Indian Journal of Natural Products and Resources Vol. 3(3), September 2012, pp. 371-385

Pharmacognostical studies on the root and rhizome of hydrophylla (Linn.) O. Kuntze –An alternate source for Tagara drug

V Madhavan1, M Jayashree1, S N Yoganarasimhan1*, M Gurudeva2, R Deveswaran3 and R Mythreyi1 1Department of Pharmacognosy, MS Ramaiah College of Pharmacy, Bangalore-560 054, India 2Department of Botany, VV Pura College of Science, Bangalore 3Department of Pharmaceutics, MS Ramaiah College of Pharmacy, Bangalore Received 13 October 2011; Accepted 19 January 2012 Tagara is an important drug used in Ayurvedic medicine for the treatment of several diseases. The accepted botanical source of Tagara is Valeriana jatamasni Jones, although different species of Nymphoides Hill are used by the physicians. The pharmacognostical evaluation of the root and rhizome of , a potential alternative source for Tagara is presented in this paper. Important details like morphology of the , macro-, microscopical characters, macerate, histochemical tests, UV studies of the root and rhizome along with physico-chemical constants, phytochemical analysis and HPTLC finger print profile are presented, all of which will be useful in the standardization of this drug. Isolation of β-sitosterol, betulinic, salicylic and tannic acids are reported for the first time from N. hydrophylla. The pharmacognostical and phytochemical studies help in the identification of N. hydrophylla from other species used as Tagara.

Keywords: Nymphoides hydrophylla, Pharmacognosy, Root, Rhizome, HPTLC, Tagara. IPC code; Int. cl. (2011.01)—A61K 36/00

Introduction (Lour.) O. Kuntze, N. indica (Linn.) O. Kuntze and Tagara is an important drug used in Ayurvedic N. macrosperumum Vasudevan3. N. indica is used as medicine for the treatment of diseases like anaemia an antiscorbutic and febrifuge, and as a substitute for (pandu), epilepsy (apasmara), fever (jwara), jaundice chiretta [Swertia chirayita (Roxb. ex Flem.) Kars.] to (kamala), mental disorders (unmada), tuberculosis treat fever and jaundice4. Literature review revealed (yakshma) and also as a general and brain tonic1. that while pharmacognostical studies have been carried Valeriana jatamansi Jones (Valerianaceae) is the out on the roots and rhizome of N. macrospermum5 and accepted botanical source of Tagara which constitutes N. indica6, no work is available on N. hydrophylla7-9. as one of the ingredients in ayurvedic formulations like Hence, the present investigation on the root and Bala taila, Bilvadi gutika, Dasanga lepa, Jatiphaladya rhizome of N. hydrophylla was undertaken. curna, Karpuradyarka, Maha kalyanaka ghrita, Maha Materials and Methods narayana taila, Puga khanda, Sahacaradi taila, 2 Plant material was collected from a water tank in the Srikhandasava, to name a few . In South India, a drug vicinity of Nagercoil, Kanyakumari district, Tamil under the name Granthika tagara (Kannada), Nadu, during November 2007, processed into botanically identified as Nymphoides macrospermum herbarium specimen10; voucher herbarium specimen Vasudevan () is used in place of (Jayashree 025) and a sample of the crude drug are Tagara (Valeriana jatamansi) for the same ayurvedic preserved at the herbarium and crude drug museum of preparations, under the same formulations supporting the Department of Pharmacognosy, M S Ramaiah the fact that Granthika tagara (N. macrospermum) may College of Pharmacy, Bangalore (MSRCP). The plant have similar therapeutic properties to that of Tagara material was identified following local floras11-13 and (V. jatamansi); furthermore, it is often found that authenticated by S N Yoganarasimhan, Plant Granthika tagara is an admixture of different species Taxonomist. For pharmcognostical studies, a small of Nymphoides Hill found in S. India, viz. quantity of rhizome and root were preserved in 70% N. aurantiacum (Dalz.) O. Kuntze, N. hydrophylla alcohol; free hand sections were taken for macro- and ______microscopical observations14,15. Photomicrographs *Correspondent author: E-mail: [email protected]; [email protected] were obtained by observing the free hand sections Phone: +91-80-23608942 under a compound binocular microscope (Olympus- 372 INDIAN J NAT PROD RESOUR, SEPTEMBER 2012

CH20i model) with a built-in analogue camera Gen. and Spec. Gent. 342. 1839; Clarke in Hook. f., Fl. (CMOF, 1.4 mega pixel). Computer Images were Brit. India, 4: 131. 1883; Gamble, Fl. Pres. Madras, 2: captured using AV Digitaliser fitted with a Grand VCD 883, 2005 (reprint); Menyanthes hydrophylla Lour., Fl. 2000–Capture Guard. Measurements of cells and Cochinch. 1:129, 1790. tissues (in µm) of the transverse section and macerate Aquatic herbs with long floating stem (stolon). were carried out using Micro Image Lite Image Rhizome short, erect, with petiole-like branches Analysis Software (Cybernetics, Maryland, USA) and reaching the surface of water and producing a node are provided in parenthesis. Physicochemical studies, from which arise a tuft of adventitious roots, a cluster ultra-violet and chromatographic studies were carried of , a single floating and a single branch, out following standard methods16-20. Vernacular names which again proceeds similarly. orbicular, are provided following Gurudeva21. Isolation of deeply cordate at base, often purplish, to 10 cm in β-sitosterol was carried out as per Gerhardt et al22. The diameter, lobes rounded, faintly crenate, pale green total phenolic compounds were determined following above, glandless and with prominent nerves beneath; Folin-Ciocalteace method23 and HPTLC was carried petioles to 3.5 cm long. Flowers white, with yellow out using a Camag HPTLC system with Linomat V centre, delicate, with a ring of white hairs round the sample applicator, a Camag 3 TLC Scanner and throat, numerous, in dense clusters; pedicels to 6 cm WINCATS 4 software for interpretation of data. An long, unequal. Calyx divided almost to the base, aluminum plate (20 × 10 cm) pre-coated with silica gel oblong-lanceolate, obtuse. Corolla white, when 60F254 (E Merck) was used as adsorbent. The plates expanded; lobes obovate, rounded at apex, glabrous, were developed using toluene:chloroform:ethanol with a broad longitudinal crest down the middle of (28.5:57:14.5) for alcohol and aqueous extracts in a each lobe, margins not ciliate. broadly ovoid Camag twin trough chamber and scanned under 254, or subglobose. Seeds numerous with prominent, small, 366 and 425 nm. The developed plate was derivatised slightly glochidiate tubercles, scabrous, pale yellowish- by dipping in Libermann Burchard’s reagent and brown (Plates 1, 2). drying at 110°C in a hot air oven for 10 min, scanned Nymphoides hydrophylla usually flowers and fruits in under all 3 wavelengths and photo documented both the winter season. In this species polystely occurs in the before and after derivatisation using a Camag 3 cortical region of the stolon and in the petiole of the Reprostar. For the identification of phenolic leaves. It is very common throughout India and is compounds, 10 µl of tannic acid solution and 5 µl of gregarious in habit, generally growing near the margins salicylic acid solution were applied and developed with of tanks and lakes. Also found in Sri Lanka, China and ethyl acetate: benzene: formic acid (9:11:0.5) and Malaysia26. scanned under 254, 366 and 425 nm. The developed Vernacular names plate was photo documented at 254 nm. For the Tamil: Akacavalli; Sanskrit: Kumudvathi, Seethali, identification of betulinic acid, both extracts were Kalanu saariva, Tagara; Kannada: Anthara thaavare; developed with n-hexane: ethyl acetate: glacial acetic Marathi: Kolare chikal; Hindi: Tagarmul, Cumuda, acid (7:3:0.03), derivatised with Anisaldehyde Ghainchu, Kumudini, Lodh, Baank; Telugu: Anthara (Ref.24) H2SO4 , scanned at 425 nm and photodocumented. thaarama, Chiri alli, Pitta kaluva; Bengali: Panchuli, For the identification of β-sitosterol, toluene:diethyl Chandmalla; Malayalam: Tsjeroea–citamber; ether (40:40) was used, derivatised with Vanillin- Manipuri: Tharo–macha. (Ref. 25) H SO dried at 105°C , scanned at 425 nm and 2 4, Macro- and microscopical characters of the root and rhizome photo documented. The Rf values and peak area of the Macroscopical characters of the root extracts and the isolated sample were interpreted by Roots arise in tufts all around the rhizome, running using a WINCATS 4 software. The developed plates vertically downwards, very slender, spongy, brownish, were photo documented under 425 nm using a Camag measuring to 32 cm long when fresh, and has no 3 Reprostar. All the solvents used were of HPLC grade. characteristic odour or taste. Young fresh roots are minutely hairy and white to brownish while dry roots Results are slender, brownish (Plates 3, 4). Plant morphology Nymphoides hydrophylla (Linn.) O. Kuntze, Rev. Macroscopical characters of the rhizome Gen. Pl. 429. 1891. Menyanthes cristata Roxb. Pl. Cor. The rhizomes are slender, running horizontally, 2: 3. t. 105, 1798; Limnanthemum cristatum Griseb. cone-like with many leaf bases, rough, brittle, brownish MADHAVAN et al: PHARMACOGNOSY OF NYMPHOIDES HYDROPHYLLA 373

without, white within, with several fragments of roots Microscopical characters of the root arising from different points, measuring to 9 cm long; A transverse section of the root is wheel-like and it possesses no characteristic odour or taste; dry exhibits an epidermis, an outer, middle and inner rhizomes are blackish with a few slender roots and cortex, a stele region with 7 vascular strands (Plate 8). covered by several leaf bases (Plates 5, 6); when Epidermis is single layered, made up of compactly transversely cut, it shows a broad whitish cortex and a arranged rectangular cells, measuring 14-18-21 × 16- narrow creamy-white central stele (Plate 7). 21-30 µm; a few root hairs arise from the epidermal

Plates 1-14Macro and microscopical characters of Nymphoides hydrophylla root and rhizomes —1. Habit , 2. , 3. Vertical roots, 4. Young roots , 5. Dried roots, 6. Dried rhizome, 7. Rhizome cut open, 8. T.S. showing different regions, 9. Portion showing epidermis and outer cortex, 10. Cortex with intercellular spaces, 11. Cortex showing plug-like structures, 12. Portion showing middle cortex and aerenchyma, 13. Portion showing inner cortex and stele, 14. Stele enlarged showing pith. AC-air chamber; AER-aerenchyma; CAM-cambium; COR-cortex; CT-conjunctive tissue; END-endodermis; EP-epidermis; HY-hypodermis; IC-inner cortex; ICS-intercellular space; MC-middle cortex; MXY-metaxylem; OC-outer cortex; PC-pericycle; PH-phloem; PI-pith; PLS-plug-like structure; PXY-protoxylem; RH-root hair; SCD- sclereid; SG-starch grain; ST-stele; VB-vascular bundle; VT-vascular trace; XY-xylem 374 INDIAN J NAT PROD RESOUR, SEPTEMBER 2012

Plates 15-18Macerate of root— 15, 16. Parenchyma cells, 17, 18. Vessels with reticulate and spiral thickenings.

cells. Next to the epidermis is the outer cortex which represent a pseudo polystele condition. Stele consists is 5 to 6 layered, consisting of compactly arranged of concentrically arranged vascular bundles, with parenchymatous cells with intercellular spaces, conjunctive tissue found in between. Vascular bundles measuring 26-49-65 × 23-36-48 µm (Plates 9, 10), are conjoint, collateral, open, endarch; metaxylem followed by a broad zone of middle cortex comprising cells measure 166-242-323 × 68-98-124 µm; of vertically arranged layers of parenchymatous cells, cambium is multilayered, cells measuring 5-10-20 × traversed by large air cavities comprising the 3-8-15 µm (Plates 24-26). Pith is large, aerenchyma, measuring 20-32-51 × 16-32-46 µm parenchymatous, cells measuring 20-27-41 × 10-15- (Plate 11); the aerenchyma cells in young roots are 18 µm (Plate 27). Starch grains are found in the interconnected by plug-like structures (Plate 12). Next cortex and pith; they are simple, solitary or to the aerenchyma lies 3-4-layered inner cortex aggregated, oval or rounded (Plates 28, 29). consisting of compactly arranged oval shaped Macerate of the rhizome exhibited: i. Epidermal parenchymatous cells measuring 27-45-66 × 17-34-33 cells which are compactly arranged, measuring 13-21- µm (Plate 13). Endodermis cells measure 16-24-33 × 28 × 10-15-28 µm (Plate 30); ii. Parenchyma cells 16-15-25 µm and pericycle cells measure 5-7-8 × 9- which are thin walled, measuring 23-33-51 × 16-22- 11-14 µm, single layered which is followed by the 24 µm (Plate 31); iii. Fibers which are long and with stele. Vascular bundles are radially arranged; xylem is pointed ends, having a broad lumen, measuring 331 × exarch, cells measure 5-10-15 × 10-14-18 µm, with a 9 µm (Plate 32); iv. Sclereids of different sizes and central parenchymatous pith, cells measuring 6-8-11 × shapes- U-, Y-, Z-, and H-shaped, some are with thick 7-11-16 µm (Plate 14). Starch is absent in pith cells. walled arms, some with fragmented arms, measure Macerate of the root exhibited: 1. Parenchyma cells 55-106-121 × 9.5-12.5-16.6 µm (Plates 33 to 39); v. which are oval and compactly arranged, measuring Tracheids which are long, narrow and with pitted 40-63-80 × 301-38-50 µm (Plates 15, 16). 2. Vessels thickenings, measure 505 × 20 µm (Plate 40); vi. with reticulate and spiral thickenings, measuring Vessels of different size and shape, linear or drum 84-105-122 × 5-9-12 µm) (Plates 17, 18). shaped, with reticulate and spiral thickenings, measure 30-72-133 × 13-16-24 µm (Plates 41 to 43). Microscopical characters of rhizome Transverse section of rhizome exhibits epidermis Histochemical tests which is one layered, made up of narrow, tangentially The sections of root and rhizome when treated with elongated parenchymatous cells, measuring 15-20-25 phloroglucinol and HCl turned pink indicating the × 21-28-40 µm. It is followed by 4-5-layered presence of lignin; with ferric chloride turned black, hypodermis, made up of compactly arranged indicating presence of tannin; with iodine solution, parenchymatous cells, measuring 20-29-38 × 29-35- rhizome sections turned blue indicating presence of 42 µm. Next to the hypodermis lies a broad starch whereas root sections did not turn blue, aerenchymatous cortex, cells measuring 14-21-33 × indicating absence of starch. 10-15-23 µm and large air cavities, measuring 13-20- 31×18-24-29 µm (Plates 19-21). Sclereids are present Powder studies in the cortex, protruding into the air cavities (Plate The powder is greenish-brown, possesses no 22). The innermost few layers of the cortex consists characteristic odour and taste. When treated with of compactly arranged cells, without air cavities; a phloroglucinol and HCl, stained with safranin, few root traces are found in the cortex (Plate 23) following elements were observed as: i. Fragments of giving the appearance of individual stele which epidermal peel showing compactly arranged cells MADHAVAN et al: PHARMACOGNOSY OF NYMPHOIDES HYDROPHYLLA 375

Plates 19 to 29Microscopical characters of rhizome —19. Portion showing epidermis, hypodermis and cortex, 20. Cortex showing aerenchyma and air cavity, 21. Cortex showing intercellular spaces, 22. Portion showing sclereid in cortical cells, 23. Portion showing vascular trace and inner cortex, 24, 25. Portion showing stele, 26. Portion showing phloem and vascular cambium, 27. Pith region, 28. Starch grains in cortical cells, 29. Starch grains in pith cells. AC-air chamber; AER-aerenchyma; CAM-cambium; COR-cortex; CT- conjunctive tissue; END-endodermis; EP-epidermis; HY-hypodermis; IC-inner cortex; ICS-intercellular space; MC-middle cortex; MXY- metaxylem; OC-outer cortex; PC-pericycle; PH-phloem; PI-pith; PLS-plug-like structure; PXY-protoxylem; RH-root hair; SCD-sclereid; SG-starch grain; ST-stele; VB-vascular bundle; VT-vascular trace; XY-xylem

(Plate 44); ii. Aerenchyma cells with intercellular spaces Ultra-violet studies (Plate 45); iii. Sclereids of different size and shape, some The powder exhibited the following fluorescence with thick walled arms, some with fragmented arms under 425 nm, 254 nm, and 365 nm, respectively when (Plates 46 to 49); iv. Fragments of vessels with reticulate treated with different reagents. Powder as such and spiral thickenings (Plate 50, 51). emitted royal crown, dark green, nickel grey; with dil. 376 INDIAN J NAT PROD RESOUR, SEPTEMBER 2012

Ammoniacal solution gave teak brown, dark green, 50% H2SO4 royal crown, walnut brown, bottle green; aquamarine; with ethanol (70%) gave brown, french with 50% HNO3 gave mid buff, walnut brown, satin blue, satin blue; with 1N methanolic NaOH gave teak brown; with 5% KOH gave teak brown, walnut brown, dark iced tea, light iced tea; with 1N ethanolic brown, satin brown; with 1N HCl gave timber brown, NaOH gave teak brown, wild purple, wild lilac; with satin grey, smoke grey; with acetone gave teak brown,

Plates 30 to 43Macerate of rhizome—30. Epidermal cells, 31. Parenchyma cells, 32. Fibre, 33 to 39. Sclereids, 40. Tracheid, 41 to 43. Vessels.

Plates 44 to 51Powder studies— 44. Epidermal peel, 45. Aerenchyma cells, 46 to 49. Sclereids, 50, 51. Vessels with reticulate and spiral thickenings. MADHAVAN et al: PHARMACOGNOSY OF NYMPHOIDES HYDROPHYLLA 377

royal blue, grey (the colours are based on the Asian phytoconstituents having Rf 0.31, 0.42 (Figure 2e) and paints limited, Mumbai). alcohol extract revealed 7 phytoconstituents having Rf 0.12, 0.15, 0.28, 0.37, 0.53, 0.68 and 0.75 (Figure Physico-chemical studies 2f). The chromatograms of both aqueous and alcohol The moisture content percent of the drug was found extracts were observed under 254 nm (Figure 3a, 3b), to be 10.35, total ash 8.5, acid insoluble ash 7.6, water 366 nm (Figure 3c, 3d) and 425 nm (Figure 3e, 3f) soluble ash 0.3, sulphated ash 10.0, alcohol soluble and Rf values of prominent spots are represented in extractive 12.0 and water soluble extractive 22.0. respective figures. Successive Soxhlet % extractive values, and the The plate was derivatised with Libermann- colour and consistency were found to be: petroleum Buchardt reagent and scanned under all the three ether (60-80ºC) (olive-green, solid, 2.2), chloroform wavelengths. At 254 nm, aqueous extract revealed 17 (walnut brown, solid, 1.48), acetone (brown, sticky constituents having Rf 0.11, 0.15, 0.16, 0.20, 0.24, mass, 4.24), ethanol (brown, solid, 3.7) and aqueous 0.28, 0.34, 0.41, 0.46, 0.48, 0.51, 0.52, 0.61, 0.65, extract (walnut brown, solid, 6.66). 0.68, 0.82, 0.86 (Figure 4a) and alcohol extract revealed 13 phytoconstituents having Rf 0.14, 0.19, Phytochemical analysis 0.23, 0.27, 0.30, 0.36, 0.38, 0.45, 0.50, 0.73, 0.81, The petroleum ether (60-80ºC), benzene, 0.87, 0.99 (Figure 4b). At 366 nm, aqueous extract chloroform, acetone, ethanol (95%) and chloroform- revealed 5 phytoconstituents having Rf 0.32, 0.33, water (2%) (aqueous) extracts were subjected to 0.40, 0.63, 0.86 (Figure 4c) and 11 phytoconstituents preliminary phytochemical analysis. It was found that in alcohol extract having Rf 0.10, 0.15, 0.23, 0.27, carbohydrates, glycosides, phenolic compounds, 0.32, 0.38, 0.45, 0.50, 0.59, 0.81, 0.87 (Figure 4d). At tannins and saponins were all present in ethanol and 425 nm, aqueous extract revealed 8 phytoconstituents aqueous extracts, coumarins in ethanol extract, fixed having Rf 0.11, 0.17, 0.21, 0.26, 0.32, 0.33, 0.42, 0.50 oils and fats in pet. ether and benzene extracts, phenols, (Figure 4e) and the alcohol extract revealed 11 tannin and flavonoids in acetone, ethanol and water phytoconstituents having Rf 0.14, 0.23, 0.28, 0.30, extracts, saponins, phytosterols in pet ether, benzene, 0.36, 0.38, 0.45, 0.50, 0.62, 0.81, 0.87 (Figure 4f). acetone and ethanol extracts whereas alkaloids, The chromatograms of both aqueous and alcohol flavonoids, gums and mucilage, proteins and amino extract were observed under 254 nm (Figures 5a, 5b), acids were absent in all extracts; besides traces of 366 nm (Figures 5c, 5d) and 425 nm (Figures 5e, 5f) volatile oil was found to be present on hyrodistillation. and Rf values of prominent spots are represented in respective figures. Determination of total phenolic compounds The total polyphenolic compounds found in the Identification of phenolic compounds aqueous extract was 1.11 (±0.318) (mg/g) and 1.80 To identify the presence of phenolic compounds in (± 0.35) (mg/g) (mean ± SEM) in alcohol extract aqueous and alcohol extracts, phenolic compounds (Figure 1). such as tannic acid and salicylic acid were used as standards. Standard tannic acid was identified having Chromatographic studies Rf 0.42 (Figure 6a) and standard salicylic acid was

HPTLC profile of aqueous and alcohol extract detected at Rf 0.57 (Figure 6b) at 254 nm. Both Before derivatisation, the chromatogram was developed in toluene:chloroform:ethanol (28.5:57:14.5). The aqueous extract revealed 10 phytoconstituents having Rf 0.13, 0.14, 0.20, 0.22, 0.28, 0.31, 0.40, 0.47, 0.58, 0.86 (Figure 2a) and the alcohol extract gave 10 phytoconstituents having Rf 0.13, 0.16, 0.25, 0.28, 0.31, 0.35, 0.40, 0.47, 0.76, 0.87 under 254 nm (Figure 2b). At 366 nm, aqueous extract revealed 1 phytoconstituent having Rf 0.39 (Figure 2c) and alcohol extract revealed 3 phytoconstituents having Rf 0.37, 0.53, 0.65 (Figure 2d). At 425 nm, aqueous extract revealed 2 Figure 1Calibration curve for total phenolic contents. 378 INDIAN J NAT PROD RESOUR, SEPTEMBER 2012

Figure 2a-fHPTLC profile before derivatisation—a & b- Aqueous and alcohol extract at 254 nm; c & d- Aqueous and alcohol extract at 366 nm; e & f- Aqueous and alcohol extract at 425 nm.

aqueous and alcoholic extracts at 254 nm resolved into 15 and 14 phytoconstituents having Rf 0.42, 0.45, 0.48, 0.50, 0.53, 0.55, 0.57, 0.59, 0.62, 0.64, 0.67, 0.70, 0.71, 0.73, 0.75 (Figure 7a) and having Rf 0.42, 0.44, 0.48, 0.54, 0.55, 0.57, 0.61, 0.62, 0.65, 0.67, 0.69, 0.70, 0.72, 0.73 (Figure 7b), respectively in ethyl acetate: benzene: formic acid (9:11:0.5) solvent system. The peaks and spots having Rf 0.42 and 0.57 corresponded to that of tannic acid and salicylic acid, respectively in both the extracts which was further confirmed by overlay spectrum of standard phenolic compounds (Figures 8-10).

Identification of betulinic acid  The R of standard betulinic acid24 is 0.59 which was Figure 3a-f Chromatograms before derivatisation—a & f b-aqueous and alcohol extract at 254 nm; c & d- aqueous and detected in both the aqueous and alcohol extracts under alcohol extract at 366 nm; e & f- aqueous and alcohol extract 425 nm (Figures 11a & b, 12). at 425 nm. MADHAVAN et al: PHARMACOGNOSY OF NYMPHOIDES HYDROPHYLLA 379

Figure 4a – fHPTLC profile after derivatisation — a & b- Aqueous and alcohol extract at 254 nm; c & d- Aqueous and alcohol extract at 366 nm; e & f- Aqueous and alcohol extract at 425 nm.

Isolation and identification of β-sitosterol The % yield of the isolated compound was found to be 24% and the melting point 136-137°C. It was subjected to IR analysis and the peaks obtained were recorded. The peaks of the isolated compound was observed to be as follows: Peak 3200-3550 and 1330- 1420/cm was due to O-H stretching , peak 2937 and 675-900/cm-was due to aromatic C-H bending, while peak 1608-1456/cm was due to aromatic C-C stretching (Figure 13). HPTLC profile of the aqueous extract gave a peak having Rf 0.56 (Figure 14a), alcohol extract (R 0.55) (Figure 14b), chloroform f  extract (R 0.56) (Figure 14 c) and isolated β-sitosterol Figure 5a-f Chromatograms after derivatisation— a & b- aqueous f and alcohol extract at 254 nm; c & d- aqueous and alcohol extract at (Rf 0.56) (Figure 14d) at 425 nm. Rf of standard 366 nm; e & f- aqueous and alcohol extract at 425 nm. (Ref. 25) sitosterol was found to be 0.55 . Rf 0.54 was detected in both aqueous and chloroform extracts and Diagnostic characters Rf 0.53 in alcohol extract which were identified as N. hydrophylla plant and the drug consisting of β-sitosterol (Figure 15) and confirmed by IR roots and rhizome are identified by the following spectra (Figure 13). characters: 380 INDIAN J NAT PROD RESOUR, SEPTEMBER 2012

Figure 6a-bHPTLC of standard Tannic acid at 254 nm (Rf 0.42) and standard Salicylic acid at 254 nm (Rf 0.57).

Figure 7a-b—HPTLC of aqueous extract at 254 nm (Rf 0.42-tannic acid) and alcohol extract at 254 nm (Rf 0.42-tannic acid and 0.57- salicylic acid).

Figure 8—Overlay spectrum of tannic acid, aqueous and alcohol extracts. MADHAVAN et al: PHARMACOGNOSY OF NYMPHOIDES HYDROPHYLLA 381

Figure 9—Overlay spectrum of salicylic acid, aqueous and alcohol extracts at 254 m.

1. Aquatic free-floating habit; 2. Presence of corolla lobes which are entire, white and seeds with glochidiate tubercles; 3. Presence of large aerenchyma; 4. Presence of different types of sclereids in the rhizome; 5. Vessels of spiral and reticulate thickenings in both the root and rhizome.

Discussion The stagnant and depleting ayurvedic materia medica is replenished by bringing into light new plant sources for some useful and established ayurvedic drugs like Tagara, whose accepted botanical sources are either becoming endangered or are not available 26 in adequate quantities for medicinal preparations . Nymphoides Hill is a genus of aquatic flowering Figure 10—Chromatogram of phenolic compounds (alcohol and belonging to the family Menyanthaceae. It aqueous extracts, standard and salicylic acid at 254 nm). consists of 20 species, out of which N. hydrophylla (Lour.) O. Kuntze, N. macrospermum Vasudevan, N. as Nymphoides macrospermum, is used as Tagara in aurantiacum (Dalz.) O. Kuntze, N. indica (Lour.) O. several therapeutic preparations. Granthika Tagara is Kuntze, N. parvifolium (Griseb.) O. Kuntze, N. often found as an admixture of above mentioned peltata (S.G. Gmel.) O. Kuntze, N. sivarajanii Joseph species of Nymphoides occurring in South India. Also, and N. krishnakesara Joseph and Sivar. are found in species like V. hardwickii Wall. var. arnottiana (Wt.) India27,28,29. The accepted botanical source of Tagara C.B. Clarke, V. beddomei C.B. Clarke, V. hookeriana is Valeriana jatamansi Jones. In South India, Wt., V. leschenaultiana DC. and Cryptocoryne Granthika Tagara (Kannada), botanically identified spiralis (Retz.) Fisch. ex Wydl., are also treated as 382 INDIAN J NAT PROD RESOUR, SEPTEMBER 2012

Figure 11—HPTLC of aqueous extract of Betulinic acid at 425 nm, alcohol extract of Betulinic acid at 425 nm.

potential candidates of Tagara30,31. The different species of Nymphoides, viz.. N. macrospermum, N. hydrophylla and N. indica does not exhibit much differences exomorphologically. The corolla is pure white in N. macrospermum and N. hydrophylla whereas it is yellow in N. indica. The seeds are larger in size in N. macrospermum while they are very small in N. hydrophylla and N. indica. The roots are of spongy and stout type in N. macrospermum while it is of only spongy type in N. hydrophylla and N. indica. The rhizome and root of all the three species show similar characters microscopically5,6,29. Ultra-Violet analysis of the powder gave characteristic fluorescence which helps to distinguish the drug from other species considered as Tagara. Preliminary phytochemical analysis of N. hydrophylla revealed the presence of carbohydrates and glycosides, coumarins, phytosterols, saponins, phenolic compounds and tannins and traces of volatile oil (present work) whereas steroids, phenols, tannins, saponins, sugars, Figure 12—Chromatogram of Betulinic acid in alcohol and aqueous extracts at 425 nm. iridoid or monoterpenic derivative, known as valepotriates, an iridoid ester glycoside and essential oils are some of the phytoconstituents present in V. N. macrospermum was also found in N. hydrophylla. jatamansi32 which is the accepted source of Tagara. Betulinic acid from the aqueous and alcohol extracts In the alternate sources of Tagara, viz. N. and β-sitosterol from aqueous and chloroform extracts macrospermum, steroids, phenols, tannins, saponins, were also detected in N. hydrophylla using HPTLC. sugars and in N. indica, glycosides, phytosterols, The sedative and anticonvulsant property in saponins, phenolic compounds, gums and mucilage N. macrospermum34 and the anticonvulvasant and traces of volatile oil have been reported33. property in N. indica35 are reported which are also assigned to Tagara in Ayurveda1. N. macrospermum gave 3 phytoconstituents in petroleum ether extract, while chloroform extract gave 4 phytoconstituents; the alcoholic extract of N. Conclusion indica gave 7 phytoconstituents, while the aqueous The taxonomical characters dealing with the extract gave 9 phytoconstituents5, 6; both aqueous and exomorphology of N. hydrophylla, a potential source alcoholic extracts of N. hydrophylla gave 10 of the drug Tagara in Ayurveda, is described to helps phytoconstituents before derivatisation (present in the identification of the plant in the field. Other work). A triterpenoid compound, β-sitosterol reported studies, viz. macro- and microscopical studies of the in the alcohol extract of roots and rhizomes of drug (root and rhizome), chromatographic studies, MADHAVAN et al: PHARMACOGNOSY OF NYMPHOIDES HYDROPHYLLA 383

Figure 13—IR spectra of β-sitosterol.

Figure 14(a-d) — HPTLC of β-sitosterol in aqueous extract at 425 nm, in alcohol extract at 425 nm, in chloroform extract at 425 nm, isolated β-sitosterol in alcohol extract at 425 nm

phytochemical analysis, etc carried out during this tannic acid and salicylic acid was carried out in study will help in evolving diagnostic characters, in alcohol and aqueous extracts and betulinic acid from determining pharmacopoeial parameters, aqueous and alcohol extracts and β-sitosterol from standardisation of therapeutic formulations, besides in aqueous and chloroform extracts were detected for the the isolation and identification of bioactive first time in this species. N. hydrophylla which is an principles/biomarkers. HPTLC fingerprinting of allied species of N. macrospermum and N. indica may 384 INDIAN J NAT PROD RESOUR, SEPTEMBER 2012

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