Picrorhiza kurroa (Kutaki) Royle ex Benth as a hepatoprotective agent--experimental & clinical studies.AB Vaidya, DS Antarkar, JC Doshi, AD Bhatt, VV Ramesh, PV Vora, DD Perissond, AJ Baxi, PM Kale
Ciba Research Centre, Goregaon, Bombay.
Picrorhiza kurroa (Pk), a known hepatoprotective plant, was studied in experimental and clinical situtations. The standardization of active principles--Picroside 1 and 2 was done with High Performance Liquid Chromatography. Picroside 1 ranged from 2.72 to 2.88 mg/capsule and picroside 2 from 5.50 to 6.00 mg/capsule. In the galactosamine-induced liver injury in rats, Pk at a dose of 200 mg/kg p.o. showed a significant reduction (p < 0.05) in liver lipid content, GOT and GPT. In a randomised, double-blind placebo controlled trial in patients diagnosed to have acute viral hepatitis (HBsAg negative), Pk root powder 375 mg three times a day was given for 2 weeks (n = 15) or a matching placebo (n = 18) was given. Difference in values of bilirubin, SGOT and SGPT was significant between placebo and Pk groups. The time in days required for total serum bilirubin to drop to average value of 2.5 mg% was 75.9 days in placebo as against 27.44 days in Pk group. The present study has shown a biological plausability of efficacy of Pk as supported by clinical trial in viral hepatitis, hepatoprotection in animal model and an approach for standardizing extracts based on picroside content.
Keywords: Acute Disease, Alanine Transaminase, blood,Animal, Aspartate Aminotransferases, blood,Bilirubin, blood,Cinnamates, chemistry,therapeutic use,Disease Models, Animal, Double-Blind Method, Drug Evaluation, Preclinical, Glycosides, chemistry,therapeutic use,Hepatitis, Viral, Human, drug therapy,metabolism,Human, Liver Diseases, drug therapy,metabolism,Male, Medicine, Ayurvedic, Random Allocation, Rats, Vanillic Acid, chemistry,therapeutic use,
Picrorhiza kurroa Royle ex Benth (Pk) has been mentioned as an important remedy by Jivek, Charak and Vagbhatt in ancient Ayurvedic literature,,. The plant has been described as very useful in jaundice, nausea anorexia, dyspepsia and periodic fevers. The Indian and colonial addendum, of 1901, to the British Pharmacopoeia (1897) included the plant as an official drug. Until recently, Indian Pharmacopoeia also listed Pk as an official drug. In 1966, Pandey had done pioneering experimental and clinical work, with Pk. in liver disorders. Hydrocholeretic effects of Pk were also demonstrated in dogs with biliary fistula.
In a double-blind trial, in the late seventies, Arogya-wardhani with its principal ingredient Pk (50%) was shown to significantly reduce serum bilirubin and transaminases in patients with viral hepatitis by some of the current authors. Around the same period, a placebo-controlled double-blind trial showed Pk to be as effective as Arogya-wardhani. The findings were presented at the Annual Conference of the Association of Physicians of India in 1981. More work was also carried out at many centres in India and some abroad,,,,. Now we present the experimental, analytical and clinical studies on Pk.
Identification, collection, standardisation and storage:
Pk roots growing in Himalayan region at the height of 2700-4500m were identified and collected under botanical supervision (SY Wagh) and pharmacognostic controls. The cleaning and pulverisation were carried out in the Development Laboratory of CIBA Research Centre. The fine root powder was stored at -20 deg. C and the active principles- picrosides were identified and quantified for specifications with high performance liquid chromatography (HPLC). The alcoholic extracts and capsules were stored in a refrigerator prior to use. Fresh samples were preferably used for the animal experiments.
Analytical HPLC conditions: HPLC (Waters Associates), with ? Bonclapak-C18 column and LIV 280 nm/ 0.05 AUFS with proper phase, flow and external calibration methods, used standards (Mls.Wilimar Schwabe, Germany) Picroside I and II. [Figure:1] The root powder was extracted with methanol which was also a component of the mobile phase, besides water. The flow rate was 2 ml/minute. The plants from different sources and the capsules were analysed for picrosides I and II for standardisation.
Experimental models for hepatoprotection: At Zyma Laboratories, Nyon, mate rats (RS 25, G-G Breeding, Basle) with a body weight of about 300g were selected for the study. Liver injury was produced by subcutaneous injection of 500 mg/kg of galactosamine hydrochloride (Roth, FRG) dissolved in normal saline (n=5). Group 1 (n=8) received 200 mg/kg of Rkurroa in 5% gum arabic at 24, 4 and 0 hour before galactosamine., Group 2 (n=8) received 50 mg/kg at same lime intervals. The control groups (n=5) received only 5% gum arabic. The animals were fasted and sacrificed 24 hours later. Blood was taken for the determination of transaminases (GOT, GPT), glutamate dehydrogenase (GLDH), alkaline phosphatase and bilirubin; liver lipids were also measured. Blood assays were carried out with an autoanalyzer (Centrifichem. Roche), using standard control sera and tissues. Total liver lipids were measured by colorimetry.
Clinical Trial Design: Pk root powder (375 mg) and matching placebo in opaque capsules were administered in a randomised double-blind manner to the patients diagnosed to have acute viral hepatitis, negative for HBsAg by counter immunoelectrophoresis at Haffkine Institute. Three capsules of placebo or Pk were given three times a day for two weeks. The design and the criteria of choice and evaluation followed the method described earlier for the double-blind trial of Arogyawardhani . The placebo group had 18 patients and 15 were on the drug - Pk.
Statistical Tests: Student's ‘t’ test was used for statistical significance of changes in the transaminases and bilirubin -both experimentally and clinically.
Standardisation results: Picroside 1 and 2 concentrations per capsule were assayed for stability at room temperature and at 40 deg. C and 50 deg. C for 3 and 6 months; the picroside 1 and 2 contents were stable at room temperature. Picroside 1 ranged from 2.72 to 2.88 mg/capsule and picroside 2 from 5.50 to 6.00. At higher temperature, there was 30-50% degradation.
Experimental results : [Table - 1] shows the effects of Pk on galactosamine - induced liver injury in the rat. At the small dose of the root powder (Pk) 50 mg/kg given three times at 24th, 4th and Oh before galactosamine, only a directional change was seen in GPT and GLDH, but at 200 mg/kg po a significant reduction (p<0.05) was observed in liver lipid content, GOT and GPT. GLDH showed a directional reduction, but alkaline phosphatase and bilirubin were not reduced significantly. The alcoholic extract of Pk was not studied in this model.
Clinical results: The placebo and the drug groups were comparable in respect of age, sex, weight, severity and duration of disease. [Table - 2] shows the values of bilirubin, SGPT and SGOT, basically and every week. The difference in values between the placebo and Pk groups was statistically significant (p<0.05). The time in days required for the total serum bilirubin to drop to the average value of 2.5 mg % was calculated from extrapolation on a semilog graph. For the placebo, the time in days was 75.9 days as against 27.44 for Pk group; the decay constants were 0.0198 and 0.8691 respectively.
Therapy-induced changes in the symptoms and signs of viral hepatitis (severity score) were observed in both the groups. Pk induced a more marked and early relief of anorexia, nausea and malaise. No side-effects were observed at this dose of Pk, including laxation sometimes noticed on its larger doses.
Picrohiza kurroa and several other Ayurvedic drugs have been studied by many investigators as hepatoprotective agents,. But studies at different levels of biological organisation with proper controls and double-blind controlled trials with a rigorous design and execution are sometimes lacking or inadequate. Central Drug Research Institute (CDRI) has now developed 'Picroliv', a standardised product from Pk and this may shortly be launched as a hepatoprotective drug. Our data on work done earlier and unpublished so far, reinforce the findings at CDRI by Dhawan and colleagues, Picrosides have been patented by Wilimar Schwabe (Offen Legunsschrift 2203884) in 1973.
Experimentally, Pk has been shown to have the following pharmacodynamic and biodynamic actions: (1) hydrocholeretic effects in rats and dogs  (2) anti-necrotic effect in carbon tetrachloride induced damage in rats and rabbits (3) reduces fatty infiltration and lipid deposits in galactosamine-induced liver damage (4) reduces paracetamol-induced hepatic damage  (5) reverses the loss in body weight in alcohol-treated rats (6) improves food intake in CCL 4-induced liver-damage  (7) enhances the levels of DNA, RNA, protein and cholesterol post-partial hepatectomy; the mitotic index was also increased (8) scavenging of super-oxide anions and inhibition of lipid peroxidation (9) antiviral effects on vaccinia viruses and (10) anti-inflammatory effects in carrageenin oedema and (11) inhibition of experimental passive cutaneous anaphylaxis. So many diverse effects hint that picrosides may be acting at some fundamental level in the cell homeostatic controls. Picrosides may then unravel new fields as reserpine did with adrenergic systems three decades back. There can possibly be endogenous ligands resembling the aglycones of the iridoid glycosides - picrosides.
The published literature and currently on-going work show the efficacy and safety of Pk in (1) acute viral hepatitis (2) treatment of drug-induced liver damage e.g. antituberculous drugs and (3) long-term prophylactic use in bronchial asthma,,,,. Picrorhiza kurroa Benth ex Royle, in form of its fine root powder, is a widespread remedy in India for fevers, anorexia, jaundice and as an adaptogen. Standardised extracts, with the tolerance specifications with the HPLC assay of the picrosides, offer a fresh opportunity for its use in viral hepatitis and alcohol or drug-induced liver damage. The present work has shown, besides other data, the biological plausibility of efficacy of the plant as shown 159 a double-blind randomised placebo-controlled trial in viral hepatitis.
We thank Prof. TR Govindachari, Prof. RS Grewal and Prof. D Subramaniam, the successive Directors of Ciba Research Centre for their support and encouragement. We also thank Sri. C Subramanyam, President of Bharatiya Vidya Bhavan for his support to SPARC. We thank Dr. (Mrs.) M Mulkherji, AV Athavale, Vd. PS Tatheo Vds. Kalpana and M Natekar for their technical and clinical assistance, during the studies
[Table - 1], [Table - 2]