Title of Invention

A SIMPLIFIED PROCESS FOR PURIFICATION OF KUTKIN FROM PICRORHIZA KURROA ROYALE EX BENTH

Abstract The present invention relates to development of a simplified process for purification of kutkin from Picrorhiza kurroa Royle ex Benth. Particularly, this invention relates to separation of kutkin (also known as picroliv R) from Picrorhiza kurroa underground parts by aqueous alcohol extraction, solvent - solvent partition, treatment with activated charcoal to remove colouring matters. The processing steps of the invention results into 2.0-3.0% yield. The process gave higher recovery of picroliv from the extract and 3.2% from plant powder (dry basis).
Full Text The present invention relates to development of a simplified process for purification of kutkin from Picrorhiza kurroa Royle ex Benth.
Particularly this invention relates to separation of kutkin (also known as picrolivR) from Picrorhiza kurroa underground parts by aqueous alcohol extraction, solvent - solvent partition, treatment with activated charcoal to remove colouring matters.
More particularly this invention relates to purification of kutkin from Picrorhiza kurroa in 2.0-3.0% yield.
The main usage of the present invention is the isolation of kutkin from Picrorhiza kurroa by a simple and convenient process.
Another usage of the present invention is the scope of commercial production of kutkin for various pharmaceutical preparations related to liver ailments.
Another usage of the present invention is complete and non-selective extraction of picroside-l and II, being major bioactive ingredients in kutkin (picrolivR) from the extract and no differential extraction was observed.
Genus Picrorhiza Royle (Scrophulariaceae) is endemic to the Himalayan region of Pakistan, Nepal, India and China. In India, P. kurroa is distributed from Kashmir to Sikkim at altitudes ranging from 2700 to 5000 meters. The common trade and vernacular name of the herb is Kutki Unregulated over-harvesting has caused it to be threatened to near extinction (Chandra PK. 2000. Status and conservation of rare and endangered medicinal plants in the Indian trans-Himalaya. Biolog. Conserv., 93: 371-379). Root extracts of P. kurroa have been used to treat liver disorders (Handa SS, Sharma A, Chakarvarti KK. 1986. Natural products and plants as liver protective drugs, Fitoterapia, 57:167-171), brought about by the ever increasing environmental pollution, exposure to industrial toxicants, food adulteration, malnutrition, injudicious use of drugs, excessive consumption of alcohol and certain infections (Gupta PP. 2001. Picroiiv. Drugs of future, 26(1): 25-31). Such disorders pose a major challenge in the health care world over. Presently, no effective therapy is available for the treatment of various liver ailments mentioned above.
Kutkin (sometimes referred to as PicrolivR) is a standardized bioactive fraction of P. kurroa with proven hepatoprotective, choleretic, anticholestatic, anti-hepatitis B virus and immunostimulant activities (Rastogi R, Srivastava AK, Srivastava M, Rastogi AK. 2000. Hepatoprotective effect of Picrorhiza and Silymarin against aflotoxin B1 induced hepatotoxicity in rats, Planta Medica, 66: 709-713; Vaidya AB, Antarkar DS, Doshi JC, Bhatt AD, Ramesh V, Vora PV, Perissond D, Baxi AJ, Kale PM. 1996. Picrorhiza kurroa (Kutki) Royle ex Benth. As a Hepatoprotective agent- experimental and clinical studies, J. Postgrad. Med., 42(4): 105-108.; Visen PKS, Saraswat B, Dhawan BN. 1998. Curative effect of Picroliv on primary cultured rat hepatocyres against different hepatotoxins: An in vitro study, Jour. Pharmaco. and Toxicolo. Meth., 40: 173-179; Anandan R, Devaki T. 1999. Hepatoprotective effect of Picrorhiza kurroa on tissue defence in D-galactosamine-induced hepatitis in rats, Fitoterapia 70: 54-57; Ansari RA, Aswal BS, Chander R, Dhawan BN, Garg NK, Kapoor NK, Kulshreshtha DK, Mehdi H, Mehrotra BN, Patnaik GK, Sharma SK. 1988. Hepatoprotective activity of kutkin-the iridoid glycoside mixture of Picrorhiza kurroa, Indian J. Med. Res. 87: 401-404; Ansari RA, Tripathi SC, Patnaik GK, Dhawan BN. 1991. Antihepatotoxic properties of picroliv: an active fraction from rhizome of Picrorhiza kurroa, J. Ethnopharmaco. 34: 61-68.), antioxidant (Russo A, Izzo AA, Cardile V, Borrelli F, Vanella A. 2001. Indian medicinal plants as antiradicals and DNA cleavage protectors, Phytomed., 8 (2): 125-132; Chander R, Kapoor NK, Dhawan BN. 1992. Picroliv, picroside-l and kutkoside from Picrorhiza kurroa are scavengers of superoxide anions, Biochem. Pharmaco., 44 (1): 180-183; Worm E van der, Beukelman CJ, Berg AJJ, van der Kroes BH, Labadie RP, Dijk H van. 2001. Effect of methoxylation of apocynin and analoges on the inhibition of reactive oxygen species promotion by stimulated human neutrophiles, Europ. J. Pharmaco., 433: 225-230), anti-allergy and anti-anaphylactic activity (Baruah CC, Gupta PP, Nath A, Patnaik GK, Dhawan BN. 1998. Anti-allergic and anti-anaphylactic activity of Picroliv-A standardized iridoid glycoside fraction of Picrorhiza kurroa,
Pharmaco. Research, 38 (6): 487-492) anti-inflammatory (Singh GB, Bani S, Singh S, Khanuja K, Sharma ML, Gupta BD, Banerjee SK. 1993. Antiinflammatory activity of the irridoids, kutkin, Picroside-I and Kutkoside from Picrorhiza kurroa, Phytoth. Res., 7:402-407), immunomodulator (Langer JG, Gupta OP, Atal CK. 1981. Clinical trials P. kurroa as immunomodulator. Indian J. Pharmaco., 13: 98-99), anti-asthamatic (Dorsch W, Stuppher H, Wagner H, Gropp M, Demolin S, Ring J. 1991. Antiasthamatic effect of Picrorhiza kurroa. Androsin prevents allergen and PAV induced bronchial obstruction in guinea pig. Int. Arch. All. Appl. Immunol., 95:128-33) and antitumor activity (Kapadia GJ, Sharma SC, Tokuda H, Nishino H, Ueda S. 1996. Inhibitory effect of iridoids on epstein-barr virus activation by a short term in vitro assay for anti tumor promoters, Cancer Letters, 102: 223-226). The hepatoprotective activity of picroliv has been evaluated against hepatic damage induced by various agents such as galactosamine, paracetamol, thioacetamide, carbon tetrachloride, lanthanum chloride, monocrotaline, ethyl alcohol, aflatoxin, Amanita phalloides toxin, cycloheximide in rats (Gupta PP. 2001. Picroliv. Drugs of future. 26(1): 25-31; Rastogi R, Srivastava AK, Srivastava M, Rastogi AK. 2000. Hepatoprotective effect of Picrorhiza and Silymarin against aflotoxin B1 induced hepatotoxicity in rats, Planta Medica, 66: 709-713; Kumar SHS, Anandan R, Devaki T, Kumar MS. 2001. Cardioprotective effect of Picrorhiza kurroa against isoproterenol-induced myocardial stress in rats, Fitoterapia, 72: 402-405; Saraswat B, Visen PKS, Patnaik GK, Dhawan BN. 1999. Ex vivo and in vivo investigations of picroliv from Picrorhiza kurroa in an alcohol intoxication model in rats, J. Ethnopharmaco., 66:263-269; Anandan R, Devaki T. 1999. Hepatoprotective effect of Picrorhiza kurroa on tissue defence in D-galactosamine-induced hepatitis in rats, Fitoterapia, 70: 54-57). The LD5o of picroliv by i p. route in mice was found to be 2026.9 mg/kg By oral route it was 2500 mg/kg in mice in mice and rats. In subacute toxicity study over 90 days, picroliv was found to be safe in rats and monkeys (Aswal BS, Chander R, Chatterji SK, Dhawan BN, Dwivedi Y, Garg NK, Jain P, Kapoor NK, Kulshreshtha DK, Mehrotra BN,
Sarin JPS, Saxena KC, Patnaik GK, Rastogi R, Shekhar C, Sharma KC,
Sharma SK, Shukla B, Visen PKS. 1992. Process for preparation and
composition of a fraction containing picroside I and kutkoside, USP
5,145,955) of the minor components, apocynin has been shown to inhibit
neurophil oxidative burst in addition to being a powerful anti-inflammatory agent
(Worm E van der, Beukelman CJ, Berg AJJ, van der Kroes BH, Labadie
RP, Dijk H van. 2001. Effect of methoxylation of apocynin and analogues on
the inhibition of reactive oxygen species promotion by stimulated human
neutrophiles, Europ. J. Pharmaco., 433: 225-230), and the curcubitacins to be
highly cytotoxic (Smit HF, van der Berg AJJ, Kroes BH, Beukelman CJ,
Quarles van Ufford HC, van Dijk H, Labadie RP. 2000. Inhibition of T-
lymphocyte proliferation by cucurbitacin from Picrorhiza
scrophulariaeflora, J. Natu. Prod., 63: 1300-1302). P. kurroa contains iridoid
glycosides (1-10) (Stuppner H, Wagner H. 1989. Minor iridoids and phenol
glycosides of Picrorhiza kurroa, Planta Medica, 55: 467-469; Stuppner H,
Wagner H. 1992. TLC and HPLC analysis of iridoids, cucurbitacins and
phenols glycosides from Picrorhiza kurroa, Scientia Phartnaceutica (Sci.
Pharm.), 60: 73-85; Jia Q, Hong MF, Minter D. 1999. Pikuroside: A novel
iridoid from Picrorhiza kurroa, J. Natu. Prod., 62: 901-903) cucurbitacins
glycosides (Stuppner H, Muiler EP. 1993. Cucurbitacins with unusual side
chain from Picrorhiza kurroa, Phytochem., 33 (5): 1139-1145; Stuppner H,
Wagner H. 1992. TLC and HPLC analysis of iridoids, cucurbitacins and
phenol glycosides from Picrorhiza kurroa, Scientia Pharmaceutica (Sci.
Pharm.) 60: 73-85) and phenolic compounds (Stuppner H, Wagner H. 1989.
Minor iridoids and Phenols glycosides of Picrorhiza kurroa, Planta Medica,
55: 467-469). Picroliv is a mixture of 50-70% of two iridoid glycosides, picroside-l
(6'-0-trans-cinnamoylcatalpol) and kutkoside (10-O-vanilloylcatalpol) present in a
ratio of 1:1.5 to 1:2, 4-5% of a mixture of cucurbitacins along with several other
uncharacterized substances (Aswal BS, Chander R, Chatterji SK, Dhawan BN,
Dwivedi Y, Garg NK, Jain P, Kapoor NK, Kulshreshtha OK, Mehrotra BN,
Sarin JPS, Saxena KC, Patnaik GK, Rastogi R, Shekhar C, Sharma KC,
Sharma SK, Shukla B, Visen PKS. 1992. Process for preparation and composition of a fraction containing picroside I and kutkoside, USP 5,145,955; Singh B, Rastogi RP. 1972. Chemical examination of Picrorhiza kurroa Benth: part VI- Reinvestigation of Kutkin, Ind. J. Chem., 10: 29-31).
A medicinal preparation obtained from the rhizomes and roots in chloroform or methanolic extract of P.kuiroa and its method for manufacture and also to a method for treatment of human beings for relieving constipation has been reported (Mitra SK, Babu UV, Ranganna MV. 2001. Herbal laxative preparation, USP Appl. No. 20020150639). The process involves selective extraction of the active extract from rhizomes or roots of P. kurroa using selective solvents The laxative preparation is gentle, safe and effective, free of undesirable side effects and capable of relieving severe constipation. Importantly serves as a herbal laxative for humans on a commercial scale. The laxative can be administered in various dosage forms as may be applicable.
A patent application for a food supplement formulation effective for improving the function of the liver comprises selenium, milk thistle seed, phosphatidyl choline, dandelion root, 1-methionine, 1-taurine, N-acetyle-cysteine, alpha lipoic acid, artichoke leaf, green tea leaf, turmeric root, belleric myrobaian fruit, Boerhavia diffusa, Eclipta alba, waldelactones, Tinospora cordifolia, Andrographis paniculata, and Picrorhiza kurroa has been filed. (Watson BF, Smith LO. 2002. Liver function improvement formulation, USP Appi. No. 20030044512)
In another invention for the treatment of hepatotoxicity in mammals and humans, a synergistic pharmaceutical composition comprising an effective amount of trans-tetracos-15-enoic acid (TCA) and apocynin (APO) has been reported. Apocynin is one of the active constituents of P. kurroa. (Handa SS, Suri OP, Gupta VN, Suri KA, Satti NK, Bhardwaj VSB, Chandan BK. 2002. Synergistic composition of trans-tetracos-15-enoic acid and Apocynin and method of treatment for hepatotoxicity, USP Appl. No. 20030157198).
Another composition for the treatment of osteoarthritis contains both apocynin and an inhibitor of inducible nitric oxide synthase such as curcumin has
been recently reported whereas other components such as boswellic acids, glucosamine, acetylcysteine and boron are reported to further enhance the beneficial effect of apocynin and curcumin (Graus IMF, Smit HF. 2002. Composition for the treatment of osteoarthritis, USP No. 6,492,429).
A novel polyherbal composition comprising essentially extracts of plants Rheum emodi Wall., Phyllanthus amarus Linn., Eclipta alba Hassk, Andrographis paniculata Nees, and Picrorhiza kurroa Royle ex Benth., and optionally Fumaria officinalis, Tinospora cordifolia Miers., Terminalia chebula Rets., Ochorium intybus Linn., Tephrosea purpurea and Boerhavia diffusa Linn, used for treating acute Hepatitis E, virus infection including acute liver failure due to HEV infection, healthy Hepatitis B virus carriers who develop superadded hepatitis E virus infection, acute hepatitis B virus infection, and animal hepadna virus, therapeutic effects on hepatitis B virus infection is used as a hepatoprotective agent (Mehrotra RK, Chandra K, Gupta AP. 2000. Hepatoprotective compositions and composition for treatment of conditions related to hepatitis B and E infection, USP No. 6,136,316).
An ayurvedic composition for prophylaxis and treatment of AIDS, flu, TB and other immuno-deficiency conditions, and for liver diseases such as hepatitis and sclerosis, includes prescribed doses of extracts or isolates of two multi-components drugs in selected proportions The first multi-component drug, LIVZON, consists of Phyllanthus niruri, Tinospora cordifolia, Phyllanthus emblica, Terminalia belerica, and Terminalia chebula; the second multi-component drug, IMMINEX, consists of Holarrhena antidysenterica, Picrorhiza kurroa and Swertia chirata. The beneficial composition may be administered in the form of aqueous extracts, hard gelatin capsules, or mixed with syrup. (Rohatgi S. 1996. Ayurvedic composition for the prophylaxis and treatment of AIDS, flu, TB and other immuno-deficiencies and the process for preparing the same, USP No. 5,529,778).
A number of processes have been described for extraction of kutkin which are discussed below:
In the patent by Aswal et. al. (Aswal BS, Chander R, Chatterji SK, Dhawan BN, Dwivedi Y, Qarg NK, Jain P, Kapoor NK, Kulshreshtha DK, Mehrotra BN, Sarin JPS, Saxena KC, Patnaik GK, Rastogi R, Shekhar C, Sharma KC, Sharma SK, Shukla B, Visen PKS. 1992. Process for preparation and composition of a fraction containing picroside I and kutkoside, USP No. 5,145,955) four extraction processes of kutkin with different yields and varying percentages of picroside-l and kutkoside have been described. In all the processes, yield of the final product is low; these being multi-step complex processes and require a number of chemicals and solvents.
In another process (Singh GB, Bani S, Singh S, Khanuja K, Sharma ML, Gupta BD, Banerjee SK. 1993. Antiinflammatory activity of the irridoids, kutkin, picroside-l and kutkoside from Picrorhiza kurroa, Phytoth. Res. 7: 402-407; Singh B, Rastogi RP. 1972. Chemical examination of Picrorhiza kurroa Benth: part VI- Reinvestigation of Kutkin, Ind. J. Chem., 10: 29-31) chloroform insoluble fraction was dissolved in ethyl alcohol and precipitated with acetone and diethyl ether (1:1). The precipitate was filtered off and the process repeated till no further precipitation occurred. The combined precipitates were dissolved in hot methanol, treated with activated charcoal, filtered and solvent evaporated to yield white amorphous powder. Yields are given mtable-1.
(Figure Removed)
Processes A-E are the existing ones (Aswal BS, Chander R, Chatterji SK, Dhawan BN, Dwivedi Y, Garg NK, Jain P, Kapoor NK, Kulshreshtha DK, Mehrotra BN, Sarin JPS, Saxena KC, Patnaik GK, Rastogi R, Shekhar C, Sharma KC, Sharma SK, Shukla B, Visen PKS. 1992. Process for preparation and composition of a fraction containing picroside I and kutkoside, USP No. 5,145,955; Singh GB, Bani S, Singh S, Khanuja K, Sharma ML, Gupta BD, Banerjee SK. 1993. Antiinflammatory activity of the irridoids, kutkin, picroside-l and kutkoside from Picrorhiza kurroa, Phytoth. Res. 7: 402-407; Singh B, Rastogi RP. 1972. Chemical examination of Picrorhiza kurroa Be nth: part VI- Reinvestigation of Kutkin, Ind. J. Chem., 10: 29-31) whereas the process-F has been developed by us. The existing extracting processes do not extract picroliv completely and differential extraction of picrosides was also observed in all the processes (Fig. 1), therefore a need was felt to develop a process that gives better yield as well as ratio of picrosides present in the extract. A comparative study of extraction processes is given below
Table-1: Yield of different fractions of extraction processes A-F

(Figure Removed)
We analyzed kutkin and purity of isolated compounds by UV and light-scattering detectors (Sedex) Picroside-l and picroside-ll exhibit different UV
spectra and extinction coefficients. Detection at 270 nm was found to be more suitable wavelength and calibration graph using pure reference compounds was plotted The quantification of major compounds in P. kurroa has also been evaluated by HPLC using UV detector (Stuppner H, Wagner H. 1992. TLC and HPLC analysis of iridoids, cucurbitacins and phenols glycosides from Picrorhiza kurroa, Scientia Pharmaceutica (Sci. Pharm.) 60: 73-85).
In process A and B the lipophilic substances along with other impurities were removed by ethyl acetate and picroliv was extracted with 10% ethyl alcohol in ethyl acetate (process- A) and 15% methyl alcohol in ethyl acetate (process-B) Organic layer was further treated with chloroform followed by decolourization with active charcoal in process A. Picroside-I has more solubility in ethyl acetate resulting in low recovery of picroliv. Process-C involves removal of fats by chloroform and extraction of aqueous layer by ethyl acetate followed by decolourization by active charcoal. The recovery of picroliv was recorded as 18%. Since picroliv is not readily soluble in ethyl acetate, the complete extraction does not take place. In processes D and E, the extract was first dissolved in 10% aqueous acetone (process-D) and ethyl alcohol (process-E). The soluble part was precipitated with mixture of diethyl ether and acetone (1.1) and the process was repeated till no further precipitation occured. Picroliv dissolves in acetone and diethyl ehter and some part of it goes with precipitate resulting to low recovery.
Fig. 1 Differential extraction of picroside-l and picroside-ll
(Figure Removed)
We have developed a process whereby the extract was defatted with chloroform and the aqueous part was fractionated with butanol saturated with water The combined organic layer was then treated with activated charcoal to remove the colouring impurities. The process gave higher recovery of picroliv from the extract and 3.2% from plant powder (dry basis). From the bar chart it is clear that the process developed by us gives 80% recovery of picroside-l and picroside-ll. Table-1 above indicates the yield of different fractions of all the processes whereas from bar chart above illustrates the differetial extraction of picroside-l and picroside-ll
Further verification of quantitative estimation by UV was carried out using a light scattering detector (Commercial name: Sedex). It detects the organic molecule by mass only and hence provides a direct estimation of the major compounds in kutkin. UV detector works on UV radiation and is more sensitive than the light scattering detector However from the results obtained in this study indicate that it is equally good with the added advantage of detecting non-UV active compounds. Kutkin can also be analyzed by TLC (thin layer chromatography coated with silica gel) eluted with a mixture of chloroform and methanol (80.20).
The main object of the present investigation is to develop a simplified process for purification of kutkin from Picrorhiza kurroa.
Another object of the present invention is to develop a simple and convenient commercial
extraction process for kutkin, a component of pharmaceutical importance from Picrorhiza
kurroa.
Still another object of the present investigation is to provide a simple and convenient
process for the isolation of kutkin from Picrorhiza kurroa in high yield 2.0-3.0%.
Accordingly, the present investigation provides a simplified process for purification of kutkin from Picrorhiza kurroa Royle ex Benth which comprises ; (a) extracting dried and powdered underground parts of the plant with aqueous low molecular weight alcohols (methanol/ethanol and water, 9:1-8:2), (b) repeating step a) 6 to 8 times, (c) removing solvent and fractionating alcohol free extract 6 to 8 times with chloroform followed by n-butanol or isopropanol saturated with water , (d) washing n- butanol/isopropanol extract containing kutkin with water saturated with butanol/isopropanol and treating with activated charcoal, (e) concentrating organic layer to yield white amorphous powder of kutkin having mp 210-212°C.
In an embodiment of the present invention kutkin was obtained from Picrorhiza kurooa by drying, grinding and extracting of underground parts with water and ethyl/methyl alcohol mixture followed by removal of organic solvent and fractionation of aqueous layer with butanol/isopropanol saturated with water Treatment of organic layer (butanol/isopropanol fraction) with activated charcoal followed by filtration through sintered funnel. Distillation of organic layer to yield kutkin as white amorphous powder. In another embodiment of the present invention kutkin was obtained in purified form having mp 210-212°C.
The following examples are given by way of illustrations of the present invention and should not be construed to limit the scope of present invention.
Example 1
One kg of dried and powdered underground parts of P. kurroa were placed in a percolator and cold extracted with 1.5-2.0 litres an aqueous low molecular weight alcohol mixture such as methanol/ethanol in the ratio of 9:1-28 (V/V). The process of extraction was repeated 6 to 8 times. The extracts were combined and after removal of organic solvent under reduced pressure, the residue was successively fractionated with chloroform and n-butanol or isopropanol saturated with water for 6 to 8 times. Combined n-butanol layer containing kutkin was washed 2-3 times with water saturated with butanol/isopropanol and then treated with activated charcoal, filtered in a sintered funnel under vacuum All the fractionations were carried out in a separating funnel with gentle shakings to avoid formation of an emulsion. The filtrate was concentrated in a rotavapour under reduced pressure to yield a white, amorphous powder in 2 0-3 0% overall yield, mp 210-212°C.
Example 2 HPLC Analysis
HPLC analysis was carried out by using Beckmann System Gold 126 solvent module dual pump, Beckmann System Gold 166 programmable UV detector attached to Epson LQ-1050 printer. Other detector used was Sedex light scattering (S.E.D.E.R. France) attached to Gilson 712 HPLC software with H.P LaserJet 5550 printer. Light scattering detector was attached to the main unit by Beckmann Analog Interface Module 406. Solvent used was isocratic system, 70% B. Solvent A: methanol/acetonitrile (1.1) solvent B: water with TFA (0.05%). Flow rate of 1ml/min at 270 nm. Temperature used for light scattering detector was 40°C at 2 bar N2 pressure Phenomenex Synergy RP Polar 80A (150x4 6mmx4µ). HPLC column was used for all the analysis. In all the sample analyses similar conditions were used. Few milligrams of extract was dissolved in HPLC grade methanol and 20 µl of it was injected in HPLC. Quantitative analysis
Determination of picroside-l and II in plant samples from different processes was performed by external standard method using the purified samples of picroside-l and picroside-ll. Calibration curves were constructed using
0 1% solution of picroside-l and picroside-ll of six different concentrations (0.5,
1 25, 2.5, 3 75, 4.5, 5 /ag) in HPLC mobile phase. Each concentration was carried out in triplicate Peak areas in conjunction with the calibration curves were used to calculate the amount of picroside-l and II by applying the linear regression equation obtained from the calibration curves. The regression equation and correlation coefficients for picroside-l and picroside-ll were y = 29.1587x + 2.28260, y = 14.994x +2.547, 9974, 0.9956 for calibration curves by UV and those by light scattering were recorded as y= 33.891x -14 0328, y=31.836x -14 487, 0 9954, 0.9944 for picroside-l, picroside-ll respectively The coefficient of variation for injections ranged from 1 1 to 10%.
Example 3
Isolation and characterization of standard compounds
In the present study, we isolated picroside-l, picroside-ll, 6-
ferullyolcatalpol, apocynin, mannitol by dry, flash, reverse column
chromatography and Prep-HPLC. Defatted methanol extract (80%) of P. kurroa
roots and rhizomes was fractionated with ethyl acetate to yield the enriched
fraction of picroside-l and picroside-ll. In dry column chromatography extract was
coated on celite in 1 15 ratio. Coated extract in powder form was loaded on silica
gel H in a sintered funnel and eluted with hexane with increasing proportion of
chloroform, chloroform and increasing amount of methanol in chloroform. The
sub-fractions were concentrated under vacuum. Celite coating of extract gave 70-
80% purification. Second major spot, appeared as single spot on normal TLC
plate was further resolved into two spots on RP-TLC plate and was separated
into picroside-ll and 6-ferulloyl catalpol on RP flash chromatography. The final
purification of picroside-l and II (>95%) was achieved by preparative HPLC. The
column used was Lichro Prep RP18 5-400, mobile phase mixture of
methanolacetonitrile.water (3:25), flow rate 4 ml/min and injection volume 60 ml
of 20% solution at 270 nm in Beckmann HPLC. Purity of each sub-fraction was
monitored by analytical HPLC as per the conditions mentioned above. The
identification of these compounds was carried out by comparison of NMR (13C
and 1H) values with those reported in the literature and standard sample of
mannitol was purchased from Fluka chemical. (Stuppner H, Wagner H. 1989.
Minor iridoids and Phenols glycosides of Picrorhiza kurroa, Planta Medica,
55: 467-469; Stuppner H, Wagner H. 1992. TLC and HPLC analysis of
iridoids, cucurbitacins and phenols glycosides from Picrorhiza kurroa,
Scientia Pharmaceutics (Sci. Pharm.) 60: 73-85; Weings K, Kloss P, Dieter
Henkels W. 1972. Natural Products from Medicinal Plants XVII- Picroside-ll,
a new 6-vannilloyl catalpol from Picrorhiza kurroa Royal and Benth. Liebigs
Ann. Cham., 759: 173-182; Kitagawa I, Hino K, Nishimura T, Iwata E.,
Yoshika 1.1971. On the constitution of Picrorhia kurroa (1) The structure of
picroside-l, a bitter principle of subterranean Part, Chem. Pharm. Bull.
19(12): 2534-2544; Singh B, Rastogi RP. 1972. Chemical examination of
Picrorhiza kurroa Benth: part VI- Reinvestigation of Kutkin, ind. J. Chem.,
10:29-31). Silica gel H was used for dry chromatography and silica gel 40-63n
flash chromatography was used for flash chromatography. All the chemicals used
were of Fluka and Sigma grade. HPLC grade solvents were used for detection
and isolation by preparative HPLC. Standard Mannitol was procured from Fluka
chemicals.
The main advantages of the present invention are:
1. Kutkin is a natural hepatoprotective, used for preparation of other pharmaceutical product with no side effect and is safe and effective nutraceutical.
2. It provides a simple process for purification of kutkin from Picrorhiza kurooa.
3. It provides an easy and convenient commercial extraction process for kutkin, a component of pharmaceutical importance.
4 Picrorhiza kurooa plant grows wild in temperate and sub-temperate regions of Himalayan belt from Jammu & Kashmir, Himachal Pradesh, Uttranchal and North-Eastern states and is endangered because of over extraction. Hence, it would provide an opportunity for hilly people to cultivate this species as an alternate cash crop.
6. Provides an opportunity to generate employment as seed gatherers for the hilly/tribal people.




We Claim:
1. A simplified process for purification of kutkin from Picrorhiza kurroa Royle ex Benth which comprises ; (a) extracting dried and powdered underground parts of the plant with aqueous low molecular weight alcohols (methanol/ethanol and water, 9:1-8:2), (b) repeating step a) 6 to 8 times, (c) removing solvent and fractionating alcohol free extract 6 to 8 times with chloroform followed by n-butanol or isopropanol saturated with water, (d) washing n- butanol/isopropanol extract containing kutkin with water saturated with butanol/isopropanol and treating with activated charcoal, (e) concentrating organic layer to yield white amorphous powder of kutkin having mp 210-212°C.
2. A process as claimed in claim 1, wherein purification of kutkin from Picrorhiza kurroa results in 2.0-3.0% yield of desired specifications.
3. A simplified process for purification of kutkin from Picrorhiza kurooa Royle ex Benth has been developed substantially as herein described with reference to the examples and drawings accompanying this specification.

Documents:

2021-DEL-2004-Abstract-(28-02-2011).pdf

2021-del-2004-abstract.pdf

2021-DEL-2004-Claims-(28-02-2011).pdf

2021-del-2004-claims.pdf

2021-DEL-2004-Correspondence-Others-(28-02-2011).pdf

2021-del-2004-correspondence-others.pdf

2021-DEL-2004-Description (Complete)-(28-02-2011).pdf

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2021-del-2004-form-1.pdf

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2021-del-2004-form-2.pdf

2021-DEL-2004-Form-3-(28-02-2011).pdf

2021-del-2004-form-3.pdf

2021-del-2004-form-5.pdf


Patent Number 247574
Indian Patent Application Number 2021/DEL/2004
PG Journal Number 17/2011
Publication Date 29-Apr-2011
Grant Date 25-Apr-2011
Date of Filing 15-Oct-2004
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110001, INDIA
Inventors:
# Inventor's Name Inventor's Address
1 BIKRAM SINGH HIMALAYAN BIORESOURCE TECHNOLOGY, PALAMPUR
PCT International Classification Number A61K 35/78
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA