl) Stapf. (Ranunculaceae) is found in southern Tibet (Liangqian and Kodata 2001), northern Bhutan (Grierson and Long 1984), Sikkim (Shrivastava 1998) and trans-Himalayan regions of Nepal (Stainton 1997; Lama et al. 2001; Bhattarai et al. 2006; Chetri et al. 2006; Ohba et al. 2008). This alpine medicinal herb has been widely used in Tibetan system of medicine against cold, fever and headache (Lama et al. 2001; Gao et al. 2004; Bista and Bista 2005; Bhattarai et al. 2006), and it is the most prioritized species in ethnomedicine of Manang – the present study area (Bhattarai et al. 2007). There has been no recorded trade of this plant in Nepal, and the use of this plant is mostly limited to Amchis (traditional herbal healer trained in Tibetan system of medicine) and local households for self medication.

Being found only in alpine Himalayan region (Stainton 1997), this species can be considered as species with „nowhere to go‟ (sensu Hawkins et al. 2008) in the context of climate change. Ecological study of this plant would be helpful to understand the potential impact of climate change, and develop management plan for the conservation of this important medicinal plant species. However, except taxonomic description in flora, any kind of research on ecology of Aconitum naviculare is lacking. Since ecology is the scientific approach to explain distribution and abundance of an organism (Kreb 1992), in present research environmental factors and life history traits of A. naviculare have been studied as an approach to explain the abundance and distribution pattern of this species. The hypothesis being tested was „life history traits can help to explain the distribution pattern of plant species.‟ The specific objectives were: 1) to document distribution and habitat range of Aconitum naviculare in the study area; and 2) to understand relationship among environmental variables, life history and abundance of this plant species in natural habitat.

3.2 Materials and Methods 3.2.1 Study Area The study area (28 41” – 28 46” N, 83 57” – 84 04” E, elevation 4050–4650 m asl) of A. naviculare was Manang valley which lies on the upper part of Manang district, central Nepal, in glacially formed U-shaped valley (3000–3400 m asl), traversed by the Marsyangdi

PhD dissertation by Bharat Babu Shrestha

river, and surrounded by high mountains (> 6000 m asl) (Figure 3.1). The valley lies on a rain shadow area of the trans-Himalayan (Annapurna) region with a mean annual precipitation of 444 mm (at 3420 m asl) and temperature 6.2° C (Miehe et al. 2001). During the study period there was no weather station in the study area. Therefore weather records of Jomsom station of Mustang district (which is the nearest meteorological station with comparable climate) have been analyzed (Figure 3.2). The mean monthly temperature ranged from 4.5 to 18.4 C with mean annual precipitation 282 mm for the period between 1997 and 2006. The study area is covered by snow from November to March. Forest is mostly confined on the moist north-facing slope of Annapurna mountain range, and the valley floor. On the north facing slope lower belt (3000–3500 m asl) has Pinus wallichiana and Juniperus indica forests while the upper belt (3500–4200 m asl) has Abies spectabilis and Betula utilis forests up to tree line (4200 m asl) (Shrestha et al. 2007b). On the southern slope there is Juniperus indica forest up to 3800 m asl (Ghimire BK et al. 2008) while the alpine scrub has dwarf and prostate junipers (Juniperus indica, J. recurva, J. squamata), Rhododendron lepidotum, Rosa spp., Berberis spp., Caragana gerardiana, Ephedra gerardiana, Cotoneaster microphyllus, etc.

The study area lies within the Annapurna Conservation Area (ACA) which has been managed by National Trust for Nature Conservation (NTNC). Generally collection of medicinal plants from wild for trade has been prohibited. Local users‟ group is responsible for monitoring conservation.

Most of the accessible areas where Aconitum naviculare occurred were sampled. According to local people, there were a few sites which were not easily accessible. Therefore, the sampling sites represented at least three fourth of the total „area of occupancy‟ of this plant in Manang valley. Here, the „area of occupancy‟ has been defined as the area within its extent of occurrence which is actually occupied by the species, excluding cases of vagrancy (IUCN 2001). The sampled sites represented the habitat range of this plant in the study area. Locations of the sampling sites of A. naviculare in the study area and their land uses have been presented in Table 3.1. All these sites lie on the southern slope between 4090 and 4650 m asl with mean slope 30.

PhD dissertation by Bharat Babu Shrestha

Figure 3.1. Map of Manang district showing study area and sampling sites of Aconitum naviculare. 29

PhD dissertation by Bharat Babu Shrestha

Figure 3.2. Monthly mean temperature and precipitation from 1997-2006 recorded at Jomsom meteorological station (Mustang district), which is the nearest meteorological station to the study site. This station lies to the west of Manang valley at 2744 m asl. (Source: Department of Hydrology and Meteorology, Government of Nepal).

3.2.2 Study Species

Aconitum naviculare (Brühl) Stapf. (Syn: Aconitum ferox Wallich var. naviculare Bruhl; Fam. Ranunculaceae, Plate 3.1) is locally called ponkar/bongkar in the study area. It is a perennial herb with fusiform biennial tubers; stems ascending, 10-30 cm; leaves mostly congested in proximal part of stems, petiolate, blade orbicular-cordate, deeply 3-5 lobed, lobes obovate, further shallowly 2- or 3- lobuled, lobules dentate, teeth obtuse, leaves of distal part of stem 1 or 2, much smaller than proximal one, sub sessile, deeply 3 lobed; flowers in loose raceme; sepals tinged dull reddish purple/violet blue, with dark purple veinlets, lower surface pubescent, persistent in fruits, helmet navicular; petals clawed, head 3-4 mm; fruit of 5 follicles, sparsely pubescent (Ohba et al. 2008). Dried aerial parts (Plate 3.2) of this plant have been widely used in traditional healthcare system in the study area.

PhD dissertation by Bharat Babu Shrestha

Table 3.1. Land uses and location of sampling sites of Aconitum naviculare in upper Manang. Sites Land uses* Grazing pressure Distance from the Elevation Slope Aspect nearest settlements (m asl) () () (hr)** Ice Lake Alpine scrubland used for grazing High 3 4400- 32±3 193±20 livestock; with >5 kharka around 4550 SW

Manang hill Alpine scrubland used for grazing High 2 4150- 30±8 237±37 livestock; with 2-3 kharka around 4450 SW

Khangsar Alpine scrubland used for grazing High 2 4400- 29±7 208±50 hill livestock; with >5 kharka around 4650 SW

Yak Kharka Alpine scrubland used for grazing Low 4 4090- 36±1 239±21 livestock; with 1-2 kharka around 4270 SW

Ledtar Alpine scrubland used for grazing Low 5 4100- 28±5 240±24 livestock; with one kharka 4500 SW

Thorong Alpine scrubland used for grazing High 6 4350- 38±7 108±13 Phedi livestock; with one kharka 4450 SE

Range/Mean 4090- 30±6 227±42 4650 SW

*Kharka: temporary site for gathering livestock during night. **Walking distance in hours from traditional settlements. New settlements with only hotels and tea shops were not considered. 31

PhD dissertation by Bharat Babu Shrestha

Plate 3.1. Aconitum naviculare A) Flowering individual from an open area, showing globular flower with opening on the lateral side, B) Underground parts showing tuberous root of previous and current years with perennating bud at the tip of current year‟s tuber (shown by arrow); horizontal black line indicates the position of soil surface, C) Flower above the canopy of juniper, D) Developing fruits covered by persistent sepals, E) Seeds.

PhD dissertation by Bharat Babu Shrestha

Plate 3.2. Traditional use of Aconitum naviculare. A) Aerial part of flowering Aconitum naviculare hanged on the wall inside the home for air drying, B) Storage of crushed A. naviculare in plastic bottle, and C) Aqueous extract of the plant prepared for immediate use by a shepherd.

3.2.3 Field Sampling

For field sampling, three field visits were made in June 2005, September 2005 and October 2006. Reconnaissance of the study species and study area were made during first field visit (June 2005). Population sampling and collection of soil and leaf samples were done during second visit (September 2005). During the third visit (October 2006), plant specimens were collected for the study of life history traits, including germination. Six sites were sampled, each of which constituted a distinct population of this species. Here, population has been defined as the group of individuals of this species in geographically distinct area. These six populations (or sampling sites) are separated from each other by river or deep gorges.

PhD dissertation by Bharat Babu Shrestha

Population and Biomass: A total of 240 sub plots (1 m × 1 m) lying within thirty large plots (10 m × 10 m) were sampled during September 2005. Due to unequal „area of occupancy‟ the number of plots studied in each site was not equal; three plots were sampled at Ice Lake area, five at each of Manang and Khangsar hills, and seventeen at Ledtar. Sampling was not done at Yak Kharka and Thorong Phedi sites for density and above ground biomass because there had been heavy collection of this plant before I reached there. Each large plot was divided into four quarters (5 m × 5 m) and two sub plots (1 m × 1 m) were studied in each quarter. In each sub plot individuals of Aconitum naviculare were counted to determine density (pl/m2). To determine aboveground biomass, all the mature individuals of A. naviculare (excluding seedling and sapling) were collected from the single most densely populated sub plot (1 m × 1 m) of each large plot. Out of thirty, five large plots (two at Manang hill and three at Khangsar) were not sampled for above ground biomass due to rarity of the plant at the sampling sites because collection of plant materials by researcher has been also viewed as a potential threat to rare medicinal plants (Dhyani and Kala 2005). Single, most vigorous individual was completely uprooted from each quarter to measure tuber and stem mass. At each site the area of occupancy of A. naviculare was visually estimated. Major associated species were recorded.

Leaves: Leaf nitrogen (N) content is the most important among the leaf traits, which is closely linked with maximum rate of photosynthesis (Cornelissen et al. 2003). Leaves for estimation of N content were collected from those individuals, which were uprooted for the estimation of tuber and stem mass. When leaf mass of the uprooted plant was low, additional leaves were collected from other individuals of the same plot. Leaf samples were air dried in shade and brought to laboratory in paper bags.

Soil: From the rooting depth (5–10 cm) of the plant which was uprooted for estimation of tuber and stem biomass, about 100 g of soil was collected for estimation of soil N and organic carbon (OC) content. On the same day of collection the soil samples were placed for air drying under shade.

Life History Traits: During third visit in October 2006, individual plants were sampled to study life history traits. Depending on the availability of the plant, undamaged and healthy 310 individuals of Aconitum naviculare were completely uprooted to determine tuber and stem mass, plant height, petiole length, number of flowers borne by each individual plants (hereafter

PhD dissertation by Bharat Babu Shrestha

referred as „flowers/plant‟) and number of seeds in each follicle (hereafter referred as „seeds/follicle‟). Mature seeds were collected from each sampling site to determine seed mass and germination behavior.

3.2.4 Laboratory Analysis

Soil and Leaf nutrient analysis: Soil samples were air dried in shade under laboratory condition and the dried soil was passed through the fine sieve (0.5 mm). Soil OC (Walkley and Black method) and total N (micro-Kjeldahl method) were determined following the methods described by Gupta (2000). Altogether 240 soil samples were analyzed from the habitats of Aconitum naviculare. Leaf N content was determined by modified micro-Kjeldahl method following the procedure describe by Horneck and Miller (1998). One hundred and twenty leaf samples of A. naviculare were analyzed for N concentration. The laboratory protocol used for the analysis of soil and leaf has been described in Appendix 1 and 2.

Seed Germination: After returning back to laboratory (Kathmandu, 1350 m asl), the seeds were stored at 4 C before germination. Germination experiments were done three times: after 10, 15 and 20 weeks of seed collection. At each time, 100 mature seeds were selected from each sampling site and divided randomly into five groups of 20 seeds each. Due to the large population, 200 seeds were taken from Ledtar for germination. After taking the mass of each group (20 seeds), the seeds were placed in petri dishes (diameter 10 cm) with double layered blotting papers saturated with distilled water. Thus each petri dish had 20 seeds equally placed, with 10 replicate dishes for Ledtar and five for the other sites. Seeds were examined in every four days until 32 days for germination response. Emergence of the radical was considered as a criterion for germination. The internal environment of petri dishes was maintained saturated with moisture by frequently adding distilled water. The experiment was done under open laboratory condition with natural photoperiod (11–12 h light/12–13 h dark), mid day temperature from 16 to 22 C and relative humidity 36 to 50%. Since most of the alpine plants need light for seed germination (Bliss 1958), the petri dishes were placed near a window; thus they received direct sun light for about four hours a day.

PhD dissertation by Bharat Babu Shrestha

3.2.5 Numerical Analysis

From the values of aspect (Ω), slope (β) and latitude (Φ), the relative radiation index (RRI) of each large plot was calculated following Ôke (1987) [RRI = {Cos(180  Ω).Sinβ.SinΦ} + Cosβ.CosΦ]. Estimated population size was calculated as the product of estimated area of occupancy and mean density for each sampling site. Here, the „population size‟ has been defined as the total number of mature individuals (IUCN 2001). Mean density of each large plot (for regression analysis) was calculated as the mean of density of all quadrats within the plot. Mean seed output was calculated as the product of mean number of flowers per plant, number of follicles per fruit and number of seeds per follicle. This estimation of seed output also included those flowers which might not set fruits. Before comparison of mean of population density, aboveground biomass, morphological traits, and reproductive output among sampling sites, the data were tested for normality (Kolmogorov-Smirnov test) and homogeneity of variance (Levene test). The density was normalized when square root transformed, but variance remained unequal. Therefore, mean across the sites were compared by analysis of variance (ANOVA), and the pairs of sites were compared by multiple comparison using Dunnett C test (variance assumed unequal). Log and square root transformations did not improve normality and homogeneity of variance in other measured character (morphological characters and reproductive output); for these data non-parametric tests (Kruskal-Wallis and Mann-Whitney U tests) were used for comparison of mean across the sites. Kruskal-Wallis test was used for comparison of means of more than two set of data, while Mann-Whitney U test was used to compare means of a pair of data set.

Morphological characters and reproductive output of individual plants occurring in juniper, other shrubs and open areas (i.e. without shrubs) were also compared by Kruskal-Wallis and Mann-Whitney U tests. Leaf N content met the assumptions of normality and homogeneity of variance; therefore ANOVA, followed by Duncan multiple range test was used for comparison of mean across sites. Regression analyses were done to understand the relationship among environmental variables (elevation, RRI, soil OC, and soil N), abundance of A. naviculare (density and biomass), vegetative growth characters (plant height, petiole length, stem mass, and tuber mass), and reproductive outputs (flowers/plant, seeds/follicles, and mass per 100 seeds). The data were checked for normality (Kolmogorov-Smirnov test) before regression

PhD dissertation by Bharat Babu Shrestha

analysis, and they transformed (log transformation) if necessary. As suggested by Sokal and Rohlf (1995), during log transformation, if the original values were <1, each of these values were multiplied by 100 to avoid negative values after transformation. All statistical analyses were done using Statistical Package for Social Sciences (SPSS 2002, Version 11.5).

3.3 Results

3.3.1 Distribution

Six populations of Aconitum naviculare, sampled in upper Manang, represented the entire range of habitat in the study area. Except at Ledtar, the populations were confined in one or a few patches. Population distribution was nearly clumped. At Ledtar, the population was spread over the large area. The species was found exclusively on south facing slope within the elevation range from 4090 to 4650 m asl (Table 3.1). When individual populations were considered, it was found within a narrow elevation band spanning 100300 m. The relative radiation index (RRI) ranged from 0.78 to 0.98 with mean value of 0.87 (Table 3.2). The soil of A. naviculare habitats had in average 7.79% organic carbon (OC), 13.57% organic matter (OM) and 0.70% total nitrogen (N).

Aconitum naviculare was often found with sclerophyllous and thorny species such as Juniperus squamata, Ephedra gerardiana, Cotoneaster microphyllus, Caragana gerardiana, Berberis spp. etc. (Table 3.2). In Ice Lake, Manang hill, Khangsar, Yak Kharka and Thorong phedi, where its population size was small (Table 3.3), A. naviculare was confined within the scrub of these sclerophyllous and thorny species. These sites, except Yak Kharka, had high grazing pressure of livestock. In Ledtar, where grazing pressure was relatively low, this species was also found in open places outside the bushes.

3.3.2 Abundance Total area of occupancy of Aconitum naviculare at sampling sites in Manang valley was estimated to be 40 ha (Table 3.3). The present sampling sites represented, at least, three fourth of the total area of occupancy of this plant in Manang valley. Therefore, the estimated total area of occupancy of this plant in Manang valley could be about 50 ha (i.e. 0.5 km2). In Manang

PhD dissertation by Bharat Babu Shrestha

hill, Khangsar and Phedi, it was found in small and fragmented patches while at the Ice lake area, it was found at single patch. In Ledtar, A. naviculare was found in a large area where the population density, estimated population size, and above ground biomass were also the highest. The density of A. naviculare was the highest at Ice Lake area and the lowest at Manang hill, the later being at the nearest distance from the densely populated Manang village (Table 3.1).

Table 3.2. Physical environmental conditions of habitats of Aconitum naviculare and associated shrub species in upper Manang.

Sites Relative Soil Organic Soil Nitrogen Associated shrub species (Populations) Radiation Carbon (%) (%) Index Ice Lake 0.980±0.015 4.96±1.66 0.40±0.11 Caragana gerardiana Manang hill 0.859±0.107 6.59±2.36 0.56±0.19 Spiraea arcuata, Rosa spp., Berberis spp., Ephedra gerardiana Khangsar hill 0.908±0.149 7.13±3.23 0.55±0.24 Cotoneaster microphyllus, Juniperus squamata Cotoneaster microphyllus, Yak Kharka 0.847±0.077 8.99±3.89 0.67±0.27 Spiraea arcuata, Berberis spp. Ledtar 0.873±0.093 8.61±3.35 0.81±0.41 Juniperus squamata, Spiraea arcuata, Berberis spp. Thorong 0.778±0.073 6.35±1.52 0.55±0.16 Spiraea arcuata, Berberis spp. Phedi Mean 0.870±0.098 7.87±3.29 0.70±0.37 -

3.3.3 Variation of abundance with environmental factors

Average density increased with increasing RRI though the relation was only marginal (Figure 3.3). No relationship was detected between population density and any of the soil variables measured in correlation and regression analysis (p>0.05). Area-based above ground biomass of A. naviculare declined with elevation and the relation was strong with 54% of variation in biomass being explained by elevation (Figure 3.4). Variation of above ground biomass was not significant with N content of soil and leaf (Figure 3.5).

PhD dissertation by Bharat Babu Shrestha

Table 3.3. Estimated area of occurrence, population density, estimated population size (number of individuals) and aboveground biomass of Aconitum naviculare at study sites. In the column of population density, values with same letter in superscript are not significantly different at p<0.05 (ANOVA with multiple comparison by Dunnett C test). na: data not available.

Sites Area Population Estimated Above ground of occupancy density population biomass (ha) (pl/m2)† size (g/m2) Ice lake 2.0 8.67c  3.37 173,200 2.67±0.24

Manang Hill 5.5 4.08a  2.45 223,850 5.02±1.32

Khangsar 1.3 6.43ab  6.35 80,250 3.06±1.4

Yak Kharka 7.3 na na na

Ledtar 21.0 8.35bc  4.47 1,751,400 5.73±2.71

Phedi 3.0 na na na

Total*/Mean** 40.1* 7.35  4.75** 2,228,700* 4.99±2.58** †Values were square root transformed before performing ANOVA

14 p = 0.03, R2 = 0.162, F = 5.23

12 Y =  4.67 + 13.1X

) 2 10

Figure 3.3. Relationship

Average density (pl/m density Average 4 between mean density of

Aconitum naviculare and 2 relative radiation index. Fitted line and the values 0 of p and R2 are based on .6 .7 .8 .9 1.0 linear regression analysis. Relative radiation index

PhD dissertation by Bharat Babu Shrestha

Figure 3.4. Relationship of above ground biomass (log transformed) of Aconitum naviculare with elevation. The fitted lines and values of p and R2 are based on linear regression analysis.

Figure 3.5. Variation of above ground biomass of Aconitum naviculare with soil (A) and leaf (B) N content. For N content, each point represents mean of four replicas sampled in each plot. The relations are not signficant.

PhD dissertation by Bharat Babu Shrestha

3.3.4 Life History

Aconitum naviculare is a semi-basal (sensu Cornelissen et al. 2003), perennial alpine herb. Generally the plant was unbranched but branching from the basal half was induced when apical part of the main stem was mechanically damaged, such as by animal trampling in open areas. Therefore, A. naviculare growing in the open areas were much more branched than the individuals growing with junipers and other shrubs. Aerial parts of the plant died at the end of every growing season but it perennated winter as tuberous root with fully developed apical bud buried within the soil at 2–3 cm depth (Plate 3.1B). Thus, A. naviculare is a cryptophyte (geophyte) according to Raunkiaer‟s classification of life forms. The mother plant produced a fusiform, tuberous white root with apical bud in the first year. In the second year, growth began with sprouting of the apical bud of the tuberous root. The plant first completed vegetative growth and then started to flower from mid September. During this period the tuber developed in the previous year shrunk gradually due to translocation of stored materials and development of new tuber. By the time fruit was mature, development of new tuber had completed and the old one dried up. Thus, the plant was perennial with biennial tuberous root and annual aerial parts. Generally single un-branched tuber is produced by each individual plant; rarely the number was two and it was distally branched (Plate 3.1B). There is always presence of meristematic tissue joining tuber and stem, thus these two parts can easily be detached from each other. When aerial parts were pulled during collection the tuberous root remained inside the soil.

The flower was violet-blue, globular with small opening on the lateral side (Plate 3.1A,C). In the first year of flowering a plant produced single flower, but the number increased in successive years (range: 1–17, Table 3.4). With the development of fruits, the sepals dried up becoming papery but remained persistent and covered the developing fruits until dehiscence. Generally five follicles developed in each flower but about 1/5th of the flowers had less than five follicles due to abortion of ovary. Seeds/follicle varied from 4 to 16 (mean: 11, Table 3.4). A single plant produced an average of 165 seeds (3×5×11=165). Seeds were small (mass: 4711 mg/100 seeds) with a lamellate (gill like structures) seed coat (Plate 3.1E). In addition to gravity dispersal, seeds could be dispersed up to several meters by strong continental wind.

PhD dissertation by Bharat Babu Shrestha

Table 3.4. Growth traits and reproductive outputs of Aconitum naviculare for entire range of sampling sites. CV: coefficient of variance.

Attributes Sample size* Range Mean CV **

Plant height (cm) 373 445 19 42

Petiole length (cm) 358 2.521.0 7.12 50

Stem mass (g/plant) 297 0.0151.528 0.164 115

Tuber mass (g/tuber) 297 0.0311.221 0.27 63

Number of flowers/plant 395 117 3 83

Number of seeds/follicle 262 416 11 19

Seed size (mg/100 seeds) 52 2775 47 23 *Sample size refers to the total number of plants or plant parts measured for the individual trait **Coefficient of variance is the standard deviation expressed as the percentage of mean.

3.3.5 Phenology

Phenological events began in June with germination of seeds and sprouting of apical bud of tuberous root after winter perennation (Figure 3.6) when temperature was increasing and the area received few showers of rain (Figure 3.2). In addition to rainfall, the soil received additional moisture due to melting of snow during this early growing season. Vegetative growth continued for more than three months and entered in to reproductive phase late in the growing season. Flowering is synchronized (mid September to early October, Figure 3.6) and the anthesis of flowers appeared to be induced by last shower of rain in mid to late September in the study area (Figure 3.2). During seed dispersal temperature and soil moisture both were low which prevented immediate germination. Within one month of seed dispersal (November), the habitats of Aconitum naviculare got covered by snow. Germination occurred in next growing season when the area become snow free in June.

PhD dissertation by Bharat Babu Shrestha

Figure 3.6 . Phenological events of Aconitum naviculare. The months start from June.

3.3.6 Variation in Growth Characters and Reproductive Output

Growth characters (plant height, petiole length, stem mass, and tuber mass) and reproductive output (flowers/plant and seeds/follicle) differed significantly (p<0.001) among the sampling sites (Figure 3.7). Most of the characters measured were lowest at Khangsar site. The largest difference among the sites was observed in stem mass of individual plants in which the largest value (Manang hill) was five times larger than the smallest one (Khangsar and Yak Kharka). The smallest difference was found in seeds/follicle in which the largest value (Manang hill) was only 1.25 times larger than the smallest (Khangsar hill).

A. naviculare was generally found in association with shrub species, most of which were thorny (Table 3.2). At Ice lake area and Manang hill the species was found only within patches of shrubs. At Ledtar, it was commonly found at post fire sites and open areas with other herbs. Trampling damage by animals and collection by humans were low within patches of shrubs, especially when the shrub species were thorny (e.g. Caragana gerardiana, Berberis spp.). Quantitative measurement at Ledtar site showed that life forms of associated species (such as junipers, other shrubs, and herbs between patches of shrubs – hereafter referred as „open‟) had a significant effect on height of A. naviculare, petiole length and number of flowers borne by each plant, and marginal effect on tuber mass and seeds/follicle (Table 3.5). Average stem mass/plant was not affected by life forms of the associated species. Plant height and petiole length of A. naviculare were the highest within juniper patches whereas tuber mass/plant, flowers/plant and seeds/follicle were the highest in individuals growing in open areas.

PhD dissertation by Bharat Babu Shrestha

Figure 3.7. Growth characters and reproductive output of Aconitum naviculare in the study sites (1: Ice lake area, 2: Manang hill, 3: Khangsar, 4: Yak kharka, 5: Ledtar, 6: Phedi). Main entries above bars are medians and values inside parentheses are interquartile range; values with same letter are not different significantly at p<0.05. Test statistics are based on pair-wise Mann-Whitney U test, and Kruskal-Wallis test. N = total sample size.

PhD dissertation by Bharat Babu Shrestha

Table 3.5. Growth characters and reproductive output of Aconitum naviculare in micro habitats at Ledtar. Main entries are medians while the values in parentheses are interquartile range; values in each row with same letter in superscript are not significantly different at p<0.05 (pair-wise Mann-Whitney U test).

Attributes Juniper Other shrubs Open Test statistics* N p value 2

Plant height (cm) 29.5c (9.25) 19.5b (9.75) 18a (9.5) 186 <0.001 42.7

Petiole length (cm) 13.5b (3.25) 7.0a (3.5) 6.5a (2.0) 188 <0.001 38.3

Stem mass/plant (g) 0.090a (0.06) 0.096a (0.112) 0.116a (0.142) 187 0.312 2.3

Tuber mass/plant (g) 0.194a (0.168) 0.225b (0.176) 0.249b (0.254) 156 0.021 7.7

No. of Flowers/plant 2.0a (1.0) 3.0b (3.0) 4.0c (5.0) 191 <0.001 18.1

No. of Seeds/follicle 10.25a (4.33) 11.14ab (2.58) 11.45b (2.05) 152 0.039 6.5 *Based on Kruskal-Wallis test; N: sample size (total number of plants or plant parts measured for each trait)

PhD dissertation by Bharat Babu Shrestha

Figure 3.8. Variation of growth characters and reproductive output of Aconitum naviculare with height of the plant. The fitted lines and equations were obtained by linear or quadratic regression analyses. Results of regression analyses have been given in table 3.6.

Among the growth and reproductive traits, petiole length, tuber mass, flowers/plant and seed mass varied significantly with plant height (Figure 3.8, Table 3.6). Petiole length and seed mass increased linearly with plant height but the tuber mass and flowers/plant showed unimodal relations with plant height. Petiole length had unimodal (but asymmetric) response with stem mass, but flowers/plant and seeds/follicles increased linearly with stem mass (Figure 3.9, Table 3.6). The flowers/plant also increased linearly with increasing tuber mass. The response of seeds/follicle was slightly unimodal with tuber mass and flowers/plant.

PhD dissertation by Bharat Babu Shrestha

Figure 3.9. Relationship among growth characters and reproductive output of Aconitum naviculare. The fitted lines and equations were obtained by linear or quadratic regression analyses. Results of regression analyses have been given in Table 3.6.

PhD dissertation by Bharat Babu Shrestha

Table 3.6. Results of regression analysis among growth characters and reproductive output of Aconitum naviculare.

Predictors (X) Responses (Y) Model d. f. R-square F value Significance

Plant height Petiole length [Log transformed ] Linear 52 0.716 128 <0.001

Mass per tuber [Log transformed] Quadratic 44 0.297 9.31 <0.001

Number of flowers per plant [Log Quadratic 44 0.292 9.09 <0.001 transformed]

Seed mass per 100 seeds Linear 45 0.114 5.76 0.001

Stem mass per plant [Log Petiole length [Log transformed] Quadratic 47 0.326 11.34 <0.001 transformed] Number of flowers per plant [Log Linear 48 0.828 232 <0.001 transformed]

Number of seeds per follicle Linear 48 0.429 36.11 <0.001

Mass per tuber [Log Number of flowers per plant [Log Linear 51 0.544 60.81 <0.001 transformed] transformed]

Number of seeds per follicle Quadratic 50 0.406 17.09 <0.001

Number of flower per plant Number of seeds per follicle Quadratic 51 0.463 22 <0.001 [Log transformed]

PhD dissertation by Bharat Babu Shrestha

Elevation Figure 3.10. Relationship of stem mass/plant (log transformed) of Aconitum naviculare with elevation. The fitted lines and values of p and R2 are based on linear regression analysis. Each point represents average value of each sampling plot.

Stem mass/plant decreased with an increase in elevation (Figure 3.10). Plant height, petiole length, tuber mass, flowers/plant and seeds/follicle did not vary significantly with elevation. Any significant relations were not found in regression analysis between growth and reproductive traits of Aconitum naviculare, and soil attributes (OC, N and C/N ratio).

3.3.7 Seed Germination

Germination was epigeal (sensu Baskin and Baskin 1998) in which elongating hypocotyle carried the cotyledons above the substrate surface, which expanded and became green to be active photosynthetically (Plate 3.3). Single seed, out of total 693 seeds germinated, produced two seedlings (possibly a case of polyembryony).

PhD dissertation by Bharat Babu Shrestha

First leaf

Cotyledonary leaf

Plate 3.3. Seed germination of Aconitum naviculare. A) Stages of seed germination showing epigeal mode of germination, B, C) Production of two radicles from single seed, E) Seedlings transplanted to soil from its natural habitat.

PhD dissertation by Bharat Babu Shrestha

Figure 3.11. Seed mass and germination pattern of Aconitum naviculare. A. Seed mass across the collection sites; B. Mean seed germination after 10 and 15 weeks of collection; C. Variation in germination of seed across the collection sites. Number of seeds used in each experiment was 200 for Ledtar and 100 for other sites.

Germination after ten and fifteen weeks of seed collection were 49 (±35 SD, n=35) and 43 % (±38 SD, n=35), respectively. Most of the seeds germinated between 8 and 16 days after

PhD dissertation by Bharat Babu Shrestha

sowing, and germination of new seeds continued until 24 days (Figure 3.11B). Seed germination varied across the sampling sites from 17 to 97 % after 10 weeks and 6 to 99 % after 15 weeks of collection (Figure 3.11C). After 20 weeks average seed germination reduced to 7%; there was no germination of seeds collected from Khangsar, and it was 24 % for seed collected from Yak Kharka.

3.3.8 Leaf Nitrogen (N) Content

Leaf nitrogen content of Aconitum naviculare ranged from 2.26 to 2.50 % with mean value of 2.44% (Table 3.7). The mean values varied significantly with the sites (ANOVA, p = 0.004). It was the lowest in samples collected from Khangsar and the highest in samples collected from Ledtar. Nitrogen content of leaf was 4.4 times higher than nitrogen content of the soil collected from rooting depth. It did not vary significantly with soil nitrogen content and elevation (Figure 3.12).

Table 3.7. Leaf nitrogen (N) content of Aconitum naviculare.

Sites Number of Mean leaf N Coefficient of Leaf to soil N samples content (%)* variation (%) ratio analyzed Ice lake 12 2.32ab 9.5 5.8

Manang Hill 20 2.46bc 13.4 4.4

Khangsar 20 2.26a 10.2 4.1

Ledtar 68 2.50c 11.2 3.1

Total/mean 120 2.44 11.9 4.4 *Values with same alphabet in superscript were not different significantly.

PhD dissertation by Bharat Babu Shrestha

Figure 3.12. Variation of leaf nitrogen content of Aconitum naviculare with elevation and soil nitrogen content. The relationships are not significant.

3.4 Discussion

3.4.1 Distribution

Aconitum naviculare has been reported from western (Dolpo) (Lama et al. 2001) and central Nepal (Mustang) (Ohba et al. 2008) including present study site (Manang), while there was a few specimens (col. no. 61658; collector: KR Rajbhandary) of this plant collected from eastern Nepal (Solukhumbu district) and deposited at National Herbarium of Nepal (KATH). These specimens were collected from Benikharka-Yurigolcha-Benikharka trekking route of Solukhumbu during joint botanical expedition of University of Tokyo

PhD dissertation by Bharat Babu Shrestha

(Japan) and Department of Plant Resources, Nepal (then Department of Medicinal Plants) in 1985. In all these districts, the plant was found at specific localities in a small population. Due to geographic isolation, the area of occupancy of A. naviculare was severely fragmented. Outside Nepal, the plant has been reported from southern Tibet (Liangqian and Kodata 2001), Sikkim (Shrivastava 1998) and northern Bhutan (Grierson and Long 1984). Therefore, distribution range of A. naviculare expands between western Nepal and Bhutan including southern part of Tibet. Altitudinal range of distribution of this species is similar between Nepal (4000 – 4800 m asl) and Bhutan (3900 – 4600 m asl) but it is very narrow in Sikkim (4600 – 4900 m asl) and very wide in Tibet (3200 – 5000 m asl). Exceptionally low lower limit (3200 m asl) of altitudinal distribution range of A. naviculare in Tibet could be due to its position to more northern latitude.

Aconitum naviculare was exclusively found on dry south-facing slope of the valley (Table 3.1). The south-facing slope warms up more than north-facing slope, due to high solar radiation (Larcher 1995). In the study area only a few nights had temperature below zero on the south-facing slope during growing season (Aase and Vetaas 2007). Alpine meadows, such as the habitat of A. naviculare, have high ground surface temperature than the subalpine forests at low elevation (Körner 2007). Permanent snow is absent on most of the mountain tops and south facing slope of the study site. Thus, soil moisture recharge from snow melt water during growing season could be low. Since annual precipitation is very low (444 mm, Miehe et al. 2001) in the valley, snow melt water is the major source of moisture for the vegetation. High evapotranspiration is coupled with low soil moisture recharge on the south facing slope, and the plants growing in this aspect are most likely subjected to drought stress during growing season. Thus, A. naviculare is adapted to high solar radiation, relatively warm temperature and mild water deficit, but it may not tolerate very low temperature. Density of A. naviculare is higher at the plots with higher RRI (Figure 3.3). Therefore, this species is completely absent from the moist north facing slope where several nights are cold with temperature below zero during growing season (Aase and Vetaas 2007).

The soil of A. naviculare habitats had 7.87% organic carbon (OC) and 0.7% total nitrogen (N) with C/N ratio 11.78 (Table 3.2). The OC of the soil in the habitats of A. naviculare lying on the relatively warm south facing slope was lower than in the soil of north facing slope from the similar elevation range in the present study area (Shrestha et al. 2007a). However, soil N was higher in the habitats of A. naviculare (i.e. on the south facing slope)

PhD dissertation by Bharat Babu Shrestha

than in north facing slope. These differences in soil properties could be attributed to contrasting topography of south and north facing slopes (Korner 2003). The average C/N ratio of the soil of A. naviculare habitat is very close to upper limit of C/N ratio (1012) for fertile soil with stable organic matter (Biswas and Mukherjee 1999). Thus it can be concluded that A. naviculare prefers relatively warm and sunny sites in mountains with soil rich in nitrogen, and similar landscape should be selected for cultivation.

A. naviculare is mostly found in association with alpine scrubs, most of which are thorny (Table 3.2). At sites with high pressure of livestock grazing (e.g. the Ice lake area, Manang hill, Khangsar, Yak Kharka and Phedi), the species was found mainly within alpine scrubs as they have protected the species against livestock damage and human collection. It appeared that natural distribution of this species was narrowing down due to anthropogenic disturbances which became a common pattern for other Himalayan medicinal plants too (Vashistha et al. 2006). The population density of this species was the highest at Ice lake area where the species was found at single locality amongst a thorny shrub Caragana gerardiana. The protective role of the scrubs is often important for the persistence of sensitive/rare species (Facelli and Temby 2002). At the sites with high grazing pressure, importance of the protective roles, as hypothesized by Bertness and Callaway (1994), was high. The protective role of associated scrubs against animal damage and human collection is a kind of facilitation, as proposed by Callaway (1995, 2007), and it is important for the persistence of this species in the study area.

3.4.2 Abundance

The average density of Aconitum naviculare (7.35 pl/m2) in the present study area (Table 3.3) was comparable to the average density of A. violaceum (6.7 pl/m2, Nautiyal et al. 2002) in the Garhwal Himalaya, India. Among three Aconitum species (A. balfourii, A. heterophyllum and A. violaceum) found in Garhwal Himalaya, highest density was recorded for A. violaceum which was attributed to habitat inaccessibility and less exploitation by humans (Nautiyal et al. 2002). Generally Aconitum species found in alpine zone (A. violaceum, A. naviculare) are smaller in size than others (e.g. A. heterophyllum, A. ferox) found in sub alpine and temperate zones (data from Stainton 1997; Polunin and Stainton 2000). The smaller size (i.e. dwarf) of Aconitum species found in alpine zone could have also contributed to their higher density than other Aconitum species found at lower elevation.

PhD dissertation by Bharat Babu Shrestha

Population density of Aconitum species found in the Himalayas was generally low; e.g. A. orochryseum in Gyasumdo valley of Manang: 4 pl/m2 (Ghimire et al. 1999); A. balfourii and A. heterophyllum in Garhwal Himalayas (India): 1.87 and 1.48 pl/m2, respectively (Nautiyal et al. 2002); A. balfourii, A. heterophyllum, A. rotundifolium and A. violaceum in northwest Indian Himalaya: 0.33–3.3 pl/m2 (Kala 2005); A. rotundifolium and A violaceum in Indian trans-Himalaya: 2–7.7 pl/m2 (Kala 2000). The average density of A. naviculare (7.35 pl/m2) in Manang was relatively higher in comparison to reported values for other Aconitum species mentioned above. The present data represented the maximum density of this species because the sampling plots were selected subjectively to represent the most densely populated areas in each study site. Using similar sampling strategy, Kala (2000) reported density up to 7.7 pl/m2 for A. violaceum. There were no previous data on population size and density of A. naviculare in Manang and elsewhere, but local people have perceived rapid decline in population of this species in the Ice Lake, Manang hill and Khangsar.

The species was found only in small areas in three of the six sampling sites and the population was either much fragmented (Yak Kharka) or confined at a single patch (Ice Lake area). Though A. naviculare was found from 4090 to 4650 m asl, when individual sites are considered it was found within a narrow elevation range of 100 m at Phedi to 400 m at Manang hill and Ledtar (Table 3.1). In Dolpo (north-western Nepal) the species is rare (Lama et al. 2001) while in Mustang (western border of Manang) it has been recorded from five sites (Ohba et al 2008). Nawang (1996) reported this species as rare in Bhutan. Present observation and other reports mentioned above showed that A. naviculare has narrow range of distribution and their availability was low in their natural habitats.

Population density of Aconitum naviculare, among the sampling sites, was the lowest in Manang hill (Table 3.3) which is close to the most populated village in the Manang valley. The lowest density at Manang hill could be attributed to high pressure of human collection and animal grazing. At this site, Ephedra gerardiana at high elevation and Spirea arcuata at low elevation were the common shrub associates of A. naviculare (Table 3.2). Due to absence of thorns, the protective effect of these two species against animal damage and human collection could be lower than of thorny Caragana gerardiana at Ice Lake area. A. naviculare was more commonly found at open areas than within patches of shrubs at Ledtar, which may indicate low animal damage. This was the least disturbed site because of its location far from traditional settlements. The high abundance (high density, above ground

PhD dissertation by Bharat Babu Shrestha

biomass, and the largest population) of A. naviculare at Ledtar might be attributed to the lowest pressure of human collection and animal damage.

3.4.3 Variation in Growth and Reproductive Traits across the Sites

Growth and reproductive traits varied significantly across the sites (Figures 3.7). Due to differences in micro habitats in alpine regions, local variation in reproductive efforts may be as great as over large geographic areas (Bliss 1956). The lowest value of most of the traits measured (plant height, petiole length, stem mass, tuber mass, flowers/plant, and seeds/follicle), together with the smallest population size (Table 3.3), the lowest seed mass (Figure 3.11B), and the lowest leaf N content (Table 3.7) at Khangsar, could be the indicative of harsh environmental conditions including low temperature (due to upper elevation limit of A. naviculare in Manang) and high anthropogenic pressure (grazing and collection). Plant height and petiole length appeared to be determined by height and canopy structure of associated species. Lower height and lower petiole length at Khangsar, Ice lake area and Yak Kharka were due to dominance of dwarf shrub species such as Cotoneaster microphyllus or Caragana gerardiana as associated species (Table 3.2). Highest flowers/plant at Ledtar (Figure 3.7) was due to dominance of the individuals from open areas in the population of A. naviculare because individuals growing in open areas produced more flowers/plant than individuals growing within patches of shrubs (Table 3.5).

Seeds/follicle was the highest at Manang hill (Figure 3.7) where density of A. naviculare was the lowest (Table 3.3). Bosch and Waser (1999) reported a positive relation between density and seed set in A. columbianum and Delphinium nuttallianum, and the decline in seed set in sparse populations was attributed to receipt of low quality pollens (inbreed) or harsh environmental conditions. But in Manang hill, despite low population density, growth vigor was high as evident from the high biomass of individual plants (Figure 3.7), hence high above ground biomass (Table 3.3). This indicates favorable physical environmental conditions; but high anthropogenic pressure had reduced population density of A. naviculare in Manang hill. Thus, it appears that seed set was determined by growth vigor of individual plants, and population density was not low enough to prevent effective pollination, fertilization and seed set at Manang hill.

Reproductive efforts such as flowers/plant, seeds/follicle and seed mass were positively related with morphological traits and biomass (Table 3.89, Table 3.6). Flowers/plant increased linearly with stem biomass. Seeds/follicle increased with increasing flowers/plant,

PhD dissertation by Bharat Babu Shrestha

but towards the higher value of flowers/plant, the seeds/follicle slightly declined (Figure 3.9). Resource limitation may reduce development of fertilized ovule to seeds (Körner 2003). Thus, if a plant produces several flowers, resource limitation may reduce seeds/follicle, especially in later developed terminal flowers. Reproductive efforts of Aconitum naviculare at Manang appeared to be determined by growth vigor (e.g. tall plant, high biomass) of individual plants and associated species (shrubs vs. open areas). Any consistent relations between population size/density and reproductive output could not be detected from the data of single year. The relations deviate sometimes from general trend due to overriding effect of environment (Morgan 1999) and/or spatial and temporal variation in pollinator activities (Utelli and Roy 2000).

3.4.4 Variation in Growth and Reproductive Traits with Life Forms of Associated Species

Variation in growth characters and reproductive outputs among the individuals of Aconitum naviculare growing in juniper, other shrubs and open areas (Table 3.5) represent growth responses to resources availability. The variation in plant height and petiole length was adaptive response to light. Plasticity of shoot helps to place the leaves in areas of high light (de Kroon and Hutchings 1995). Herbaceous dicot plants growing in open habitats often respond to shading by elongating internodes (thereby increasing plant height) and petiole length (Huber 1996), and improves access to light (Falster and Westoby 2003). Long stem and petiole of A. naviculare growing in shrubs enabled the plant to overtop the associated species, thus the leaf lamina were exposed to full sun light and flowers to pollinators. Variation in plant height and petiole length allowed A. naviculare to grow and reproduce in open areas as well as dense shrubs of juniper and other thorny species.

Due to dense and closed canopy, attenuation of light is expected to be higher in juniper than in other shrubs which have relatively loose and open canopy (Larcher 1995). Thus all leaves of Aconitum naviculare should overtop the juniper canopy for light interception which was achieved by the long petiole. But with other shrubs, due to loose and open canopy adequate amount of light could reach inside the canopy, and leaf lamina lying within the canopy could receive adequate light. This explains the lack of significant difference in petiole length of A. naviculare growing in open area and other shrubs (Table 3.5).

Stem mass of Aconitum naviculare did not vary significantly among the three habitats though plant height was the highest in juniper scrub (Table 3.5). There appeared tradeoff

PhD dissertation by Bharat Babu Shrestha

between height and thickness of stem. In shaded condition the stem are thinner than in full sunlight (Huber 1996). These thin stems are biomechanically weak but thick shrubs associated with A. naviculare could protect the plant from mechanical damage by strong wind. The lowest tuber biomass of this plant growing within juniper scrubs could be the result of „inevitable effect of resource limits‟ (sensu Sultan 2003) rather than adaptive response. To position the leaves and flower in the upper layer of canopy relative allocation of biomass to stem increases in shade condition (Huber 1996), which reduced tuber biomass of A. naviculare growing in juniper scrubs. Relatively low stem and tuber biomass of A. naviculare growing in juniper scrub was also the indicative of stressful environment such as low light condition. Only a few of the proximal leaves can overtop the canopy of scrub, thereby reducing carbon assimilation and biomass allocation in comparison to individuals growing in open areas where plants were exposed to full sun light. This low resource accumulation could be responsible for the lowest reproductive effort (flowers/plant) of A. naviculare growing in juniper scrub (Table 3.5).

3.4.5 Seed Size and Germination

Seed size of Aconitum naviculare (mean: 47 mg/100 seeds = 470 μg/seed) was very close to mean seed size (467 μg/seed) of alpine forbs from various latitudes compiled by Körner (2003). Seeds collected from Khangsar had the smallest size and lost germination after 20 weeks of storage (Figure 3.11B), which could be an indicative of harsh environmental conditions at that site (also discussed in next section). Seed mass has been considered as taxonomic character (Cornelissen et al. 2003), but similar data of other Aconitum species from the Himalaya is not available for comparison.

Germination was epigeal (sensu Baskin and Baskin 1998) in which elongating hypocotyle carried the cotyledons above which it expanded and became green to be active photosynthetically (Plate 3.3). There was wide variation in germination percentage of seeds across the sampling sites and it appears to be determined by multiple factors such as elevation, density, population size and associated species. Seed viability declined with storage and there was almost no germination (0-1%) of seeds collected from Khangsar, Ledtar and Phedi after 20 weeks of storage (Figure 3.11B). Any consistent pattern in the relationship between population size and seed germinability of Aconitum naviculare could not be detected. Some earlier studies by Widen (1993), Oostermeijer et al. (1994), Ouborg and van Treuren (1995) and Morgan (1999) also failed to detect the effect of population size

PhD dissertation by Bharat Babu Shrestha

on germination, possibly because populations have not been small for long enough (i.e. several generations) for genetic erosion to have occurred (Ouborg and van Treuren 1995; Morgan 1999). Sometimes environmental factors may have more overriding impact on reproductive output than the population size (Morgan 1999).

Low seed germination of Khangsar site, lying at the upper elevation limit, could be attributed to low temperature, and fragmented and the smallest population size. In late flowering alpine plants like Aconitum naviculare, low temperature may result in underdeveloped seeds with low seed mass, poor germination and short viability (Molau 1993). Relatively high temperature at the Ice lake area due to the highest relative radiation index among the sampling sites (Table 3.2) allows complete seed and fruit development in a short period before the plant died. The habitat of A. naviculare at Yak Kharka represented the lower elevation limit (4090–4270 m asl) of distribution in Manang valley which could be relatively warm. Seed development could be complete there too, leading to the highest seed germination (~ 98%) (Figure 3.11B). Pollination behavior of A. naviculare could not be examined, but Aconitum species are exclusively insect pollinated, especially by bumble bees (Utelli and Roy 2000, and references therein). In fragmented and small populations, flowers of insect pollinating plants receive low quality pollens (i.e. inbreed) leading to significantly low fitness in natural population (Mavraganis and Eckert 2001). Though the largest population of A. naviculare was found at Ledtar site (Table 3.3), seed germination was the lowest until 15 weeks of storage and seed viability lost virtually in 20 weeks of storage (Figure 3.11B). There could be two plausible reasons for this. Firstly, population and/or activities of pollinators were insufficient to provide high quality pollens (i.e. out breed) to flowers in adequate amount, leading to low fitness (i.e. germination). Secondly, greater proportion of A. naviculare population was found in between alpine scrub along with other herbaceous species flowering at the same time. This synchronous flowering leads to inadequate pollination service due to inter-specific competition (Kudo 1993), resulting in low seed germination and short seed viability. These two assumptions could also explain high germination (~ 90%) of seeds collected from Ice lake area where the density was the highest but confined in a small area (Table 3.3), and the plant mostly grew within the alpine scrub of early flowering Caragana gerardiana.

PhD dissertation by Bharat Babu Shrestha

3.4.6 Leaf Nitrogen (N) Content

Leaf N content is the most commonly used index of nutrient status in plant body (Chapin and Cleve 1989). Leaf N content of Aconitum naviculare (2.44 %, Table 3.7) lies within the range (0.2–6.4%) of global data set for 2548 species compiled by Wright et al. (2004). It was higher than in some tropical herbs in Nepalese grasslands (0.9–2.3 %, Jha 2003), but comparable to leaf N content of Neopicrorhiza scrophulariiflora (1.31–2.73 %) growing on the northern slope of the Manang valley at the similar elevation range (Shrestha et al. 2007a). Generally leaf N content increases with elevation (Korner 1989), and mass-based maximum photosynthetic rate (carbon assimilation) increases with leaf N content (Reich et al. 1997; Garnier et al. 1999). High leaf N content in A. naviculare can be considered as an adaptive response to short growing season in alpine region whereby plants can maximize carbon assimilation by exploiting high light availability as in arid regions (Cunningham et al. 1999). Leaf N content from 1.5 to 2.0 % (15–20 mg/g) has been considered as adequate for the growth of most plants (Chapin and Cleve 1989). Leaf N content of A. naviculare was more than this range and the difference between N content of leaves and soil was low (i.e. low leaf to soil N ratio) (Table 3.7). Therefore, it can be inferred that growth of A. naviculare in present study sites was not limited by N supply. However, the lowest value of leaf N content at Khangsar is the indicative of relatively hostile environment. This could be the reason for having the lowest values of the measured growth characters and reproductive outputs at Khangsar.

3.4.7 Plant Response to Natural Environment

Among the environmental factors, temperature appeared to be important for determining abundance and distribution of Aconitum naviculare. Area based above ground biomass (Figure 3.4) and stem biomass of individual plant (Figure 3.10) declined with elevation. At higher elevation, temperature is low which reduces plant height and hence biomass. Population density increases with increasing relative radiation index (Figure 3.3) and it was highest at Ice lake area (Table 3.3) where relative radiation index was the highest (Table 3.2). The highest density might also be due to high seed germination at Ice lake area (Figure 3.11B). Seed germination as well as leaf N content (Table 3.7), and growth characters and the reproductive outputs (Figure 3.7) were lower at Khangsar, which lies at the upper elevation limit of A. naviculare distribution in Manang, and where plants were exposed to lowest temperature.

PhD dissertation by Bharat Babu Shrestha

Aconitum naviculare showed a number of adaptive features to alpine environments with strong wind. The flower did not open completely, and was globular with a small opening on the lateral side (Plate 3.1A). A globular shape has been suggested as an adaptation to minimize mechanical damage (including pollen loss) by strong wind in alpine environment (Garg and Husain 2003). Temperature inside the blue flower is higher than in flowers of other colors (Tikhomirov et al. 1960 as cited by Bliss 1962). This could compensate, to some extent, the problem of short favorable air temperature for late flowering plants to develop seeds and fruits completely as opined by Molau (1993). Persistent sepals (Plate 3.1D) could help to maintain relatively high temperature inside for rapid development of fruits, and protect developing fruits from desiccating effect of strong wind and solar radiation. The tuberous root of A. naviculare (Plate 3.1B) functions predominantly as storage organ rather than clonal spread, which is common among geophytes (Cornelissen et al. 2003). In open areas, this plant assumed cushion form with meristem of shoots and leaves buried inside the soil. Burial of perennating bud and meristem below the soil surface may prevent damage from low temperature (Larcher 1995; Körner 2003). A. naviculare had a well developed winter perennating bud in tuberous root, used food reserve of previous year for early growth during growing season, shoot and leaf meristem inside the soil, blue flower, and persistent sepals. These traits have been considered important adaptive features of plants to alpine environments (Bliss 1962; Larcher 1995; Körner 2003).

3.4.8 Life History Traits vs. Distribution

Since there was significant reduction in seed viability after 20 weeks of storage at 4 C with no germination of seeds collected from some sites (Figure 3.11B), it could be possible that no perennial soil seed bank of Aconitum naviculare is developed in its natural habitats. The plant had gravity dispersed seeds without active mechanism of dispersal. It has prevented long-range seed dispersal of this plant. Generally seeds without long-range dispersal exhibited dormancy (Barbour et al. 1999), but this situation was not observed in this plant. Early loss of seed viability and absence of long-range seed dispersal could explain the clumped dispersion of individuals, and narrow range of distribution of population of A. naviculare. Plasticity in plant height and petiole length has enabled the plant to colonize a range of micro-habitats from thick scrubs of junipers to open areas without any scrubs.

In conclusion, Aconitum naviculare is naturally rare, and exclusively found on sunny and dry south facing slope between 4000 and 4700 m asl. This species was often found in

PhD dissertation by Bharat Babu Shrestha

association with alpine scrubs, which had protective role against livestock damage and collection by human. This protective role appears to be critical for the persistence of this plant. Seeds of A. naviculare were non dormant but environmental factors inhibited germination immediately after seed dispersal. Any consistent pattern in relationship between population size and seed germinability of A. naviculare was not detected. Most of the growth and reproductive traits varied significantly with sampling sites and life forms of associated species. Mean density of the plant increased with relative radiation index while mass of stem of individual plant and area based above ground biomass both declined with elevation. A. naviculare has evolved a number of adaptation features to alpine environment and biotic disturbance (including anthropogenic) which includes bluish globular flower with small opening on lateral side, persistent sepal covering developing fruits, winter perennating bud inside soil (geophyte), and presence of intercalary meristem between stem and tuber. Early loss of seed viability, lack of long-range seed dispersal, and plasticity in plant height and petiole length are some of the plant traits which explained observed habitat range and distribution pattern of this species. This supported the assumption that life history traits are helpful in understanding the distribution patterns of plant species.

PhD dissertation by Bharat Babu Shrestha

4. ECOLOGY OF CURCULIGO ORCHIOIDES

4.1 Introduction

Curculigo orchioides Gaertn. (Hypoxidaceae) is found in tropical to subtropical regions of Asia and Australia (Press et al. 2000; Zhanhe and Meerow 2000). Rootstock of this plant, which contains glycosilated phenolics, triterpenic saponins and aliphatic ketones, has been used in traditional medicine as aphrodisiac, tonic to restore vigor, and against asthma, bronchitis, jaundice, dysentery, diarrhea, leucorrhoea and male sterility (Shrestha et al. 2008). In recent time, research interest on C. orchioides has been increasing as this plant species has been emerging as a source of new bioactive natural products with potential use in drug (Kapoor 2008). In Indian herbal market, this species has been mainly supplied from wild collection, and there is higher demand than supply (Kala et al. 2006). In Nepal, the trade status of this plant has been controversial; the plant material being marketed from Nepal under the trade name “musali” was confirmed not to be C. orchioides (Shrestha et al. 2008).

Medicinal uses of Curculigo orchioides have been well documented in ethnobotanical literatures, and nearly two dozens of peer reviewed references are available on phytochemistry and pharmacognosy (Shrestha et al. 2008). Paradoxically, ecological study of this plant is virtually lacking. Though empirical data on population status was absent, this plant has been assigned to various threat categories from vulnerable (Bhattarai et al. 2001), threatened (Manandhar 2002) to endangered (Suri et al. 1999; Jasrai and Wala 2001; Prajapati et al. 2003; Dennis and Alphonsa 2004; Francis et al. 2007) based on general observations and experts‟ perceptions. In present research, ecological studies of C. orchioides pertaining to distribution, abundance, and reproductive biology were undertaken. The hypothesis being tested was „life history traits can help to explain the distribution pattern of plant species.‟ The specific objectives were: 1) to document distribution and habitat range of C. orchioides in the study area; and 2) to understand relationship among environmental variables, life history, and abundance of this plant species in natural habitat.

PhD dissertation by Bharat Babu Shrestha

4.2 Materials and Methods

4.2.1 Study Area

Population of Curculigo orchioides was sampled from riparian Shorea robusta forest (410 m asl) of inner tarai to Schima-Castanopsis forest (1018 m asl) of hills, including grassy slope and abandoned agriculture land, to cover entire vertical range of distribution of this species (Figure 4.1, Table 4.1). All forests in sampling sites have been managed by local people under community forestry. The „abandoned agriculture land‟ is a terraced slope which has been abandoned for about 10 years for growing herbaceous fodder. The „grassy slope‟ is a piece of land prepared after forest clearing for growing herbaceous fodder, and lies between terraced agriculture land and natural forest. Abandoned agriculture land and grassy slope, though both are open areas, have 10-15% canopy cover of isolated trees.

Table 4.1. Land uses and physical locations of sampling sites of Curculigo orchioides.

Land uses Elevation Aspect Slope ( ) Locations (m) Chitwan (Barandabhar) Tarai Shorea 410 Plain 0 robusta forest Nawalparasi (Rajahar Foot hill Shorea 425-660 SE 0-30 and Mukundapur VDCs) robusta forest Syanja (Putalibazaar-10) Hill S. robusta 980-1000 SE 20-35 forest Grassy slope 845-860 E 30-35 Lamjung (Gaunshahar Schima-Castanopsis 1009-1018 NE 15-25 VDC) forest Abandoned 877-903 E 15-20 agriculture land Range/Mean 410-1018 0-35

PhD dissertation by Bharat Babu Shrestha

Figure 4.1. Map of Nepal showing sampling sites of Curculigo orchioides.

PhD dissertation by Bharat Babu Shrestha

Table 4.2. Some features of sampling sites in tarai Shorea robusta forest (Barandabhar) and foot hill Shorea robusta forest (other four sites). All these sampling sites lied within community managed forests (Community Forests).

Sampling Elevation Slope Soil characters Litter cover Livestock Tree Shrub sites (m asl) () (%) Grazing canopy canopy cover (%) cover (%)

Barandabhar 410 0 Dark brown, sandy loam 10 Banned† 60 50

Dhuseri 450 0-10 Brown to reddish brown, 55 Banned 65 45 coarse to fine

Chautari 660 10-20 Brown; abundant 50 Goat grazing 35 30 pebbles on the surface

Sundari 450 10-20 Reddish brown, fine 64 Banned 65 55

Sansari 425 20-30 Brown, coarse to fine 27 Goat grazing 30 65

†But, wild animals like rhino, deer, wild boar, etc. come to this forest in significant number from adjoining Chitwan National Park.

PhD dissertation by Bharat Babu Shrestha

Figure 4.2. Monthly mean temperature and precipitation from 1997-2006 recorded at Rampur Meteorological Station, Chitwan. The station lies between sampling sites of Chitwan (tarai Shorea robusta forest at Barandabhar) and Nawalparasi (foot hill S. robusta forests) at 256 m asl. (Source: Department of Hydrology and Meteorology, Government of Nepal)

Phenological pattern of Curculigo orchioides and plant-environment interaction were examined in tarai Shorea robusta forest (Barandabhar of Chitwan district) and foot hill S. robusta forest (Mukundapur-Rajahar area of Nawalparasi district) (Table 4.2). In Nawalparasi, the sampling sites spread across four community managed forests (Dhuseri, Chautari, Sundari and Sansari). The study area (27 37–42 N, 84 13–26 E) lies in tropical region of central Nepal with mean temperature from 15 to about 30 C in different months (Figure 4.2). The area receives monsoon rain from June to September, which accounts about 80% of the total annual rainfall (2244 mm). Rest of the months are dry with few showers of winter rain. The area has deciduous to semi-deciduous tropical forests with Shorea robusta forest as dominant type, and riverine Acacia-Dalbergia forest as a minor component (Stainton 1972).

PhD dissertation by Bharat Babu Shrestha

4.2.2 Study Species

Curculigo orchioides Gaertn. (synonyms: Curculigo malabarica Wight, family: Hypoxidaceae, Plate 4.1) is called Kalo musali in Nepali; Xian Mao in Chinese; Kali musli in Hindi; Hemapushpi in Sanskrit; black musale, golden eye grass in English. C. orchioides is a perennial herb, with vertical rootstock and bundle of fleshy roots at the base of rootstock; leaf blade narrow to broadly lanceolate; inflorescence spike, appearing almost at the ground level, first 1-2 flowers bisexual, others male; perianth lobes yellow, lanceolate; berry hidden in leaf bases, irregular, oblong to ovoid, tapered into beak; seeds few, shiny black with hook like projection (Noltie 1994; Siwakoti and Verma 1999; Zhanhe and Meerow 2000).

Rootstock is the medicinally important part of this plant. In addition to reported uses (review in Shrestha et al. 2008), the plant has been fed to male buffalo as aphrodisiac in one of the study area (Syanja). In study sites of Chitwan (tarai Shorea robusta forest) and Nawalparasi (foot hill Shorea robusta forest) the rootstock of this plant has been extensively foraged by wild boar during September-October.

4.2.3 Field Sampling

Field sampling was done between 2005 and 2007. Preliminary information on habitat range of Curculigo orchioides was obtained by expert consultation and examining herbarium specimens deposited at National Herbarium (KATH) and Tribhuvan University Central Herbarium (TUCH). Then potential areas were visited to select sampling sites. Four localities and six habitat types were identified for the study of soil nutrients, population density and rootstock yield (Table 4.1). Two localities (Chitwan and Nawalparasi) were selected for reproductive biology (Table 4.2).

Population Density, Rootstock Biomass and Soil Nutrients: In each habitat type, a number of 10 m × 10 m plots were subjectively chosen to represent the sites with relatively high density of C. orchioides. Since the abundance and population size varied widely with habitat types, the number of sampling plots also varied from two to sixteen (four in tarai Shorea robusta forest, sixteen in foot hill S. robusta forest, two in hill S. robusta forest, and three in each of Schima- Castanopsis forest, abandoned agriculture land and grassy slope). In each plot, 10 quadrats (1 m × 1 m) were sampled randomly. Total number of individuals of C. orchioides was recorded. In each quadrat, single best looking mature

PhD dissertation by Bharat Babu Shrestha

Plate 4.1. Curculigo orchioides. A) mature individual with flowers and fruits, B) flowering individual tagged for monitoring phenology, C) rootstock with cluster of fleshy roots at the base, D) fruits, E) seeds, and F) seedling.

PhD dissertation by Bharat Babu Shrestha

individual of this plant was completely uprooted for estimation of rootstock biomass (g/plant) and area based yield of rootstock (kg/ha). During that process, about 200 g soil sample was also collected from 10-15 cm depth to determine soil organic carbon and nitrogen. Soon after collection, the soil samples were placed for air drying under shade.

Phenology: To record phenology, four plots (10 m × 10 m) in tarai Shorea robusta forest (Chitwan) and 16 plots in foot hill S. robusta forest (Nawalparasi, four plots in each of the four community forests) were subjectively chosen and marked permanently. The plots were relatively undisturbed and had high density of Curculigo orchioides. In each plot, ten quadrats (1 m × 1 m) were located randomly. The number of individuals in each phenophase – seedling, vegetative (mature but without flower and fruit), flowering and fruiting – were recorded in each quadrat. Tree canopy and ground vegetation (shrubs layer) cover were estimated visually. Twenty, best looking flowering individuals in each plot were tagged using aluminum plate (Plate 4.1B). Altogether, 200 quadrats lying within 20 plots were sampled; and 400 individuals tagged for regular monitoring of phenological changes. First sampling and tagging was done in June 2007, and sampling at the marked plots was continued until October 2007 when the plant began to senesce. Sampling could not be done in September, due to harsh weather condition (continuous heavy rainfall). In repeated observation, the phenophases of tagged individuals were recorded as vegetative, flowering and fruiting. In October, litter cover (%) on the ground was estimated.

Transplantation: For close monitoring of phenological events, mature individuals (in reproductive stage) were transplanted in August 2007 from foot hill Shorea robusta forest (Nawalparasi), Schima-Castanopsis forest (Lamjung) and abandoned agriculture land (Lamjung) to Kathmandu (1350 m asl at Central Department of Botany, Tribhuvan University, Kirtipur) and grown in earthen pots (mean diameter: 20 cm) filled with garden soil. Two to four individuals were grown in each pot maintained under ambient environment with partial shade and frequent watering during dry months. Starting from last week of May 2009, number of mature individuals that had sprouted from underground rootstock, seedling, individual in flowering and fruiting stage, and fruit dehiscence were recorded in every five days until November 2009.

4.2.4 Laboratory Analysis

Soil Analysis: Soil samples were air dried in shade under laboratory condition and the dried soil was passed through the fine sieve (0.5 mm). Soil organic carbon (OC) (Walkley and

PhD dissertation by Bharat Babu Shrestha

Black method) and total nitrogen (N) (micro-Kjeldahl method) were determined following the methods described by Gupta (2000). Altogether 310 soil samples were analyzed from the habitats of Curculigo orchioides. The laboratory protocol used for the analysis of soil has been described in Appendix 1.

Seed Germination: Seeds collected for germination experiment were stored at 4 C. In preliminary experiments, seeds of this plant did not germinate when placed between moist blotting paper for three months, indicating dormancy. Viability of seeds was tested using 0.1% solution of 2,3,5 triphenyl-tetrazolium chloride. Seeds with seed coat removed were placed between blotting papers saturated with tetrazolium solution for 24 hours. To test permeability of seed coat the methods followed by Hidayati et al. (2001) was adopted with some modifications. Three replicas of twenty seeds each (total seeds = 60) were weighed to nearest 1 mg after soaking with distilled water for about two minutes and subsequent surface-drying by blotting paper. This initial measurement was considered as dry mass

(M0). The seeds were then placed between three layers of blotting paper placed in petri dishes and saturated with distilled water. After one hour, the seeds were surface-dried by blotting paper, and reweighed to get wet mass (Mt). The process was repeated in every one hour for first ten hours and in every 24 hours for next one week. For each measurement time,

Table 4.3. Various treatments given to seeds of Curculigo orchioides during germination experiments.

SN Treatments 1 Intact seeds + 24 h soaking in distilled water 2 Intact seeds + 24 h soaking in 100 ppm GA3 solution 3 Intact seeds + 24 h soaking in 250 ppm GA3 solution 4 Intact seeds + 24 h soaking in 500 ppm GA3 solution 5 Seed coat partly removed + 24 h soaking in distilled water 6 Seed coat partly removed + 24 h soaking in 100 ppm GA3 solution 7 Seed coat partly removed + 24 h soaking in 250 ppm GA3 solution 8 Seed coat partly removed + 24 h soaking in 500 ppm GA3 solution 9 Seed coat partly removed + 24 h soaking in 100 ppm GA3 solution + MS medium 10 Seed coat partly removed + 24 h soaking in 250 ppm GA3 solution + MS medium 11 Seed coat partly removed + 24 h soaking in 500 ppm GA3 solution + MS medium

PhD dissertation by Bharat Babu Shrestha

the percentage increase in mass (M) was calculated as: M = (Mt – M0)  100/M0. For germination experiment, 98 seeds were selected from the stock of freshly collected seeds and they were subjected to various treatments as summarized in Table 4.3.

For treatment 1-8, ten seeds were used in each and the seeds were transferred to petri dishes (five seeds in each of two petri dishes) lined with double layer of moist blotting paper. These petri dishes were placed in the laboratory under the same conditions as for Aconitum naviculare. For treatments 911, seeds were inoculated to MS medium after surface sterilized by 70% ethanol (1 minute) and then 1% sodium hypochlorite (5 minutes).

4.2.5 Numerical Analysis

Rootstock yield (kg/ha) of C. orchioides was calculated from density (plant/m2) and rootstock biomass (g/plant). Based on field observations about 50% of total individuals in the population were assumed to be mature. Therefore half of density (density/2) was multiplied with rootstock biomass/plant of each sampling plot to get yield. Because the variances of different set of data were not equal (Levene test), the non-parametric tests (Kruskal-Wallis and Mann-Whitney U tests) were used for comparison of average of density, rootstock biomass, and rootstock yield among habitats. Values of density, rootstock biomass and rootstock yield across the habitats were pooled together, and their variation with soil OC and N were analyzed. Regression analysis was avoided because some of these data set did not show normal distribution even after transformation (log, square root and acrsine). Therefore, the relationships were shown in scattered diagram.

From the data of quadrat sampling for phenology, percentage of individuals in various phenophases were calculated for each sampling month for each sampling site as well from combined data. Data from tagged individuals was also used to calculate percentage of individuals in flowering, fruiting and vegetative for each sampling month and site. The plant species was found phenologically the most active during July. Therefore, frequency of individuals in each phenophase has been calculated for each plot from the July data of quadrat sampling and tagging. Average values of soil OC and total N were calculated for each plot. In regression analysis, phenological characters were used as response variables and environmental variables (litter cover, canopy, soil OC, soil N) as explanatory. Before regression, the data were checked for normality (Kolmogorov-Smirnov test), and log transformed, if necessary, to meet the assumption of normality.

PhD dissertation by Bharat Babu Shrestha

The percentage of individuals in flowering and fruiting were also calculated for the data collected in transplant experiment. Highest number of seedlings was recorded during first three weeks of August (6th to 21st August). Therefore, the ratio between the number of seedlings and mature individuals for each plot with at least single seedling was calculated from four consecutive data recorded during that period. All statistical analyses were done using Statistical Package for Social Sciences (SPSS 2002, Version 11.5).

4.3 Results

4.3.1 Distribution

Direct field observation and review of herbarium specimens showed that Curculigo orchioides was found throughout Nepal (eastern, central and western) from <200 to about 2100 m asl in a wide range of habitats from riparian Shorea robusta forest of tropical region to dry sunny grassy slopes in upper subtropical region (Table 4.4). Population distribution of C. orchioides in Shorea robusta forests was nearly random but in Schima- Castanopsis forest it showed clumped distribution because it was mainly found at forest gaps. The sunny grassy slopes, where C. orchioides was found, were the pieces of land developed after forest clearing along the margin of cultivated land. Above ground biomass of these grasslands was annually harvested for stall feeding of livestock. Soil OC across the habitats of C. orchioides ranged from 0.85 to 5.25 % (mean: 2.21) while soil N from 0.058 to 0.211 % (mean: 0.113%, Table 4.5).

4.3.2 Abundance

Population density, rootstock biomass and estimated rootstock yield varied with habitat types (Figure 4.3). Mean values of these parameters across the habitat range were 7 pl/m2, 1.32 g/plant and 49 kg/ha, respectively. Highest density was shared by foot hill Shorea robusta forest and abandoned agriculture land in hill, but rootstock biomass and yield were both highest in foot hill S. robusta forest. Rootstock biomass varied significantly among the forest habitats but it was similar between open lands. When compared between pairs of land uses in the same study area (hill S. robusta forest vs. grassy slope in Syanja and Schima- Castanopsis forest and abandoned agriculture land in Lamjung), forests had lower density and rootstock yield as compared to corresponding open areas.

PhD dissertation by Bharat Babu Shrestha

Table 4.4. Collection sites and habitats of Curculigo orchioides specimens deposited at National Herbarium (KATH), Godawari, Lalitpur and Tribhuvan University Central Herbarium (TUCH), Kirtipur, Kathmandu. All specimens were collected within Nepal. Some information from personal collection and references has been also presented.

SN District Locality Elevation Ecological Habitat Herbaria (m asl) region* 1 Banke Mahadevpuri 200 W Mallotus forest TUCH 2 Bardia Brindapuri 168 W - TUCH 3 Dadeldhura Jogbudha 574 W Mixed sal forest § 4 Dang Garhwa 250 W Open field KATH 5 Dang Phedi 440 W Shady and rocky KATH land 6 Kailali Attaria 300 W Sal forest KATH 7 Kanchanpur Daijaee 265 W Sal forest § 8 Kanchanpur Suklaphant 150 W sal forest KATH 9 Kapilvastu Nardasagar 170 C - TUCH 10 Palpa Dovan 300-1100 C Churia sal forest §§ 11 Nawalparasi Rajahar 500 C Foot hill sal forest TUCH 12 Nawalparasi Rajahar, 425-660 C Foot hill sal forests ** Mukundapur 13 Syanja Putalibazaar - C Hill sal forest, ** 10 Grassy slopes 14 Syanja Raniswara 1700 C Moist ground KATH 15 Chitwan Tikauli 450 C - TUCH 16 Chitwan Barandabhar 410 C Riparian sal forest ** 17 Kaski Deurali 2080 C Shady place KATH 18 Kaski Lumle 1600 C - TUCH 19 Lamjung Gaunshahar C Schima-Castanopsis ** forest, Abandoned agriculture land 20 Gorkha Bungkot 600 C Hill sal forest ** 21 Dhanding Maidi 400-500 C Hill sal forest ** 22 Kathmandu Nagarjun 1500 C Forest gap of ** Schima-Castanopsis forest 23 Lalitpur Goth danda 1219 C Sunny place KATH 24 Makawanpur Kamle 1000 C Open land KATH 25 Kavrepalanchock Baluwa - C Forest KATH 26 Kavrepalanchock Khardorpati 1550 C Moist land KATH 27 Kavrepalanchock Dapcha 1740 C Shady place KATH 28 Dolakha Malephu 970 E Open place KATH Tamakoshi along trail 29 Dolakha Kirantichhap 1110 E Pine forest KATH basti 30 Morang Rajarani 570 E Shady slope KATH *W: Western Nepal, C: Central Nepal, E: Eastern Nepal. **Collection of Bharat B Shrestha. §Collection of PK Jha and Narayan Ghimire. §§Gubhaju and Ghimire 2009; sal: Shorea robusta

PhD dissertation by Bharat Babu Shrestha

Table 4.5. Organic carbon and nitrogen contents in the soil of habitats of Curculigo orchioides

Sampling sites Soil organic Soil nitrogen carbon (%) (%) Tarai Shorea robusta 0.850.19 0.0580.009 forest Foot hill Shorea robusta 1.280.48 0.0880.041 forest Hill S. robusta forest 4.711.07 0.1410.035 Schima-Castanopsis forest 3.630.30 0.2110.032 Abandoned agriculture 2.830.65 0.1640.046 land Grassy slope 5.250.51 0.1560.021 Mean 2.211.58 0.1130.058

PhD dissertation by Bharat Babu Shrestha

2.00 p<0.001, chi-square = 91.94, N = 310 Barsp<0.001, show Medians chi-square = 98.42, N = 308 Bars show Medians d 8.0 1.55 d (2.5) 8.0 7.0 d (1.37) 6.5 c (6.0) 1.50 (7.0) 1.10 c 5.1 c 6.0 (0.65) (3.4) 4.0 b 1.00 0.78b 0.81 b (3.0) 4.0 0.66 b (0.93) (0.50) (0.62)

Density (pl/sq m) (pl/sq Density 0.47 a (0.46) 1.5 a 0.50

2.0 (1.0) Rootstock biomass (g/plant) biomass Rootstock

0.00 1 2 3 4 5 6 1 2 3 4 5 6 Habitats Habitats

p<0.001, chi-square = 126.6 Bars show Medians 56.4d N = 310 60.0 (74.3)

40.0 31.9c (53.0)

22.6bc (35.4) 14.9b 20.0 (19.4) Rootstock yield (kg/ha) yield Rootstock 8.5 a 7.7 a (6.6) (10.9)

0.0 1 2 3 4 5 6 Habitats

Figure 4.3. Population density, rootstock biomass and estimated yield of Curculigo orchioides across the habitats (1: Tarai Shorea robusta forest, 2: Foot hill S. robusta forest, 3: Hill S. robusta forest, 4: Schima-Castanopsis forest, 5: Abandoned agriculture land, and 6: Grassy slope). Main entries above bars are medians and values inside parentheses are interquartile ranges; values with same letter are not different significantly at p<0.05. Test statistics are based on pair-wise Mann- Whitney U test, and Kruskal-Wallis test. N = total sample size.

PhD dissertation by Bharat Babu Shrestha

4.3.3 Environmental Variables and Abundance

Density, rootstock biomass and yield of C. orchioides declined with increasing soil OC and N across the habitats (Figures 4.4-6). In S. robusta forests of tarai and foot hills, density of C. orchioides declined with increasing tree canopy (Figure 4.7).

Habitats

6 DENSITY 5 10 4

)

2 2

0 1 0 1 2 3 4 5 6 7

Soil organic carbon (%)

Density (pl/m Density 30

Habitats

6 DENSITY 5 10 4

0 1 0.0 .1 .2 .3

Soil nitrogen (%) Figure 4.4. Variation of density of Curculigo orchioides with soil organic carbon and nitrogen using data pooled from across the sampling habitats (1: Tarai Shorea robusta forest, 2: Foot hill S. robusta forest, 3: Hill S. robusta forest, 4: Schima- Castanopsis forest, 5: Abandoned agriculture land, and 6: Grassy slope). Each point represents a sampling quadrat.

PhD dissertation by Bharat Babu Shrestha

Habitats 3 6

ROOTSTOC 5 2 4

) 2

0 1 0 1 2 3 4 5 6 7

Soil organic carbon (%) 6

5 Rootstock biomass (g/pl Rootstock biomass

Habitats 3 6

ROOTSTOC 5 2 4

3 1 2

0 1 0.0 .1 .2 .3

Soil nitrogen (%) Figure 4.5. Variation of rootstock biomass of Curculigo orchioides with soil organic carbon and nitrogen using data pooled from across the sampling habitats (1: Tarai Shorea robusta forest, 2: Foot hill S. robusta forest, 3: Hill S. robusta forest, 4: Schima- Castanopsis forest, 5: Abandoned agriculture land, and 6: Grassy slope). Each point represents a sampling quadrat.

PhD dissertation by Bharat Babu Shrestha

300

200

Habitats

6 RTSTKYD 5 100 4

0 1 0 1 2 3 4 5 6 7

Soil organic carbon (%)

300 Rootstock yield (kg/ha) Rootstock yield

200

Habitats

6 RTSTKYD 5 100 4

0 1 0.0 .1 .2 .3

Soil nitrogen (%)

Figure 4.6. Variation of rootstock yield of Curculigo orchioides with soil organic carbon and nitrogen using data pooled from across the sampling habitats (1: Tarai Shorea robusta forest, 2: Foot hill S. robusta forest, 3: Hill S. robusta forest, 4: Schima- Castanopsis forest, 5: Abandoned agriculture land, and 6: Grassy slope). Each point represents a sampling quadrat.

PhD dissertation by Bharat Babu Shrestha

Figure 4.7. Variation of density of Curculigo orchioides with tree canopy. Each point for density represents the mean of 10 quadrats sampled in tarai and foot hill Shorea robusta forests. Fitted lines were obtained by two order polynomial regression.

4.3.4 Life History

Curculigo orchioides is a perennial herb with modified, vertical, underground stem called rootstock. Leaves are annual, which remained alive for 78 months from late summer to early winter. The rootstock is a perennating organ with shoot apex (perennating bud) near the soil surface. Therefore, the plant is hemi-cryptophyte according to Raunkiaer‟s classification of life forms. The rootstock is generally unbranched, but it may produce more than one adventitious shoots when primary shoot apex is mechanically damaged. Dry mass of a single rootstock was measured up to 5.1 g (mean: 1.32 g, n = 308). Length of rootstock was measured up to 22.5 cm with mean value of 10.8 cm (n = 148) while the maximum diameter measured was 1.5 cm.

With the onset of growing season, the dormant bud produced new leaves. The inflorescence was produced together with new leaves or after unfolding of first few leaves. In rare cases, inflorescence was produced before the unfolding of new leaves. During a growing season

PhD dissertation by Bharat Babu Shrestha

some individuals of this plant produced up to four bouts of inflorescences (Figure 4.8). Average number of inflorescences/plant was 1.63 (n = 579), and the number of flowers/inflorescence was six (n = 24) with five male flowers and single bisexual flower. If an individual plant produced more than one inflorescence/plant during a growing season, they did in two ways. They produced inflorescences in a subsequent order such that a plant would have more than one inflorescence at a time but at different developmental stages (Plate 4.1A). Alternatively, they produced inflorescences with interval of a few weeks to a month. There was asynchrony in flowering within an inflorescence. The first flower produced was always bisexual and it opened 3-5 days before subsequent flowers did. A few individuals produced more than one bisexual flower/ inflorescence; therefore average number of bisexual flowers/inflorescence was 1.13 (n =31). In such case, the time gap between opening of first and second flowers could be shorter. But in any case, the bisexual flowers mostly withered before anthesis of male flowers.

Figure 4.8. Frequency of tagged individuals of Curculigo orchioides producing one, two, three or four bouts of inflorescences across the sampling sites.

Fruits were berry with soft wall. Seed dispersal was by the disintegration of fruit wall while remaining attached to mother plant. A fruit contained 115 seeds (mean: 7 seeds/fruit, n = 263) with seed size 5 mg/seed (n = 3 with 100 seeds each). Mean seed output per flowering

PhD dissertation by Bharat Babu Shrestha

plant per year was 12 (product of mean inflorescences/plant, bisexual flowers/plant and seeds/fruit with the assumption that all bisexual flowers developed fruits). Immature seeds were white and fleshy while the mature ones were black, hard and non-wettable (Plate 4.1E). Longitudinal section of seed showed a small and undifferentiated embryo surrounded by copious endosperm (Plate 4.2A,B). Seeds were dispersed by ants as well as by surface runoff water during rainy season. Immediately after the wall ruptured, seeds were often carried away by ants. When the fruits were not sufficiently exposed to the soil surface, seeds were not dispersed. In that situation, seedlings were crowded very close to mother plant (Plate 4.2D). Seeds remained dormant over the winter and germinated during the first half of the next growing season. Seedling density was negatively affected by litter, and it declined with increasing litter cover (Figure 4.9). In addition to reproduction by seeds, this plant also propagated, though less frequently, by plantlets produced from mid-rib region of leaves after mild damage (See „Clonal Propagation‟ below for detail).

Figure 4.9. Relationship between seedling density of Curculigo orchioides and litter cover. Each point represents mean of ten replicate samples. Fitted line, equation and other values are based on linear regression.

PhD dissertation by Bharat Babu Shrestha

Plate 4.2. A and B) Longitudinal section of seed of Curculigo orchioides (emb: embryo, end: endosperm); C) Development of two seedlings from single seed; D) Seedlings crowded near the mother plants.

4.3.5 Phenological Patterns

Phenological events started with emergence of new leaves from apical bud of underground rootstock in May (Figure 4.10A) when temperature was the highest and soil was moistened by few showers of rain (Figure 4.2). At moist but sunny sites, plants produced inflorescence together with new leaves. June-July was phenologically the most active period when there was flowering, fruiting and seed germination. During that phenologically active period,

PhD dissertation by Bharat Babu Shrestha

27% of individuals in June and 18% in July were in reproductive stage (flowering and fruiting) while 73 and 82%, respectively, were in non reproductive stages (seedlings and vegetative) (Figure 4.10B). When only mature individuals were used in analysis excluding seedlings, 74% in June and 80% in July were vegetative. Frequency of individuals in reproductive stages was always the highest at Dhuseri except in July when it was the highest at Barandabhar (Figure 4.11). In forest gaps of Schima-Castanopsis forest of Lamjung, 74% in June and 55 % in July were vegetative, but it was 57% in June and 51 % in July, respectively, in abandoned agriculture land of the same area. In transplantation, percentage of individuals in reproductive stage (flowering and fruiting) was the highest during mid August (Figure 4.12). During this period about 60% of the mature individuals were in reproductive stage.

There was single flowering peak in June (Figure 4.10B) when monsoon just began. However flowering was not synchronized and it continued until October at some sites (Figure 4.11). Fruiting occurred from June to October across the sites, but there was a single fruiting peak during July (Figure 4.10B). Fruiting occurred earlier at lowland moist site (Barandabhar) where frequency of fruiting individuals was always the highest except in October (Figure 4.11). Fruiting continued until October at Dhuseri, while there was no fruiting individual at other sites in that month. But in transplantation, individual retained fruits until mid November (Figure 4.12). About 50% (204 out of 410) of flowering individuals in June bore fruits during July. However percentage of individuals in fruiting varied across the sites; it was 89% at Barandabhar while at other sites it varied between 40 and 55% (Figure 4.13). Seed maturation nearly took one to two months, being slower towards the end of growing season. In transplantation, fruit dehiscence commenced from third week of July and continued until the end of November (Figure 4.12). Leaf senescence began from early September and plant senescence from mid October. Seeds did not germinate in the current year. It remained dormant until June of next year when rainy season began. Seed germinated in July (Figures 4.10A, 4.12).

PhD dissertation by Bharat Babu Shrestha

Figure 4.10. Phenological patterns of Curculigo orchioides based on field observation and quadrat sampling. A) Phenological events in one year starting from May; vertical lines in flowering and fruiting indicate peak frequency of the respective phase. B) Changes in frequency of individuals in various phenophases during growing season across the sampling sites.

PhD dissertation by Bharat Babu Shrestha

Figure 4.11. Percentage of individuals of Curculigo orchioides in various phenophases from June through October across the sampling sites.

PhD dissertation by Bharat Babu Shrestha

Figure 4.12. Changes in the total number of mature individuals (that sprout from perennating rootstocks), seedlings, and percentage of individuals in flowering, fruiting and reproductive phase (flowering + fruiting) of Curculigo orchioides grown in earthen pots. Arrow on the x-axis marked the first event of fruit dehiscence.

PhD dissertation by Bharat Babu Shrestha

Figure 4.13. Percentage of tagged individuals of Curculigo orchioides in different phenophases. At each site, 80 flowering individuals were tagged in June.

PhD dissertation by Bharat Babu Shrestha

4.3.6 Environmental Factors and Phenological Patterns at Population Level

Flowering was influenced by canopy cover and soil characters (Figure 4.14, Table 4.6). In June, percentage of individuals in flowering increased with increasing soil nitrogen content, tree canopy and tree×shrub canopy. In July, flowering increased with organic matter but the relation was only marginally significant (Table 4.6). There was unimodal relation between fruiting in July and tree×shrub canopy with highest fruiting at intermediate level of canopy cover (Figure 4.14). Similar pattern of variation, though not significant, was observed between tree×shrub canopy and the percentage of flowering individuals in June that borne fruits in July (Figure 4.14). The canopy and soil variables (organic carbon and nitrogen) had no effect on percentage of individuals in seedling stage, and the percentage of flowering individuals that borne fruits.

4.3.7 Seed Germination

Germination was hypogeal (sensu Baskin and Baskin 1998). Some of the seeds produced two seedlings which were unequal in size at early stage (Plate 4.2).In natural habitats seeds of Curculigo orchioides germinated after 1012 months of dispersal. In transplantation seeds of previous year began germination before the first event of dehiscence of current year‟s fruits (Figure 4.12). The number of seedlings for each reproductive individual (i.e. that flowered) was the highest for the individuals which were transplanted from abandoned agriculture land (Table 4.7). It was six times higher for abandoned agriculture land than for forests.

Freshly collected seeds did not germinate when placed between moist filter papers for more than 90 days. These seeds remained viable because the embryos were stained pink by tetrazolium solution. When placed between moist filter papers, there was increase in seed mass by 30.5% over air dry mass (Figure 4.15). GA3 treated seeds also did not germinate in moist filter paper as well as MS medium. In short, the environmental condition to break seed dormancy of Curculigo orchioides could not be identified in the present work.

PhD dissertation by Bharat Babu Shrestha

Figure 4.14. Variation of the percentage of individuals of Curculigo orchioides in different phenophases and the environmental variables. The fitted lines and the equations were obtained by linear or quadratic regression analyses. The relationship between fruiting in tagged individuals and treeshrub canopy was not significant, and therefore dotted line was fitted to the points. The restuls of regression analyses have been given in table 4.6

PhD dissertation by Bharat Babu Shrestha

Table 4.6. Results of regression analyses between some reproductive (responses) and environmental (predictors) variables of Curculigo orchioides populations in tarai Shorea robusta and foot hill S. robusta forests.

Predictors (X) Responses (Y) Model d. f. R-squared F-value Signi- ficance Soil organic Flowering (%) in July Linear 18 0.21 4.7 0.04 carbon (%) Soil nitrogen Flowering (%) in June Linear 18 0.19 4.34 0.05 content (%) (log transformed) Tree canopy Linear 18 0.57 23.55 <0.001 Tree  Shrub Flowering (%) in June Linear 18 0.46 15.59 0.001 canopy (%) (log transformed) Fruiting (%) in July Quadratic 15 0.54 8.77 0.003 (log transformed) Fruiting (%) in tagged Quadratic 16 - 2.49 0.114 individuals

Table 4.7. The ratio between the number of seedlings and mature individuals of Curculigo orchioides in transplantation. Values inside parantheses (n) are sample size.

Source of transplant Percentage of pots Mean of seedling to with seedlings mature ratio Foot hill Shorea robusta forest 44 (n=41) 0.84 (n=18)

Schima-Castanopsis forest 33 (n=15) 0.96 (n=5)

Abandoned agriculture land 94 (n=17) 5.51 (n=16)

PhD dissertation by Bharat Babu Shrestha

Figure 4.15. Increase in seed mass of Curculigo orchioides over time when placed between moist filter paper. Each value is the mean of three replicates with 20 seeds in each.

4.3.8 Clonal Propagation

Underground rootstock was mainly a mean of winter perennation. When upper part of the rootstock was damaged by human collection or animal foraging, there was often development of more than one shoot (Plate 4.3A). These adventitious shoots detached from each other in few years and became two individual ramets.

Clonal propagation from leaves (Plate 4.3B-D) was observed during second half of growing season (September-October). When mid-ribs of mature leaves were mildly damaged and the injured part came in direct contact with moist surface soil few whitish swellings were formed along midrib which resembled tiny callus. Calli were also observed at the proximal end of completely detached leaves. Wild animals, especially the wild boar, were the main biotic factors for mechanical damage to the leaves, or whole plant. During September- October, wild boar selectively foraged on the rootstock of this plant. These calli like structures that appeared at damaged parts of leaves developed into globular bulbils which produced leaves and roots. A complete plantlet was formed while being still attached to leaf mid rib. This phenomenon was observed at three sampling sites (Barandabhar, Dhuseri and

PhD dissertation by Bharat Babu Shrestha

Sundari) which were relatively moister than other sites. Among them, frequency of leaves with such plantlets was higher at Barandabhar, the wettest site.

New planlets

Basal part of old leaf

Plate 4.3. Clonal propagation in Curculigo orchioides. A) Branching in rootstock after removal of uppermost part, B-D) Clonal propagation from leaves.

PhD dissertation by Bharat Babu Shrestha

4.4 Discussion

4.4.1 Distribution

Curculigo orchioides has wide distribution in terms of elevation (<200 to about 2100 m asl), habitat types (riparian Shorea robusta forest of tarai to pine forest and dry sunny grassy slopes in hills) and physiography (eastern to western Nepal; tarai plain to mid hills of Mahabharat range in Nepal) (Tables 4.1, 4.4). It was also reported from wetland systems of tarai region (BPP 1995). Semi-deciduous dry tropical forests of tarai plain (including inner tarai) and foot hills of Mahabharat range dominated by Shorea robusta were the main habitats of this plant (Behera and Mishra 2006; Kandel and Shrestha, unpublished data). This species has also been reported from moist deciduous tropical forest in India with Xylia xylocarpa, Lagerstroemia lanceolata and Terminalia spp. as dominant tree species (Bhat and Murali 2001) and tropical dry mixed evergreen forest in Sri Lanka (Solangaarachchi and Perera 1993). In S. robusta forests, the population dispersion of this plant was nearly random. In Schima-Castanopsis forest of subtropical region in Nepal, this plant had clumped dispersion where it was mainly confined to forest gaps. It appears that deep shade within closed canopy in Schima-Castanopsis forest was not a suitable microhabitat for C. orchioides. The sunny grassy slopes, where C. orchioides was found, were the pieces of land developed after forest clearing along the margin of cultivated land. In these grassy slopes, density of C. orchioides was higher than in nearby hill S. robusta forest (Figure 4.3). Thus distribution of this plant in subtropical region appeared to be determined by light availability.

4.4.2 Abundance

Population density, rootstock biomass and estimated rootstock yield varied with habitat types (Figure 4.3). Given the highest rootstock biomass and rootstock yield and the second highest density in Shorea robusta forests of the foot hills of Mahabharat range, this forest can be considered as the most suitable habitats for the growth of this plant under natural conditions. S. robusta forests are naturally poor in species richness (Stainton 1972) and light attenuation may be relatively low, as compared to Schima-Castanopsis forests, due to it multilayered and elliptical canopy structure (Singh and Singh 1992). This creates partial shade condition to forest floor and the ground vegetation received adequate light. In such S. robusta forests, density of C. orchioides was high when tree canopy was low (Figure 4.7).

PhD dissertation by Bharat Babu Shrestha

Experimental shading has shown that rootstock yield of Curculigo orchioides was the highest under partial shading condition (Joy et al. 2004). When compared between pairs of land uses in the same study area (hill Shorea robusta forest vs. grassy slope in Syanja, and Schima-Castanopsis forest and abandoned agriculture land in Lamjung), forests had lower density of C. orchioides and rootstock yield as compared to corresponding open areas (Figure 4.3). Comparable data on population and abundance of this species is not available from Nepal and elsewhere (Shrestha et al. 2008), but this species has been reported as one of the three most frequent herbaceous plant species in Shorea robusta forests of churia range in Palpa district by Guhbaju and Ghimire (2009).

Density, rootstock biomass and yield of Curculigo orchioides declined with increasing soil organic carbon and nitrogen (Figures 4.46). When we examine spatial distribution of sampling units in the, sampling units of individual habitat type are narrowly distributed with low to no overlapping between them. For example, there was no overlapping in soil organic carbon between tarai Shorea robusta forest (1) – foot hill Shorea robusta forest (2) and hill Shorea robusta forest (3) – Schima-Castanopsis forest (4) – grassy slope (6). It appears that there was no direct „cause and effect‟ relationship between measured soil attributes (organic carbon and nitrogen) and abundance (density, rootstock biomass and yield) of C. orchioides. The relationship predicted from the present data might be due to effect of habitat types on both soil attributes and abundance of C. orchioides, rather than due to direct „cause and effect‟ relationship between latter two. In Aconitum naviculare too, population density did not vary significantly with soil variables (Section 3.3.3). However, by experimental transplanting of C. orchioides, Joy et al. (2005) have shown that type of manure had significant effect on rootstock yield. There could be two reasons for lack of direct/significant relationship, which is not expected theoretically, between soil variables (organic carbon and nitrogen) and abundance of these two species. Firstly, effect of soil nutrients on abundance under natural condition might be overridden by other environmental variables such as light, temperature and moisture. Secondly, subjective selection of sampling plots may not be a suitable sampling strategy to test the relations between them. Control experiments in ex situ condition or completely randomized sampling strategies in natural habitats may be suitable to assess the relationship between soil variable and abundance of these two species.

PhD dissertation by Bharat Babu Shrestha

4.4.3 Phenological Patterns

Phenological patterns are the result of a compromise between a variety of selective pressures, such as seasonal climatic changes, resource availability, and the presence of pollinators, predators and seed dispersers (Fenner 1998). In Curculigo orchioides, flowering began when soil was moistened by few showers of pre-monsoon rain and temperature was increasing, with flowering peak during early monsoon (Figures 4.10, 4.12). This pattern is common in seasonally dry tropical forests, such as of present study area, where flowering is induced by rainfall and often concentrated in the transition from the late dry to the early wet season (Rathcke and Lacey 1985; van Schaik et al. 1993; Murali and Sukumar 1994). Resource availability influences the timing of phenological events (Rathcke and Lacey 1985). Flowering during pre- to early monsoon in C. orchioides, together with leaf flushing, could be possible due to presence of adequate assimilate in the perennial rootstock. Moisture, temperature and the presence of adequate reserved food could have imposed bottom-up selective pressure on the timing of flowering (Elzinga et al. 2007).

Duration of flowering in C. orchioides was intermediate between mass flowering (synchrony) and steady-state-flowering. There was clear flowering peak in June (Figure 4.10, 4.12) but it continued from May to October depending upon the micro-habitats (Figure 4.11). In transplantation too, flowering occurred until early October (Figure 4.12). This is a type of phenotypic plasticity in flowering phenology. Spreading of flowering over the longer period in population level was due to production of more than one bouts of inflorescence by single individuals (Figure 4.8), with interval of few weeks to a month.

Curculigo orchioides had short (about one month) flower to fruit duration which could possibly due to utilization of reserved food for fruit development (Murali and Sukumar 1994). Fruiting during early wet season is a common tendency of plants in seasonal dry tropical forest which minimize seedling mortality in the next dry season (van Schaik et al. 1993). However, early fruiting in C. orchioides could have no effect on the seedling mortality. Seed dormancy postponed germination until early in the next wet season (July, Figures 4.10, 4.12) which ensured establishment of seedlings before next dry season began. Seed dormancy in this plant is an adaptive mechanism to time the germination so as to avoid unfavorable weather condition for subsequent establishment (Finch-Savage and Leubner-Metzger 2006). During late growing season (September-October) the plant was damaged extensively by wild boar (and other animals) while foraging the rootstock which is

PhD dissertation by Bharat Babu Shrestha

nutrient rich with calorific value 1630 KJ/100g dry mass (Suresh 2008) and aphrodisiac (Ramawat et al. 1998). Therefore, early flowering and fruiting ensured successful completion of seed production before the plant was damaged by wild animals. Any phenotype that would flower and produce fruits late in the growing season could most likely be damaged before the fruits mature.

Seeds of Curculigo orchioides had no active mechanism of dispersal. It was dispersed by ants and surface runoff water during rainy season. It is not clear whether there is presence of chemical attractants in seed to attract ants, which has been considered as an adaptive modification in ant dispersed seeds (Howe and Smallwood 1982; Bennett and Krebs 1987). But ants foraged seeds for nutritious cotyledons and fleshy pericarp. Though most of the understory herbs of moist tropical forests has fruiting peak during post-monsoon (Bhat and Murali 2001), C. orchioides had fruiting peak during mid monsoon. Since seed coat of C. orchioides was unwettable, this could help seeds to float on the surface of runoff water in rainy season (Howe and Smallwood 1982). It appears that fruiting in C. orchioides had been shaped by seed dispersal mode and potential damage by animals during late growing season.

Curculigo orchioides showed spatial variation in timing of phenological events. In tarai Shorea robusta forest at Barandabhar, a moist site, this plant started flowering and produced fruits earlier than at other sites which are relatively dry (Figure 4.11). At dry sites, soil moisture is limiting for development of new tissues. This pattern of difference between moist and dry sites was a common feature in tropical forests at community as well as species level (Newstrom et al. 1994). At community level, there was flowering peak during late dry season at moist sites while it was during early wet season at dry sites in dry tropical forests (Murali and Sukumar 1994). Individual species of tropical region also followed the same pattern (Newstrom et al. 1994).

4.4.4 Sexual Reproduction and Environmental Factors

Less than one-fourth of the mature individuals entered into reproductive phase (Figure 4.10) and about half of the flowering individuals could develop fruits (Figure 4.13). This indicates a relatively low seed output at population level and hence low regenerative potential of Curculigo orchioides through sexual reproduction in forest habitats. This low seed output could be a reason for lack of adequate regeneration of this plant in natural habits (Gupta and Chadha 1995). But in forest gaps of Schima-Castanopsis forest, and abandoned agriculture

PhD dissertation by Bharat Babu Shrestha

lands, nearly half (45 and 49 %, respectively) of the mature individuals were in reproductive stage in July. When transplanted to earthen pots, about 60% of the mature individuals were in reproductive phase (Figure 4.12). Reproductive success of this plant appeared to be determined by canopy and litter cover. Though flowering frequency increased linearly with increasing tree×shurb canopy, fruiting frequency was lower in the plots with the highest tree×shrub canopy (Figure 4.14). Reproductive success and population density of understory herbaceous monocots increased due to canopy opening after selective logging in Amazonian tropical forests (Costa et al. 2002). Effect of tree canopy on regeneration is modified by litter accumulation (Dupuy and Chazdon 2008). Among the sampling sites, highest percentage of flowering individuals (89%) in June bore fruits in July at Barandabhar (Figure 4.13), where litter accumulation was the lowest (10% at Barandabhar and 27-64% at sampling sites of foot hill Shorea robusta forest). Litter accumulation had negative impact on the seedlings density of C. orchioides (Figure 4.9), which is a common effect of litter accumulation on herbaceous species (Berendse 1999). Negative effect of litter accumulation has been also reported for seedlings of small-seeded trees (Dupuy and Chazdon 2008). The combined result of these two effects was the decline in total density of this plant with increasing tree canopy (Figure 4.7). Reproductive success was high in plots with partially open canopy and accumulation of thin layer of litter.

4.4.5 Seed Germination

Seed germination of Curculigo orchioides is hypogeal (sensu Baskin and Baskin 1998). In transplantation, seed germination began from last week of June and continued until first week of August, with exponential increase in seedling number during that period (Figure 4.12). First event of seedling emergence was observed about three weeks earlier than the first event of fruit dehiscence. This clearly showed that seeds of C. orchioides germinate in nature after at least one year, and corroborate our earlier field observations. Freshly collected, untreated seeds did not germinate when placed between moist blotting paper for ninety days. Tetrazolium test showed that these seeds were viable. This clearly indicates dormancy in seeds of this plant. There was increase in seed mass by 30.5% over dry mass when placed between moist filter paper (Figure 4.15). This increase in seed mass is typical for permeable seed coat (Carol C Baskin, University of Kentucky, USA, per. com. Nov. 20, 2009). This excluded the possibility of physical dormancy. Seed did not germinate when treated with GA3 (100, 250 and 500 ppm) for 24 h and placed in between moist filter paper in petri dishes as well as in MS medium. Longitudinal section of mature seed revealed a

PhD dissertation by Bharat Babu Shrestha

small and undifferentiated embryo surrounded by copious endosperm (Plate 4.2.A, B). Undifferentiated embryo was also reported in mature seeds of another species, Curculigo crassofolia (Dutt 1970, as cited by Johri et al. 1992). These evidences showed that seed of C. orchioides has deep morphophysiological dormancy (Baskin and Baskin 1998; Carol Baskin, per. com. Nov 20, 2009).

4.4.6 Clonal Propagation

Clonal propagation in Curculigo orchioides from leaves is not a common phenomenon but it was frequent at moist sites of Shorea robusta forests (Plate 4.3). Propagation from leaves was not observed in forest gaps of Schima-Castanopsis forests and open lands (grassy slopes and abandoned agriculture lands). It was the combined effect of mechanical damage, availability of adequate moisture in soil, and probably the high humidity. High humidity slows down the drying rate of detached or damaged leaves, and provides sufficient time for the development of plantlets. Level of clonal propagation through leaf was high in tarai Shorea robusta forest at Barandabhar where the level of damage to this plant by wild animals was also high. Therefore, frequency of clonal propagation through leaves of C. orchioides appeared to be determined by level of damage to leaves. The indiscriminate damage to adult plants by animals might have induced regeneration capacity from leaf midrib. To our knowledge, this is the first report of natural (in vivo) regeneration of C. orchioides from leaves. Earlier, Augustine and D‟Souza (1997) reported direct plantlet regeneration from mid rib of leaf explants on basal MS medium without any growth substances. Suri et al. (1999) and Prajapati et al. (2003) also reported that in vitro direct regeneration of C. orchioides plantlet was the best from mid rib region of leaf. Identification of the specific environmental conditions essential for natural regeneration of C. orchioides from leaves can help to improve the techniques of in vitro micro-propagation in large scale.

There was temporal separation between sexual reproduction and clonal propagation by leaf. Fruiting and seed germination peaked in July (Figure 4.10) while plantlets from leaves developed in September, when rainfall was the highest. Villegas (2001) also reported such seasonality in clonal propagation in a tropical understory herb (Aechmea magdalenae) which was attributed to variation in abiotic factors such as light and moisture. But in C. orchioides, seasonality appeared to be due to variation in activity of wild animals damaging the plant.

100

PhD dissertation by Bharat Babu Shrestha

4.4.7 Life History Traits vs. Distribution

Curculigo orchioides showed mixed mode of reproduction (polymorphic reproductive mode). Offspring were produced from sexually produced seeds and asexually produced bulbils from leaves. In addition to origin, seeds differed from bulbils in timing of producing offspring. Offspring from seeds were produced in the next year while offspring from bulbils from leaves were produced in the same year. Thus the population is the mixture of individuals derived from sexually produced seeds and asexually produced bulbils. The relative proportion of sexually and asexually produced offspring is determined by the complex interplay between genetic and environmental factors (Ceplitis 2001). The role of genetic variation in selecting mode of reproduction in C. orchioides is not known at present, but some of the environmental factors such as high humidity and soil moisture appeared to favor bulbils formation from damaged leaves. There appeared trade off in regenerative strategies between moist/shady and dry/open habitats. Sexual reproductive success was low at sites with high canopy cover (high shade and thereby high moisture) where frequency of clonal propagation from leaves was high. Clonal propagation from leaves was virtually absent in the dry sites with low or no tree canopy cover, where sexual reproductive success was high. This was also supported by the highest ratio of the number of seedlings and mature individuals which were transplanted from abandoned agriculture land (Table 4.7). It appears that relative importance of different regenerative strategies (sexual vs. clonal) varied with habitat types. This could be the reason for having wide range of distribution in the landscape from dense tropical Shorea robusta forests of tarai to open grassy slopes of the hills.

In conclusion, Curculigo orchioides has a wide range of habitats from tropical riparian Shorea robusta forest to pine forests and grassy slopes in subtropical region between altitude range of 200 and 2100 m asl. In terms of density, rootstock biomass/plant and area- based rootstock yield, S. robusta forest with partially open canopy of foot hills was the most suitable natural habitat of C. orchioides. Flowering was extended, and it was partly due to production of up to four bouts of inflorescences by some individuals and partly due to different timing of flowering among the individuals in the population. Fruiting frequency among individuals in forest habitats showed unimodal relationship with treeshrub canopy. Seeds showed deep morphophysiological dormancy, and the condition to break dormancy

101

PhD dissertation by Bharat Babu Shrestha

could not be identified in present work. Seeds were dispersed by ants and surface runoff water; active mechanism of seed dispersal was not observed. Germination of seeds was postponed until early monsoon of the next growing season due to dormancy. Germination during early monsoon ensured seedling establishment during the same rainy season. Regenerative potential of C. orchioides through sexual reproduction was relatively low in forest habitats because less than one-fourth of the mature individuals entered into reproductive phase and only one-half of the flowering individuals developed fruits. It was partly compensated by clonal propagation from the mid rib region of leaves which was induced by mild mechanical damage to the mature leaves. Spatial and temporal variation in regenerative strategies, and mode of seed dispersal partly explained the observed abundance and distribution pattern of this species. This supported the hypothesis that life history traits are helpful in understanding the abundance and distribution of patterns of plants.

102

PhD dissertation by Bharat Babu Shrestha

5. PHYTOCHEMISTRY

5.1 Introduction

Plant-based formulations are the major components of traditional healthcare system, and relative importance of these formulations is likely to increase in modern medicine for a foreseeable future (Craker 2007). Using traditional ethnomedicinal knowledge as starting point, scientists were able to isolate bioactive natural products from plants and used them successfully as drugs (Dahanukar et al. 2000; Cordell 2000; Ji et al. 2009), saving lives of millions of people every year (Roberson 2008). Medicinal properties of plants are mainly due to presence of bioactive secondary metabolites such as alkaloids, saponins, glycosides, flavonoids, etc. (Balunas and Kinghorn 2005; Phillipson 2007; Ji et al. 2009). Phytochemical analyses involve the screening of the plant materials for the presence of various secondary metabolites, their isolation, and characterization. These phytochemical analyses are pre-requisite for the pharmacognosy of medicinal plants (Balunas and Kinghorns 2005), which provides scientific validation to traditional uses of medicinal plants.

Research on Nepalese medicinal plants has been largely focused to ethnobotanical inventory; the phytochemical and pharmacological works constituted only 17% of the total research works related to medicinal plants (Ghimire 2008). Some initiatives were taken by the then „Royal Drug Research Laboratory‟ early in 1970s for phytochemical analysis of medicinal plants (Singh et al. 1979). However, the development of laboratory infrastructure appears to be an important limiting factor for the advanced phytochemical analysis in Nepal. Foreign researchers have significantly contributed to advance phytochemical analyses of Nepalese medicinal plants with isolation of several new compounds (e.g. Kizu et al. 1994; Matsuura et al. 1994; Tomimori 2000; Suzuki et al. 2009). Due to lack of well equipped laboratories in national research institutions (e.g. Department of Plant Resources, Chemistry Department of Tribhuvan University, Nepal Academy of Sciences and Technology, etc), phytochemical researches of medicinal plants in Nepal have been limited to „primary screening‟ (e.g. Shrestha et al. 1997, 2002; Karanjit and Singh 2003). On this ground, using secondary data from published literature and primary data from laboratory analysis, we hypothesized that „Medicinal plants in Nepal can be an important source of novel plant-based natural products‟. The specific objective of present work was to perform phytochemical analysis of two medicinal plants from contrasting habitats; Aconitum

103

PhD dissertation by Bharat Babu Shrestha

naviculare (Brühl) Stapf. from trans-Himalayan alpine region, and Curculigo orchioides Gaertn. from tropical lowland.

5.2 Materials and Methods

5.2.1 Survey of Literatures

References (research articles, book chapters, proceedings publications) on phytochemical and pharmacological studies which used medicinal plants collected from Nepal were consulted. Any references which used plant materials collected from outside Nepal, even if the species are native to Nepal, were excluded. Review of phytochemical works in Nepal (e.g. Watanabe et al. 2005), internet sites (PubMed, Scirus, and Google) and personal as well as library collections were the sources of information. References, published between 1973 and April 2010 were surveyed.

5.2.2 Study Species

In search of new compound, phytochemical analyses of two medicinal plants, Aconitum naviculare and Curculigo orchioides, were under taken.

Aconitum naviculare (Brühl) Stapf. (Ranunculaceae) is an alpine medicinal herb which has been widely used in Tibetan system of medicine against cold, fever and headache (Lama et al. 2001; Gao et al. 2004; Bista and Bista 2005; Bhattarai et al. 2006). Phytochemical analyses of this plant done so far (excluding present work) have isolated and characterized eight alkaloids; six alkaloids by Gao et al. (2004) and two by Cao J-X et al. (2008). Three, out of these six alkaloids, have not been reported in other plants.

Curculigo orchioides Gaertn. (Hypoxidaceae) is found in tropical to subtropical regions of Asia and Australia. Rootstock of this plant has been used in traditional medicine as aphrodisiac, tonic to restore vigor, and against asthma, bronchitis, jaundice, dysentery, diarrhea, leucorrhoea and male sterility (Shrestha et al. 2008). The rootstock of C. orchioides contains glycosilated phenolics, triterpenic saponins and aliphatic ketones. Altogether thirteen derivatives of glycosilated phenolics (Tiwari and Misra 1976; Kubo et al. 1983; Garg et al. 1989; Chen et al. 1999; Wu et al. 2005; Gupta et al. 2005; Valls et al. 2006), sixteen triterpenic ketones (Misra et al. 1990; Xu et al. 1992a, b; Xu and Xu 1992;

104

PhD dissertation by Bharat Babu Shrestha

Chen et al. 1999) and four aliphatic ketones (Misra et al. 1984a, b) have been isolated and characterized from this plant species.

5.2.3 Collection of Plant Materials

Aconitum naviculare: Aerial parts of Aconitum naviculare were collected during September 2004 and October 2005 from Ledtar area (4100-4200 m asl) of upper Manang. The collected material included stem, leaves, flowers and immature fruits. Simulating the collection method of local people, the tuberous root was not collected for phytochemical analysis. Fresh plant material was spread in a shaded, windy environment for two days, and was then packed in thin cotton bags and hung in the shade until it was dry.

Curculigo orchioides: The rootstocks of Curculigo orchioides were collected from Nawalparasi district (500 m asl) in August 2006. Fibrous roots were completely removed. The rootstocks were washed in tap water to remove soil particles. It was cut in to small pieces and placed for air drying in shade.

5.2.4 Laboratory Analysis

Methanol extract of plant materials was used for phytochemical analysis. The methanol extract was subjected to repeated fractionation with solvents of different polarity. Column (CC) and thin layer chromatography (TLC) were used to get crude compounds. Purification of these compounds was done using high performance liquid chromatography (HPLC). Structures of isolated compounds were elucidated by spectroscopic methods using ultraviolet (UV), infra red (IR), nuclear magnetic resonance (NMR) and mass spectroscopy (MS). Extraction and fractionation of plant materials of both species were done at Central Department of Botany, Tribhuvan University, Nepal, while identification and structural elucidation were done at Department of Pharmaceutical Sciences, University of Padua, Italy.

Aconitum naviculare: Air-dried powdered aerial parts of Aconitum naviculare were exhaustively extracted in a Soxhlet apparatus with methanol. The solvent was evaporated under reduced pressure (230–250 mbar) and a semisolid methanol extract was obtained. The methanol (MeOH) extract was suspended in a mixture of 9:1 H2O–MeOH (200 mL) and the pH was adjusted to pH 2 using 5% aqueous hydrochloric acid. The solution was then partitioned first with chloroform (CHCl3) and then with ethyl acetate (EtOAc). Solvents were removed under vacuum obtaining the CHCl3 fraction (CL-I) and EtOAc fraction (EA-

105

PhD dissertation by Bharat Babu Shrestha

I). The pH of the residual aqueous layer was then adjusted to 8.0 using diluted ammonia.

The aqueous layer was partitioned with CHCl3 and EtOAc. Solvents were removed under vacuum yielding the CHCl3 fraction (CL-II) and EtOAc fraction (EA-II). The pH of the aqueous layer was then adjusted to 7.0, and the solvent was removed by freeze-drying, giving aqueous fraction (AQ).

TLCs of the fractions CL-II and EA-II in several solvents (toluene/diethylamine/EtOAc

80:5:20; v/v CHCl3/MeOH/NH3 85:15:0.5; v/v) showed spots that gave a positive reaction to Dragendorf reagent. Fraction CL-II was repeatedly subjected to preparative thin layer chromatography (PTLC) with toluene/diethylamine/EtOAc (80:5:20 v/v),

CHCl3/MeOH/NH3 (85:15:0.5 v/v) as eluents, yielding compounds 1 and 2. Fraction EA-II was subjected to silica plate chromatography using chloroform/methanol (4:1, v/v) as eluent. Further purification was achieved by semi-preparative HPLC with aqueous 0.1% HCOOH (A) and AcCN (B) as eluents. Gradient elution was used from 90% (A) to 85% (A) in 4 min, and then to 40% (A) in 22 min, yielding compound 3.

The AQ fraction was subjected to several semi preparative HPLC steps (Spherisorb C-18, 1 x 300 mm, 10 μm) with aqueous 0.1% HCOOH (A) and methanol (B) as eluents. Gradient elution used for the isolation of compounds 4-7 was as follows: from 90% (A) to 85% (A) in 10 min, and then to 70% (A) in 15 min, yielding compounds 4 and 5; from 75% (A) to 45 % A in 25 min for the isolation of compounds 6 and 7.

In another series of experiments, the AQ fraction was applied to a Sephadex LH-20 column (300 mL) and eluted with MeOH. The eluted fractions were analyzed by TLC and pooled on the basis of their chromatographic behavior into eight groups. Further chromatographic steps on TLC (10:5:1 CHCl3–MeOH–H2O) and semi preparative HPLC yielded three new compounds (8-10).

Curculigo orchioides: The dry rootstock was ground. The powder was then extracted with methanol at room temperature for 48 hours. After filtration, the solvent was removed under reduced pressure (230–250 mbar) and the residue was used for the phytochemical analysis. The methanol extract was dissolved in 2000 ml of a mixture methanol/water (1/9) and partitioned with petroleum ether (3 times × 500 ml each) and chloroform (3 times × 500 ml each). The solvents were removed under vacuum. The volume of the aqueous layer was reduced by evaporation under vacuum followed by freeze-drying yielding a solid residue. The residue was applied on a silica gel column (250 mL) and eluted with a mixture of

106

PhD dissertation by Bharat Babu Shrestha

chloroform/methanol/ water (10/5/1). Eluted fractions were collected on the basis of their chromatographic behavior in four groups. Group 3 was subjected to semi preparative HPLC yielding in the isolation of compounds 1 and 2.

5.3 Results

5.3.1 Phytochemical Research of Nepalese Medicinal Plants

Altogether 118 references have reported phytochemical composition and biological activities of Nepalese medicinal plants. Phytochemical compositions have been reported by 58% of the total references; 13% reported both phytochemical composition and bioactivities, and 29% reported only bioactivities. In 52.5% of the references, Nepalese researchers have a major role (either single author, or first author in references with multiple authorship); foreign researchers have major roles in remaining 47.5% of the references. About 400 medicinal and aromatic plants of Nepal have been examined at least once for phytochemical composition (screening, or isolation and characterization of secondary metabolites) and biological activities. However, secondary metabolites have been isolated and characterized only from 78 species of Nepalese medicinal and aromatic plants.

5.3.2 Isolation and Characterization of Secondary Metabolites

Aconitum naviculare

Phytochemical analysis of aerial part of Aconitum naviculare led to the isolation of two new diterpenoid alkaloids (navirine B and navirine C, Figure 5.1) and three new flavonoid glycosides (Figure 5.2) along with previously known one alkaloid (chelespontine), two flavonol glycosides and two phenylpronanoid glycosides (Table 5.1). The previously known compounds were also the first report from this plant. 1H and 13C NMR data of new compound 1-2 are given in the Table 5.2 and of compounds 8-10 in Table 5.3. Properties of these new compounds have been given bellow. For interpretation of NMR data and detailed properties of these compounds, refer papers I and III in appendix.

Compound 1 (Navirine B): Amorphous solid. HRAPI-TOF MS: m/z [M+H)]+

calculated for C30H40N2O3: 477.3117; found 477.3122.

107

PhD dissertation by Bharat Babu Shrestha

Compund 2 (Navirine C): Amorphous solid. HRAPI-TOF MS: m/z [M+H)]+

calculated for C31H44N2O: 461.3532; found: 461.3512. Compund 8: Yellow amorphous powder. HRAPI-TOF MS: m/z [MH)]

calculated for C54H66O31H: 1209.3510; found 1209.3555. Compund 9: Yellow amorphous powder. HRAPI-TOF MS: m/z [MH)]

calculated for C54H66O32H: 1225.3459; found: 1225.3515. Compound 10: Yellow amorphous powder. HRAPI-TOF MS: m/z [MH)]

calculated for C27H30O16H: 609.1456; found: 609.1485.

Figure 5.1. Structure of new diterpenoid alkaloid isolated from the aerial part of Aconitum naviculare. 1: Navirine B, and 2: Navirine C.

Figure 5.2. Structure of new flavonoid glycosides isolated from the aerial part of Aconitum naviculare. 1: 3-O-[-D-glucopyranosyl-(13)-(4-O-trans-p-coumaroyl)--L- rhamnopyranosyl-(16)--D-glucopyranosyl]-7-O-[-D-glucopyranosyl-(13)-- L-rhamnopyranosyl] kaempferol; 2: 3-O-[-D-glucopyranosyl-(13)-(4-O-trans-p- coumaroyl)--L-rhamnopyranosyl-(16)--D-lucopyranosyl]-7-O-[-D- glucopyranosyl-(13)--L-rhamnopyranosyl]quercetin; and 3: 7-O-[-D- glucopyranosyl-(13)--L-rhamnopyranosyl]quercetin.

108

PhD dissertation by Bharat Babu Shrestha

Table 5.1. Secondary metabolites (compounds 1-10) isolated from the aerial part of Aconitum naviculare.

SN Chemical name Compound type Remarks

1 Navirine B [5-hydroxy navirine] Diterpenoid New alkaloid compound

2 Navirine C Diterpenoid New alkaloid compound

3 (+) Chellespontine Alkaloid Known in other plants

4 Kaempferol-7-O-β-D-glucopyranosyl(1→3)α-L- Flavonol Known in rhamnopyranoside glycoside other plants

5 Kaempferol-7-O-α-L-rhamnopyranoside,3-O-β- Flavonol Known in D-glucopyranoside glycoside other plants

6 p-coumaric-4-O-β-D-glucopyranoside acid Phenylpronanoid Known in glycoside other plants

7 Ferulic-4-O-β-D-glucopyranoside acid Phenylpronanoid Known in glycoside other plants

8 3-O-[-D-glucopyranosyl-(13)-(4-O-trans-p- Flavonoid New coumaroyl)--L-rhamnopyranosyl-(16)--D- glycoside compound glucopyranosyl]-7-O-[-D-glucopyranosyl- (13)--L-rhamnopyranosyl] kaempferol

9 3-O-[-D-glucopyranosyl-(13)-(4-O-trans-p- Flavonoid New coumaroyl)--L-rhamnopyranosyl-(16)--D- glycoside compound lucopyranosyl]-7-O-[-D-glucopyranosyl- (13)--L-rhamnopyranosyl]quercetin

10 7-O-[-D-glucopyranosyl-(13)--L- Flavonoid New rhamnopyranosyl]quercetin glycoside compound

109

PhD dissertation by Bharat Babu Shrestha

Table 5.2. 1H NMR and 13C NMR data for three diterpenoid alkaloids (Navirine B and

Navirine C) isolated from Aconitum naviculare. (300 and 75 MHz, CD3OD,  in ppm, number in parenthesis are J in Hz).

Position Navirine B Navirine C H C H C 1 1.65-1.78 m 27.9 1.74-2.08 m 27.8 2 1.30 m 30.1 1.62 m 28.5 3 1.75-1.98 m 30.9 1.25-1.78 m 34.1 4  44.9  41.7 5  72.6 1.26 m 53.5 6 1.87-2.02 m 30.9 1.83-2.14 m 33.9 7 1.26-1.58 m 20.9 1.80 m 34.1 8  43.8  44.4 9 1.64 m 47.1  44.5 10  41.0  44.0 11 1.42-1.88 m 43.2 1.34-1.68 m 28.1 12 2.54 m 31.7 2.87 m 31.1 13 1.56-1.91 m 43.5 1.33 m 44.1 14  69.8  45.0 15 5.67 brs 130.8 5.63 brs 132.4 16  146.8  145.8 17 4.55 brs 68.8 4.52 brs 68.7 18 1.05 s 19.1 1.00 s 27.8 19 7.43 brs 169.9 2.30 m 58.0 20 3.57 brs 80.6 2.53 m 76.7 21    22    1  150.2  158.8 2, 6 6.85 d (8.0) 114.9 6.84 d (8.0) 115.1 3, 5 7.12 d (8.0) 129.2 7.10 d (8.0) 129.7 4  125  128.0 7 2.99 brt 33.3 2.89 m 33.2 8 3.02 brt 52.5 2.89 m 60.5 9 2.40 s 34.3 2.55 s 44.3

110

PhD dissertation by Bharat Babu Shrestha

Table 5.3. 1H NMR and 13C NMR data for three flavonoid glycosides (compounds 8-10) from Aconitum naviculare. Serial number of the compounds corresponds to Table 16. (300 and 75 MHz, MeOD,  in ppm, number in parenthesis are J in Hz).

Position Compound 8 Compound 9 Compound 10 H C H C H C 1       2  158.9  158.0  149.9 3  135.3  135.2  135.0 4  178.8  180.0  176.2 5  163.0  161.7  163.3 6 6.50 d (1.8) 100.8 6.43 d (1.5) 99.6 6.46 d (1.7) 100.3 7  163.2  162.4  164.0 8 6.64 d (1.8) 95.7 6.60 d (1.5) 94.2 6.75 d (1.7) 95.6 9  157.4  156.5  158.0 10  107.4  106.9  107.0 1  121.8  133.8  124.6 2 8.20 d (8.5) 132.7 7.89 d (1.6) 116.7 7.77 d (1.5) 116.2 3 6.96 d (8.5) 116.3  144.6  148.0 4  161.6  149.0  147.4 5 6.96 d (8.5) 116.3 6.90 d (8.0) 115.7 6.89 d (7.8) 116.4 6 8.20 d (8.5) 132.7 7.67 d (8.0; 1.6) 122.0 7.67 dd (7.8; 1.5) 122.4 RhaI (7) 1 5.59 d (0.9) 99.7 5.51 d (1.0) 98.2 5.58 d (0.9) 99.9 2 4.37 m 71.0 4.30 m 69.6 4.31 m 71.2 3 3.99 m 82.7 3.93 m 80.9 3.95 m 83.3 4 3.67 m 71.0 3.60 m 71.0 3.66 m 71.8 5 3.71 m 71.3 3.65 m 69.0 3.70 m 71.5 6 1.31 d (6.0) 18.3 1.25 d (7.0) 18.1 1.28 d (6.0) 18.5 GlcI 1 4.69 d (7.8) 105.7 4.64 d (8.0) 105.0 4.63 d (8.0) 106.4 2 3.41 m 75.5 3.36 m 77.1 3.28 m 75.2 3 3.50 t (9.0) 77.6 3.40 m 71.0 3.37 m 77.8 4 3.37 m 70.9 3.36 m 71.0 3.64 m 71.9 5 3.38 m 77.6 3.48 m 78.0 3.47 m 78.2 6 3.99-3.65 m 62.3 3.93 dd (12.0; 2.0)- 62.0 3.72-3.86 m 62.7 3.72 dd (12.0; 5.0) GlcII (3) 1 5.15 d (7.5) 105.6 5.14 d (7.8) 105.8 2 3.88 m 72.7 3.81 m 75.2 3 3.69 m 75.0 3.69 m 73.7 4 3.30 m 77.0 3.34 m 77.0 5 3.20 m 77.5 3.61 m 71.0 6 3.80-3.57 m 68.8 3.87 m-3.76 m 68.5 RhaII 1 4.63 d (0.8) 102.3 4.61 d (0.9) 101.9 2 3.95 dd (8.8; 3.3) 71.7 3.95 m 71.8 3 3.85 m 79.1 3.86 m 79.0 4 5.05 dd (8.0; 8.2) 73.6 5.00 dd 72.6 5 3.85 m 67.9 3.80 m 68.2 6 0.98 d (6.5) 17.9 0.95 d (6.5) 18.0

111

PhD dissertation by Bharat Babu Shrestha

Table 5.3 (contd.)

Position Compound 8 Compound 9 H C H C GlcIII 1 4.24 d (8.0) 105.7 4.21 d (7.6) 105.2 2 3.16 brt (9.0) 74.7 3.14 m 73.7 3 3.31 m 77.5 3.26 m 77.2 4 3.35 m 75.5 3.28 m 76.0 5 3.26 m 71.0 3.30 m 71.0 6 3.60 dd 62.2 3.70-3.80 m 62.1 (11.2;5.0)-3.80 dd (11.8; 2.0) Coumaric 1  127.4  126.8 2-6 7.54 d (8.0) 131.4 7.50 d (8.0) 129.8 3-5 6.89 d (8.0) 117.1 6.84 d (8.9) 115.7 4  161.3  161.1 7 7.61 d (15.95) 147.0 7.59 d (15.5) 149.6 8 6.31 d (15.95) 115.5 6.29 d (15.5) 114.1 9  168.9  167.4

Curculigo orchioides

Two new glycosides – curculigoside E and orchioside D – were isolated and characterized from the rootstock of Curculigo orchioides (Table 5.4, Figure 5.3). 1H NMR and 13C NMR of these two compounds have been given in Table 5.5 and their properties have been given below:

Curculigoside E: Colorless amorphous solid. HRAPI-TOF MS: m/z [M–H]−

calculated for C34H46O21–H; 789.2453; found: 789.2445. Orchioside D: White powder. HRAPI-TOF MS: m/z [M–H]− calculated for

C23H24O10–H: 459.1291; found: 459.1307.

For interpretation of NMR data and detailed properties of these compounds, refer paper V in appendix.

112

PhD dissertation by Bharat Babu Shrestha

Figure 5.3. Structure of two new phenolic glycosides isolated from the rootstock of Curculigo orchioides. 1: Curculigoside E; and 2: Orchioside D.

Table 5.4. Two new phenolic glycosides isolated from rootstock of Curculigo orchioides

SN Trivial Name Chemical Name

1 Curculigoside E 3,5-Dimethoxy-4-O-[β-D-glucopyranosyl-(1→6)-β-D- glucopyranoside]-benzoic acid 5-methyl-2-O-β-D- glucopyranoside-phenyl ester

2 Orchioside D 2-(3,4-Dihydroxy-phenyl)-3-[3-(3,4-dihydroxy-phenyl)-prop-2- ynyl]-6-hydroxymethyl-hexahydro-pyrano[2,3-β][1,4]dioxine- 7,8-diol

113

PhD dissertation by Bharat Babu Shrestha

Table 5.5. 1H NMR and 13C NMR data for curculigoside E and orchioiside D (300 and 75 MHz, MeOD,  in ppm, number in parenthesis are J in Hz).

Curculigoside E Orchioiside D C H C C H C 1 - 128.5 1 4.64 m 76.1 2 7.34 s 108.9 2 4.00 82.3 3 - 154.3 3 2.28 dd (16.9; 5.46)- 22.7 2.58 dd (16.9; 4.48) 4 - 138.5 4 - 85.6 5 - 154.3 5 - 84.6 6 7.34 s 108.9 1 - 133.7

OCH3 3.88 s 57.8 2 6.88 d (1.80) 116.0

OCH3 3.88 s 57.8 3 - 147.7 9 - 172.6 4 - 147.2 1 - 160.9 5 6.68 d (8.11) 116.9 2 - 161.0 6 6.72 dd 120.4 3 6.28 d (1.8) 110.0 1 - 134.0 4 - 140.3 2 6.79 d (1.90) 120.3 5 6.37 dd (1.8;8.0) 111.7 3 - 147.8 6 6.41 d (8.0) 110.8 4 - 146.4 7 2.13 s 22.2 5 6.64 d (J=9.0) 116.3 GlcI:1 5.04 d (7.5) 105.0 6 6.74 125.3 2 3.52 m 74.9 1 4.58 d (7.43) 102.8 3 3.39 m 77.1 2 3.31 m 74.1 4 3.74 m 71.2 3 3.40 m 77.5 5 3.51 m 76.7 4 3.28 m 71.8 6 4.13 dd (11.8; 2.0) 69.8 5 3.34 m 77.8 3.86 dd (11.8; 5.1) GlcII:1 4.46 d (7.5) 104.1 6 3.86dd (11.25;1.50) 62.7 3.64 dd (11.25; 4.05) 2 3.50 m 74.9 3 3.40 m 77.4 4 3.19 m 72.1 5 3.30 m 78.0 6 3.77 dd (11.2; 1.59) 62.1 3.77 dd (11.2; 4.4) GlcIII:1 4.80 d (7.7) 110.5 2 3.51 m 74.6 3 3.61 m 77.1 4 3.40 m 72.7 5 3.25 m 77.7 6 3.72 dd (11.1; 2.0) 62.2 3.66 dd (11.1; 5.5)

114

PhD dissertation by Bharat Babu Shrestha

5.4 Discussion

In phytochemical analysis of aerial parts of Aconitum naviculare, two new diterpenoid alkaloids and three flavonoid glycosides together with previously known one alkaloid and four phenol glycosides were isolated and characterized (Table 5.1, Figures 5.12). The structural elucidation of these compounds were done by employing extensive spectroscopic techniques including 2D NMR methods such as COSY, HMQC, HMBC and ROESY as well as high resolution mass. Earlier, Gao et al. (2004) reported a new diterpenoid alkaloid (navirine) along with five previously known alkaloids (isoatisine, hordenine, atisine, hetisinone and delfissinol). Recently Cao J-X et al. (2008) characterized two new diterpenoid alkaloids – naviculine A and naviculine B. Therefore, the total number of secondary metabolites known from A. naviculare becomes eighteen, with eleven alkaloids, three flavonoid glycosides and four phenol glycosides. Out of these eighteen phytochemicals, five alkaloids and three flavonoid glycosides have not been reported from any other plants. This fact indicates the high possibility of isolating new compounds in further phytochemical analysis of A. naviculare.

Thirteen phenolic glycosides, sixteen triterpenic saponins and four aliphatic ketones, totalling 33 derivatives, have been previously isolated and characterized from Curculigo orchioides (reviewed in Shrestha et al. 2008). Recently, four new phenolic glycosides (orcinosides D, E, F and G) from the rootstock of this plant have been reported by Zuo et al. (2010). With two new compounds reported in present study (Figure 5.3, Table 5.4), the number of phenolic glycosides isolated from this plant increased to nineteen and the total derivatives to 39.

While Aconitum naviculare is among the least studied species (two references available besides present work), the Curculigo orchioides has been studied extensively with over 17 references published on its phytochemical analysis. Nevertheless, phytochemical analysis of Nepalese specimen of C. orchioides has not been reported. In present study, five new derivatives from A. naviculare and two from C. orchioides have been isolated and characterized. It appears that chance of isolating new derivatives is still high in both of these Nepalese medicinal plants.

Review of literatures showed that phytochemical analysis Nepalese medicinal plants leading to isolation and characterization of at least single secondary metabolite was limited to 78

115

PhD dissertation by Bharat Babu Shrestha

species. This number accounts only 4.8 % of the total [1633 species = 1614 species vascular plants (Ghimire 2008) + 11 species fungi + 8 species lichens (Baral and Kurmi 2006)] medicinal plants that are native to Nepal. Phytochemical analysis of only a small percentage of Nepalese medicinal plants too, led to the isolation of several new compounds/derivatives. Some of these reports are as follows. Tomimori (2000) reported 121 new compounds from 21 medicinal plants of Nepal. Nine new compounds were isolated from single Nepalese medicinal plant Oroxylum indicum (Kizu et al. 1994) and seven from Ocimum sanctum (Suzuki et al. 2009). Some of the other Nepalese medicinal plants, from which new compounds were isolated, include Scutellaria repens (Matsuura et al. 1994), Sarcococca coriacea (Kalauni et al. 2001), Leucosceptum canum (Choudhary et al. 2004), Sarcococca hookeriana (Devkota et al. 2008), etc. These works, together with the present study, indicate that Nepalese medicinal plants could have an array of novel natural products with a high potential for bioprospecting.

In conclusion, review of literature showed that phytochemical analysis of medicinal plants collected from Nepal, leading to isolation and characterization of secondary metabolites, was limited to 78 species; it accounts only 4.8 % of the total medicinal plants (vascular plants, fungi and lichens) which are native to Nepal. Several new secondary metabolites have been reported from the phytochemical analysis of the limited number of Nepalese medicinal plants. In present phytochemical analysis of aerial parts of Aconitum naviculare, two new diterpenoid alkaloids and three flavonoid glycosides together with previously known one alkaloid and four phenol glycosides were isolated and characterized. Similarly, two phenolic glycosides have been added to the list of phytochemicals known from the rootstocks of Curculigo orchioides. These findings, together with earlier reports, indicate the high possibility of isolating new compounds in further phytochemical analysis of Nepalese medicinal plants. This has supported the hypothesis that medicinal plants in Nepal can be important source of novel plant-based natural products and deserve detail phytochemical screening for bioprospecting.

116

PhD dissertation by Bharat Babu Shrestha

6. CONSERVATION STATUS

6.1 Introduction

Factors related to expanding human population and changing feeding behaviors have been considered as the root causes of plant loss worldwide (Hamilton and Hamilton 2006). Out of estimated 70,000 medicinal plants worldwide, about 21% of them are considered under threat (Shippmann et al. 2006). At global scale, habitat destruction, biopiracy, and over harvesting are the major factors that have put the medicinal plants under threat (Roberson 2008). In Europe, over-exploitation, destructive harvesting techniques, habitat change and decrease in genetic diversity have been identified as major threats to medicinal plants (Lange 1998). At regional and national levels, lack of clear policy on common property rights, illegal trade, destructive harvest trends, high dependence on exclusive wild form, and unavailability of quality information on abundance, distribution, trade, level of sustainable harvest from wild and biological information (regenerative strategies, viable population size) are additional threats to medicinal plants in the Himalaya (Dhar et al. 2000; Larsen and Olsen 2007; Mulliken and Crofton 2008).

Conservation of medicinal plants has been hampered by lack of good quality information on their abundance in natural habitats, market trend, collection methods, and threats (Hawkins 2008). Several assumptions made on the conservation issues of Nepalese medicinal plants are based on inadequate and poor empirical data without scientific validation, leading to misguided conservation efforts (Larsen and Olsen 2007). Conservation status of species largely depends on expert opinions (Mulliken and Crofton 2008), without objective assessment of threats (Dhar et al. 2000). For example, while assessing status of Curculigo orchioides, a medicinal plant of tropical to subtropical region, during Conservation Assessment and Management Plan (CAMP) workshop in India, expert participants used data based on „general field studies‟ and „informal field sighting‟ (Molur and Walker 1998). Such approach has given confused scenarios of species in question such as C. orchioides. This species has been assigned threat categories from vulnerable (Bhattarai et al. 2001), threatened (Manandhar 2002) to endangered (Suri et al. 1999; Jasrai and Wala 2001; Prajapati et al. 2003; Dennis and Alphonsa 2004). Therefore, generation of species-specific baseline data on population status in wild is a research priority for development of medicinal plant sector (Hawkins 2008) and prioritization for conservation (Dhar et al. 2000). In this section, conservation status of two Nepalese medicinal plants, Aconitum

117

PhD dissertation by Bharat Babu Shrestha

naviculare and Curculigo orchioides has been assessed by using both primary and secondary data. The specific objective was to identify threat status of these two plant species using IUCN guidelines for threat categories.

6.2 Materials and Methods

Both primary and secondary data were used for threat categorization of Aconitum naviculare and Curculigo orchioides. Primary data pertaining to anthropogenic pressure (e.g. collection, livestock damage, etc.) was obtained from direct observation and consultation with local people in the study area (Manang valley for A. naviculare, and Nawalparasi, Chitwan, Lamjung and Syanja for C. orchioides). [For detail of study area, refer section 3.2.1 for A. naviculare and 4.2.1 for C. orchioides].

Both species (Aconitum naviculare and Curculigo orchioides) were evaluated against IUCN threat categories (IUCN 2001). Information pertaining to geographic range (criteria B) is the „best‟ of all available information and this was used for the assessment. As required by IUCN guidelines for the application of threat categories (IUCN Standards and Petitions Working Group 2008), a „location‟ has been defined as the area within which similar regulations apply, the collectors can reach different sites within the area, and change in demand at any point alters the collection pressure within the area. For example, Manang valley (one of the present study sites) is one „location‟ while upper mustang another. This definition of „location‟ is based on the initial perception that collection and habitat degradation are the major threats to the population size and abundance of these two medicinal plants. Both of these species has not been evaluated for IUCN Red List Categories at global scale. In this assessment the area within the political boundary of Nepal has been considered as a „region‟ and the assessment „regional assessment‟ (IUCN 2003).

The „area of occupancy‟ (sensu IUCN 2001) of Aconitum naviculare at six sites in Manang valley was visually estimated during field sampling. Mean value of area of occupancy at single site was calculated. Sites of collection of this plant in other parts of Nepal were obtained from references (e.g. Ohba et al. 2008; Ghimire SK et al. 2008a) and the information sheet of herbarium specimens deposited at KATH. The total number of sites (16), from where A. naviculare was collected, was multiplied by mean area of occupancy (0.067 km2, determined for Manang valley) to obtain total area of occupancy of this species

118

PhD dissertation by Bharat Babu Shrestha

in Nepal. The area of occupancy was not estimated for Curculigo orchioides because any criteria that uses „area of occupancy‟ was not applicable for this species.

Table 6.1. Collection sites of Aconitum naviculare in Nepal.

SN Collection Area of Elevation Sources of information District Sites Occupancy (m asl) (km2)* 1 Dolpo Dho-Tarap na 4200 Ghimire et al. 2008 2 Dolpo Tsharka na 4230 Ghimire et al. 2008 3 Dolpo Mukut Himal Camp na Ghimire et al. 2008 4 Mustang Above Chabarbu, na 4010 Ohba et al. 2008 Thorongphedi 5 Mustang Chabarbu na 4120 Ohba et al. 2008 6 Mustang Ghemi-Jhaite na 4350 Ohba et al. 2008 7 Mustang Arround Thanti na 4520 Ohba et al. 2008 8 Mustang Kungale na 4771 Ohba et al. 2008 9 Mustang Above Sandak na 4500 **

10 Manang Ice Lake area 0.020 4400-4550 Present study

11 Manang Manang Hill 0.055 4150-4450 Present study

12 Manang Khangsar 0.013 4400-4650 Present study

13 Manang Yak Kharka 0.073 4090-4270 Present study

14 Manang Ledtar 0.210 4100-4500 Present study

15 Manang Thorong Phedi 0.030 4350-4450 Present study 16 Solu Benikharka na 4000 KR Rajbhandari, 61658, khumbu Aug 23, 1985, KATH *na: data not available; ** Dr. SK Ghimire, Central Department of Botany, Tribhuvan University, Kathmandu; per. com. May 3, 2010.

6.3 Results

6.3.1 Aconitum naviculare

No recorded trade of Aconitum naviculare has been documented from Manang valley. However, according to local people, particularly the shepherds, the abundance and the area

119

PhD dissertation by Bharat Babu Shrestha

of occupancy of this plant has been declining due to collection for traditional uses. The plant has not been included in any of the conservation categories, neither by Annapurna Conservation Area Project (ACAP) nor by the local users‟ groups. The A. naviculare in Manang valley was under anthropogenic pressure. The major threats identified were human collection during peak flowering (i.e. before seed set), and the mechanical damage to aerial parts due to trampling by livestock. The populations of this plant at Ice lake area, Manang hill and Khangsar hills have faced high pressure of collection and livestock damage because these sites were nearer to traditional settlements (Table 3.1). The Phedi site is far from the settlement, but there was kharka within the area of occupancy of this plant, leading to high anthropogenic pressure.

When assessed against IUCN threat categories, Aconitum naviculare belonged to „endangered‟ (EN) based on criteria „geographic range size, and fragmentation, decline or fluctuation‟ (criteria: B2ab(iii)). Estimated area of occupancy in Nepal was 1.072 km2 which was less than 500 km2. The regional population and area of occupancy was severely fragmented and known to occur at less than five locations. Outside Manang Valley, A. naviculare was known to occur at three „locations‟ in Nepal (Dolpo, Mustang and Solukhumbu). Continuing decline in area, extent and quality of habitats was observed in Manang, and it had been inferred at other locations.

6.3.2 Curculigo orchioides

There was no collection of Curculigo orchioides for trade in the study areas. Abundance of this species was the highest in foothill Shorea robusta forest of Nawalparasi (section 4.3.2), but the people living around did not know the medicinal uses of this plant. Most of the people residing around the sampling sites of Chitwan and Nawalparasi, who were mostly migrants from the various hill districts, were not aware of the medicinal uses of this plant. In study areas of Syanja and Lamjung, people have been using this plant for indigenous healthcare of human and animals, but the amount of plant materials they collected was insignificant. Therefore collection by human was not a threat to population of C. orchioides in the study areas. However, degradation of habitat has occurred due to some of the silvicultural activities in community managed forests such as litter collection from the forest floor.

When evaluated against IUCN threat categories, Curculigo orchioides in Nepal did not qualify for any of the threatened categories (critically endangered, endangered and

120

PhD dissertation by Bharat Babu Shrestha

vulnerable). However, given the trends of habitat degradation, and the population status outside Nepal ( reported as „endangered‟ in India), the plant can be considered as „near threatened‟ (NT) in Nepal which would qualify for threatened category in the near future.

6.4 Discussion

6.4.1 Aconitum naviculare

In present evaluation against IUCN threat category, Aconitum naviculare belonged to „endangered‟ (EN) because this plant has small (<500 km2) and severely fragmented „areas of occupancy‟ where area, and the quality of habitats have been declining. Thus it is recommended that this species be included in the list of threatened species of Nepal, and collection from wild be monitored. Any kind of threat assessment for this plant species was not available outside Nepal, though it has been reported as „rare‟ in Bhutan (Nawang 1996).

For sustainable management of medicinal plants in their natural habitats, harvesting pattern (parts used, intensity and time of collection) and life history traits (e.g. life form and growth pattern) should be considered together (Ghimire et al. 2005). In addition, modes of reproduction are also equally important. In most of the Aconitum species (8 out of 12), the tuberous root is used as medicine or poison (Baral and Kurmi 2006). The entire plant has been used in alpine Aconitum species (e.g. A. violaceum, A. naviculare and A. gammiei). Although whole plant of A. naviculare is medicinally important, people of Manang (the study area) have been collecting only aerial parts. This method is less destructive in terms of parts removed, but collection of plant at flowering stage did not allow regeneration by seeds. In A. naviculare, the tuberous root is only the mean of winter perennation. Increase in number of individuals depends on production of seeds. Collection of aerial parts of this plant before seed set has negative impact to increase or maintain population size, as has been reported by Ghimire et al. (1999). The negative impact could be more critical for areas with small population size and/or low density such as Ice Lake area, Manang hill and Khangsar.

Aconitum naviculare is habitat specific with narrow geographic distribution (in trans- Himalayan alpine region only), and it is being destructively harvested for whole plant. Therefore, this plant can be considered as one of the most susceptible medicinal plants to over-harvest (Shippmann et al. 2006). A. naviculare has gravity dispersed seeds, and such

121

PhD dissertation by Bharat Babu Shrestha

plant is vulnerable to habitat degradation (McLachlan and Bazely 2001). In the context of climate change, the plants found only in alpine region of the Himalaya appears to be more sensitive to increasing temperature, and subsequent shrinkage in natural habitat (Cavaliere 2009). Being found only in alpine regions of the Himalaya (Stainton 1997) with „nowhere to go‟ and having narrow distribution range (see section 3.3.1), A. naviculare, along with other similar species, may experience greater risk of extinction. Such species are of grave conservation concern (Hawkins et al. 2008). Extinction or any decline in abundance of this plant will have adverse effect to healthcare system of people in such remote mountains as Manang, where the species is the most prioritized in terms of efficacy and frequency of uses (Bhattarai et al. 2007). Since the plant has key role in local health care, and has been qualified for „endangered‟ category of IUCN, I recommend to developing conservation management plan to implement immediately in the areas of its natural distribution. Based on the present work, three recommendations have been made for conservation management of this plant in Manang to be implemented immediately: collection only for local use, domestication, and rotational harvest from wild. A. naviculare has been collected not only for local uses in Manang but also to send Manangis who have migrated to urban areas of Nepal, where modern medicine is easily accessible. Local people should be made aware of the future scenario when there will be no more A. naviculare in Manang, and be asked not to collect this plant to send migrated Manangis. Some of the settlements like Manang village, Tanki Manang and Khangsar lie very close to natural habitats of A. naviculare where people can be requested to grow this plant in their home gardens so as to meet their household demand. Some attempts have been made for domestication by transplanting it to herbal gardens by Khamso Nature Product (Pvt.) in Manang and the Himalayan Amchi Association in Mustang. To increase population size and density of A. naviculare in localities with very low abundance such as Ice lake area, Manang hill and Khangsar, harvesting of aerial parts should be employed once in an interval of 3-5 years. This may ensure adequate regeneration of the plant by seeds. In an area with large population and high density as in Ledtar site, the population can be divided into five sub populations and harvesting can be done in rotation with the interval of five years. However, impact of rotational harvest on recruitment and biomass yield should be monitored and evaluated to improve future management plans. First two recommended strategies will reduce collection pressure from the wild, and the third one will increase regeneration and hence abundance in the wild.

122

PhD dissertation by Bharat Babu Shrestha

6.4.2 Curculigo orchioides

As mentioned in the introduction, Curculigo orchioides has been assigned various threat categories, from vulnerable to endangered, but without empirical data on distribution and abundance in its natural habitats. It is one of the most frequent herbaceous ground flora of Shorea robusta forests in churia (Gubhaju and Ghimire 2009) and foot hill of Mahabharat range (Kandel and Shrestha, unpub. data), and found throughout Nepal from east to west (Table 4.4). To the best of my knowledge the plant has not been collected from wild for trade, neither there is any attempt for cultivation in Nepal (Shrestha et al. 2008). Commercial collection of this plant from wild may not be profitable because harvesting is labor intensive and too time consuming (personal observation/experience). This could be a reason for why herbal collectors in Nepal have not been collecting this plant from wild, though there is higher demand than supply in Indian market (Kala et al. 2006), the main destination of Nepalese MPs in trade. With wide distribution and no commercial collection at present, C. orchioides cannot be considered as threatened in Nepal. When life history trait is considered in the context of commercial collection and other anthropogenic disturbances to its habitats, together with the earlier reports related to population status in and outside Nepal (Suri et al. 1999; Bhattarai et al. 2001; Jasrai and Wala 2001; Manandhar 2002; Prajapati et al. 2003; Francis et al. 2007) the species can be assigned to „near-threatened‟ (NT) category of IUCN as discussed below.

Curculigo orchioides showed mixed mode of reproduction (polymorphic reproductive mode) from seeds, leaves and rootstocks. Regenerative potential of this plant from seeds is quite low. Clonal propagation from leaves is limited to moist sites only and needs mild mechanical damage, which is only accidental. Rootstock is a mean of winter perennation, not for multiplication. Despite wide range of distribution in landscape, above mentioned traits explicitly indicate a low reproductive (multiplicative) success of this plant in natural habitats. Another important trait is the lack of active seed dispersal mechanism. Seeds are either buried closely around the mother plant or dispersed to limited distance by ants and surface runoff water. Plants with ant or gravity dispersed seeds are the most vulnerable to habitat degradation and fail to re-colonize during restoration (McLachlan and Bazely 2001). Re-colonization of such species would be possible only if they are actively reintroduced. Thus habitat degradation could be an important reason for declining natural population size of C. orchioides in India as suggested by Jasrai and Walla (2001). This is also evident from the observation of some Shorea robusta forests in mid hill of Nepal (e.g. forests at

123

PhD dissertation by Bharat Babu Shrestha

Panchkhal, Kavre district, per. obs.), where litter collection is intensive with virtually no litter on the forest floor; in this forest, less than 10 individuals were observed in an area of about one hectare. Therefore, given the high anthropogenic disturbance and habitat degradation, this plant appeared to be a „near threatened‟ species due to low reproductive success and limited seed dispersal range.

Curculigo orchioides has been emerging as a source of new bioactive natural products with potential therapeutic applications. Due to a wide range of beneficial effects of this plant on human health, further biomedical researches and their applications have been urged (Kapoor 2008). It is likely that market demand of this plant will further increase in near future with positive impact on collector‟s price. This price hike may encourage herbal collectors to harvest C. orchioides from wild. When this potential commercial collection from wild is combined with poor regenerative potential and continued habitat degradation the scenario will be that the plant will shortly qualify for threatened category of IUCN. Therefore it has been recommended that appropriate agro technology be developed as soon as possible for large scale commercial cultivation of this plant to meet the future market demand before the plant becomes „new rare‟. Some attempts have been made in this direction elsewhere (e.g. Augustine and D‟Souza 1997; Suri et al. 1999; Prajapati et al. 2003; Wala and Jasrai 2003; Dennis and Alphonsa 2004; Joy et al. 2004, 2005; Francis et al. 2007; Nema et al. 2008; Sharma et al. 2009), but in Nepal it has not been initiated yet.

Some interesting issues have emerged from the conservation assessment (described above) of two medicinal plant species from contrasting ecological regions. While comparing the traditional knowledge of local people about the medicinal uses of Aconitum naviculare and Curculigo orchioides, it is apparent that mountain communities have conserved traditional knowledge of medicinal plants but the communities in peri-urban areas (e.g. Nawalparasi and Chitwan sites) of tropical regions have failed to do so. Though C. orchioides has been assigned various threat categories from vulnerable to endangered, information pertaining to its‟ biology and abundance in natural habitats is virtually non-existent. Therefore, reliability of earlier conservation assessment of this species (e.g. Molur and Walker 1998; Bhattarai et al. 2001) would be questionable.

In conclusion, based on the population status and geographic distribution, Aconitum naviculare qualifies to „endangered‟ and Curculigo orchioides to „near threatened‟

124

PhD dissertation by Bharat Babu Shrestha

categories of IUCN. Inherently narrow distribution range, collection before seed dispersal and high livestock pressure in the areas of occupancy of A. naviculare have been identified as the major threat to its existence. Given the key role of A. naviculare in local health care system and endangered status of its wild population, formulation and immediate implementation of conservation management plan have been recommended. This should include limiting collection to local use only, domestication to home garden, and rotational harvest from the wild. In case of C. orchioides, low regenerative success, and degradation of natural habitats have been identified as the major existing threats, and high market demand as potential threat. The recommended conservation management plan for C. orchioides should include development of appropriate agro technology and subsequent cultivation to meet the market demand.

125

PhD dissertation by Bharat Babu Shrestha

7. CONCLUSIONS AND RECOMMENDATIONS

7.1 Conclusions

Based on new empirical data (qualitative as well as quantitative) and the review of literatures, both hypotheses of the present study have been justified. Some of the life history traits could explain observed distribution pattern of both of these plant species. Clumped dispersion and narrow range of distribution of A. naviculare can be attributed to early loss of seed viability and lack of long-range seed dispersal while the plasticity in plant height and petiole length enabled this plant to colonize from thick alpine scrubs to open areas. Spatial and temporal variation in relative importance of different regenerative strategies (sexual vs. clonal), along with seed dormancy, could be responsible for wide altitudinal and habitat range of distribution of C. orchioides. Low regenerative potential through sexual reproduction in its major habitats (i.e. Shorea robusta forests) and limited seed dispersal can be attributed to high vulnerability to habitat degradation, and rarity of this plant reported from elsewhere. These findings supported the assumption that life history traits are helpful in understanding the abundance and distribution of patterns of plants. Phytochemical analysis of less than 5% of medicinal plants of Nepal, exciting trend of isolating new secondary metabolites from these medicinal plants, and identification of seven new secondary metabolites from these two medicinal plants (five in A. naviculare and two in C. orchioides) supported the hypothesis that medicinal plants in Nepal can be important source of novel plant-based natural products.

Aconitum naviculare is naturally rare with natural distribution from west Nepal (Dolpo) to Bhutan including southern Tibet. In Manang valley this plant was found exclusively on sunny and dry south facing slope between 4000 and 4700 m asl, often in association with alpine scrubs. These alpine scrubs had protective role against livestock damage and collection of this plant by human. In the context of declining area of occupancy of this plant due to its apparent disappearance from open places (outside scrubs) at four of the six sampling sites, this positive interaction appears to be critical for the persistence of this plant. A. naviculare is a perennial plant with annual aerial parts and biennial tuber. Seeds were non dormant but environmental factors inhibited germination immediately after seed dispersal. In laboratory (at 1350 m asl), the percentage of seed germination declined with storage (4 C) from about 50% after 10 weeks of storage to 7% after 20 weeks. Any consistent pattern in relationship between population size and seed germinability of A.

126

PhD dissertation by Bharat Babu Shrestha

naviculare was not detected. Most of the growth and reproductive traits varied significantly with sampling sites and life forms of associated species. Mean density of the plant increased with relative radiation index while mass of stem of individual plant and area based above ground biomass both declined with elevation. Population density and life history traits did not vary significantly with soil nitrogen and organic carbon. High leaf nitrogen content (2.44 %) in A. naviculare can be considered as an adaptive response to short growing season in alpine region. Based on leaf nitrogen content and the difference in nitrogen content between leaves and soil, growth of A. naviculare in present study sites was not limited by nitrogen supply. A. naviculare has evolved a number of adaptation features to alpine environment and biotic disturbance (including anthropogenic) which includes bluish globular flower with small opening on lateral side, persistent sepal covering developing fruits, winter perennating bud inside soil (geophyte), and presence of intercalary meristem between stem and tuber.

Curculigo orchioides has a wide range of habitats from tropical riparian Shorea robusta forest to pine forests and grassy slopes in subtropical region between altitude range of 200 and 2100 m asl. In terms of density, rootstock biomass/plant and area-based rootstock yield, S. robusta forest with partially open canopy of foot hills was the most suitable natural habitat of C. orchioides. Density, rootstock biomass and yields varied significantly with types of habitats. Flowering phenology was intermediate between synchrony and steady- state-flowering. Extended flowering was partly due to production of up to four batches of inflorescences by some individuals and partly due to different timing of flowering among the individuals in the population. An inflorescence has single bisexual flower and an average of five male flowers, but there is less chances of pollen transfer from male flowers to female flower of the same inflorescence due to different timing of anthesis. Fruiting frequency among individuals in forest habitats showed unimodal relationship with treeshrub canopy. Seeds were non-wettable and hard to touch but seed coat was permeable to water. They showed deep morphophysiological dormancy, and the condition to break dormancy could not be identified in present work. Seeds were dispersed by ants and surface runoff water, and had no active mechanism of seed dispersal. Germination of seeds was postponed until early monsoon of the next growing season due to dormancy. Germination during early monsoon ensured seedling establishment during the same rainy season. Regenerative potential of C. orchioides through sexual reproduction was relatively low in forest habitats because less than one-fourth of the mature individuals entered into

127

PhD dissertation by Bharat Babu Shrestha

reproductive phase and only one-half of the flowering individuals developed fruits. It was partly compensated by clonal propagation from the mid rib region of leaves which was induced by mild mechanical damage to the mature leaves.

In phytochemical analysis of aerial parts of Aconitum naviculare, two new diterpenoid alkaloids and three flavonoid glycosides together with previously known one alkaloid and four phenol glycosides were isolated and characterized. Similarly, two phenolic glycosides have been added to the list of phytochemicals known from the rootstocks of Curculigo orchioides. These findings indicate the high possibility of isolating new compounds in further phytochemical analysis of Nepalese medicinal plants. This has supported the hypothesis that medicinal plants in Nepal can be important source of novel plant-based natural products and deserve detail phytochemical screening for bioprospecting.

Based on the population status and geographic distribution, Aconitum naviculare qualifies to „endangered‟ and Curculigo orchioides to „near threatened‟ categories of IUCN. Inherently narrow distribution range, collection before seed dispersal and high livestock pressure in the areas of occupancy of A. naviculare have been identified as the major threat to its existence. Given the key role of A. naviculare in local health care system and endangered status of its wild population, formulation and immediate implementation of conservation management plan have been recommended. This should include limiting collection to local use only, domestication to home garden, and rotational harvest from the wild. In case of C. orchioides, low regenerative success, and degradation of natural habitats have been identified as the major existing threats, and high market demand as potential threat. The recommended conservation management plan for C. orchioides should include development of appropriate agro technology and subsequent cultivation to meet the market demand.

128

PhD dissertation by Bharat Babu Shrestha

7.2 Recommendations

Following recommendations have been made based on empirical data generated during the present study, and predicted future scenarios:

 The study of medicinal plants should be expanded beyond inventory and documentation to the level of life history and demographic studies including understanding the plant‟s response to natural and anthropogenic disturbances.

 Due to high chances of finding new natural products with potential therapeutic applications, intensive and organized pharmacological researches of Nepalese ethnomedicinal plants have been recommended.

 Aconitum naviculare and Curculigo orchioides be included in the list of threatened species of Nepal, and A. naviculare as „endangered‟ in the regional redlist of IUCN threat category and C. orchioides as near threatened.

 Collection only for local use by rotational harvest from wild, and domestication are recommended conservation management plans for Aconitum naviculare while it is the development of agro technology and subsequent cultivation for Curculigo orchioides.

129

PhD dissertation by Bharat Babu Shrestha

REFERENCES

Aase TH and OR Vetaas. 2007. Risk management by communal decision in trans- Himalayan farming: Manang valley in central Nepal. Human Ecology 35:453–460. Anonymous 2009. Conditions and cures. World Conservation 31:18–19. Augustine AC and L D‟Souza. 1997. Regeneration of anticarcinogenic herb, Curculigo orchioides Gaertn. In Vitro Cellular and Developmental Biology – Plant 33:111– 113. Bafna AR and SH Mishra. 2006. Immunostimulatory effects of methanol extract of Curculigo orchioides on immunosuppressed mice. Journal of Ethnopharmacology 104:1–4. Balunas MJ and AD Kinghorn. 2005. Drug discovery from medicinal plants. Life Sciences 78:431–441. Bannerman RH. 1982. Traditional medicine in modern health care. World Health Forum 3:8–13. Barakoti TP. 2002. Germination of chiraito (Swertia chirata) seeds tested through various methods under different conditions in the eastern hills of Nepal. In: T Watanabe, A Takano, MS Bista and HK Saiju (eds.) The Himalayan Plants: can they save us? Proceedings of Nepal-Japan Joint Symposium on Conservation and Utilization of Himalayan Medicinal Resources, November 6-11, 2000, Kathmandu. Pp. 271–278. Baral SR and PP Kurmi. 2006. A Compendium of Medicinal Plants in Nepal. Kathmandu: R. Sharma. 534 p. Barbour MG, JH Burk, WD Pitts, FS Gilliam and MW Schwartz. 1999. Terrestrial Plant Ecology. 3rd edition. California (USA): Benjamin/Cummings. 700p. Baskin CC and JM Baskin. 1998. Seed: Ecology, Biogeography, and Evolution of Dormancy and Germination. California: Academic Press. 666p. Behera SK and MK Misra. 2006. Aboveground tree biomass in a recovering tropical sal (Shorea robusta Gaertn. f.) forest in Eastern Ghats, India. Biomass and Bioenergy 30:509–521. Bennett A and J Krebs. 1987. Seed dispersal by ants. TREE 2:291–292. Berendse F. 1999. Implications of increased litter production for plant biodiversity. TREE 14:4–5. Bertness MD and RM Callaway. 1994. Positive interactions in communities. Trends in Ecology and Evolution 9:191–193.

130

PhD dissertation by Bharat Babu Shrestha

Bevill RL and SM Louda. 1999. Comparison of related rare and common species in the study of plant rarity. Conservation Biology 13:493–498. Bhat DM and KS Murali. 2001. Phenology of understorey species of tropical moist forest of Western Ghats region of Uttara Kannada district in South India. Current Science 81:799–805. Bhattarai KR and B Basnet. 2002. Study on seed germination of Swertia chirayita: a step towards its domestication. In: T Watanabe, A Takano, MS Bista and HK Saiju (eds.) The Himalayan Plants: can they save us? Proceedings of Nepal-Japan Joint Symposium on Conservation and Utilization of Himalayan Medicinal Resources, November 6-11, 2000, Kathmandu. Pp. 198–200. Bhattarai KR and M Ghimire. 2006. Commercially important medicinal and aromatic plants of Nepal and their distribution pattern and conservation measure along the elevation gradient of the Himalayas. Banko Janakari 16:3–13. Bhattarai N, V Tandon and DK Ved. 2001. Highlights and outcomes of the Conservation Assessment and Management Planning (CAMP) workshop, Pokhara, Nepal. In: N Bhattarai and M Karki (eds.) Sharing Local and National Experiences in Conservation of Medicinal and Aromatic Plants in South Asia. Proceedings of Regional Workshop at Pokhara, Nepal. Kathmandu: HMG/N, IDRC and MAPPA. Pp. 46–53. Bhattarai S, RP Chaudhary and RSL Taylor. 2006. Ethnomedicinal plants used by the people of Manang district, central Nepal. Journal of Ethnobiology and Ethnomedicine 2:41 doi: 10.1186/1746-4269-2-41. http://www.ethnobiomed.com/content/2/1/41 Bhattarai S, RP Chaudhary and RSL Taylor. 2007. Prioritization and trade of ethnomedicinal plants by the people of Manang district, central Nepal. In: RP Chaudhary, TH Aase, OR Vetaas and BP Subedi (eds.) Local Effects of Global Changes in the Himalayas: Manang, Nepal. Kathmandu, Nepal: Tribhuvan University (Nepal) and University of Bergen (Norway). Pp. 151–169. Bhuju UR, PR Shakya, TB Basnet and S Shrestha. 2007. Nepal Biodiversity Resource Book: Protected Areas, Ramsar Sites and World Heritage Sites. Kathmandu: International Center for Integrated Mountain Development (ICIMOD) and Ministry of Environment, Science and Technology. 794 p. Bista T and G Bista. 2005. Himalayan Doctors and Healing Herbs: The Amchi Tradition and Medicinal Plants of Mustang. Mustang, Nepal: Lo-Kunphen Mensikhang. 96 p.

131

PhD dissertation by Bharat Babu Shrestha

Biswas TD and SK Mukherjee. 1999. Text Book of Soil Science. 2nd ed. New Delhi, India: Tata McGraw-Hill Publishing Company Limited. 433 p. Bliss LC. 1956. A comparison of plant development in microenvironment of arctic and alpine tundras. Ecological Monograph 26:303–337. Bliss LC. 1958. Seed germination in arctic and alpine species. Arctic 11:180–188. Bliss LC. 1962. Adaptation of arctic and alpine plants to environmental conditions. Arctic 15:117–144. Bosch M and NM Waser. 1999. Effect of local density on pollination and reproduction in Delphinium nuttallianum and Aconitum columbianum (Ranunculaceae). American Journal of Botany 86:871–879. BPP (Biodiversity Profile Project). 1995. Biodiversity Assessment of Terai Wetlands. Biodiversity Profile Project Technical Publication No. 1. Kathmandu: Department of National Park and Wildlife Conservation, Ministry of Forest and Soil Conservation. ..p. Brower V. 2008. Back to nature: extinction of medicinal plants threatens drug discovery. Journal of National Cancer Institute 100:838–839. Buenz EJ, BA Bauer, HE Johnson, G Tavana, EM Beekman, KL Frank and CL Howe. 2006. Searching historical herbal texts for potential new drugs. British Medical Journal 333:1314–1315. Callaway RM. 1995. Positive interactions among plants. The Botanical Review 61:306–349. Callaway RM. 2007. Positive Interactions and Interdependence in Plant Communities. The Netherlands: Springer. 416 p. Canter PH, H Thomas and E Ernst. 2005. Bringing medicinal plants into cultivation: opportunities and challenges for biotechnology. TRENDS in Biotechnology 23:180– 185. Cao DP, YN Zheng, LP Qin, T Han, H Zhang, K Rahman and QY Zhang. 2008. Curculigo orchioides, a traditional Chinese medicinal plant, prevents bone loss in ovariectomized rats. Maturitas 59:373–380. Cao J-X, L-B Li, J Ren, S-P Jiang, R-R Tian, X-L Chen, S-L Peng, J Zhang and H-J Zhu. 2008. Two new C20-diterpernoid alkaloids from the Tibetan medicinal plant Aconitum naviculare Stapf. Helvetica Chimica Acta 91:1954–1960. Cavaliere C. 2009. The effect of climate change on medicinal and aromatic plants. HerbalGram 81:44–57.

132

PhD dissertation by Bharat Babu Shrestha

Ceplitis A. 2001. Genetic and environmental factors affecting reproductive variation in Allium vineale. Journal of Evolutionary Biology 14:721–730. Chapin FS III and KV Cleve. 1989. Approaches to studying nutrient uptake, use and loss in plants. In: RW Pearcy, JR Ehleringer, HA Mooney and PW Rundel (eds.) Plant Physiological Ecology: Field Methods and Instrumentation. London: Chapman and Hall. Pp. 185–202. Chauhan NS and VK Dixit. 2008. Spermatogenic activity of rhizome of Curculigo orchioides Gaertn in male rat. International Journal of Applied Research in Natural Products 1:26–31. Chauhan NS, CV Rao and VK Dixit. 2007. Effect of Curculigo orchioides rhizomes on sexual behaviors of male rats. Fitoterapia 78:530–534. Chen C, W Ni and W Mei. 1999. The Glycosides from Curculigo orchioides. Acta Botanica Yunnanica 21:521–524. Chetri M, NR Chapagain and BD Neupane. 2006. Flowers of Mustang: A Pictorial Guide Book. Kathmandu, Nepal: National Trust for Nature Conservation, Annapurna Conservation Area Project, Upper Mustang Biodiversity Conservation Project. 196 p. Chhetri DR, D Basnet, PF Chiu, S Kalikotay, G Chhetri and S Parajuli. 2005. Current status of ethnomedicinal plants in Darjeeling Himalaya. Current Science 89:264–268. Choudhary MI, R Ranjit, AU Atta-Ur-Rahman, TM Shrestha, A Yasin and M Parvez. 2004. Leucosceptrine – a novel sesterterpene with prolylendopeptidase inhibitory activity from Leucosceptrum canum. Journal of Organic Chemistry 69:2906–2909. Cordell GA. 2000. Biodiversity and drug discovery – a symbiotic relationship. Phytochemistry 55:463–480. Cordell GA. 2002. Natural products in drug discovery - creating a new vision. Phytochemistry Review 1:261–273. Cornelissen JHC, S Lavorel, E Garnier, S Diaz, N Buchmann, DE Gurvich, PB Reich, H ter Steege, HD Morgan, MGA van der Heijden, JG Pausas and H Poorter. 2003. A handbook of protocols for standardized and easy measurement of plant functional traits worldwide. Australian Journal of Botany 51:335–380. Costa FRC, C Senna and EM Nakkazono. 2002. Effects of selective logging on populations of two tropical understory herbs in an Amazonian forest. Biotropica 34:289–296.

133

PhD dissertation by Bharat Babu Shrestha

Craker LE. 2007. Medicinal and aromatic plants: future opportunities. In: J Janick and A Whipkey (eds.) Issues in New Crops and New Uses. Alexandria VA (USA): ASHS Press. Pp. 248–257. Cunningham AB, P Shanley and S Laird. 2008. Health, habitats and medicinal plants use. http://www.peopleandplants.org/storage/Cunningham%20Shanley%20Laird_2008.p df (Access: Nov 15, 2009) Cunningham SA, B Summerhayes and M Westoby. 1999. Evolutionary divergences in leaf structure and chemistry, comparing rainfall and soil nutrient gradients. Ecological Monograph 69:569–588. Dahanukar SA, RA Kulkarni and NN Rege. 2000. Pharmacology of medicinal plants and natural products. Indian Journal of Pharmacology 32:S81–S118. de Kroon H and MJ Hutchings. 1995. Morphological plasticity in clonal plants: the foraging concept reconsidered. Journal of Ecology 83:143–152. Dennis T and J Alphonsa. 2004. Direct somatic embryogenesis of Curculigo orchioides Gaertn., an endangered medicinal herb. Journal of Plant Biotechnology 6:193–193. Department of Forest. 2009. HAMRO BAN [Our Forest] [in Nepali]. Kathmandu: Department of Forest, Ministry of Forest and Soil Conservation. 190 p. Devkota KP, BN Lenta, JD Wansi, MI Choudhary, DP Kisangau, Q Naz, Samreen and N Sewald. 2008. Bioactive 5alpha-pregnane-type steroidal alkaloids from Sarcococca hookeriana. Journal of Natural Product 71:1481–1484. Dhar U, RS Rawal and J Upreti. 2000. Setting priorities for conservation of medicinal plants – a case study in the Indian Himalaya. Biological Conservation 95:57–65. Dhyani PP and CP Kala. 2005. Current research in medicinal plants: five lesser known but valuable aspects. Current Science 88:335. Dobson A. 1995. Biodiversity and human health. Trends in Ecology and Evolution 10:390– 391. Dubey NK, R Kumar and P Tripathi. 2004. Global promotion of herbal medicine: India‟s opportunity. Current Science 86:37–41. Dupuy JM and RL Chazdon. 2008. Interacting effects of canopy gap, understory vegetation and leaf litter on tree seedling recruitment and composition in tropical secondary forests. Forest Ecology and Management 255:3716–3725. Dutt BSM. 1970. Hypoxidaceae [Symposium paper on comparative embryology of angiosperm]. Indian National Science Academy Bulletin 41:365–367.

134

PhD dissertation by Bharat Babu Shrestha

Elvin-Lewis M. 2001. Should we be concerned about herbal remedies? Journal of Ethnopharmacology 75:141–164. Elzinga JA, A Atlan, A Biere, L Gigord, AE Weis and G Bernasconi. 2007. Time after time: flowering phenology and biotic interactions. Trends in Ecology and Evolution 22:432–439. Facelli JM and AM Temby. 2002. Multiple effects of shrubs on annual plant communities in arid lands of South Australia. Austral Ecology 27:422–432. Falster DS and M Westoby. 2003. Plant height and evolutionary game. Trends in Ecology and Evolution 18:337–343. Fenner M. 1998. The phenology of growth and reproduction in plants. Perspectives in Plant Ecology, Evolution and Systematics 1:78–91. Ferner RE and K Beard. 2005. Regulating herbal medicines in UK. British Medical Journal 331:62–63. Finch-Savage WE and G Leubner-Metzger. 2006. Seed dormancy and the control of germination. New Phytologist 171:501–523. Francis SV, SK Senapati and GR Rout. 2007. Rapid clonal propagation of Curculigo orchioides Gaertn., an endangered medicinal plant. In Vitro Cellular and Developmental Biology – Plant 43:140–143. Gao L, X Wei and L Yang. 2004. A New diterpenoid alkaloid from a Tibetan medicinal herb Aconitum naviculare Stapf. Journal of Chemical Research No. 4:307–308. Garg A and T Husain. 2003. Strategies adopted by the alpine genus Pedicularis L. (Scrophulariaceae) to overcome environmental stress. Current Science 85:1413– 1414. Garg SN, LN Misra and SK Agarwal. 1989. Corchioside A: an orcinol glycoside from Curculigo orchioides. Phytochemistry 28:1771–1772. Garnier E, J-L Salager, G Laurent and L Sonie. 1999. Relationship between photosynthesis, nitrogen and leaf structure in 14 grass species and their dependence on the basis of expression. New Phytologist 143:119–129. Ghimire BK, HD Lekhak, RP Chaudhary and OR Vetaas. 2008. Vegetation analysis along an altitudinal gradient of Juniperus indica forest in southern Manang valley, Nepal. International Journal of Ecology and Development 9:20–29. Ghimire SK, D McKey and Y Aumeeruddy-Thomas. 2005. Conservation of Himalayan medicinal plants: harvesting patterns and ecology of two threatened species,

135

PhD dissertation by Bharat Babu Shrestha

Nardostachys grandiflora DC. and Neopicrorhiza scrophulariiflora (Pennell) Hong. Biological Conservation 124:463–475. Ghimire SK, JP Sah, KK Shrestha and D Bajracharya. 1999. Ecological study of some high altitude medicinal and aromatic plants in the Gyasumdo valley, Manang, Nepal. Ecoprint 6:17–25. Ghimire SK, IB Sapkota, BR Oli and RR Parajuli. 2008a. Non-Timber Forest Products of Nepal Himalaya: Database of Some Important Species Found in the Mountain Protected Areas and Surrounding Regions. Kathmandu: WWF Nepal. 186p. Ghimire SK, O Gimenez, R Pradel, D McKey and Y Aumeeruddy-Thomas. 2008b. Demographic variation and population viability in a threatened Himalayan medicinal and aromatic herb Nardostachys grandiflora: matrix modeling of harvesting effects in two contrasting habitats. Journal of Applied Ecology 45:41–51. Ghimire SK. 2008. Medicinal plants in the Nepal Himalaya: current issue, sustainable harvesting, knowledge gaps and research priorities. In: PK Jha, SB Karmacharya, MK Chettri, CB Thapa and BB Shrestha (eds.) Medicinal Plants in Nepal: An Anthology of Contemporary Research. Kathmandu: Ecological Society (ECOS). Pp. 25–42. Grierson AJC and DG Long. 1984. Flora of Bhutan. Vol 1 Part 2. Edinburgh: Royal Botanical Garden. Pp. 188–462. Gubhaju MR and SK Ghimire. 2009. Diversity and population status of non-timber forest products (NTFPs) in community forest of Dovan, Palpa, Nepal. Journal of Natural History Museum 24:21–46. Guo R, PH Canter and E Ernst. 2007. A systematic review of randomized clinical trial of individualized herbal medicine in any indication. Postgraduate Medical Journal 83:633–637. Gupta M, B Achari and BC Pal. 2005. Glucosides from Curculigo orchioides. Phytochemistry. 66:659–663. Gupta PK. 2000. Methods in Environmental Analysis: Water, Soil and Air. New Delhi, India: Agrobios (India). 409 p. Gupta R and KL Chadha. 1995. Medicinal and aromatic plant research in India. In: KL Chadha and R Gupta (eds.) Advances in Horticulture, Medicinal and Aromatic Plants, Vol 11. New Delhi: Malhotra House. pp. 1–43. Gurung K. 2009. Essential Oils in Nepal: A Practical Guide to Essential Oils and Aromatherapy. Kathmandu: Himalayan Bio Trade Pvt. Ltd. 147p.

136

PhD dissertation by Bharat Babu Shrestha

Hamilton A. 2003. Medicinal plants and conservation: Issues and approaches. Surrey (UK): International Plant Conservation Unit, WWF-UK. 51p. Hamilton A and P Hamilton. 2006. Plant Conservation: An Ecosystem Approach. London: Earthcan. 324p. Haque N, SAR Chowdhury, MTH Nutan, GMS Rahman, KM Rahman and MA Rashid. 2000. Evaluation of antitumor activity of some medicinal plants of Bangladesh by potato dish bioassay. Fitoterapia 71:547–552. Hawkins B, S Sharrock and K Havens. 2008. Plants and Climate Change: Which Future? Richmond (UK): Botanic Gardens Conservation International. 98 p. Hawkins B. 2008. Plants for Life: Medicinal Plant Conservation and Botanic Garden. Richmond (UK): Botanic Garden Conservation International. 50 p. Hidayati SN, JM Baskin and CC Baskin. 2001. Dormancy breaking and germination requirements for seeds of Symphoricarpos orbiculatus (Caprifoliaceae). American Journal of Botany 88:1444-1451. Horneck DA and RO Miller. 1998. Determination of total nitrogen in plant tissue. In: YP Kalra (ed.) Hand Book of Reference Methods for Plant Analysis. Boca Raton, Florida (USA): CRC Press. Pp. 75–83. Hostettmann K and A Marston. 2002. Twenty year of research into medicinal plants: results and perspectives. Phytochemistry Review 1:275–285. Howe HF and J Smallwood. 1982. Ecology of seed dispersal. Annual Review of Ecology and Systematics 13: 201–228. Huber H. 1996. Plasticity of internodes and petioles in erect and prostrate Potentilla species. Functional Ecology 10:401–409. IUCN 2006. 2006 Red List of Threatened Species. www.iucnredlist.org. [accessed on December 1, 2006]. IUCN Standards and Petitions Working Group. 2008. Guidelines for Using the IUCN Red List Categories and Criteria. Version 7.0. Prepared by the Standards and Petitions Working Group of the IUCN SSC Biodiversity Assessments Sub-Committee in August 2008. Downloadable from http://intranet.iucn.org/webfiles/doc/SSC/RedList/RedListGuidelines.pdf. IUCN. 2001. IUCN Red List Categories and Criteria: Version 3.1. Gland (Switzerland) and Cambridge (UK): IUCN Species Survival Commission. 30 p.

137

PhD dissertation by Bharat Babu Shrestha

IUCN. 2003. Guidelines for Application of IUCN Red List Criteria at Regional Levels: Version 3.0. Gland (Switzerland) and Cambridge (UK): IUCN Species Survival Commission. 26p. Jasrai YT and B Wala. 2001. Curculigo orchioides Gaertn (Kali musli): an endangered medicinal herb. In: IA Khan and A Khanum (eds.) Role of Biotechnology in Medicinal and Aromatic Plants, vol. IV. Hyderabad (India): Ukaaz Publication. Pp. 89–95. Jha S. 2003. Ecological Study of Some Selected Grasses and Forbs in Morang District of Nepal. Ph. D. Dissertation. Kathmandu: Central Department of Botany, Tribhuvan University. 167 p. Ji H-F, X-J Li and H-Y Zhang. 2009. Natural products and drug discovery. EMBO Reports 10:194–200. Jiao L, D-P Cao, L-P Qin, T Han, Q-Y Zhang, Z Zhu and F Yan. 2009. Antiosteoporotic activity of phenolic compound from Curculigo orchioides. Phytomedicine 16:874– 881. Johri BM, KB Ambegaokar and PS Srivastava. 1992. Comparative Embryology of Angiosperm. Vol 2. Berlin: Springer Verlag. Pp. 615-1221. Joy PP, KE Savithri, S Mathew and BP Skaria. 2004. Effect of shade and spacing on growth, yield and quality of black musli (Curculigo orchioides). Journal of Medicinal and Aromatic Plant Sciences 26:707–716. Joy PP, KE Savithri, S Mathew, J Thomas and CT Abraham. 2005. Effect of mulch and sources of nutrients on growth, yield and quality of black musli (Curculigo orchioides). Journal of Medicinal and Aromatic Plant Sciences 27:646–656. Kala C. 2000. Status and conservation of rare and endangered medicinal plants in the Indian Trans-Himalaya. Biological Conservation 93:371–379. Kala C. 2005. Indigenous uses, population density and conservation of threatened medicinal plants in protected areas of the Indian Himalayas. Conservation Biology 19:368– 376. Kala CP, PP Dhyani and BS Sajwan. 2006. Developing the medicinal plant sector in northern India: challenges and opportunities. Journal of Ethnobiology and Ethnomedicine 2:32 doi: 10.1186/ 1746-4269-2-32 http://www.ethnobiomed.com/content/2/1/32.

138

PhD dissertation by Bharat Babu Shrestha

Kalauni SK, MI Choudhary, F Shaheen, MD Manandhar, AU Atta-ur-Rahman, MB Gewali and A Khalid. 2001. Steroidal alkaloids from the leaves of Sarcococca coriacea of Nepalese origin. Journal of Natural Products 64:842–844. Kapoor S. 2008. Osteoporosis prevention and beyond: the systemic beneficial effects of Curculigo orchioides. Maturitas 61:371. Karanjeet N and S Singh. 2003. Phytochemical screening of flavonoid containing medicinal plants of Nepal. Plant Resources (Bulletin of Department of Plant Resources, Ministry of Forest and Soil Conservation, Kathmandu). 22:22-24. Kizu H, S Habe, M Ishida and T Tomimori. 1994. Studies on the Nepalese crude drugs. XVII. On the Naphthalene related compounds from the root bark of Oroxylum indicum Vent. [Article in Japanese] [Abstract]. Yakugaki Zasshi 114:492–613. Klein JA. J Harte and X-Q Zhao. 2008. Decline in medicinal and forage species with warming is mediated by plant traits on the Tibetan plateau. Ecosystem 11:775–789. Körner Ch. 1989. The nutritional status of plants from high altitudes: a worldwide comparison. Oecologia 81:379–391. Körner Ch. 2003. Alpine Plant Life: Functional Plant Ecology of High Mountain Ecosystems. 2nd edition. Berlin Heidelberg: Springer-Verlag. 344 p. Körner Ch. 2007. Climate treelines: conventions, global patterns, causes. Erdkunde 61:316– 324. Koul O and S Wahab. 2004. Neem: Today and in the New Millennium. Boston and London: Kluwer Academic Publishers. 296 p Kreb CJ. 1994. Ecology: The Experimental Analysis of Distribution and Abundance. 4th edition. London: Addison-Wesley Educational Publishers Inc. 801 p. Kubo M, K Namba, N Nagamoto, T Nagao, J Nakanishi, H Uno and H Nishimura. 1983. A new phenolic glucoside, curculigoside from rhizomes of Curculigo orchioides. Planta Medica 47:52–55. Kudo G. 1993. Relationship between flowering time and fruit set of entomophilous alpine shrub, Rhododendron aureus (Ericaceae), inhabiting snow patches. American Journal of Botany 80:1300–1304. Kunwar RM. 2006. Non Timber Forest Products of Nepal: A Sustainable Management Approach. Kathmandu: Center for Biological Conservation, and Tokyo: International Tropical Timber Organization. 423 p. Lakshmi V, K Pandey, A Puri, RP Saxena and KC Saxena. 2003. Immunostimulant principles from Curculigo orchioides. Journal of Ethnopharmacology 89:181–184.

139

PhD dissertation by Bharat Babu Shrestha

Lama YC, SK Ghimire and Y Aumeeruddy-Thomas. 2001. Medicinal Plants of Dolpo: Amchis' Knowledge and Conservation. Kathmandu: WWF Nepal Program. 150 p. Lange D. 1998. Europe’s Medicinal and Aromatic Plants: Their Use, Trade and Conservation. Cambridge (UK): TRAFFIC International. 107 p. Larcher W. 1995. Physiological Plant Ecology, 3rd ed. Berlin (Germany): Springer Verlaag. 493 p. Larsen HO and CS Olsen. 2007. Unsustainable collection and unfair trade? Uncovering and accessing assumptions regarding central Himalayan medicinal plant conservation. Biodiversity and Conservation 16:1679–1697. Larsen HO. 2005. Impact of replanting on regeneration of medicinal plant Nardostachys grandiflora DC. (Valerianaceae). Economic Botany 59:213–220. Leaman D. 2009. Easing the pressure. World Conservation 31:8. Liangqian L and Y Kodata. 2001. Aconitum L. In: W Zhengyi, PH Raven and H Deyuan (eds.) Flora of China, vol. 6, Caryophyllaceae through Lardizabalaceae. Beijing: Science Press; and St. Louis: Missouri Botanical Garden Press. Pp. 149–222. Manandhar NP. 1998. Native phytotherapy among the Raute tribes of Dadeldhura district, Nepal. Journal of Ethnopharmacology 60:199–206. Manandhar NP. 2002. Plants and People of Nepal. Timber Press Inc. Portland, Oregon, USA. 599 p. Marks N. 2004. PR coup for herbal cancer drug. British Medical Journal 329:804. Matsuura Y, Y Miyaichi and T Tomimori. 1994. Studies on the Nepalese crude drugs. XIX. On the flavonoid and phenylethanoid constituents of the root of Scutellaria repens Buch.-Ham. ex D. Don. [Article in Japanese] [Abstract]. Yakugaki Zasshi 114:775– 778. Mavraganis K and CG Eckert. 2001. Effect of population size and isolation on reproductive output in Aquilegia canadensis (Ranunculaceae). Oikos 95:300–310. McLachlan SM and D Bazely. 2001. Recovery patterns of understory herbs and their use as indicators of deciduous forest regeneration. Conservation Biology 15:98–110. Miehe G, M Winiger, J Böhner and Y Zhang. 2001. The climate diagram map of High Asia: purpose and concepts. Erdkunde 55:94–95. Misra TN, RS Singh and DM Tripathi. 1984a. Aliphatic compounds from Curculigo orchioides rhizomes. Phytochemistry 23:2369–2371. Misra TN, RS Singh, DM Tripathi and SC Sharma. 1990. Curculigol, a cycloartane triterpene alcohol from Curculigo orchioides. Phytochemistry 29:929–931.

140

PhD dissertation by Bharat Babu Shrestha

Misra TN, RS Singh, J Upadhyay and DM Tripathi. 1984b. Aliphatic hydroxy-ketones from Curculigo orchioides rhizomes. Phytochemistry 23:1643–1645. Molau U. 1993. Relationship between flowering phenology and life history strategies in tundra plants. Arctic and Alpine Research 25:391–402. Molur S and S Walker (eds.). 1998. Report of the workshop ‘Conservation Assessment and Management Plan (CAMP) for selected medicinal plant species of northern, northeastern and central India’. Coimbatore, India: Conservation Breeding Specialist Group of Zoo Outreach Organization. 62 p. Morgan JW. 1999. Effects of population size on seed production and germinability in an endangered, fragmented grassland plant. Conservation Biology 13:266–273. Mulliken T and P Crofton. 2008. Review of the Status, Harvest, Trade and Management of Seven Asian CITES-listed Medicinal and Aromatic Plant Species. Bonn (Germany): Federal Agency for Nature Conservation (BfN) (www.bfn.de). 144 p. Murali KS and R Sukumar. 1994. Reproductive phenology of a tropical dry forest in Mudumalai, southern India. Journal of Ecology 82:759–767. Murray BR, PH Thrall, AM Gill and AB Nicotra. 2002. How plant life history and ecological traits relate to species rarity and commonness at varying spatial scales. Austral Ecology 27:291–310. Nautiyal BP, V Prakash, R Bahuguna, U Maithani, H Bisht and MC Nautiyal. 2002. Population study for monitoring the status of rarity of three aconite species in Garhwal Himalaya. Tropical Ecology 43:297–303. Nawang R. 1996. Medicinal Plants. In: FAO Non-Wood Forest Products of Bhutan. Bangkok, Thailand: Food and Agriculture Organization (FAO) of United Nations. URL: http://www.fao.org/docrep/X5335E/X5335E00.htm (accessed on Dec 19, 2008) Nema RL, KG Ramawat and JM Merillon. 2008. Rapid micro propagation of Curculigo orchioides in shake flask culture. Pharmacognosy Magazine 4:315–319. Newstrom LE, GW Frankie and HG Baker. 1994. A new classification for plant phenology based on flowering patterns in lowland tropical rain forest trees at La Selva, Costa Rica. Biotropica 26:141–159. Noltie HJ. 1994. Flora of Bhutan. Vol. 3, part 1. Edinburgh: Royal Botanical Garden. 456 p. Ohba H, Y Iokawa and LR Sharma (eds.). 2008. Flora of Mustang, Nepal. Tokyo: Kodansha Scientific Ltd. 506 p.

141

PhD dissertation by Bharat Babu Shrestha

Ôke TR. 1987. Boundary Layer Climates. New York: Metheuen and Co. 435 p.

Olsen CS and F Helles. 2009. Market efficiency and benefit distribution in medicinal plant markets: empirical evidence from South Asia. International Journal of Biodiversity Science and Management 5:53–62. Oostermeijer JGB, MW van Eijck and JCM den Nijs. 1994. Offspring fitness in relation to population size and genetic variation in the rare perennial plant species Gentiana pneumonanthe (Gentianaceae). Oecologia 97:289–296. Ouborg NJ and R van Treuren. 1995. Variation in fitness related characters among small and large populations of Salvia pratensis. Journal of Ecology 83:369–380. Pandit P, A Singh, AR Bafna, PV Kadam and MJ Patil. 2008. Evaluation of antiasthmatic activity of Curculigo orchioides Gaertn rhizome. Indian Journal of Pharmaceutical Sciences 70:440–444. Phillipson JD. 2007. Phytochemistry and pharmacognosy. Phytochemistry 68:2960–2972. Phoboo S, A Devkota and PK Jha. 2008. Medicinal plants in Nepal: an overview. In: PK Jha, SB Karmacharya, MK Chettri, CB Thapa and BB Shrestha (eds.) Medicinal Plants in Nepal: An Anthology of Contemporary Research. Kathmandu (Nepal): Ecological Society (ECOS). Pp. 1–24. Pimprikar GD and M Haque. 2002. “Plant for Life” an attempt to develop a sustainable source of medicinal plants in Himalayas. In: T Watanabe, A Takano, MS Bista and HK Saiju (eds.) The Himalayan Plants: can they save us? Proceedings of Nepal- Japan Joint Symposium on Conservation and Utilization of Himalayan Medicinal Resources, November 6-11, 2000, Kathmandu. Pp. 284–287. Polunin O and A Stainton. 2000. Flowers of the Himalayas. 4th Impression. New Delhi: Oxford University Press. 518 p + 128 plates. Prajapati HA, DH Patel, SR Mehta and RB Subramanian. 2003. Direct in vitro regeneration of Curculigo orchioides Gaertn., an endangered anti-carcinogenic herb. Current Science 84:747–749. Press JR, KK Shrestha and DA Sutton. 2000. Annotated Checklist of the Flowering Plants of Nepal. London: The Natural History Museum. 430 p. Rajbhandary M, R Mentel, PK Jha, RP Chaudhary, S Bhattarai, MB Gewali, N Karmacharya, M Hipper and U Lindequist. 2007. Antiviral activity of some plants used in Nepalese traditional medicine. eCAM doi:10.1093/ecam/nem156. Ramawat KG, S Jain, S Suri and DK Arora. 1998. Aphrodisiac plants of Aravalli hills with special reference to safed musali. In: IA Khan and A Khanum (eds.) Role of

142

PhD dissertation by Bharat Babu Shrestha

Biotechnology in Aromatic and Medicinal Plants. Vol. 1. Hyderabad (India): Ukaaz Publications. Pp. 210–223. Rathcke B and EP Lacey. 1985. Phenological patterns of terrestrial plants. Annual Review of Ecology and Systematics 16:179–214. Reich PB, MB Walters and DS Ellsworth. 1997. From tropics to tundra: Global convergence in plant functioning. Proceedings of National Academy of Science 94:13730–13734. Rijal A. 2008. Living knowledge of the healing plants: Ethnophytotherapy in the Chepang communities from the mid-hills of Nepal. Journal of Ethnobiology and Ethnomedicine 4:23, doi:10.1186/1746-4269-4-23 (http://www.ethnobiomed.com/content/4/1/23). Roberson E. 2008. Medicinal Plants at Risk. Tucson, AZ: Center for Biological Diversity. 19 p. Sharma D, R Kapoor and AK Bhatnagar. 2009. Differential growth response of Curculigo orchioides to native arbuscular mycorrhizal fungal (AMF) communities varying in number and fungal components. European Journal of Soil Biology 45:328–333. Sharma RS, V Mishra, R Singh, N Seth and CR Babu. 2008. Antifungal activity of some Himalayan medicinal plants and cultivated ornamental species. Fitoterapia 79:589– 591. Shippmann U, D Leaman and AB Cunningham. 2006. A comparison of cultivation and wild collection of medicinal and aromatic plants under sustainability aspects. In: RJ Bogers, LE Craker and D Lang (eds.) Medicinal and Aromatic Plants. The Netherlands: Springer. Pp. 75–95. Shrestha BB, BK Ghimire, HD Lekhak and PK Jha. 2007b. Regeneration of treeline birch (Betula utilis D.Don) forest in a trans-Himalayan dry valley in Central Nepal. Mountain Research and Development 27:259–267. Shrestha BB, MB Gewali and PK Jha. 2007a. Ecology of Neopicrorhiza scrophulariiflora (Pennell) Hong. growing under different land uses in a trans-Himalayan dry valley of central Nepal. International Journal of Ecology and Environmental Sciences 33:233–241. Shrestha BB, S Dall‟Acqua, MB Gewali, PK Jha and G Innocentii. 2008. Biology and phytochemistry of Curculigo orchioides Gaerten. In: PK Jha, SB Karmacharya, MK Chettri, CB Thapa and BB Shrestha (eds.) Medicinal Plant of Nepal: An Anthology of Contemporary Research. Kathmandu: Ecological Society (ECOS). pp. 50–67.

143

PhD dissertation by Bharat Babu Shrestha

Shrestha KK, SR Rajbhandary, NN Tiwari, RC Powdel and Y Uprety. 2004. Ethnobotany in Nepal: Review and Perspectives. An unpublished report submitted to WWF Nepal Program by Ethnobotanical Society of Nepal. 271 p. Shrestha PM. R Pradhan and DM Shakya. 1997. Screening of essential oil bearing plants of Nepal. In: Proceedings of Second National Conference on Science and Technology, June 8-11, 1994. Kathmandu: Royal Nepal Academy of Science and Technology. pp. 472–489. Shrestha PM and SS Dhillion. 2003. Medicinal plant diversity and use in the highlands of Dolakha district, Nepal. Journal of Ethnopharmacology 86:81–96. Shrestha TM, N Karanjeet and NB Pradhan. 2002. Phytochemical screening/study of acute toxicity (LD50) of the plants from Langtang area (I). In: T Watanabe, A Takano, MS Bista and HK Saiju (eds.) The Himalayan Plants: can they save us? Proceedings of Nepal-Japan Joint Symposium on Conservation and Utilization of Himalayan Medicinal Resources, November 6-11, 2000, Kathmandu. Pp. 254–256. Shrestha UB and S Shrestha. 2004. Major Non-Timber Forest Products of Nepal (in Nepali). Kathmandu: Bhundipuran Prakashan. 441 p. Shrivastava RC. 1998. Flora of Sikkim (Ranunculaceae – Moringaceae). Dehradun (India): Oriental Enterprises. 309 p. Singh JS and SP Singh. 1992. Forest of Himalaya: Structure, Functioning and Impact of Man. Nainital (India): Gyanodaya Prakashan. 294 p. Singh MP, SB Malla, SB Rajbhandari and A Manandhar. 1979. Medicinal Plant of Nepal: retrospects and prospects. Economic Botany 33:185–198. Siwakoti M and SK Verma. 1999. Plant Diversity of Eastern Nepal: Flora of Plains of Eastern Nepal. Dehra Dun (India): M/S Bishen Singh Mahendra Pal Singh. 491p. Sokal RR and FJ Rohlf. 1995. Biometry. New York: WH Freeman and Company. 887p. Solangaarachchi SM and BMS Perera. 1993. Floristic composition and medicinally important plants in the understory of tropical dry mixed evergreen forest at the Hurulu Reserve of Sri Lanka. J. Natn. Sci. Count. Sri Lanka 21:209–226. SPSS. 2002. Statistical Package for Social Sciences (SPSS), version 11.5. USA: SPSS Inc. Stainton A. 1997. Flowers of the Himalayas: A Supplement. New Delhi: Oxford University Press. 86 p+128 plates. Stainton JDA. 1972. Forests of Nepal. New York: Hafner Publishing Company. 174 p. Subedi KR and NN Tiwari. 2000. Shausruta Nighantu [in Sanskrit]. Dang (Nepal): Mahendra Sanskrit University.

144

PhD dissertation by Bharat Babu Shrestha

Sultan SE. 2003. Phenotypic plasticity in plants: a case study in ecological development. Evolution and Development 5:25–33. Suresh S. 2008. Studies on the Wild Edible Plants of Paliyans and Pulayans in Palani Hills, Tamil Nadu. Ph. D. dissertation. Tirunelveli (India): Manonmaniam Sundaranar University. 119p. Suri SS, S Jain and KG Ramawat. 1999. Plantlet regeneration and bulbils formation in vitro from leaf and stem ex-plant of Curculigo orchioides, an endangered medicinal plant. Scientia Horticulturae 79:127–134. Suzuki A, O Shirota, K Mori, S Sekita, H Fuchino, A Takano and M Kuroyanagi. 2009. Leishmanicidal active constituents from Nepalese medicinal plant Tulsi (Ocumum sanctum L.). Chemical Pharmaceutical Bulletin 57:245–251. Tang SY, M Whiteman, JF Peng, A Jenner, EL Yong and B Halliwell. 2004. Characterization of antioxidant and antiglycation properties and isolation of active ingredients from traditional Chinese medicines. Free Radical Biology and Medicine 36:1575–1587. Taylor RSL, NP Manandhar and GHN Towers. 1995. Screening of selected medicinal plants of Nepal for antimicrobial activities. Journal of Ethnopharmacology 46:153– 159. Taylor RSL, NP Manandhar, JB Hudson and GHN Towers. 1996. Antiviral activities of Nepalese medicinal plants. Journal of Ethnopharmacology 52:157–163. Tikhomirov BA, VF Shamurin and VS Shtepa. 1960. Temperature of arctic plants. Academy of Science USSR Biology Series 3:429–442. [in Russian] Tiwari RD and G Misra. 1976. Structural studies of the constituents of the rhizomes of Curculigo orchioides. Planta Medica 29:291–294. Tomimori T. 2000. On the constituents and biological activities of some Nepalese medicinal plants [Article in Japanese] [Abstract]. Yakugaku Zasshi 120:591–606. UNEP-WCMC. 2009. UNEP-WCMC Species Database: CITES-Listed Species. http://www.unep- wcmc.org/isdb/CITES/Taxonomy/country_list.cfm/isdb/CITES/Taxonomy/country_list.cfm

?col=all&country=NP&source=plants&displaylanguage=eng [accessed on July 24, 2009]. Utelli AB and BA Roy. 2000. Pollinator abundance and behavior on Aconitum lycoctum (Ranunculaceae): an analysis of quantity and quality components of pollination. Oikos 89:461–470.

145

PhD dissertation by Bharat Babu Shrestha

Valls J, T Richard, F Larronde, V Leblais, B Muller, JC Delaunay, JP Monti, KG Ramawat and JM Merrilon. 2006. Two new benzylbenzoate glucosides from Curculigo orchioides. Fitoterapia 77:416–419. van Schaik CP, JW Terborgh and SJ Wright. 1993. The phenology of tropical forests: adaptive significances and consequence for primary consumers. Annual Review of Ecology and Systematics 24:353–377. Vashistha R, BP Nautiyal and MC Nautiyal. 2006. Conservation status and morphological variation between populations of Angelica glauca Edgew. and Angelica archangelica Linn. in Garhwal Himalayas. Current Science 91:1537–1542. Venkatesh P, PK Mukherjee, SN Kumar, NK Nema, A Bandhyopadhyay, H Fukui and H Mizuquchi. 2009. Mast cell stabilization and antihistaminic potentials of Curculigo orchioides rhizomes. Journal of Ethnopharmacology 126: 434–436. Vijayanarayanan K, RS Rodrigues, KS Chandrashekhar and EVS Subrahmanyam. 2007. Evaluation of estrogenic activity of alcoholic extract of rhizome of Curculigo orchioides. Journal of Ethnopharmacology 114:241–245. Villegas AC. 2001. Spatial and temporal variability in clonal reproduction Aechmea magdalenae, a tropical understory herb. Biotropica 33:48–59. Wala BB and YT Jasrai. 2003. Micropropagation of an endangered medicinal plant: Curculigo orchioides Gaertn. Plant Tissue Culture 13:13–19. Watanabe T, SR Rajbhandary, KJ Malla and S Yahara. 2005. A Handbook of Medicinal Plants of Nepal. Kanagawa (Japan): Non-Profit Organization AYUR SEED, Life Environmental Institute. 262 p. Widen B. 1993. Demographic and genetic effects on reproduction as related to population size in a rare, perennial herb, Senecio integrifolius (Asteraceae). Biological Journal of the Linnaean Society 50:179–195. Willcox ML and G Bodeker. 2004. Traditional herbal medicines for malaria. British Medical Journal 329:1156–1159. World Bank. 2004. Sustaining Forestry: A Development Strategy. Washington: The World Bank. 80 p. Wright IJ, PB Reich, M Westoby, DD Ackerly, et al. 2004. The worldwide leaf economics spectrum. Nature 428:821–827. Wu Q, DX Fu, AJ Hou, GQ Lei, ZJ Liu, JK Chen and TS Zhou. 2005. Antioxidative phenols and phenolic glycosides from Curculigo orchioides. Chemical Pharmaceutical Bulletin 53:1065–1067.

146

PhD dissertation by Bharat Babu Shrestha

Wu Q, DX Fu, AJ Hou, GQ Lei, ZJ Liu, JK Chen and TS Zhou. 2005. Antioxidative phenols and phenolic glycosides from Curculigo orchioides. Chemical Pharmaceutical Bulletin 53:1065–1067. Xu J and R Xu. 1992. Cycloartane-type sapogenins and their glycosides from Curculigo orchioides. Phytochemistry 31:2455–2458. Xu J, R Xu and X Li. 1992a. Glycosides of a cycloartane sapogenin from Curculigo orchioides. Phytochemistry 31:233–236. Xu J, R Xu and X Li. 1992b. Four new cycloartane saponins from Curculigo orchioides. Planta Medica 58:208–210. Zhanhe J and AW Meerow. 2000. Amaryllidaceae. In: W Zhengyi and PH Raven (eds.) Flora of China vol. 24. Beijing: Science Press; and St Louis: Missouri Botanical Garden Press. Pp. 264–273. Zuo A-X, Y Shen, X-M Zhang, X-Y Jiang, J Zhou, J Lu and J-J Chen. 2010. Four new trace phenolic glycosides from Curculigo orchioides. Journal of Asian Natural Products Research 12:43–50.

147

PhD dissertation by Bharat Babu Shrestha

APPENDICES

Appendix 1. Laboratory Protocol for Soil Analysis

Organic Carbon: Walkley and Black method was used to determine organic carbon and organic matter in soil. Dry and fine (passed through 0.5 mm sieve) soil (0.5 g) was taken in a conical flask (500 ml) and added 10 ml potassium dichromate (1 N) with gentle swirling. Concentrated sulphuric acid (20 ml) was added and the mixture was allowed to cool down. After 30 minutes, 200 ml of distilled water was added to the mixture. After adding 10 ml phosphoric acid and 1 ml diphenylamine indicator, the mixture was titrated with freshly prepared ferrous ammonium sulphate (0.5 N) until the color changed from blue-violet to green. In every batch of 10 soil samples, a single blank (without soil) was run. Soil OC was calculated as follows:

Organic carbon (%): 1.30.003100N (B-C) M

Where, N = Normality of ferrous ammonium sulphate B = volume of ferrous ammonium sulphate consumed in blank titration (ml) C = volume of ferrous ammonium sulphate consumed with soil sample (ml) M = mass of soil (g)

Nitrogen: Estimation of total N by micro-Kjeldahl method included three steps: digestion, distillation and titration. In digestion, 1 g air dried and sieved (passed through 0.5 mm sieve) soil was taken in a clean and dry kjeldahl digestion flask (300 ml). As a catalyst, mixture of 3.5 g potassium sulphate and 0.4 g copper sulphate was mixed with the soil sample, and 6 ml concentrated sulphuric acid was added to the mixture with gentle shaking. The flask was heated at low temperature until the bubbles disappeared from the black mixture. Then the temperature was raised to about 350 C and heating was continued until the mixture turned to turquoise (greenish-blue). The flask was removed from the heating mantle and allowed to cool down for about 15 minutes. Then 50 ml distilled water was added to the digest with shaking. For distillation, the digest mixture was transferred to distillation flask (300 ml). Boric acid indicator (10 ml) was taken in a small beaker (100 ml) and placed below the noggle of condenser in such a way that the noggle was dipped into the indicator solution. When the mixture in the distillation flask was slightly warm, 30 ml of sodium hydroxide solution (40%) was added. When distillate began to accumulate in the beaker with boric acid indicator, color of the indicator changed from pink to green.

148

PhD dissertation by Bharat Babu Shrestha

Distillation was continued until the volume of distillate reached to about 50 ml. Then the distillate was titrated with hydrochloric acid (0.1 N) and the volume of acid consumed was recorded. With each batch of soil samples, single blank (without soil) was run. Following formula was used to determine total nitrogen content of the soil samples: Soil N (%) = 14  N  (S – B)  100 M

Where, N = normality of hydrochloric acid S = volume of hydrochloric acid consumed with sample (ml) B = volume of hydrochloric acid consumed with blank (ml) M = mass of soil taken (mg)

Appendix 2. Laboratory Protocol for Estimation of Leaf Nitrogen (N) Content

Estimation of leaf N content also included three steps: digestion, distillation and titration. Oven dried (60 C for 72 h) leaf sample (250 mg) was taken in a clean and dry kjeldahl digestion flask (300 ml), and 2 g catalyst (mixture of potassium sulphate, copper sulphate and selenium powder in the ratio of 100:10:1) was added to it. After adding 6 ml of concentrated sulphuric acid to the mixture of leaves and catalyst, the flask was heated at low temperature until the bubbles ceased to ooze out. Then temperature was raised to about 350 C and heating was continued until the color changed from black to yellowish and finally to turquoise. Once the final color appeared the flask was immediately removed from the heating mantle and allowed to cool down. After about 15 minutes, 50 ml of distilled water was added gradually to the mixture. With each batch of leaf samples, single blank (without leaf) was run. Further steps of distillation and titration were same to nitrogen estimation in soil (Appendix 1).

149

PhD dissertation by Bharat Babu Shrestha

Appendix 3. List of Publications

Research

I. Shrestha BB, S Dall‟Acqua, MB Gewali, PK Jha and G Innocenti. 2006. New flavonoid glycosides from Aconitum naviculare (Bruhl) Stapf, a medicinal herb from the trans-Himalayan region of Nepal. Carbohydrate Research 341:21612165. II. Shrestha BB, PK Jha and MB Gewali. 2007. Ethomedicinal use and distribution of Aconitum naviculare (Bruhl) Stapf in upper Manang central Nepal. In: RP Chaudhary, TH Aase, OR Vetaas and BP Subedi (eds.) Local Effects of Global Changes in the Himalayas: Manang, Nepal. Tribhuvan University, Nepal and University of Bergen, Norway. Pp 171181. III. Dall‟Acqua S, BB Shrestha, MB Gewali, PK Jha, M Carrara and G Innocenti. 2008. Diterpenoid alkaloids and phenolic glycosides from Aconitum naviculare (Brühl) Stapf. Natural Product Communication 3(12):19851989. IV. Shrestha BB and PK Jha. 2009. Habitat range of two alpine medicinal plants in a trans-Himalayan dry valley, central Nepal. Journal of Mountain Science 6:6677. V. Dal‟Acqua S, BB Shrestha, S Comai, G Innocenti, MB Gewali and PK Jha. 2009. Two phenolic glycosides from Curculigo orchioides Gaertn. Fitoterapia 80 (5): 279282. doi:10.1016/j.fitote.2009.03.002 VI. Shrestha BB, PK Jha and DR Kandel. 2011. Reproductive ecology and conservation prospects of threatened medicinal plant Curculigo orchioides Gaertn. in Nepal. Tropical Ecology 53 (1): 91-101. VII. Shrestha BB and PK Jha. Life history and population status of Himalayan endemic Aconitum naviculare. Mountain Research and Development 30 (4): 353-364. doi:10.1659/MRD-JOURNAL-D-10-00003.1

Review

I. Shrestha BB, S Dall‟Acqua, MB Gewali, PK Jha and G Innocenti. 2008. Biology and phytochemistry of Curculigo orchioides Gaerten. In: PK Jha, SB Karmacharya, MK Chetti, CB Thapa and BB Shrestha (eds.) Medicinal Plant of Nepal: An Anthology of Contemporary Research. Kathmandu: Ecological Society (ECOS). pp. 50–67.

150

Medicinal Plants in Nepal: An Anthology of Contemporary Research, 2008, pp. 50-67. Eds: P.K. Jha, S.B. Karmacharya, M.K. Chettri, C.B. Thapa and B.B. Shrestha Publisher: Ecological Society (ECOS), P.O. Box 6132, Kathmandu, Nepal

BIOLOGY AND PHYTOCHEMISTRY OF CURCULIGO ORCHIOIDES GAERTN.

Bharat B. Shrestha, S. Dall’Acqua*, M.B. Gewali**, P.K. Jha and G. Innocenti* Central Department of Botany Tribhuvan University, Kirtipur, Kathmandu *Department of Pharmaceutical Sciences Padova University, Padova, Italy **Central Department of Chemistry Tribhuvan University, Kirtipur, Kathmandu For correspondence: Email: [email protected], [email protected]

ABSTRACT

Curculigo orchioides is a vulnerable medicinal herb distributed throughout Nepal from tropical to subtropical regions. Ethnomedicinal uses of rootstock of Curculigo orchioides have been well documented and it has been used in a wide range of health problems; most commonly as aphrodisiac, tonic to restore vigor and against asthma and jaundice. This plant reproduces sexually by seeds and vegetatively by rootstock and leaves. Techniques have been developed for in vitro mass propagation from leaf explants. Secondary metabolites so far isolated and characterized from rootstock mainly belong to three classes: glycosilated phenolics, triterpenic saponins and aliphatic ketones. At least thirty secondary metabolites belonging to these classes have been isolated and characterized but quantitative estimation has been done only for a few of these compounds. Few of the secondary metabolites evaluated for bioactivity showed antioxidative, immunostimulatory and estrogenic activities. Polyherbal medicines containing rootstock of Curculigo orchioides showed neuronal differentiating, anticarcinogenic and antihepatotoxic activities. Though the market demand of rootstock of this plant is higher than supply, development of agrotechnology for commercial cultivation is at infancy.

Present review identifies the research gap in the fields of demography, population status, resource estimation, phenology, pollination and germination ecology, response to harvest in wild, and agrotechnology development for commercial cultivation. For exploitation of medicinal values, secondary metabolites should be quantitatively estimated and their bioactivity should be assessed following the lead provided by its ethnomedicinal uses.

Key words: Bioactivity, cultivation, distribution, ethnomedicinal uses, regeneration.

INTRODUCTION phenology, growth pattern and adaptation) and Understanding the distribution pattern, habitat population dynamics are pre-requisite for planning requirements, life history traits (e.g. reproduction, conservation and management of medicinal plants.

50 Phytochemical analysis and assessment of Earlier this genus was placed in another family bioactivity of phytoconstituents are essential for Amaryllidaceae. The term ‘Curculigo’ was derived evaluating medicinal efficacy of ethnomedicinal from Latin ‘curulio (gurgulio)’ meaning ‘a weevil, plants. Ethnomedicinal uses of Nepalese plants a corn worm, the grain weevil’ referring to the have been well documented (e.g. Manandhar beaked ovary or to the appearance of mature seed 2002, Baral and Kurmi 2006), however of Curculigo orchioides (Quattrocchi 2000). In information related to ecology (life history traits, Nepal, Hypoxidaceae is represented by two population dynamics, etc.), phytochemical genera, Curculigo and Hypoxis, and the genus composition and bioactivity of the Curculigo is represented by four species (C. phytoconstituents are lacking for a large fraction capitulata (Lour.) Kuntze, C. crassifolia (Baker) Hook. f., C. gracilis (Kurz) Wall. ex Hook. f. and of ethnomedicinal plants. The situation becomes C. orchioides Gaertn.) (Press et al. 2000). more serious when these information are lacking Curculigo orchioides is called Kalo musali in for highly traded species which are mostly Nepali; Xian Mao in Chinese; Kalimusli, collected from wild population. Muslikand in Hindi; Hemapushpi, In this review we have surveyed literatures for Kanchanapushpika in Sanskrit; Kin-bai-zasa in ecology, phytochemistry and bioactivity of Japanese; jool-lum, uoba, undora, kom-mol and phytoconstituents of Curculigo orchioides, a yuara in Australia; black musale, golden eye grass, medicinal herb of tropical to subtropical region in ground palm in English. A brief morphological Nepal. We have also highlighted the information description below follows Noltie (1994): tuber gap and research opportunities in this plant. For vertical, with bundle of fleshy roots at the base; distribution in Nepal, we examined herbarium leaf blade barrow to broadly lanceolate, 8 – 38 × 1 specimens deposited at National Herbarium – 3 cm, usually glabrous, petiolate, sometimes (KATH), Godawari, Kathmandu, and Tribhuvan flowers before leaf developed; inflorescence spike, University Central Herbarium (TUCH), Kirtipur, upper flowers male only, lower bisexual, bract Kathmandu. Except few ethnomedicinal narrowly lanceolate, membranous; perianth lobes documentations (Manandhar 1991, Rajbhandari et yellow, lanceolate, ca. 1 × 0.2 cm, outer three al. 2001, Panthi and Chaudhary 2003, Shrestha slightly wider, usually hairy; capsule sessile, and Dhillion 2003) and evaluation for antiviral hidden in leaf bases, irregular, oblong to ovoid, tapered into beak; seeds few, smooth, shiny black activity (Rajbhandari et al. 2001), all references with hook like projection. Leaves of the genus were based on studies outside Nepal. Ecological Curculigo mostly has tetracytic (61%) and tricytic information is awfully lacking for its whole range (21%) stomata (Shah and Gopal 1970). of distribution. There is not a single work on phytochemical analysis of Nepalese specimens of ETHNOMEDICINAL USES Curculigo orchioides. Underground rootstock of Curculigo orchioides, commonly known as kali musali, has a TAXONOMIC DESCRIPTION wide range of ethnomedicinal uses (Table 1). Curculigo orchioides Gaertn. (synonyms: Various terms like ‘root’, ‘rootstock’ and Curculigo malabarica Wight, Hypoxis orchioides ‘rhizome’ have been used to indicate the Kurz) is a member of family Hypoxidaceae. medicinally important part of this plant. The better

51 term is ‘rootstock’ because the part in use is applied twice a day to treat ringworm infection underground vertical stem with leaf scars. The (Chendurpandy 2006). cluster of fleshy root at the base of the rootstock is Table 1. Ethnomedicinal uses of Curculigo also used in ethnomedicine. Ethnomedicinal uses orchioides. of this plant have been covered in most of the SN Disorders/Uses References 1 Asthma Kirtikar and Basu 1994, books on ethnobotany and medicinal plants of Bajpai and Mishra 1998, Nepal (e.g., Rajbhandari et al. 1995, Joshi and Rajbhandari et al. 2001, Singh et al. 2002, Kala et al. Joshi 2001, Rajbhandary 2001, Manandhar 2002, 2006 LISP 2004, Shrestha and Shrestha 2004, Baral and 2 Jaundice Chopra et al. 1956, Kirtikar and Basu 1994, Bajpai and Kurmi 2006, Bhattarai and Ghimire 2006, Mishra 1998, Rajbhandari et Adhikari et al 2007). However, some information al. 2001 like altitude range of distribution (1000 – 2080 m 3 Pruritis (itching), Rajbhandari et al. 2001 skin diseases asl), dimension of rootstock (1 cm long) and 4 Dysentery Manandhar 1985, photograph given in Medicinal Plants of Nepal for Shrestha and Dhillion Ayurvedic Drugs by Rajbhandari et al. (1995) 2003, Kala et al. 2006 does not match with Curculigo orchioides. 5 Piles, haemorrhoides Singh et al. 2002 6 Diarrhoea Kirtikar and Basu 1994 Rootstock of this plant has alterative, demulcent, 7 Tonic, restore vigour Chopra et al. 1956, Rao et diuretic, restorative, coolant and aphrodisiac and vitality al. 1978, Tandon and Shukla 1995, Singh et al. effects on human (Chopra et al. 1956, Kirtikar and 2002, Lakshmi et al. 2003, Basu 1994, Ramawat et al. 1998 and Singh et al. Panthi and Chaudhary 2003, Jain et al. 2004, Kala et al. 2002). A few of references have described detail 2006 methods of uses of Curculigo orchioides in 8 Stop bleeding in cut, Chopra et al. 1956, Shah ethnomedicine. Barley and black pepper seeds are healing and drying 1978, Pattanaik et al. up wounds 2008 mixed in equal amount which in turn are mixed 9 Leucorrhoea Singh et al. 2002, with the rootstock of Curculigo orhioides and Shrestha et al. 2004 given by Tharus of Dang district, Nepal in three 10 Bronchitis, white Singh et al. 2002 discharge in urine, nose doses per day to treat dysentery (Manandhar 1985, bleeding, epilepsy cited in Rajbhandary 2001). The decoction of 11 Ring worm infection Chendurpandy 2006 pounded rootstock along with crushed fruit of 12 Peptic ulcer Manandhar 1991 Trachyspermum ammi (Umbelliferae) is given to 13 Eye tonic, Jain et al. 2004 gonorrhoea, syphilis unconscious children (Parrotta 2001). According 14 Aphrodisiac Chopra et al. 1956, Kirtikar to Shrestha and Dhillion (2003), the rootstock is and Basu 1994, Ramawat et al. 1998, Singh et al. 2002, chewed and swallowed for dysentery in Dolakha Panthi and Chaudhary 2003, district of Nepal. In southern Rajasthan of India Shrestha et al. 2004 100 g dried and powdered rootstock is mixed with 15 Male sterility Bajpai and Mishra 1998 16 Diabetes, Parrotta 2001 1 kg semi-solid milk (prepared after extensive unconsciousness boiling of 5 litter buffalo milk) and taken early in 17 Stop over-bleeding BB Shrestha et al. the morning for seven days as an eye tonic and to during pregnancy in (unpublished data) women, as aphrodisiac increase potency (Jain et al. 2004). About 10 g of to male buffalo the fresh or dried rootstock paste is externally 18 Enhance lactation in Shrestha et al. 2004 cattle

52 In many Ayurvedic formulations it has been to 2100 m asl) (Table 3). Historically the plant was used as metabolic enhancer and aphrodisiac along collected from Kirentechap (Dolakha) in 1963 by with Asparagus racemosus, Chlorophytum Japanese Expedition team of Chiba University borivilianum and Withania somnifera (Ramawat et (Murata 1983). Malla et al. (1986) have described al. 1998). The rootstock is one of the constituents this plant from Kathmandu valley. Out of 17 Ayurvedic formulations such as ‘Talamuli herbarium specimens examined, 10 were collected churna’, ‘Shaktivardak yoga’ and ‘Vajikara from central Nepal, four from western Nepal and Shakti’ (Sapkota and Adhikari 2001). It is one of three from eastern. Curculigo orchioides the constituents in ‘Ghandak Rasayan’, a product commonly grows in sal (Shorea robusta) forests in of Singha Durbar Baidhykhana (Nepal) (Kanai tropical belts (Behera and Misra 2006, Kandel 2006), and ‘Dhatupaustic Churnas’, a product of 2007), and Schima-Castanopsis forest and grassy Baidyanath Ayurved Bhawan Pvt. Ltd. (India) slopes in subtropical regions. This species has (Chandel et al. 1996). It is also a constituent of also been reported from moist deciduous tropical several modern polyherbal medicines, some of forest with Xylia xylocarpa, Lagerstroemia which are presented in Table 2. lanceolata and Terminalia spp. as dominant tree species (Bhat and Murali 2001). Review of Table 2. Proportion of Curculigo orchioides herbarium specimens deposited at two herbaria of rootstock in some polyherbal medicines. Nepal (KATH and TUCH) showed the occurrence SN Herbal Percentage Manufacturer References of Curculigo orchioides from a wide range of medicine of (trade Curculigo habitats from sunny slopes to moist places and name) orchioides forest floor to open places (Table 3). This species rootstock is also found in wetland system of Nepal’s terai 1 Maharishi 2.0 Maharishi Ramawat et Amrit Ayurved al. 1998 (BPP 1995). Kalash, Corporation Ltd, MAK-5* Delhi REPRODUCTION 2 Shivtone 25.0 Shiv Herbal Ramawat et Research Lab al. 1998 Pvt. Ltd., Curculigo orchioides is a geophilous, perennial Nagpur, India herb and mainly reproduces by seeds. A mature 3 Shilaforte 5.2 Deccan Ramawat et Ayurvedashram al. 1998 plant produces generally a single inflorescence Pharmacy Ptd., with a single (rarely two) bisexual flower at the Hyderabad base and several male flowers above. The bisexual 4 Manoll 0.04 Charak Ramawat et Pharmaceuticals, al. 1998 flower matures and withers earlier than the male Mumbai flowers mature. Thus, the male flowers apparently 5 Kamilari 15.0 Nupal Remedies Rajesh and Private Limited, Latha 2004 have no role for the pollination of the bisexual Cochin, India flower of the same individual plant. A fruit * Marketed in Europe by Global Trading Group, developed from the bisexual flower contains six Verona, Italy (Rodella 2004, Penza et al. 2007) seeds on average. The seeds over-winter in the soil DISTRIBUTION AND HABITAT and germinate in the next growing season. The flowering, fruit production and seed germination Curculigo orchioides is distributed in China (Peng et al. 2006), Bangladesh (Hasanuzzaman are negatively affected by thick litter and deep 2006), Himalayas (Kashmir to Assam), Nepal, shade in dense forest. Poor seed set and India, Sri Lanka, east to Japan, Malaysia and germination associated with over-exploitation are Australia (Press et al. 2000). In Nepal this plant is responsible for its declining wild population in distributed from tropical to subtropical region (150 India (Gupta and Chadha 1995).

53 Table 3. Collection sites and habitats of Curculigo orchioides specimens deposited at National Herbarium (KATH), Godawari, Lalitpur and Tribhuvan University Central Herbarium (TUCH), Kirtipur, Kathmandu. All specimens were collected within Nepal SN District Locality Elevation Collection Habitat Herbaria (masl) region* 1 Banke Mahadevpuri 200 W Mallotus forest TUCH 2 Bardia Brindapuri 168 W - TUCH 3 Chitwan Tikauli 450 C - TUCH 4 Dang Garhwa 250 W Open field KATH 5 Dang Phedi 440 W - KATH 6 Dolakha Malephu 970 E Open place KATH Tamakoshi along trail 7 Dolakha Kirantichhap basti 1110 E Pine forest KATH 8 Kapilvastu Nardasagar 170 C - TUCH 9 Kaski Deurali 2080 C Shady place KATH 10 Kaski Lumle 1600 C - TUCH 11 Kavrepalanchock Baluwa - C Forest KATH 12 Kavrepalanchock Khardorpati 1550 C Moist land KATH 13 Kavrepalanchock Dapcha 1740 C Shady place KATH 14 Lalitpur Goth danda 1219 C Sunny place KATH 15 Makawanpur Kamle 1000 C Open land KATH 16 Morang Rajarani 570 E Shady slope KATH 17 Nawalparasi Rajahar 500 C Sal forest TUCH 18 Syanja Putali Bazaar - 10 1000 C Sal forest ** 19 Lamjung Gaunshahar - C Schima- ** Castanpsis forest, grassy slope 20 Gorkha Bungkot 600 C Sal forest ** 21 Kathmandu Nagarjun 1500 C Forest gaps of ** Schima- Castanpsis forest *W: Western Nepal, C: Central Nepal, E: Eastern Nepal; ** Collection of B.B. Shrestha.

The plant survives the winter as underground data). When mid rib part of mature leaves are rootstock. The rootstock has high regenerative damaged and immediately come in contact with potential. When upper part of rootstock along with moist soil, one to few bulbils are formed at the shoot apical meristem is removed, the cut end of beginning which latter develop into complete the rootstock can regenerate by producing 1-3 new plantlets. Thus, population of Curculigo shoots. In moist places, the plant can also orchioides in such moist habitat has a mixture of regenerate by leaves from mid rib region during sexually reproduced individuals and vegetatively rainy season (BB Shrestha et al., unpublished regenerated individuals.

54 IN VITRO REGENERATION Prajapati et al. (2003) inoculated rootstock and leaf explants on MS medium supplemented with In vitro propagation by tissue culture has been MS vitamins, 0.2 % (w/v) activated charcoal, 1.5 considered as an important tool for conservation and 3.0 % (w/v) sucrose and plant growth and propagation of rare and threatened plants. Suri regulators. Rootstock explants did not show any et al. (1999, 2000) and Prajapati et al. (2003) have significant response with the combination tried. reported direct in vitro regeneration of plantlets of Leaf explants inoculated in MS basal medium with Curculigo orchioides from midrib region of its 1.5% sucrose showed direct organogenesis, and leaf. Suri et al. (1999) reported a maximum entire plantlet with single shoot and well number of shoots (10 per explants) from rootstock developed roots were obtained after nine weeks in explants on B5 medium supplemented with 4.4 culture. Similarly leaf explants inoculated in MS µM BAP (6-benzyl-aminopurine). B5 medium basal medium with low concentration (0.2 – 1.0 supplemented with 4.4 µM BAP was also optimal mg/l) of BAP showed single shoot regeneration for leaf growth and produced four shoots per with rooting. But high concentration (1.5 – 2.0 explants from the midrib region. On B5 basal mg/l) of BAP in MS medium induced callus medium supplemented with 4.4 µM BAP the stem formation which died after 60 days without explants directly produced multiple shoots but organogenesis. Leaf explants inoculated in MS when the basal medium was supplemented with medium supplemented with different concentration 2.25 µM 2,4 – D (2,4- Dichlorophenoxy acetic (0.5 – 2.5 mg/l) of 2,4 – D showed differentiation acid) and 1.15 µM kinetin the stem explants of multiple shoots and number of leaf explants produced callus. When these calli were transferred with multiple shoot differentiation increased with to different concentrations and combinations of increasing concentration of 2,4 – D. These BAP and sucrose, maximum number of bulbils (10 multiple shoots obtained in MS medium per inoculum) were produced on B5 medium supplemented with 2,4 – D produced roots after 60 supplemented with 35.2 µM BAP and 0.18 M days. The in vitro regenerated plantlets had 96.27 sucrose. Isolated in vitro produced bulbils as well % and 82.57 % survival in polyhouse and field as shoot directly obtained from leaf explants condition, respectively. developed into complete plantlets when grown on B5 medium without plant growth regulators. The Since leaf explants provide contamination free plantlets were successfully transferred to soil culture, they can be the most suitable materials to which showed 90% survival with the vigor establish in vitro culture and for rapid micro- comparable to that of naturally propagated plants. propagation. Direct regeneration of plantlets from leaf and stem explants without callus formation, as Suri et al. (2000) developed a method for large mentioned above, can help to preserve true-to-type scale multiplication of Curculigo orchioides from traits in the propagation and facilitates rapid leaf explants in shake flask culture. The leaf multiplication (Suri et al. 1999). explants were cultured in B5 liquid medium supplemented with potassium nitrate, ammonium PHYTOCHEMICAL COMPOSITION sulphate, benzyl adenosine, adenine, indole butyric acid and polyvinyl pyrrolidone. The shake flask Due to various uses in traditional medicine of culture yielded 2737 bulbils/l medium where as several Asiatic countries (Table 1) the rootstock of the static culture yielded 624 bulbils/l medium. Curculigo orchioides has been studied from The bulbils germinated (90.26%) into plantlets and phytochemical viewpoint. The rootstock is rich in survivality rate in soil was 90%. free sugars (7.56 %, mainly xylose and glucose),

55 polysaccharides (17.01%), hemicellulose (20.15%) anacardoside (Garg et al. 1989, Wu et al. 2005). and mucilage (8.12%) (Tiwari and Misra 1976). Chen et al. (1999) reported the presence of a Secondary metabolites reported in the rootstock of dichlorinated and methoxy derivative of the C. orchioides can be grouped into three main orcinol called curculigine A. classes, namely glycosilated phenolics, triterpenic Another simple phenol derivatives are the saponins and aliphatic ketones. benzyl benzoates. Orchioside A and the curculigosides A-D are characterized by different Cl Cl substituents on the aromatic rings. These derivatives have glucopyranoside unit linked at the R glc-glc HO O O O position 2 of the aromatic ring (Kubo et al. 1983, Orcinol aglycone curculigine A O R1 Fig. 1. Structures of orcinol aglycone and R3 R2 curculigine A. Sugars linked to orcinol O aglycone are described in table 4. O O HO O Table 4. Sugars (R) linked to the orcinol aglycone. OH HO Sugars (R) References HO

β-D-Xylopyranosyl-(1→6)- β-D- Garg 1989 Fig. 2. Structure of Curculigosides A-D. lucopyranoside Descriptions of R1, R2 and R3 are presented in table 5. β-D-glucopyranoside Chen 1999, Wu 2005 Table 5. Description of R1, R2 and R3 of figure 2. β-D-apiofuranosyl-(1→6)- β-D- Wu 2005 R1 R2 R3 Reference glucopyranoside s

β-D-glucopyranosyl-(1→6)-β-D- Wu 2005 Curculigoside A OCH3 H OH Kubo et al. glucopyranoside 1983, Chen et al. Among the phenolic derivatives, the rootstock 1999 is rich in simple compounds as syringic acid, 2,6 Curculigoside B OH H OH Wu et al. dimethoxybenzoic acid, and a number of orcinol 2005 (5-methyl resorcinol) glycosides (Wu et al. 2005, Curculigoside C OCH3 OH OH Wu et al. Garg et al. 1989). The sugars linked to the orcinol 2005 aglycone are units of xylopyranosyl (1→6) glucopyranoside, glucopyranoside, apiofuranosyl Orchioside A or OCH3 OH H Gupta et Curculigoside D al. 2005, (1→6) glucopyranoside, and glucopyranosyl Valls et al. (1→6) glucopyranoside (Table 4). These orcinol 2006 glycosides are indicated by some authors as corchiosides. The orcinol 1-O-β-D- Chen et al. 1999, Gupta et al. 2005). Structures of glucopyranosyl (1→6) glucopyranoside is called the benzyl benzoate are presented in Fig. 2 and

56 Table 5. Recently some authors reported the isolated and characterized (Xu and Xu 1992; Xu et isolation of two new benzyl benzoate glycosides al. 1992a,b). HO H O from in vitro cultures of the aerial parts of H Curculigo orchioides (Valls et al. 2006) but the two derivatives, called by these authors HO O O curculigoside C and D, have been previously H OH reported (Wu et al. 2005, Gupta et al. 2005). The OH H HO orchioside A (Gupta et al. 2005) is also called O curculigoside D (Valls et al. 2006). OH OH Fig. 4. Structure of orchioside B (Gupta et al. OH 2005).

H3CO O In addition Misra et al. (1990) reported the OH structure of curculigol. Among the cycloartane derivatives one cycloartane alcohol (curculigol, R= Fig. 5) (Misra et al. 1990) and several saponins OR have been isolated from the rootstock.

OCH3 O R = xylopyranosyl-glucopyranoside OH Fig. 3. Myricetin derivative isolated from C. orchioides (Tiwari and Misra 1976).

Tiwari and Misra (1976) reported the isolation of the 5,7-dimethoxy myricetin 3-O-α-L- xylopyranosyl 4-O-β-D-glucopyranoside but considering the published structure (Fig. 3) the HO aglycone seems to be the 2,3 dihydromiricetin (ampelopsin) with the two methoxy groups at the positions 5 and 7. Fig. 5. Structure of curculigol (Misra et al. 1990). Other phenolic derivatives are compounds characterized by the presence of two aromatic The saponins isolated from C. orchioides are rings linked by a five member chain containing characterized by three different aglycones, one is two ether linked to a sugar unit. Some derivatives the 3β, 11α, 16β, -trihydroxycycloartan-24-one. have been reported in other Curculigo species; for This is called curculigenin A (Fig. 6) and is C. orchioides the orchioside B was isolated by present in the curculigosaponins A-J (Table 6). Gupta et al. (2005) (Fig. 4). Two other aglycones called curculigenin B (Fig. 7) and C (Fig. 8, Table 8) are the (24S)-3β, 11α, 16β, Another important class of compounds isolated 24-tetrahydrocycloartane and 3β, 11α, 16β- from this plant belong to triterpenoids of the cyclo- trihydroxycycloartan-24 (25)-en, respectively. The oartane type. Fifteen derivatives have been curculigenin A and C based saponins are

57 characterized by the presence of glycosidic OH substituents at position 3 or 3 and 16 while the O HO

OH HO

OR2

R1O

R O 1 Fig. 7. Structure of curculigenin B. Fig. 6. Structure of curculigenin A. Description of R1 is given in the table Description of R1 and R2 are given in 7. the table 6.

Table 6. Sugar substituents in curculigosaponins A-J. Table 7. Sugar substituents in curculigo- Curculigo R1 R2 References saponin saponins K-L. For the compounds A β-D- H Xu et al. indicated with * there is no trivial glucopyranoside 1992a name published. B H α-L- Xu et al. 1992a Arabinopyranoside Curculigosaponin R1 References C β-D- α-L- Xu et al. glucopyranoside Arabinopyranoside 1992a K β-D-glucopyranosyl Xu and Xu D β-D- H Xu et al. (1→3)-β-D- 1992 glucopyranosyl 1992a glucopyranosyl (1→2)-β-D- glucopyranoside (1→2)-β-D- E β-D- α-L- Xu et al. glucopyranoside glucopyranosyl Arabinopyranoside 1992a (1→2)-β-D- L α-L- Xu and Xu glucopyranoside rhamnopyranosyl 1992 F β-D- H Xu et al. glucopyranosyl 1992a (1→2)-β-D- (1→3)-β-D- glucopyranoside glucopyranosyl (1→2)-β-D- * β-D-glucopyranosyl Chen et al. glucopyranoside (1→3)-β-D- 1999 G α-L- H Xu et al. glucopyranoside rhamnopyranosyl 1992b (1→2)-β-D- * α-L- Chen et al. glucopyranoside rhamnopyranosyl 1999 H α-L- α-L- Xu et al. rhamnopyranosyl Arabinopyranoside 1992b (1→2)-β-D- (1→2)-β-D- glucopyranoside glucopyranoside I β-D- H Xu et al. glucopyranosyl 1992b curculigenin B derivatives possess the glycosidic (1→3)- α-L- rhamnopyranosyl residue only at the position 3. Sugars linked to the (1→2)-β-D- position 3 are glucose and rhamnose while only glucopyranoside J β-D- α-L- Xu et al. the arabinopyranose is found at the position 16 glucopyranosyl Arabinopyranoside 1992b (Xu et al. 1992a,b, Xu and Xu 1992). Chen et al. (1→3)- α-L- rhamnopyranosyl (1999) isolated and characterized two more (1→2)-β-D- saponins based on the curculigenin B, and glucopyranoside

58 considered one derivative a new triterpenoid al. (2006) optimized solvent system, column compound (Table 7). temperature, flow rate and revolution speed in high-speed counter-current chromatography (HSCCC) to isolate and separate two glycosides HO (curculigoside and curculigoside B) from

OR2 rootstock of this plant. Thus HPLC and HSCCC methods can be used for quality control of herbal medicines containing rootstock of Curculigo orchioides as a constituent. R1O BIOACTIVITY OF PHYTOCHEMICALS Fig. 8. Structure of curculigenin C. Scientific basis of medicinal uses of Description of R1 and R2 are given in table 8. ethnomedicinal plants can be assessed by evaluating the biological activities of their phytoconstituents. Curculigo orchioides is Table 8. Sugar substituents in curculigo- hypoglycemic, spasmolytic and anticancer saponin M (Xu and Xu 1992). (Chandel et al. 1996). It showed antidiabetic R R 1 2 activity (hypoglycemic effect) in normal as well as β-D- α-L- alloxan-treated animals (Rahman and Zaman glucopyranosyl arabinopyranoside Curculigosaponin 1989). Alcoholic extracts of the rootstock showed M (1→3)-β-D- glucopyranosyl antioxidative (Tang et al. 2004, Wu et al. 2005), (1→2)-β-D- immunostimulatory (Lakshmi et al. 2003, Bafna glucopyranoside and Mishra 2006) and estrogenic (Vijayanarayana et al. 2007) activities. The rootstock did not show Another class of phytoconstituents isolated antiviral activity against influenza virus A and from Curculigo orchioides rootstock are aliphatic herpes simplex virus type 1 (Rajbhandari et al. derivatives. These includes 27-hydroxytriacontan- 2001). MAK and Kamilari, polyherbal medicines 6-one, the 23-hydroxytriacontan-2-one (Misra et with Curculigo orchioides as one of the al. 1984a), 21-hydroxytetracontan-10-one and 4- constituents, have been also tested for their methylheptadecanoic acid (Misra et al. 1984b). bioactivity. MAK showed neuronal differentiating (Rodella et al. 2004) and anticarcinogenic (Penza There are a few publications on the analytical et al. 2007) activities whereas the Kamilari methods for assessing extract quality. Yamasaki et showed antihepatotoxic activities (Rajesh and al. (1994) reported an HPLC method for the Latha 2004). measurement of the curculigosides contents in curculigins rhizome (rootstock). The authors Methanolic extract of rootstock showed weak proposed an indirect method measuring the inhibitory effects on biosynthesis of prostaglandin amount of 2,6-dimethoxybenzoic acid obtained by and production of nitric oxides (Hong et al. 2002). the hydrolysis of the curculigosides. Lu et al. The inhibitors of the prostaglandin synthesis and (2002) also used HPLC method to estimate nitric oxide production are considered as potential curculigoside content in the rootstock of Curculigo anti-inflamatory and cancer chemopreventive orchioides; they found 0.11 – 0.35% of agents. Phenolic compounds present in Curculigo curculigosides in the rootstock. Recently, Peng et

59 orchioides showed antioxidant activity which has and enhanced sexual performance in ethanol potential to be used for protection of biological extract treated rat support the traditional use of this molecules including DNA from oxidative damage plant as aphrodisiac. (Tang et al. 2004). Wu et al. (2005) evaluated Rodella et al. (2004) showed differentiation antioxidative activity, based on scavenging effect and proliferation activity of MAK-5 on PC12 on hydroxyl radicals and superoxide anion cells. This finding indicates the presence of radicals, of isolated phenols and phenolic neuronal differentiating agents in this drug and glycosides from rootstock of Curculigo potentiates its use in pharmacological therapy of orchioides. All eight compounds examined the neuro-degenerative disorders. showed scavenging effects on the above MAK supplemented diet inhibits liver mentioned radicals, thereby indicating the carcinogenesis in urethane-treated mice, possibly antioxidative activity. through prevention of excessive oxidative damage and the up regulation of connexin expression Phenolic glycosides (Orcinol-3-O-β-D- (Penza et al. 2007). Kamilari protects carbon glucoside, Orchioside A and purified glycoside tetrachloride-induced liver dysfunction in albino fraction) present in Curculigo orchioides rat (Rajesh and Latha 2004). However, neuronal stimulates immune response by acting on differentiating and anticarcinogenic agents of macrophages and lymphocytes (Lakshmi et al. MAK and antihepatotoxic agent of Kamilari have 2003). Methanol extract of its rootstock also not been isolated and characterized. showed immunostimulatory effect on mice and TRADE AND CONSERVATION STATUS holds potential as a protective agent against cytotoxic drugs (Bafna and Mishra 2006). Thus Trade related information of Curculigo the active compounds present in the methanol orchioides in Nepal is blurred. Tuberous root of a extract have the potentiality to be used as plant has been in trade from Nepal under the name complimentary therapeutic agents. Water soluble Musuli and the largest amount of this plant has fractions of the rootstock have systemic effect on been traded from Gorkha district. In a fiscal year bone histomorphology and formation as well as 2005/06 the Government of Nepal collected royalty (@ NRs 5.0/kg) for 47,512 kg of Musuli local bone healing of mice (Wong et al. 2007). (Department of Forest 2006). Nepal Gazette The aqueous extract increased the trabecular (2005) clearly mentioned Kalo Musuli as number by 10% and induced new bone formation Curculigo orchioides and proposed the royalty of on the margin of the defect. NRs 6/kg. One of the authors (BBS) collected Ethanolic extract of rootstock of Curculigo specimens of Musuli from a trader in Gorkha orchioides significantly increased percentage which was neither Curculigo orchioides nor the vaginal cornification, uterine wet weight, uterine Chlorophytum spp. The Musuli has been collected glycogen content and proliferative changes in from upper temperate regions (2500 – 3000 m asl) uterine endometrium in bilaterally ovariectomized of Gorkha district from wild (Deepak Pande, young albino rat, thus showing the estrogenic medicinal plant trader in Gorkha, per comm. July activity (Vijayanarayana et al. 2007). Ethanolic 22, 2005). Some people have considered the extract of rootstock increased sexual performance Musuli as Curculigo orchioides and this mistake of male rat as evident by increased mount has entered into the references (e.g. Amatya 2002). frequency, attractability towards female and penile In our study area (part of Syangja, Gorkha, erection (Chauhan et al. 2007). Estrogenic activity Kathmandu, Chitwan and Nawalparasi districts)

60 Curculigo orchioides has not been collected for by tissue culture (Suri et al. 2000). However there trade. However, Shukla (2001) listed this species has been no report of production of marketable as an over harvested medicinal plant from Nepal’s rootstock from these plantlets. Terai and according to Shrestha et al. (2004) it has In an experimental shading and spacing, dry moderate trade value in Dang-Deukhuri area, mid- matter production and yield of Curculigo west Nepal. orchioides were the highest at 25 percent shade In India, market demand is more than supply (Kala et al. 2006). In Bangladesh the market and 10 cm × 10 cm spacing due to improved plant demand was 10,000 kg/year (Hasanuzzaman height, number of leaves and chlorophyll content 2006). Poor seed set and germination of Curculigo (Joy et al. 2004). By transplanting individuals of orchioides associated with over-exploitation wild population to experimental plots, Joy et al. (Gupta and Chadha 1995) and extensive (2005) evaluated the yield and chemical denudation of forest floor due to cattle grazing and composition of rootstock in various nutrient litter collection (Jasrai and Wala 2001) are sources. Poultry manure at 2.7 t/ha (without responsible for its declining wild population. Thus, mulch) was found to be the best for optimum the species has been described as endangered yield, quality and soil health. The rootstock yield species in India (Suri et al. 1999, Jasrai and Wala was 407 kg/ha of dry rootstock with 59.11 % 2001, Prajapati et al. 2003). In Nepal it has been starch, 3.04 % crude fibre, 11.87 % crude protein, classified as vulnerable species with broad 1.71 % crude fat and 11.32 ppm curculigosides. distribution (Bhattarai et al. 2001) as well as Joy et al. (2005) had not considered economic threatened species (Manandhar 2002). Although aspect of cultivation but this is very important for the species has been included in different threat commercial production. categories, there is no legal provision, both in India and Nepal, to monitor wild collection and CONCLUSIONS trade. In India a small population has been maintained in some of the herbal gardens as an Despite long list of references on Curculigo effort to conserve this plant (Maiti 2006). orchioides, which mostly focused on CULTIVATION phytochemistry, ethnomedicine and bioactivity, there is no data on seed germination, habitat Cultivation is the best option for the requirements, demography (population dynamics), conservation of declining wild population of resource estimation, etc. Poor seed set and habitat medicinal plants. Cultivation ensures both quality control and sustainable supply of raw materials to degradation have been considered as the causes of commercial sectors. Since the supply of rootstock population decline in wild, but there is no of Curculigo orchioides is less than the market empirical data available for this plant from across demand in India (Kala et al. 2006), there is good the habitats. Market supply of this plant mainly opportunity for cultivation at commercial scale. comes from wild collection but impacts of harvest There has been no report of cultivation of this on population dynamics and yield have not been plant but about 100 ha land was under cultivation assessed. in Kerala, Tamil Nadu, Madhya Pradesh and Karnataka states of India (Maiti 2006). However, For conservation perspective future research is development of agrotechnology for cultivation of intended in the areas of demography, population Curculigo orchioides is at infancy. Methods have status, resource estimation, phenology, pollination been developed for mass production of plantlets and germination ecology, response to harvest in

61 wild, and agrotechnology development for Baral, S.R. and P.P. Kurmi. 2006. A Compendium commercial cultivation. For exploitation of of Medicinal Plants of Nepal. Mrs Rachana medicinal values, secondary metabolites should be Sharma, Kathmandu, Nepal. 534 pp. quantitatively estimated and their bioactivity should be assessed following the lead provided by Behera, S.K. and M.K. Misra. 2006. Aboveground its ethnomedicinal uses. tree biomass in a recovering tropical sal (Shorea robusta Gaertn. f.) forest in Eastern ACKNOWLEDGEMENTS Ghats, India. Biomass and Bioenergy. 30:509- We are thankful to National Herbarium and 521. Plant Laboratory, Godawari, Lalitpur for Bhat, D.M. and K.S. Murali. 2001. Phenology of permission to consult herbarium specimens, and understorey species of tropical moist forest of Uttam B Shrestha for sending some old references. Western Ghats region of Uttara Kannada district in South India. Current Science. REFERENCES 81:799-805.

Adhikari, M.K., D.M. Shakya, M. Kayastha, S.R. Bhattarai, K.R. and M.D. Ghimire. 2006. Baral and M.N. Subedi (eds.). 2007. Medicinal Cultivation and Sustainable Harvesting of Plants of Nepal [Revised]. Bulletin No. 28. Commercially Important Medicinal and Department of Plant Resources, Kathmandu, Aromatic Plants of Nepal [in Nepali]. Heritage Nepal. 402 pp. Research and Development Forum, Amatya, K.R. 2002. Utilization of Himalayan Kathmandu, Nepal. 394 pp. medicinal plant resources: status, problems and Bhattarai, N., V. Tandon and D.K. Ved. 2001. prospects. In: The Himalayan Plants, Can they Highlights and outcomes of the Conservation Save Us? Proceedings of Nepal-Japan Joint Assessment and Management Planning Symposium on Conservation and Utilization of (CAMP) workshop, Pokhara, Nepal. In: Himalayan Medicinal Resources. (eds.) Sharing Local and National Experiences in Watanabe, T., A. Tanako, M.S. Bista and H.R. Conservation of Medicinal and Aromatic Saiju. Society for the Conservation and Development of Himalayan Medicinal Plants in South Asia. Proceedings of Regional Resources, Japan. pp. 331-352. Workshop at Pokhara, Nepal. (eds.) Bhattarai, N. and M. Karki. HMG/N, IDRC and MAPPA. Bafna, A.R. and S.H. Mishra. 2006. pp. 46-53. Immunostimulatory effects of methanol extract of Curculigo orchioides on immunosuppressed BPP (Biodiversity Profile Project). 1995. mice. Journal of Ethnopharmacology 104:1-4. Biodiversity Assessment of Terai Wetlands. Biodiversity Profile Project Technical Bajpai, H.R. and M. Mishra. 1998. Indigenous Publication No. 1, Department of National medical practices of Hills Korwas of Madhya Park and Wildlife Conservation, Ministry of Pradesh. Journal of Human Ecology. 9:295. Forest and Soil Conservation, Kathmandu.

62 Plants. Vol 11. (eds.) Chadha, K.L. and R. Chandel, K.P.S., G. Shukla and N. Sharma. 1996. Gupta. Malhotra House, New Delhi. pp. 1-43. Biodiversity in Medicinal and Aromatic Plants in India: Conservation and Utilization. Hasanuzzaman, S.M. 2006. Medicinal and National Bureau of Plant Genetic Resources, Aromatic Plants of Bangladesh. In: Guide on Pusa Campus, New Delhi. 239 pp. Medicinal and Aromatic Plants of SAARC Countries. SAARC Agricultural Information Chauhan, N.S., C.V. Rao and V.K. Dixit. 2007. Center, Dhaka, Bangladesh. pp. 3-158. Effect of Curculigo orchioides rhizome on Hong, C.H., S.K. Hur, O. Oh, S.K. Kim, K.A. sexual behavior of male rats. Fitoterapia. Nam and S.K. Lee. 2002. Evaluation of natural 78:530-534. products on inhibition of inducible Chen, C., W. Ni and W. Mei. 1999. The cyclooxygenase (COX-2) and nitric oxide Glycosides from Curculigo orchioides. Acta synthase (iNOS) in cultured mouse Botanica Yunnanica. 21:521-524. macrophage cells. Journal of Chendurpandy, P. 2006. Studies on Ethnopharmacology. 83:153-159. ethnomedicinal plants of Kanyakumari district, Jain, A., S.S. Katewa, B.L. Chaudhary and P. Tamilnadu with special reference to skin Galav. 2004. Folk herbal medicines used in diseases. Ph.D. dissertation, Manonmanian birth control and sexual diseases by tribal of Sundaranar University, Tirunelveli, India. 123 southern Rajasthan, India. Journal of pp. Ethnopharmacology. 90:171-177. Chopra, R.N., S.L. Nayar and I.C. Chopra. 1956. Jasrai, Y.T. and B. Wala. 2001. Curculigo Glossary of Indian Medicinal Plants. Council orchioides Gaertn (Kali musli): an endangered for Scientific and Industrial Research (CSIR), medicinal herb. In: Role of Biotechnology in New Delhi, India. Medicinal and Aromatic Plants. Vol. IV. (eds.) DOF. 2006. Hamro Ban (Our Forest) [in Nepali]. Khan, I.A. and A. Khanum. Ukaaz Publication, Department of Forest, Ministry of Forest and Hyderabad (India). pp. 89-95. Soil Conservation, Kathmandu, Nepal. Joshi, K.K. and S.D. Joshi. 2001. Genetic Heritage Garg, S.N., L.N. Misra and S.K. Agarwal. 1989. of Medicinal and Aromatic Plants of Nepal Corchioside A: an orcinol glycoside from Himalayas. Buddha Academic Publishers Curculigo orchioides. Phytochemistry. and Distributors Pvt. Ltd., Kathmandu, Nepal. 28:1771-1772. 239 pp.

Gupta, M., B. Achari and B.C. Pal. 2005. Joy, P.P., K.E. Savithri, S. Mathew and B.P. Glucosides from Curculigo orchioides. Skaria. 2004. Effect of shade and spacing on Phytochemistry. 66:659-663. growth, yield and quality of black musli (Curculigo orchioides). Journal of Gupta, R. and K.L. Chadha. 1995. Medicinal and Medicinal and Aromatic Plant Sciences. 26: aromatic plant research in India. In: Advances 707-716. in Horticulture, Medicinal and Aromatic

63 Local Initiatives Support Program (LISP), Joy, P.P., K.E. Savithri, S. Mathew, J. Thomas and Tansen, Palpa, Nepal. 196 pp. C.T. Abraham. 2005. Effect of mulch and sources of nutrients on growth, yield and Lu, H.W., B.H. Zhu and Y.K. Liang. 2002. quality of black musli (Curculigo orchioides). Determination of curculigoside in crude Journal of Medicinal and Aromatic Plant medicine Curculigo orchioides by HPLC Sciences. 27:646-656. (article in Chinese). China Journal of Chinese Materia Medica. 27:192-194. Kala, C.P., P.P. Dhyani and B.S. Sajwan. 2006. Developing the medicinal plant sector in Maiti, S. 2006. Medicinal and aromatic plants of northern India:challenges and opportunities. India. In: Guide on Medicinal and Aromatic Journal of Ethnobiology and Ethnomedicine. Plants of SAARC Countries. SAARC 2:32 doi: 10.1186/ 1746-4269-2-32 Agricultural Information Center, Dhaka, http://www.ethnobiomed.com/content/2/1/32. Bangladesh. pp. 229-320. Kandel, D.R. 2007. Vegetation structure and Malla, S.B., S.B. Rajbhandari, T.B. Shrestha, P.M. regeneration of sal (Shorea robusta Gaertn. f.) Adhikari, S.R. Adhikari and P.R. Shakya in community managed forests of inner Terai, (eds.). 1986. Flora of Kathmandu Valley. central Nepal. M.Sc. dissertation, Central Bulletin No. 11. Department of Medicinal Department of Botany, Tribhuvan University, Plants, Thapathali, Kathmandu. 962 pp. Kirtipur, Kathmandu. Manandhar, N.P. 1985. Ethnomedicinal notes on Kaini, B.R. 2006. Medicinal and Aromatic Plants certain medicinal plants used by Tharus of of Nepal. In: Guide on Medicinal and Dang-Deukhuri District, Nepal. Journal of Aromatic Plants of SAARC Countries. SAARC Crude Drug Research. 23 (4): 153-159. Agricultural Information Center, Dhaka, Bangladesh. pp. 321-414. Manandhar, N.P. 1991. Medicinal plant-lore of Tamang tribe of Kabhrepalanchok district, Kirtikar, K.R. and B.D. Basu. 1994. Indian Nepal. Economic Botany. 45:58-71. Medicinal Plants. Vol IV. 4th reprint edition. Lalit Mohan Basu, Allahabad. pp. 2395-2761. Manandhar, N.P. 2002. Plants and People of Nepal. Timber Press Inc. Portland, Oregon, Kubo, M., K. Namba, N. Nagamoto, T. Nagao, J. USA. Nakanishi, H. Uno and H. Nishimura. 1983. A new phenolic glucoside, curculigoside from Misra, T.N., R.S. Singh and D.M. Tripathi. 1984a. rhizomes of Curculigo orchioides. Planta Aliphatic compounds from Curculigo Medica. 47:52-55. orchioides rhizomes. Phytochemistry. 23:2369- 2371. Lakshmi, V., K. Pandey, A. Puri, R.P. Saxena and K.C. Saxena. 2003. Immunostimulant Misra, T.N., R.S. Singh, J. Upadhyay and D.M. principles from Curculigo orchioides. Journal Tripathi. 1984b. Aliphatic hydroxy-ketones of Ethnopharmacology. 89:181-184. from Curculigo orchioides rhizomes. Phytochemistry. 23:1643-1645. LISP. 2004. Bikas ka Pailaharoo – 6: Medicinal Plants Promotion Special Issue (In Nepali). 64 diet inhibits liver carcinogenesis in mice. BMC Misra, T.N., R.S. Singh, D.M. Tripathi and S.C. Complementary and Alternative Medicine. Sharma. 1990. Curculigol, a cycloartane 7:19. doi: 10.1186/1472-6882-7-19. triterpene alcohol from Curculigo orchioides. http://www.biomedcentral.com/1472- Phytochemistry. 29:929-931. 6882/7/19

Murata, G. 1983. A list of plants collected by the Prajapati, H.A., D.H. Patel, S.R. Mehta and R.B. Chiba University Rolwaling Himal Expedition Subramanian. 2003. Direct in vitro 1963. In: Biota and Ecology of Eastern Nepal, regeneration of Curculigo orchioides Gaertn., (ed.) Numata, M. Himalayan Committee of an endangered anti-carcinogenic herb. Current Chiba University, Japan. pp. 160-167. Science. 84:747-749.

Nepal Gazette. 2005 (2062, Nepali calendar). Press, J.R., K.K. Shrestha and D.A. Sutton. 2000. Released by then His Majesty Government of Annotated Checklist of the Flowering Plants of Nepal on September 2005. Nepal. The Natural History Museum, London, Noltie, H.J. 1994. Flora of Bhutan. Vol. 3, part 1. UK and Central Department of Botany, Royal Botanical Garden, Edinburgh, UK. 456 Tribhuvan University. 430 pp. pp. Quattrocchi, U. 2000. CRC World Dictionary of Panthi, M.P. and R.P. Chaudhary. 2003. Plant Names. Vol. 1 (A-C). CRC Press, Boca Ethnomedicinal plant resources of Raton, Florida, USA. 714 pp. Arghakhanchi district, west Nepal. Ethnobotany. 15:71-86. Rahman, A.U. and K. Zaman. 1989. Medicinal plants with hypoglycemic activity. Journal of Parrotta, J.A. 2001. Healing Plants of Peninsular Ethnopharmacology. 26: 1-55. India. CABI Publishing, Oxon (UK) and New York (USA). 917 pp. Rajbhandari, M., U. Wegner, M. Julich, T. Schopke and R. Mentel. 2001. Screening of Pattanaik, C., C.S. Reddy and M.S.R. Murthy. Nepalese medicinal plants for antiviral activity. 2008. An ethnobotanical survey of medicinal Journal of Ethnopharmacology. 74:251-255. plants used by the Didayi tribe of Malkangiri district of Orissa, India. Fitoterapia. 79:67-71. Rajbhandari, T.K., N.R. Joshi, T. Shrestha, S.K.G. Joshi and B. Acharya (eds.). 1995. Medicinal Peng, J., Y. Jiang, G. Fan, B. Chen, Q. Zhang, Y. Plants of Nepal for Ayurvedic Drugs. Natural Chai and Y. Wu. 2006. Optimization suitable Products Development Division, Department condition for preparative isolation and of Plant Resources, Thapathali, Kathmandu, separation of curculigoside and curculigoside Nepal. 387 pp. B from Curculigo orchioides by high-speed counter-current chromatography. Separation Rajbhandary, K.R. 2001. Ethnobtany of Nepal. and Purification Technology. 52:22-28. Ethnobotanical Society of Nepal, Kathmandu. 189 pp. Penza, M., C. Montani, M. Jeremic, G. Mazzoleni, W.L.W. Hsiao, M. Marra, H. Sharma and D.D. Rajesh, M.G. and M.S. Latha. 2004. Preliminary Lorenzo. 2007. MAK-4 and -5 supplemented evaluation of the antihepatotoxic activity of 65 Kamilari, a polyherbal formulation. Journal of National Workshop at Kathmandu. Ethnopharmacology. 91:99-104. IDRC/MAPPA. pp. 190-213.

Ramawat, K.G., S. Jain, S. Suri and D.K. Arora. Shrestha, P.M. and S.S. Dhillion. 2003. Medicinal 1998. Aphrodisiac plants of Aravalli hills with plant diversity and use in the highlands of special reference to safed musali. In: Role of Dolakha district, Nepal. Journal of Biotechnology in Aromatic and Medicinal Ethnopharmacology. 86:81-96. Plants. Vol. 1. (eds.) Khan, I.A. and A. Shrestha, U.B. and S. Shrestha. 2004. Major Non- Khanum. Ukaaz Publications, Hyderabad Timber Forest Products of Nepal (in Nepali). (India). pp. 210-223. Bhundipuran Prakashan, Kathmandu, Nepal. Rao, P.V.K., N. Ali and M.N. Reddy. 1978. 441 pp. Occurrence of both sapogenins and alkaloid Shukla, R.N. 2001. Conservation and lycorine in Curculigo orchioides. Indian commercialization of medicinal plants of the Journal of Pharmaceutical Sciences. 40:104- Terai region, Nepal (Abstract). In: Sharing 105. Local and National Experiences in Rodella, L., E. Borsani, R. Rezzani, R. Lanzi, C. Conservation of Medicinal and Aromatic Lonati and R. Bianchi. 2004. MAK-5 treatment Plants in South Asia. (eds.) Bhattarai, N. and enhances the nerve growth factor-mediated M. Karki. Proceedings of regional workshop at neurite outgrowth in PC12 cells. Journal of Pokhara, Nepal. HMG/N, IDRC and MAPPA. Ethnopharmacology. 93:161-166. pp. 207-208.

Sapkota, C.R. and S.M. Adhikari. 2001. Ayurvedic Singh, A.K., A.S. Raghubansi and J.S. Singh. Farmacology (Bheshaja Guna-Vijnana). 2002. Medicinal ethnobotany of tribal of Singhadurbar Vaidyakhana Vikas Samiti, Sonaghati of Sonbhadra district, Uttar Pradesh, Kathmandu, Nepal. 154 pp. India. Journal of Ethnopharmacology. 81:31- 41. Shah, G.L. 1978. Flora of Gujarat State – III. Sardar Patel University Press, Vallab, Suri, S.S., D.K. Arora and K.G. Ramawat. 2000. A Vidyanagar, India. method for large scale multiplication of Curculigo orchioides through bulbil formation Shah, G.L. and B.V. Gopal. 1970. Structure and from leaf explant in shake flask culture. Indian development of stomata on the vegetative and Journal of Experimental Biology. 38:145-148. floral organs of some Amaryllidaceae. Annals of Botany. 34: 737-749. Suri, S.S., S. Jain and K.G. Ramawat. 1999. Plantlet regeneration and bulbil formation in Shrestha, K.K., S. Rajbhandari and N.N. Tiwari. vitro from leaf and stem explant of Curculigo 2004. Non timber forest products and orchioides, an endangered medicinal plant. community development in Dang-Deukhuri, Scientia Horticulturae. 79:127-134. mid west Nepal. In: Local Experience –based National Strategy for Organic Production and Tandon, M. and Y.N. Shukla. 1995. Management of MAPs/NTFPs in Nepal. (eds.) Phytoconstituents of Asparagus adscendens, Bhattarai, N. and M. Karki. Proceedings of the Chlorophytum arundinaceum and Curculigo 66 orchioides: A review. Current Research in curculiginis and Rhizoma drynariae extracts on Aromatic and Medicinal Plants. 17:42-50. bone. Chinese Medicine. 2:13. doi:10.1186/1749-8546-2-13 Tang, S.Y., M. Whiteman, J.F. Peng, A. Jenner, (http://www.cmjournal.org/content/2/1/13). E.L. Yong and B. Halliwell. 2004. Characterization of antioxidant and Wu, Q., D.X. Fu, A.J. Hou, G.Q. Lei, Z.J. Liu, antiglycation properties and isolation of active J.K. Chen and T.S. Zhou. 2005. Antioxidative ingredients from traditional Chinese medicines. phenols and phenolic glycosides from Free Radical Biology and Medicine. 36:1575- Curculigo orchioides. Chem. Pharm. Bulletin. 1587. 53:1065-1067.

Tiwari, R.D. and G. Misra. 1976. Structural Xu, J. and R. Xu. 1992. Cycloartane-type studies of the constituents of the rhizomes of sapogenins and their glycosides from Curculigo orchioides. Planta Medica. 29:291- Curculigo orchioides. Phytochemistry. 294. 31:2455-2458. Valls, J., T. Richard, F. Larronde, V. Leblais, B. Xu, J., R. Xu and X. Li. 1992a. Glycosides of a Muller, J.C. Delaunay, J.P. Monti, K.G. cycloartane sapogenin from Curculigo Ramawat and J.M. Merrilon. 2006. Two new orchioides. Phytochemistry. 31:233-236. benzylbenzoate glucosides from Curculigo orchioides. Fitoterapia. 77:416-419. Xu, J., R. Xu and X. Li. 1992b. Four new cycloartane saponins from Curculigo Vijayanarayana, K., R.S. Rodrigues, K.S. orchioides. Planta Medica. 58:208-210. Chandrashekhar and E.V.S. Subrahmanyam. 2007. Evaluation of estrogenic activity of Yamasaki, K., A. Hashimoto, Y. Kokusenya, T. alcoholic extract of rhizome of Curculigo Miyamoto, M. Matsuo and T. Sato. 1994. orchioides. Journal of Ethnopharmacology. Determination of Curculigoside in curculigin 114:241-245. rhizome by High Performance Liquid Chromatography. Chem. Pharm. Bull. 42:395- Wong, R.W.K., B. Rabie, M. Bendeus and U. 397. Hagg. 2007. The effect of Rhizoma