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2 RESEARCH ARTICLE Impact Factor (2014) by International Scientific Indexing (ISI) UAE A STUDY OF VARIOUS MARKET SAMPLES OF PICRORHIZA/ HELLEBORE (Picrorhiza kurroa Royle Ex Benth) WITH SPECIAL REFERENCE TO ITS PHARMACOGNOSTIC & PHYTOCHEMICAL ASPECTS Das Sangram Keshari 1*, Mohanty Bishnupriya 2, Pradip Kumar Panda 3, Joshi Arun B. 4, Binorkar Sandeep V Associate Professor & Head, Dept. of Dravyaguna vijnana, Gomantak Ayurveda Mahavidyalaya & Research Centre, At/Po- Shiroda, Dist- North Goa, Goa, India Contact No , - skeshari@ymail.com 2. Professor & Head, Dept. of Sanskrit Samhita Siddhanta, Gomantak Ayurveda Mahavidyalaya & Research Centre, At/Po- Shiroda, Dist- North Goa, Goa, India Professor & Head, Department of Nidana, Govt. Ayurveda College, Bolangir, Odisha. 4. Professor & Head, Dept of Pharmacognosy,Goa college of Pharmacy, Govt of Goa, Panaji, Goa. 5. Assistant Professor, Dept. of Agadatantra & Vyavahar Ayurveda, Government Ayurveda College, Vazirabad, Nanded , (MS), Contact No , dr.sandeepb@gmail.com Article Received on - 19 th Jan 2015 Article Revised on - 3 rd Feb 2015 Article Revised on - 14 th Feb 2015 Article Accepted on - 23 rd Feb 2015 All articles published in IJAAM are peer-reviewed and can be downloaded, printed and distributed freely for non commercial purpose (see copyright notice below). (Full Text Page IJAAM This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( which permits unrestricted non commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

3 RESEARCH ARTICLE A STUDY OF VARIOUS MARKET SAMPLES OF PICRORHIZA/ HELLEBORE (Picrorhiza kurroa Royle Ex Benth) WITH SPECIAL REFERENCE TO ITS PHARMACOGNOSTIC & PHYTOCHEMICAL ASPECTS *Corresponding Author Das Sangram Keshari Associate Professor & Head, Dept. of Dravyaguna vijnana, Gomantak Ayurveda Mahavidyalaya & Research Centre, At/Po- Shiroda, Dist- North Goa, Goa, India Contact No , - skeshari@ymail.com QR Code IJAAM ABSTRACT: Picrorhiza / Hellebore is a well known herb in Ayurvedic system of medicine mentioned by Charaka Samhita and Vagbhatta in the name of Katuka, Katurohini, Katwi, Tikta, Tiktarohini etc. Rhizomes with root are useful parts in medicine. Botanical source is Picrorhiza Kurroa Royle Ex Benth & belongs to family Scrophulariaceae. Rhizomes are bitter, acrid, cooling, laxative, carminative, antiinflammatory and hence useful in constipation, gastric disorders, flatulence, hiccough, fever, inflammation, diseases of spleen and liver including jaundice, anemia, hemorrhoids etc. Its demand in market increases with about 2.9% of average growth rate. However Picrorhiza has included under negative listed drugs. Therefore its export of plant parts, other derivatives and extracts as such is prohibited. But its prepared formulation can be exported. Market supply is less according to demand so it is substituted and adulterated with many other species. These substitutes and adulterants have very less potency. Practice of such medicine never shows the efficacy and causes failure in health care system. Therefore a study has been conducted by procuring different market samples of Picrorhiza. Then these were analyzed on basis of Pharmacognostic & Phytochemical parameters. The reported data also compared with standard parameters of Ayurvedic Pharmacopoeia. Key Words: Picrorhiza/ Hellebore, Picrorhiza kurroa, Market Sample, Pharmacognostical study & Phytochemical study. INTRODUCTION: In Brihatrayi (Charaka Samhita, Susruta Samhita & Astanga Samgraha) and other classics mentioned Picrorhiza useful for constipation, flatulence, colic, anorexia, inflammation, burning sensation, skin diseases, fever, diabetes, spleen and liver problems including anemia, jaundice, hemorrhoids and general debility etc. Rhizomes and roots are used in medicinal formulations. Its botanical identity is Picrorhiza Kurroa Royle Ex Benth & family is Scrophulariaceae. Rhizome contains Kutkinol, Kutikisterol, D-manitol, Kutkin, Picroside-I, Picroside-II, Picroside-III, Kutkoside, Picrorhizin, Cucurbitacin, Apocyanin, Minecoside and Verinicoside etc. This drug is a small perennial herb found in alpine Himalayas from Kashmir to Sikkim. In Himachal Pradesh it is found in Dainasar, Thamsar, Bara & Chota bhangal in Kangra district, Pangi Bharmour of Chamba district, Rohtang pass in Kullu, Shimla districts etc. especially in exposed rocky slopes. Manual harvesting of the plant takes place in October to December. This plant is self regenerating but unregulated over harvesting has caused it to be threatened to near extinction. Insignificant commercial cultivation along with the decline natural resources, increased therapeutic applications create an imbalance in between the demand and supply of Picrorhiza. This prepared a platform for intentional practice of substitution and adulteration. Therefore a study has been conducted on different market samples of Picrorhiza were analyzed on basis of Pharmacognostic & Phytochemical parameters. The reported data also compared with standard parameters of Ayurvedic Pharmacopoeia. [1-7] Characteristic of Picrorhiza The fresh and commercial samples of rhizome are sub-cylindrical, straight, usually apical buds, which are surrounded by a tufted crown of leaves.the roots are thin, cylindrical in pieces, 5 to 10 cms long, 0.5 to1.0 mm in diameter, slightly curved with a few longitudinal wrinkles dotted scars and dusky grey in color. Fracture is tough, inner surface black with white wood. The odor is pleasant & the taste is bitter. SUBSTITUTE & ADULTERANTS: - Picrorhiza is substituted with several other drugs like Gentiana kurroo Royle, Gentiana decumbens Linn., Gentiana tenella Fries and Hellebours niger Linn etc. It also adulterated with Picrorrhiza scrophularia Pennell, Actaea spicata, Cimcifuga foetida, Coptis teeta, Coscinium fanestratum, Page34

4 Swertia chirata and Latotis cashmiriana, Royle ex Benth etc. [8-12]. MATERIALS & METHODS:- Four different market samples were purchased from the markets of Vadodara, Delhi, Belgaum & Kolkata. These four samples were named as Sample A, Sample B, Sample C & Sample D respectively. All the Pharmacognostic & Phytochemical studies were carried out in Department of Pharmacognosy, Goa College of Pharmacy, Panaji, Goa as per the standard procedures. [13-18] Organoleptic characteristics:- The organoleptic characters of Picrorhiza were analyzed on basis of criteria i.e, Physical appearances, Color, Smell &Taste etc. Table No-1: Organoleptic examination Character Sample-A Sample-B Sample-C Sample-D Sub cylindrical, Sub cylindrical, curved Cylindrical pieces curved Cylindrical pieces curved Slightly curved rough pieces longitudinal longitudinal wrinkles and longitudinal wrinkles and due to longitudinal wrinkles. Roots are Physical annulations at the tip. annulations at the tip. Roots wrinkles, circular longitudinally appearances Roots are longitudinally are longitudinally wrinkles scars and scales are wrinkles with wrinkles with transverse with transverse cracks, grey present in root and transverse cracks, light cracks, grey brown in color. brown in color. dark brown color. grey in color. Color Rhizomes are grayish brown externally. Rhizomes are deep grayish brown externally and blackish internally. Rhizomes are yellowish brown externally. Rhizomes are pale grayish brown externally and blackish internally. Smell Slightly unpleasant. Slightly unpleasant. Unpleasant. Unpleasant. Taste Bitter. Much bitter. Much bitter. Bitter. Common identification tests for Picrorhiza:- 1. Powder treated with NaOH in methanol gives brownish under UV light. 2. Powder treated with NaOH in methanol and mounted in nitrocellulose gives yellowish green florescence. 3. Powder mounted in nitrocellulose gives dirty green. 4. Powder as such gives dirty green. Table No-2: Common identification of all Samples Test Sample-A Sample-B Sample-C Sample-D Powder treated with NaOH in methanol Slightly brownish Brownish green Deep brownish Light brownish Powder as such Light green Dirty green Dirty green Slightly dirty green Powder mounted in Dirty green Slightly dirty green Dirty green Dirty green Nitrocellulose Treating powder in NaOH in methanol and mounted in nitrocellulose Light yellowish Yellowish green Light yellowish green Yellowish green In Physical constant values, quantitative standards like-foreign matter, total percent of moisture, total ash, acid insoluble ash, water soluble ash, water soluble extractive, alcohol soluble extractive, total percentage of volatile oil, specific gravity of aqueous extractive value, ph of aqueous extractive value, total percentage of resin etc. were determined. Qualitative chemical tests were done for aqueous extracts to analyze & identification of the phytoconstituents present in it. Study for carbohydrate, starch, proteins, steroids, alkaloids, tannins & phenolic compounds, fat, glycosides, flavonoids, saponin, gum, mucilage & volatile oil etc. were done. Chemical tests for detection of inorganic matters were conducted for iron, chloride, sulphate, calcium, magnesium, aluminium, sodium & potassium. Page35

5 Table No-3:Physico- chemical analysis Name of the Test Sample-A Sample-B Sample-C Sample-D Standard Pharmacopoeia Value (%) Foreign matter NMT 2% Total % of moisture NMT 4% Total ash value NMT 7 % Acid insoluble ash value NMT 1% Water soluble ash value Water soluble extract value NLT 20 % Alcohol soluble extractive value NLT 10% Total % of volatile oil w/v Specific gravity of aqueous extractive value ph of aqueous extractive value Total % of resin Table No-4: Preliminary Phytochemical study Test for Sample-A Sample-B Sample-C Sample-D Carbohydrates (Molish s test) Positive Positive Positive Positive Reducing test (Fehling s test) Positive Positive Negative Negative Reducing test (Benedict s test) Negative Positive Negative Positive Tannic test for starch Negative Negative Negative Positive Proteins (Biuret test) Negative Positive Positive Positive Proteins (Million s test) Negative Positive Positive Positive Steroids(Liebermann-Burchard s reaction) Negative Negative Negative Positive Steroids (Salkowski) reaction Negative Negative Negative Positive Alkaloids (Dragendorff s test) Negative Negative Negative Negative Alkaloids (Mayer s test) Negative Negative Negative Negative Tannin and phenolic compounds(fecl3) Negative Negative Negative Negative Tannin&phenolic comp.(lead acetate) Negative Negative Negative Negative Fat Negative Negative Negative Negative Glycosides(Legal s test ) Positive Positive Positive Negative Glycosides(Liebermann-Burchard ) Positive Positive Positive Negative Flavonoids(Shinoda test) Negative Positive Positive Positive Flavonoids(lead acetate solution) Negative Positive Positive Positive Saponin (Foam test) Negative Negative Negative Negative Saponin(Haemolytic test) Negative Negative Negative Negative Gum Negative Negative Negative Negative Mucilage(Ruthenium red solution) Negative Negative Negative Negative Mucilage(KOH) Negative Negative Negative Negative Volatile oil (Sudan III solution) Negative Negative Negative Negative Volatile oil ( Tincture alkana) Negative Negative Negative Negative Table No-5: Chemical Test For Detection Of In-Organic Constituents Test For Sample-A Sample-B Sample-C Sample-D Iron Negative Negative Positive Negative Chloride Positive Negative Negative Negative Sulphate Negative Negative Negative Negative Calcium Negative Positive Negative Positive Magnesium Negative Negative Positive Negative Aluminium Negative Negative Negative Negative Sodium Negative Negative Negative Negative Potassium Negative Negative Negative Negative Page36 RESULT & DISCUSSION:- Raw material samples of Picrorhiza were purchased from four different markets i.e Vadodara, Delhi, Belgaum & Kolkata have variations in physical appearances. The rates of samples were also different. The samples purchased from Delhi (sample B) satisfy all the physical constant values/standards of Ayurvedic pharmacopoeia of India. Macroscopic and Organoleptic observations:- Rhizome of Sample A was sub cylindrical, slightly curved & rough due to longitudinal wrinkles. It was having circular scars of dark brown color root and scales; rhizomes were grayish brown externally; slightly unpleasant in smell and bitter in taste.

6 Rhizome of Sample B was morphologically cylindrical, curved longitudinal wrinkles and having annulations at the tip. Grey brown color roots having longitudinal wrinkles with transverse cracks were attached to the rhizomes. Rhizomes were deep grayish brown in color externally and blackish internally; slightly unpleasant in smell and much bitter in taste. Sample C was sub cylindrical, curved having longitudinal wrinkles. Light grey color roots which were attached to it also having longitudinal wrinkles with transverse cracks. Rhizomes were yellowish brown in color externally; unpleasant in smell and much bitter in taste. Rhizome of Sample D was cylindrical, curved, having longitudinal wrinkles and annulations at the tip. Grey brown color roots were having longitudinal wrinkles with transverse cracks. Rhizomes were pale grayish brown color externally and blackish internally, unpleasant in smell and bitter in taste. These suggest that Sample-B procured from Delhi was more genuine and not adulterated. Common identification test :- Brownish green color obtained after the powder treated with NaOH in methanol, dirty green color in powder as such, dirty green obtained when powder mounted in Nitrocellulose; yellowish green color obtained when powder treated in Na OH in methanol and mounted in nitrocellulose. These observations made from Sample B which shows maximum genuinity. On Physico-chemical analysis:- Less percentage of foreign matter was obtained in Sample B i.e 2.2% which is very near to the standard pharmacopeial limit i.e. 2%.The values were gradually higher in other three samples like 2.8%, 3.5 % & 4 % respectively in D, A & C. Moisture content in Sample B was less i.e. 7.8 % in comparison to others; where as the standard pharmacopeial limit is not more than 4 %. The values were gradually higher in (comparison to the standard limit) other three samples like 8.6%, 9.5 % & 10.2 % respectively in C, D & A. The values of total ash were observed within standard pharmacopeial limit in all four samples i.e, 5.8 %,4.2 %, 4.7 % & 6.6 % respectively in Sample A,B,C and D. The standard pharmacopeial limit is not more than 7 %. The values of acid insoluble ash in all four samples were nearly close to the standard pharmacopeial limit i.e, not more than 1 %. The values of water soluble ash were 3.8 %, 4.1%, 2.8 % & 2.4% respectively in Sample A, B, C & D. Sample B was having higher value. The standard pharmacopeial limit of water soluble extract is not less than 20 %. Higher value of water soluble extract was obtained in Sample B i.e 78.8 % in comparison to others. Alcohol soluble extractive value was higher in Sample B i.e 67.6 %; in comparison to others. The standard pharmacopeial limit is not less than 10 %. Volatile oil and resin were totally absent in all four samples. Specific gravity of aqueous extractive value was higher in Sample B i.e % in comparison to others. ph of aqueous extractive value was higher in Sample B i.e, 5.8 %. Above analysis obtained data shows the maximum genuinity in Sample B which was procured from Delhi. On Preliminary phytochemical screening:- Positive result observed in Sample B under the test like test for carbohydrates by Molish s test, test for reducing sugar by Fehling s test / Benedict s test, proteins by Biuret test / Million s test, glycosides by Legal s test / Liebermann-Burchard test and test for Flavonoids by Shinoda test / lead acetate solution. This Sample B shows negative result in Tannic test for starch, test for Steroids. By Liebermann- Burchard s / Salkowski reaction, test for alkaloids by Dragendorff s / Mayer s test, test for Tannin and phenolic compounds with FeCl3 / lead acetate solution, test for fat, test for saponin by Foam test / Haemolytic test, test for gum, test for mucilage with ruthenium red solution/ KOH and test for volatile oil with Sudan III solution or tincture alkana.the results obtained in other three samples i.e, A, C & D were also merely same as per the textual reference. But Sample B is comparatively posses maximum genuinity. On Chemical Test For Detection Of In-Organic Constituents :- Negative result observed in all tests under this phytochemical study except test for chloride in Sample A. Sample B shows negative result in all tests except test for calcium. In Sample C negative result observed in all tests except test for iron and Magnesium. Sample D was having negative result in all tests except test for calcium. This does not show any significant difference among the procured Samples. Page37

7 CONCLUSION:- This study gives clear evidence about the practices of adulteration in available market samples of Picrorhiza. When such raw material used in medicinal preparations it will have less efficacy to combat the diseases. It may produce several health problems. Further studies for adulteration can be confirmed by TLC, HPTLC techniques. AKNOWLEDGEMENTS:- Authors are thankful to the Principal & all the concerned staff of Dept of pharmacognosy, Goa college of Pharmacy, Panaji, Goa for providing Pharmacognosy Lab.& other necessary facilities. REFERENCES:- 1. Billore K.V, Database of Medicinal plants used in Ayurveda, CCRAS, New Delhi, Vol. VII, 2005; p Chopra R.N., Chopra s Indigenous drug of India, 2nd reprint, Academic publishers, Kolkata, 1994, p Raghunathan K, Pharmacognosy of indigenous drugs; Vol.I, reprint, CCRAS; New Delhi, 1999: p Bhāvaprakāśa Nighaṅtu, Ȃchārya Bhāvamiśra, Hindi commentary by G.S. Pandey, 6th edition, Reprint 2006, Chaukhambha bharati academy, Varanasi : p Pharmacological investigation of certain medicinal plants used in Ayurveda and Siddha, edited by Dr. S.C.Malhotra, 1st edition 1996, CCRAS, New Delhi: p Rastogi R.P., Compendium of Indian medicinal plants, Vol. II, , CSIR publication, New Delhi ; p Satyavati G.V., Indian medicinal plants, ICMR Publications, New Delhi, Vol. II: p Gogte V.M, Ayurvedic pharmacology and therapeutic uses of medicinal plants, Bharatiya vidya bhavan, Mumbai, 1st English edition, 2000; p Bapalal Vaidya, Nighantu Adarsa, Chaukhambha Bharati Academy, Varanasi, 1st edition 1994, Vol. I; p Misra Banwarilal, Dravyaguna Hastāmlaka, Hindi, edited by Vd. S.K.Sharma, Publication scheme, Jaipur, revised 4th edition, 2004, p. 207, Handa S.S, Pharmacognosy, Vallabh Prakashan, Delhi, 2nd edition, 11th reprint, 2000; p Warrier.P.K, Indian medicinal plants, Vol. IV, reprint 1996, Orient and Longman Ltd, Madras; p Iyengar M.A, Pharmacognosy, 8th edition, 1995, Manipal power press: p Wallis T.E, Textbook of Pharmacognosy, 5th edition, Reprint 1999, CBS Publishers and distributors, New Delhi; p Kokate C.K., Pharmacognosy, 39th edition, 2007, Nirali publication, Pune, p The Ayurvedic pharmacopoeia of India, part 1, Vol. II, 1st edition, New Delhi, Govt. of India, Ministry of health and family welfare, Dept. of Indian system of medicine, 1986: p Gokhale S. B., Practical Pharmacognosy, Nirali Prakashan, Pune, 12th edition, 2008: p Khandelwal K.R, Practical pharmacognosy techniques and experiments, 22nd edition, 2012, Nirali Publication, Pune, p. 23.2, CITE THIS ARTICLE AS Das Sangram Keshari et.al., A Study of Various Market Samples of Picrorhiza/ Hellebore (Picrorhiza Kurroa Royle Ex Benth) with Special Reference to its Pharmacognostic & Phytochemical Aspects, Int. J. Ayu. Alt. Med., 2015; 3(1):33-38 Source of Support Nil Conflict of Interest None Declared Page38

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