Evaluation of Picrorhiza Kurrooa Accessions for Growth and Quality in North Western Himalayas

Full Length Research Paper

Evaluation of Picrorhiza kurrooa accessions for growth and quality in north western Himalayas

Rakesh Kumar1*, Pamita Bhandari2, Bikram Singh1 and P. S. Ahuja1

1CSIR-Institute of Himalayan Bioresource Technology (Council of Scientific and Industrial Research) Palampur 176 061 (HP), India. 2Natural Product Department, NIPER, Mohali, Punjab, India.

Accepted 9 January, 2012

Different accessions of Picrorhiza kurrooa were evaluated for growth and marker compound accumulation pattern under field conditions at village Chuner, Sub Tehsil, Holi, Distt. Chamba (HP) located at an elevation of 2538 m during 2006 to 2010. Six accessions with higher Picroside content and vegetative growth were identified for further multiplication. Accession IHBT-PK-8 recorded higher leaf numbers/plant (250), length of 6th leaf (5.4 cm) stolon girth (7.5 mm) and plant spread in N-S direction (50.0 cm). Picroside content in leaf and rhizome of different accessions was determined by high- performance liquid chromatography (HPLC). It was found that leaves of P. kurrooa are good source of picrosides. Picroside-I (P-I) content in leaf was higher in IHBT-PK-2 (3.89%) followed by IHBT-PK-11 (3.72%) and IHBT-PK-21 (3.70%). Picroside -II (P-II) content in leaf was higher in IHBT-PK-5 (4.82%). P-I content in rhizomes of P. kurrooa varied from 0.20 to 4.14% and P-II varied from 0.83 to 7.29% in different accessions. Higher P-I in rhizome was found in IHBT- PK-16 (4.14%). Rhizomes showed higher amount of P- II as compared to P-I.

Key words: Picrorhiza kurrooa, accessions, picroside I and II, growth.

INTRODUCTION

The genus Picrorhiza (Scrophulariaceae) comprises of their population have declined in some parts of their two species Picrorhiza kurrooa Royle ex Benth and range owing to overharvest for medicinal use and trade Picrorhiza scrophulariiflora Pennel which are extensively by the traders. used in traditional medicine system of India, China, Tibet, P. kurrooa, commonly known as ―Kutki‖ is an important Nepal and Sri Lanka for the treatment of various immune medicinal plant used in traditional as well in modern related diseases (Jain and DeFillips, 1991; Varier, 1995; medicines for the treatment of liver disorders, fever, Sharma, 1996). P. Kurrooa is predominant in Western asthama and jaundice (Ansari et al., 1988; Chaturvedi Himalayan regions of Northern India whereas P. and Singh 1996). It is also useful in gastrointestinal and scrophulariiflora is mainly found in the Himalayan regions urinary disorders, leukoderma, snake bite, scorpion sting of Sikkim, Nepal and Tibet (Bantawa et al., 2010, 2011). and inflammatory affections (Weinges et al., 1972; P. kurrooa, a perennial creeping herb, which spreads by Halliwell and Gutteridge 1999; Visen et al., 1998; Verma stolon grown naturally between 3,000 to 5,000 m asl. In et al., 2009). It is also reported to possess Himachal Pradesh it is found in the higher reaches of hepatoprotective (Dhawan, 1995; Vaidya et al., 1996), Chamba, Kangra, Mandi, Shimla, Kinnaur and Lahaul anti inflammatory (Del Carmen Recio et al., 1994; Singh and Spiti districts (Uniyal et al., 2006). Though the plant et al., 1993), immunomodulatory (Puri et al., 1992), free is widely distributed in the Himalayas occurring in radical scavenging (Chander at al., 1992), gastric ulcer Pakistan, India, Nepal, Bhutan and Southern China but (Ray et al., 2002; Banerjee et al., 2008), anti allergic and anti-anaphylactic activities (Baruah et al., 1998). P. kurrooa was also found to have promising anti-hepatitis B surface antigen activity (Mehrotra et al., 1990). P. *Corresponding author. E-mail: [email protected]. Tel: scrofloriiflora is widely used for the treatment of damp +91 1894 233341. Fax: +91 1894 230433. heat, dysentery, jaundice and steaming of bones (Singh

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et al., 2005). Its roots are used as a tonic, cathartic, The plants of each accession were spaced at 30 × 30 cm2 in 1 × 1 stomachic and purgative (Stuppner and Wagner, 1989). m plots. Well decomposed farmyard manure (FYM) @ 15t/ha was The aforementioned activities are attributed due to the incorporated into the soil every year. Five plants were selected in each treatment plot and averaged for recording different yield presence of iridoid glycosides (mainly picroside-I and attributes viz., plant height, plant spread, number of branches per picroside-II) in the plant. Kutkin, (an active fraction of P. plant, number of leaves per plant. After fourth year of plantation, kurrooa is a mixture of picroside-I and picroside- II) rhizome and leaf were taken from each accession and were showed significant hepatoprotective activity in hepatic analysed for picroside I and picroside II. damage induced by glucosamine in rats and Plasmodium berghei (mastomys) (Singh et al., 2005). Solvents and chemicals Over exploitation and consequent degradation of natural habitat are reported to be a major threat to this High-performance liquid chromatography (HPLC) grade solvents plant. Over 90% of the market demand for this species is (acetonitrile-water) were purchased from J. T Bakers (USA) and met from the wild. Uniyal et al. (2011) reported that to get analytical reagent-grade chemicals were purchased from Merck 1 kg dry weight of P. kurrooa plant, as many as 300 to (Darmstadt, Germany). The reference compounds, picroside-I and picroside-II, were isolated in our laboratory and characterization 400 individual plants are uprooted. Due to narrow was established by 1H and 13C-NMR spectral analysis, mass distribution range, small population size and high use spectral data and comparison with earlier reports (Singh and value, the species figure among the 37 identified as top Rastogi, 1972; Bhandari et al., 2009). priority species for conservation and cultivation in Western Himalaya. Indiscriminate, unscientific harvesting and lack of organized cultivation of the plant has Plant material threatened its status in wild and listed as ‗endangered‘ Mature plants of P. kurrooa showing variability in leaf size were species by International Union for Conservation of Nature collected from the field. The plants, containing rhizome and leaves and Natural Resources (Nayar and Sastri, 1990). Though were also collected. The plants were gently uprooted, washed with this plant grows naturally at altitude above 3000 m asl but distilled water and blotted dry. Leaf and rhizome tissues of it can be cultivated at lower altitudes (Chandra, 2004; individual plants were separated independently. Subsequently, the tissues were dried at 60°C in a hot air oven and data were taken Nautiyal et al., 2001). when concordant values were obtained. Each part of all the Increasing demand for the kutki drug has prompted samples were air-dried separately and ground to a fine powder in many researchers to search for sources of genotypes of mixer grinder. The plant material was stored in an airtight glass P. kurrooa rich in picroside content (Katoch et al., 2011). bottle in a desiccator at 30°C and powdered to 40 mesh. Variation in growth and picroside content in plants from different area is a serious question that needs to be Preparation of sample solutions addressed. Considering the economic importance of this plant and increasing demand of herbal drugs and its The powdered material (100 mg) was percolated with ethanol:water assessment as an endangered plant species, it felt (1:1) for 3 h at 25 ± 2°C. The aqueous ethanolic extracts were important to initiate the steps for screening P. kurrooa filtered and dried under reduced pressure at 50 ± 5°C. The dried accessions with higher picroside content and growth at filtrate (20 to 25 mg) was re-dissolved in mobile phase (1 ml), lower altitude and in vicinity of its natural habitat. filtered through 0.45 m membrane and an aliquot (10 l) of the filtrate was injected into HPLC for analysis. The objective of this study was to assess the growth and picroside content in P. kurrooa accessions collected from different regions of Himalayas and grown at lower Preparation of standard solutions altitude to identify superior cultivars for further exploitation and breeding programme. Stock solution of standards, picroside-I and picroside-II (1.0 mg/ml) was prepared in acetonitrile-water and diluted to a series of appropriate concentrations with the same solvent. An aliquot (10 µl) of the diluted solutions was injected into the HPLC for the MATERIALS AND METHODS construction of six point calibration curves. The calibration curves were constructed by plotting the peak areas versus amount (µg) of Experimental site each analyte.

Field experiment was conducted during 2006 to 2010 at village Chuner, Sub Tehsil, Holi, Distt. Chamba (HP). The experimental HPLC analysis site is located at an elevation of 2538m amsl, 32° 16‘ 30.8‖ N latitude and 76° 39‘ 40.6‖ E longitude. The area is covered with HPLC analysis was performed on a Shimadzu Prominence HPLC snow from November to March. The soil of the area was silty loam system equipped with LC-20AT quaternary gradient pump, in texture, acidic in reaction (pH 5.9), high in organic carbon (1.1%), photodiode array detector (PDA), CBM-20A communication bus medium in available nitrogen (357.5 kg/ha), high in available module, CTO-10AS vp column oven, auto sampler and Shimadzu phosphorus (34.1%), and available potassium (296.2 kg/ha). P. LC solution software (ver. 1.21 SP1). The chromatographic kurrooa accessions were collected from, Kashmir, north east, resolution was achieved on a Zorbax C-18 column (250 × 4.6 mm, Uttrakhand and Himachal Pradesh with altitudinal variation ranging 5 μm particle size) from Agilent. The mobile phase used for the from 3134 m to 4152 m asl. These accessions were planted in the analysis was water (0.1% TFA): acetonitrile (75:25) in isocratic experimental field at Chuner for four years (2006 to 2010). elution with flow rate 0.7 ml/min at detection wavelength 270 nm.

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Table 1. Biometric observations of different accessions of P. kurrooa.

Plant spread Plant height No. of leaves/ Leaf length Leaf width Stolon no/ Stolon girth S/N Accession no. (cm) (cm) plant (cm) (cm) plant (mm) (N-S) E-W 1 IHBT-PK-1 6 58 3.0 1.5 6 3.9 13.0 8.0 2 IHBT-PK-2 11 68 3.1 1.4 6 5.6 17.0 11.7 3 IHBT-PK-3 8 37 3.0 1.7 4 4.1 11.3 6.2 4 IHBT-PK-5 6 13 1.0 0.7 1 4.2 5.0 4.0 5 IHBT-PK-7 8 150 4.3 2.4 23 7.5 21.0 23.0 6 IHBT-PK-8 12 250 5.4 1.7 22 3.4 50.0 28.0 7 IHBT-PK-9 16 89 2.0 0.9 8 4.4 15.0 20.0 8 IHBT-PK-10 17 195 4.0 1.8 11 4.3 22.0 23.0 9 IHBT-PK-11 11 55 2.0 1.1 8 4.5 9.0 20.0 10 IHBT-PK-12 6 35 2.4 1.9 3 6.3 5.0 11.0 11 IHBT-PK-13 10 65 4.0 1.7 13 3.7 19.0 23.0 12 IHBT-PK-14 6 60 3.2 1.6 5 7.3 18.0 14.0 13 IHBT-PK-15 16 164 4.8 2.1 9 4.9 20.0 32.0 14 IHBT-PK-16 11 75 4.8 2.2 4 6.7 9.0 19.0 15 IHBT-PK-17 8 130 3.2 1.7 6 5.6 17.0 18.0 16 IHBT-PK-18 7 30 2.1 1.2 8 4.7 15.2 10.7 17 IHBT-PK-20 12 37 3.5 1.9 4 3.9 13.0 19.0 18 IHBT-PK-21 9 25 3.5 1.4 3 6.6 8.0 6.0 SEm± 0.86 12.58 0.18 0.06 0.73 0.14 0.89 0.78

The marker compounds were quantified by using external standard (0.7 cm), stolon number/ plant (1.0), plant spread in N-S method. (5.0 cm) and E-W (4.0 cm) directions. The developed high-performance liquid chromatography-diode-array detection (HPLC-DAD) RESULTS AND DISCUSSION method was subsequently applied for determination of picroside contents (P-I and P-II) in different accessions of An extensive literature survey revealed that Picrorhiza P. kurrooa. HPLC results revealed variations in the genus is well characterized chemically as well as distribution and relative content of P-I and P-II extracted pharmacologically (Dwivedi et al., 1997; Sturm and from different accessions (Table 2). The identification of Stuppner, 2001; Kumar et al., 2004, 2005; Singh et al., investigated compounds was carried out by comparison 2005; Bhandari et al., 2008; Sood and Chauhan, 2010; of their retention time and UV spectra with standard Rathee et al., 2011; Katoch et al., 2011), however, few compounds and also by spiking the samples with studies were undertaken to select chemically superior standard stock solution. The typical chromatograms of plants among the existing population. Therefore, we sample and standard were shown in Figure 1. The results aimed to identify the chemically superior plants for their revealed that picroside- I (P-I) content in leaf was higher cultivation from wild /cultivated population. in IHBT- PK-2 (3.89%) followed by IHBT-PK-11 (3.72%) A perusal of data presented in Table 1 on biometric and IHBT-PK-21 (3.70%) while picroside-II (P-II) content observations of P. kurrooa accessions showed variation was higher in IHBT-PK-5 (4.82%) followed by IHBT-PK-9 for different attributes among the accessions. Plant height (4.33%), IHBT-PK-12 (4.28%) and IHBT-PK-11 (4.20%). of different accessions varied from 6 to 17 cm. IHBT-PK- The total picrosides (P-I and -II) content was found to be 10 produced plant with higher plant height (17 cm) higher in IHBT-PK-11 (7.92%). The previous reports followed by IHBT-PK-9 and 15, whereas, IHBT-PK 14 (Singh et al., 2011; Sood and Chauhan 2010) suggested recorded the lowest height among all the accessions. that leaves are good source of picrosides and can be Number of leaves/ plant varied from 13 to 250. Accession used as a resource for picrosides, hence, not essential to IHBT-PK-8 recorded higher leaf numbers/plant (250), uproot the whole plant and our results are also in length of 6th leaf (5.4 cm) stolon girth (7.5 mm) and plant accordance to earlier reports that leaves contain good spread in N-S direction (50.0 cm). Whereas, IHBT-PK-5 amount of picrosides. Sood and Chauhan (2010) recorded the lowest plant height (6 cm), leaf number/ reported that accumulation of P-I is developmentally plant (13), leaf length of 6th leaf (1.0), width of 6th leaf regulated in different morphologic stages of P. kurrooa,

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Table 2. Marker compound in leaf of different accessions of P. kurrooa.

S/N Accession no. PI PII Total (P-I & P-II) 1 IHBT-PK-1 1.42 1.33 2.75 2 IHBT-PK-2 3.89 1.46 5.35 3 IHBT-PK-3 1.45 1.47 2.92 4 IHBT-PK-5 3.23 4.82 8.05 5 IHBT-PK-7 2.73 3.07 5.80 6 IHBT-PK-8 1.30 1.48 2.78 7 IHBT-PK-9 1.63 4.33 5.96 8 IHBT-PK-10 1.89 3.90 5.79 9 IHBT-PK-11 3.72 4.20 7.92 10 IHBT-PK-12 3.00 4.28 7.28 11 IHBT-PK-13 2.24 0.24 2.48 12 IHBT-PK-14 3.15 4.13 7.28 13 IHBT-PK-15 1.54 3.22 4.76 14 IHBT-PK-16 0.08 0.00 0.08 15 IHBT-PK-17 2.09 3.91 6.00 16 IHBT-PK-18 2.21 2.57 4.78 17 IHBT-PK-20 1.50 1.05 2.55 18 IHBT-PK-21 3.70 2.17 5.87 SEM± 0.24 0.36 0.52

mAU 270nm4nm (1.00)

250 270 nm, 4 nm (1.00) Picroside-I

Picroside-II (A) 200

150

100

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

mAU 270nm,4nm (1.00)

(B)

750 Picroside-II

500 Picroside-I

250

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

Figure 1. HPLC-DAD chromatograms (λmax 270 nm) of standard picrosides (A) and sample PKL-15 (B).

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Table 3. Marker compound in rhizomes of different accessions of P. kurrooa.

S/N Accession no. PI PII Total (P-I & P-II) 1 IHBT-PK-1 1.58 2.93 4.51 2 IHBT-PK-2 1.73 7.29 9.02 3 IHBT-PK-3 2.06 5.43 7.49 4 IHBT-PK-5 0.20 2.71 2.91 5 IHBT-PK-7 1.17 2.40 3.57 6 IHBT-PK-8 2.38 5.37 7.75 7 IHBT-PK-9 1.56 5.90 7.46 8 IHBT-PK-10 0.87 4.10 4.97 9 IHBT-PK-11 0.41 1.05 1.46 10 IHBT-PK-12 1.09 3.96 5.05 11 IHBT-PK-13 0.53 1.10 1.63 12 IHBT-PK-14 3.35 4.38 7.73 13 IHBT-PK-15 0.42 4.60 5.02 14 IHBT-PK-16 4.14 0.83 4.97 15 IHBT-PK-17 0.40 4.34 4.74 16 IHBT-PK-18 1.10 1.06 2.16 17 IHBT-PK-20 1.78 2.92 4.70 18 IHBT-PK-21 0.01 3.72 3.73 SEM± 0.26 0.44 0.52

while Singh et al. (2011) reported that P-I content was Comparative analysis of picrosides in leaf and rhizome higher in leaf tissue as compare to root and rhizomes and revealed that P-I content was higher in leaves, whereas, also the total picrosides content in roots was less as PII content was higher in rhizomes of different accessions compare to leaves. However, Katoch et al. (2011) (Tables 2 and 3). Singh et al. (2011) have also reported reported that though leaves of P. kurrooa contain similar findings. picroside but their concentration is lower than the picroside of rhizome and root and this accumulation depends on altitude. Conclusion

Both picrosides, P-I and P-II were present in rhizomes Out of 18 accessions maintained at higher altitude region in comparable amounts. P-I content in rhizomes of P. of HP, 6 accessions with higher Picroside content and kurrooa varied from 0.20 to 4.14% and P-II varied from vegetative growth were identified for further multiplication. 0.83 to 7.29% in different accessions. Higher P-I in The present results revealed the variation in growth and rhizome was found in IHBT-PK-16 (4.14%) followed by picrosides (P-I and P-II) content in different accessions of IHBT-PK-14 (3.35%), IHBT-PK-8 (2.38%) and IHBT-PK-3 P. kurrooa collected from the north-western Himalayas (2.06%). In remaining accessions P-I was lower than and grown at lower altitude. 2.0%. P-II content in rhizomes was higher in IHBT-PK-2 The accession IHBT-PK-5, IHBT-PK-11, IHBT-PK-12, (7.29%) followed by IHBT-PK-9 (5.90%), IHBT-PK-3 having the higher P-II content in leaf and these could be (5.43%), IHBT-PK-8 (5.37%), IHBT-PK-15 (4.60%) and further exploited for cultivation of marker-specific IHBT-PK-14 (4.38%). Rhizomes showed higher amount populations, if higher P-II content is required compared to of P- II as compared to P-I. Total picroside content in P-I. rhizomes was higher in IHBT-PK-2 (9.02%) followed by IHBT-PK-8 (7.75%) and IHBT-PK-14 (7.73%). Kawoosa et al. (2010) have reported that light and low temperature ACKNOWLEDGEMENT favor picrosides accumulation in natural population of kutki, wherein it has been reported that picrosides The authors are grateful to the Director, IHBT, Palampur content increased by 135% in the plants growing at high for providing necessary facility during the course of study. (4,145 m) as compared to the low (1,350 m) altitude The authors are also thankful to Mr. Sushil Kumar (Singh et al., 2005). Increase in altitude accompanies technical assistant for Field Management. The financial decrease in temperature and increase in light quanta assistance from Council of Scientific and Industrial (Streb et al., 1998). This might be due to the fact that Research, New Delhi, India for undertaking this study is these are cultivated plants, and thus their picroside I gratefully acknowledged. This is IHBT Publication No. content was reduced after cultivation at lower altitude. 2306.

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