Picrosides content in the rhizomes of Picrorhiza kurroa Royle ex Benth. traded for herbal drugs in the markets of North India

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1 , ISSN Vol. 3, No. 2, pp , June 2013 RESEARCH ARTICLE Picrosides content in the rhizomes of Picrorhiza kurroa Royle ex Benth. traded for herbal drugs in the markets of North India Kirti SHITIZ, Saurabh PANDIT, Rajinder Singh CHAUHAN, Hemant SOOD* Department of Biotechnology & Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan , Himachal Pradesh, India. Article History: Received 4 th April 2013, Revised 29 th April 2013, Accepted 30 th April Abstract: Picrorhiza kurroa Royle ex. Benth (Kutki) is an important medicinal plant used in various herbal drug formulations possessing hepatoprotective activity. Herbal drug industries purchase the raw plant material (dried rhizomes) of P. kurroa from the markets by judging the quality mainly on the basis of physical appearance of the rhizomes rather than the contents of active constituents. This study assessed the current status of trade and quality of P. kurroa plant material being sold in major herbal drug markets of north India (Delhi, Amritsar, Manali) on the basis of major chemical constituents i.e. P-I (Picroside-I) and P-II (Picroside-II) by using Reverse Phase HPLC (High Pressure Liquid Chromatography). The P. kurroa rhizomes sold in the Amritsar market contained highest picrosides content (10.9%) whereas sample from China (sold in Delhi market) showed the least amount of picrosides (2.8%). Various herbal drug formulations, containing P. kurroa as one of the constituents, were also analysed for picrosides content. The study highlights the importance of quality of raw material used in the preparation of herbal drug formulations. Keywords: Picrorhiza kurroa; Picroside-I; Picroside-II; HPLC; Herbal Drug Formulations. Introduction Picrorhiza kurroa Royle ex Benth. is a small perennial herb from the family Scrophulariaceae, found in the Himalayan region growing at elevations of 3,000-5,000 meters (Chettri et al. 2005). Indiscriminate and extensive harvesting and lack of organized cultivation of the plant has threatened its status in wild and listed as endangered species by International Union for Conservation of Nature and Natural Resources (Nayar and Sastri 1990 ). Picrorhiza kurroa has been included in the Appendix II of CITES list. CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) is an international agreement between governments. Its aim is to ensure that international trade in specimens of wild animals and plants does not threaten their survival. As there are certain rules and regulations for the trade of this medicinal plant/plant material, this will lead to increase in illegal trade and finally the price. Kutkin is the active principal of Picrorhiza kurroa and is comprised of kutkoside and the iridoid glycosides Picroside I and II. The herbal drug, in the form of dried roots and rhizomes, is prescribed in the treatment of several ailments of the liver and spleen, and in cases of fever and asthma (Chaturvedi and Singh 1996, Dorsch et al. 1991, Langer et al. 1981, Rajaram 1976, Yegnarayan et al. 1982). Picroliv, which was launched as a herbal drug formulation, is prepared from a standardized iridoid fraction containing 60 % of Picroside-I and Kutkoside in a 1: 1.5 ratio ( Dwivedi et al. 1989, Ansari et al. 1991, Dhawan 1995). There are several commercially available herbal formulations of Picrorhiza kurroa (Table1) which contain Picroside-I and Picroside-II in different concentrations (Bhandari et al. 2009). It is one of the top 15 plant species traded in India in terms of the economic value of traded materials (Malaisamy and Ravindran 2003; Ved and Goraya 2008). With an annual growth rate of 20% in herbal medicines in recent years, demand for medicinal plants has increased by 11.1% (Subrat 2002 ). Annual transaction of more than 10,000 kg of Picrorhiza kurroa takes place only in Delhi market (Uniyal et al. 2011). During the period , the prices of *Corresponding author: ( ) hemant.sood <@> juit.ac.in 2013 Copyright by the Authors, licensee Open Access Science Research Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC- ND 3.0) License (

2 Picrorhiza plant material varied from Rs. 220 to 340 per kg (Uniyal and Uniyal 2008 ) though prices are much higher today. The price increases as it enters the market away from collection site. Material collected in the months of July 227 and August is supposed to have high moisture content and is generally rated low, while material collected in September is rated high because of low moisture content ( Uniyal and Uniyal 2008). Table 1: Total picrosides content in different herbal preparations of P. kurroa (Bhandari et. al 2009). Sample Picroside-I (%) Picroside-II (%) Total picrosides content (%) Katuki, Zandu Pharma Works Ltd. (Mumbai, India) Arogya, Zandu Pharma Works Ltd. (Vapi, GJ, India) Kutaki, Tansukh Herbal Pvt. Ltd. (Lucknow, UP, India) Livocare, Dindayal Aushdi Pvt. Ltd. (Gwalior, MP, India) Livomap, Maharishi Ayurveda Pvt. Ltd. (New Delhi, India) Livomyn, Charka Pharma Pvt. Ltd. (Mumbai, India) Livplus, BACFO Pharmaceuticals Ltd. (Noida, UP, India) Pravekliv, Pravek Kalp Herbal products Pvt. Ltd. (Noida, UP, India) Vimliv, OM Pharma Ltd. (Bangalore, India) Traces Global supply (excluding China and Pak i- stan) of Picrorhiza kurroa is around 375 tonnes, with India contributing around 70 tonnes. In India annual demand for P. kurroa is more than 5000 tonnes; however, its supply is less than 100 tonnes (Kumar 2006). Picrorhiza kurroa is largely collected from Jammu and Kashmir, Himachal Pradesh, Uttarakhand and Sikkim to fulfil the demand of herbal drug industries. Picrorhiza is also imported from Nepal, Bhutan and China. The plant material is largely collected from the wild, cleaned, dried and transported to the herbal drug market and supplied to herbal drug industries. However, what parameters determine the quality of plant material is not known. It may be only the physical appearance (thickness of rhizomes, presence of dust or other unwanted material, etc.) rather than the contents of marker compounds being used as selection criteria of plant material. Therefore, determining the quality of plant material available in different herbal drug markets on the basis of major chemical compounds is necessary. The present study was undertaken with an aim of (i) seeing whether the Picrorhiza raw material sold in the north Indian markets for the preparation of herbal drug formulations is of desired quality w.r.t. marker compounds and to present the actual status of major chemical constituents in the raw material. This analysis would also provide authenticity of plant material used in the preparation of herbal drug formulations. (ii) Quantification of picrosides in herbal drug formulations available in the market to find out the authenticity of preparations and amount of active ingredients. We also wanted to see whether morphological appearance of rhizomes is an appropriate criteria for evaluating the quality of raw plant material since that is used by most of the industry buyers. Materials and method Dried rhizomes of Picrorhiza kurroa were purchased from herbal drug markets in Delhi, Amritsar, Manali for quantification of picrosides. Various herbal drug suppliers were also interviewed from Delhi and Amritsar herbal drug markets to assess the current status of Picrorhiza trade. Different herbal fornulations (Capsules and tablets) containing Picrorhiza kurroa were also purchased from the market for estimation of picrosides content. Sample Preparation Rhizome Dried rhizomes were ground to a fine powder in a grinder. 100mg of powdered material was percolated with 10ml 80% Methanol overnight at room temperature. The methanolic extract was filtered through 0.22µm membrane

3 filter and 1:10 dilution of filtered extract was made using 80% Methanol. Herbal formulations (Capsules and Tablets) Capsule shells were removed and powder was used for HPLC analysis. Tablets were ground in a grinder and powder was used for analysis. HPLC Analysis Quantification of picrosides was performed by following the protocol developed by (Sood and Chauhan 2010) on Waters HPLC System equipped with Waters 515 HPLC pumps, Waters 717 autosampler, Waters 2996 photodiode array detector and Empower software. Waters Spherisorb reverse phase C18 column (4.6mm x 250mm, 5µm) was used and 20µl of sample was injected into the column for analysis. The mobile phase used for the analysis was solvent A (0.05% trifluoro- acetic acid in water) and Solvent B (1:1 methanol/acetonitrile mixture). Solvent A and B were used in the ratio of 70:30 (v/v). The column was eluted in isocratic mode with a flow rate of 1ml/min at detection wavelength of 270nm. The cycle time of analysis was 30 min at 30 C. The compounds were identified on the basis of their retention time and comparison of UV spectra with the authentic standards procured from ChromaDex, Inc. The chromatograms of standards P-I and P-II are shown in (Figure 1). Results P. kurroa rhizome samples purchased from Delhi, Amritsar and Manali markets were from different locations namely China, Nepal, Uttarakhand (purchased from Delhi market), 228 Himachal Pradesh (purchased from Amritsar market), Manali (purchased from Manali ma r- ket). The prices ranged from Rs per kg; though price was much higher (upto Rs. 770 per kg) for plant material imported from China and Nepal. It was found that annual transaction of about tonnes of Picrorhiza kurroa takes place from Amritsar market whereas from Delhi market annual transaction of about 100 tonnes takes place as it is a major herbal drug market of north India. Various herbal drug formulations containing Picrorhiza kurroa namely, Purim, Arogyawardhini Bati, Cirrholiv and Pigmento were also purchased from the market for estimation of picrosides. HPLC analysis showed that the Picrorhiza kurroa plant material from India and Nepal showed higher picrosides content in comparison to plant material from China. The sample from Amritsar market contained the highest picrosides content (10.9%) whereas China sample purchased from Delhi market showed least content (2.8%). Samples from Nepal, Manali and Uttarakhand showed picrosides contents of (7.9%), (8.6) and (6.4%), respectively (Table 2 and Figure 2). The chromatograms of these samples are shown in Figure 3 along with standards. Therefore, large variation in the total contents of picrosides was observed, which would certainly affect the quality of herbal drug formulations based on P. kurroa. HPLC analysis of herbal formulations showed total picrosides content of 0.3%, 0.41% and 0.27% in Purim, Arogyawardhini Bati and Cirrholiv respectively (Table 3) whereas no picrosides content was detected in the formulation Pigmento. Figure 4 shows chromatograms of these herbal formulations along with standards. Table 3: HPLC analysis of herbal drug formulations containing Picrorhiza kurroa. Formulation Weight of Amount of P-I P-II Total Name each tablet/ Capsule (mg) Picrorhiza kurroa (mg) content (%) content (%) picrosides content Purim Arogyawardhini Bati Cirrholiv Pigmento Not Detected Not Detected -

229 Table 2: Picroside-I and Picroside-II

kurroa rhizomes purchased from herbal drug

4 229 Table 2: Picroside-I and Picroside-II contents in different samples of P. kurroa rhizomes purchased from herbal drug markets of North India. Sample Name Price (Rs. per kg) P-I (%) P-II (%) Total (%) Manali (from Manali Market) Himachal Pradesh (from Amritsar Market) Nepal (from Market) Delhi Uttarakhand (from Delhi Market) China (from Market) Delhi

A U A U 0. 1 0 0 0. 0 9 0 0. 0 8 0 0. 0 7 0 0. 0 6 0 0. 0 5 0 0. 0 4 0 0. 0 3 0 0. 0 2 0 0. 0 1 0 0. 0 0 0 0. 1 0 0 0. 0 9 0 0. 0 8 0 0. 0 7 0 0. 0 6 0 0. 0 5 0 0. 0 4 0 0. 0 3 0 0. 0 2 0 0. 0 1 0 0. 0 0 0 M in u t e s 1 8.

5 A U A U M in u t e s M in u t e s 230 P-I P-II Figure 1: HPLC elution profile of picroside-i (P-I) and picroside-ii (P-II) standards. Discussion The sample from Himachal Pradesh showed highest picrosides content, whereas inspite of the good physical appearance, the plant material from China showed least picrosides content. Proper concentration of P-I and P-II is required for the preparation of herbal drug formulations from P. kurroa, therefore, it is necessary to judge the quality of plant material so that herbal drug formulations with desired pharmacological efficacy can be produced. As all these investigations are not specified in the standard literature, the authors are of the view that these parameters may be helpful in standardisation, authentication and evaluation of other formulations containing picrosides. The manufactured products were tested to check the amount as well as variation in P-I and P-II in different herbal formulations. Picrorhiza kurroa plant material is added in herbal formulations in different amounts (on weight basis) and not on the basis of content of marker compounds. Through analysis of manufactured products we made a conclusion that these formulations contain varying amounts of P-I and P-II and these marker compounds were absent in one of the formulations inspite it was mentioned in the constituents of the formulation. Picrorhiza kurroa has received increased scientific and commercial attention; hence there is increased pressure on the wild population of this medicinal plant due to which it has been listed as endangered. To fulfil the increasing commercial demand of Picrorhiza kurroa plant material its price has also increased in recent years. Therefore, the study and conservation of this medicinal plant has become necessary. Figure 2: Picroside-I and Picroside-II content in rhizome samples of Picrorhiza kurroa purchased from different herbal drug markets.

6 231 Figure 3: HPLC Elution Profiles of Rhizome Samples of P. kurroa Purchased from Market along with Standards P-I and P-II.

7 232 Figure 4: HPLC Elution Profiles of Herbal Formulations along with Standards P-I and P-II. Conclusion The present study concludes that Picrorhiza kurroa market samples tested varied in quality in respect to major chemical compounds i.e. P-I and P-II. As the plant material from Himachal Pradesh is of better quality in terms of major chemical compounds, this study would be beneficial for bringing the focus of pharmaceutical industry to purchase the quality raw material from Indian markets rather than importing the plant material. Also, physical appearance should not be the only criteria for determining the quality of plant material but amount of chemical compounds should also be considered. Acknowledgements: The authors are thankful to the Department of Biotechnology, Govt. of India for providing financial support to RSC in the form of a programme support on high value medicinal plants. References Ansari, R.A., Tripathi, S.C., Patnaik, G.K., Dhawan, B.N Antihepatotoxic properties of picroliv: an active fraction from rhizomes of Picrorhiza kurrooa. Journal of Ethnopharmacology, 34: Bhandari, P., Kumar, N., Singh, B., Gupta, A.P., Kaul, V.K., Ahuja, P.S Stability-

8 233 Indicating LC PDA Method for Determination of Picrosides in Hepatoprotective Indian Herbal Preparations of Picrorhiza kurroa. Chromatographia, 69: Chaturvedi, G.N., Singh, R.H Jaundice of infectious hepatitis and its treatment with an indigenous drug, Picrorhiza kurrooa. Journal of Research in Indian Medicine, 1: Chettri, N., Sharma, E., Lama, S.D Nontimber forest produces utilization, distribution and status in a trekking corridor of Sikkim, India. Lyonia, 8: Dhawan, B.N Picroliv A new hepatoprotective agent from an Indian medicinal plant Picrorhiza kurroa. Medicinal Chemistry Research, 5: Dorsch, W., Stuppher, H., Wagner, H., Gropp, M., Demolin, S., Ring, J Antiasthmatic effect of Picrorhiza kurrooa: Androsin prevents allergen and PAFinduced bronchial obstruction in guinea pigs. International Archives of Allergy and Immunology, 95: Dwivedi, A.K., Chaudry, M., Seth, R.K., Sarin, J.P.S Simultaneous micro determination of Picroside-I and kutkoside in Picroliv (kutkin) by High Performance Liquid Chromatography. Indian Journal of Pharmaceutical Sciences, Kumar, P Medicinal plants in India: Conservation and sustainable utilization in the emerging global scenario. Bishen Singh Mahendra Pal Singh, Dehradun, India. Langer, J.G., Gupta, O.P., Atal, C.K Clinical trials Picrorhiza kurrooa as immunomodulator. Indian Journal of Pharmacology, 13: Malaisamy, A., Ravindran, C Medicinal plants: Where do we stand globally? Science Tech Entrepreneur Magazine, 2(5): Nayar, M.P., Sastri, A.R.K Red data plants of India. CSIR Publication, New Delhi, p 271. Rajaram, D A preliminary clinical trial of Picrorhiza kurrooa in bronchial asthma. Bombay Hospital Journal, 18: Sood, H., Chauhan, R.S Biosynthesis and accumulation of a medicinal compound, Picroside-I, in cultures of Picrorhiza kurroa Royle ex Benth. Plant Cell Tissue and Organ Culture, 100: Subrat, N Ayurvedic and herbal products industry: An overview. Paper presented at the Workshop on Wise Practices and Experimental Learning in the Conservation and Management of Himalayan Medicinal Plants. Kathmandu, Nepal, Uniyal, A., Uniyal, S.K., Rawat, G.S Commercial Extraction of Picrorhiza kurrooa Royle ex Benth. in the Western Himalaya. Mountain Research and Development, 31(3): Uniyal, A., Uniyal, S.K Distribution, Status and Conservation of Picrorhiza kurrooa in the Himalayan region. In: Rawat GS, Editor. Special Habitats and Threatened Plants of India, Wildlife Institute of India, Dehradun, India, 11(1). Ved, D.K., Goraya, G.S Demand and Supply of Medicinal Plants in India, Bishen Singh, Mahendra Pal Singh, Dehra Dun & FRLHT, Bangalore, India. Yegnarayan, R., Dange, S.V., Vaidya, S.D., Balwani, M Study of Picrorhiza kurrooa (PK300) in case of bronchial asthma. Bombay Hospital Journal, 24:

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