CHRONIC TOXICITY STUDY OF GARBHPAL RAS, AN AYURVEDIC MEDICINE

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1 Journal of Herbal Medicine and Toxicology 3 (1) (2009) ISSN : Original Article CHRONIC TOXICITY STUDY OF GARBHPAL RAS, AN AYURVEDIC MEDICINE D. MISHRA 1, M. SINHA 1, M. KUMAR 2 AND V. KUMAR 3 * 1 Department of Prasuti Tantra, 2 Department of Pathology, Institute of Medical Sciences, Banaras Hindu University 3 Pharmacology Research Laboratory, Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi , UP. vikas.phe@itbhu.ac.in Received: 18 th Sept., 2008, Accepted : 2 th Dec., 2008 Abstract: Garbhpal Ras, a well known ayurvedic herbo-mineral formulation used during antenatal period in cases of habitual abortions or miscarriages and for ensuring better nourishment to foetus. In the present study, safety of Garbhpal Ras was evaluated on its chronic administration in rats. Charles Foster albino rats of either sex were distributed equally in four groups including control. The Garbhpal Ras was administered orally for 180 consecutive days in doses of 12, 0 and 80 mg /kg i.e. 3, 10 and 20 times higher than the recommended dose. Body weight, feed and water consumption were monitored fortnightly. On day 181, blood was collected by retroorbital technique for performing haematological and biochemical tests. After collection of blood, animals were sacrificed for isolation of liver and kidney to observe histopathological changes, if any. The levels of glucose, cholesterol, alkaline phosphatase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, blood urea nitrogen, creatinine, total protein and albumin were not altered significantly in Garbhpal Ras treated rats compared to control rats. The level of hemoglobin and WBC count were not changed significantly. There was no sign of haemopoitic, hepatic or renal toxicity. Histopathological examination of control and experimental rats revealed absence of any gross pathological lesion in either liver or kidney, except for ballooning degeneration of hepatocytes in liver mainly in centrilobular area in samples at 80 mg/kg dose. All animals were found healthy during entire study period and devoid of deleterious effect like reduction of body weight. Garbhpal Ras did not reveal any sign(s) of toxic manifestation. Key words : Garbhpal Ras, pregnancy, chronic toxicity, herbomineral. INTRODUCTION Garbhpal Ras is a herbomineral preparation. It contains Hingula (Cinnabar, HgS), Nag (Lead, Pb), Vanga (Tin, Sn) and Loh Bhasma (Iron, Fe). Herbal contents of Garbhpal Ras include Dalchini (Cinnamomum zeylanicum), Tejpatra (Cinnamomum tamala), Ela (Eletaria cardamomum), Shunthi (Zingiber officinale), Marich (Piper nigrum), Pippali (Piper longum), Dhaniya (Coriandrum sativum), Chavya (Piper retrofractum), Krishna jeerak (Carum bulbocastanum), Draksha (Vitis vinifera), Devdaru (Cedrus deodara) and Vishnukranta (Clitoria ternatea). It is widely used from a very long time by Ayurvedic physicians and also mentioned in the classical text book of Ayurveda of the 17th century named as Ras-Chandanshu [1]. It is used for the protection and preservation of pregnancy, in cases of habitual abortions or miscarriages. Garbhpal Ras is claimed to have better nourishment to foetus. Earlier, we have reported safety profile of Garbhpal Ras investigated through acute and subchronic toxicity studies in rodents [2]. We have also observed that 13

2 Journal of Herbal Medicine & Toxicology Garbhpal Ras is safe during pregnancy when investigated through teratological study in rodents (unpublished findings). There is no report available on the toxicological profile of Garbhpal Ras on chronic administration; therefore, present study was conducted to evaluate the safety profile and toxic effect of Garbhpal Ras in rats on chronic treatment. MATERIALS AND METHODS 6 adult Charles Foster albino rats (150±10 gm) of either sex were distributed into groups (,, and control). The rats were housed in groups in polypropylene cages at an ambient temperature of 25 0 C±1 0 C and 5-55 relative humidity, with a 12:12 h light/dark cycle. Animals were provided with commercial food pellets and water ad libitum throughout the study. Garbhpal Ras was administered orally for 180 consecutive days in the doses of 12, 0 and 80 mg/kg (3, 10 and 20 times higher than the recommended dose respectively) in the form of suspension in 0.3 CMC. Body weight, food and water consumption were monitored for 15 consecutive days after every 15 days interval for six months. Blood samples were collected by retro-orbital technique on the day 181 st of the study for haematological (haemoglobin, total leukocyte count, differential leukocyte count) and biochemical (glucose, cholesterol, alkaline phosphatase, aspartate transaminase, alanine transaminase, blood urea nitrogen, creatinine, total protein and albumin) estimation [3, ]. All animals were fasted overnight prior to blood collection. After collection of blood, animals were sacrificed for isolation of liver and kidney to observe histopathological changes, if any [5]. The liver and kidney were dissected out and were fixed in 10 formalin solution. Paraffin sections were made and stained with haematoxylin and eosin for detailed histopathology study. Statistical analysis: Data generated in the study were analysed statistically. Treated and control groups were compared using a one-way analysis of variance (ANOVA), followed by Dunnett Multiple Comparisons Test. A 95 confidence level was used to determine statistically significant differences between treated and control groups. GraphPad (Version 3.06) software was used for all statistical analysis. Table 1. Effect of Garbhpal Ras on body weight Body weight (gm) (Dose) Day 0 Day Day 6-60 Day Day Day Day Control ± ± ± ± ± ± ±3. (0mg/kg) ± ± ± ± ± ± ± ± ±.23* ± ±2.6* 207.2±3.1** ±2.6** ±.6** ± ± ± ± ± ± ±3.6 Values are mean ± SEM. n=16 in each group. * p < 0.05, ** p < 0.01 compared to control group. Table 2. Effect of Garbhpal Ras on daily food intake (Dose) Average daily feed intake (gm) Day Day 6-60 Day Day Day Day Control ± ± ± ± ± ± ± ± ± ± ± ±2.3 (0mg/kg) ± ± ± ± ± ±.2* 30.8± ± ± ± ± ±2.89 Values are mean ± SEM. n=16 in each group. * p < 0.05 compared to control group. 1

3 Mishra D. et al. Table 3. Effect of Garbhpal Ras on daily water intake (Dose) Average daily water intake (ml) Day Day 6-60 Day Day Day Day Control ± ± ± ± ± ± ± ± ± ± ± ±3.61 (0mg/kg) ±.6* ±5.3* 669.2±3.68* ±2.39* ±.21* ±3.39* 60.12± ± ± ± ± ±5.32 Values are mean ± SEM. n=16 in each group. * p < 0.01 compared to control group. Table. Effect of Garbhpal Ras on haematology parameters of rats (Dose) Haemoglobin Total WBC (X10³/µl) Neutrophils Lymphocytes Differential Leukocyte Counts Eosinophil Monocyte Basophil Control 11.88± ± ± ± ±0.15 1± ± ± ± ±1.17 1±0 1±0 0 (0mg/kg) 11.31± ± ± ± ±0.15 1± ± ± ±3.8 6.± ± ± No statistically significant difference was found between control and treated groups. n=16 in each group. Table 5. Effect of Garbhpal Ras on biochemical parameters of rats (Dose) Glucose Cholestero l AST (U/ml) ALT (U/ml) Alkaline Phospha tase (U) Blood Urea Nitrogen Creatinin e Albumin Total Protein Control 86.55± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.17 (0mg/kg) 83.36± ± ± ± ± ± ± ± ± ± ±3.5 6 RESULTS AND DISCUSSION 2.3± ± ± ± ± ± ±0.16 No statistically significant difference was found between control and treated groups. n=16 in each group. The criteria for the assessment of effect of Garbhpal Ras administration in rats was based on the appearance of any kind of abnormal signs and symptoms, feed and water intake and growth pattern [3]. The haematological, biochemical and biopsy investigations were also taken into consideration for assessing the toxicity of Garbhpal Ras. Body weight: The mean body weight changes of rats of all four groups are depicted in Table 1. There was no statistically significant difference between control 15

4 Journal of Herbal Medicine & Toxicology and group. However, there is a significant increase in body weight gain of group compared to control rats during the entire period of study. Food and water consumption: The average daily food consumption of Garbhpal Ras treated group and was comparable with control animals. Average daily water intake in group did not differ significantly compared to control however, group show significant increase in daily water consumption compared to control group. Results of food and water intake are presented in Tables 2 and 3 respectively. Effect of Garbhpal Ras on haematology of rats: It was observed that there was no significant alteration in all hematology parameters. The oral administration of Garbhpal Ras for consecutive 180 days did not depress the haemoglobin and blood cell values. There was no major shift in white cell population of Garbhpal Ras treated rats compared to control animals. There was no leukocytosis or leucopenia. The results are shown in Table. Effect of Garbhpal Ras on liver: Garbhpal Ras did not cause any disturbance in hepatic functions as compared to control rats. The excretory function of liver of Garbhpal Ras treated animals was not disturbed because alkaline phosphatase (ALKP) values were not elevated in comparison to control rats. Plasma transaminases levels of rats in control and Garbhpal Ras administered group did not reveal any abnormal hepatotoxic effect. The alanine transaminase (ALT) and aspartate transaminase (AST) values are very sensitive indicators of hepatic 16

5 Mishra D. et al. injury because liver cells are very rich in these enzyme and they are released in plasma when hepatocytes are damaged. In our experiments, it is evident that there is no statistically significant increase in values of these enzymes even when the rats were administered up to twenty times higher than the recommended dose. Further, biochemical parameters showed that Garbhpal Ras did not cause any disturbance in synthesis of albumin even at high dose. The total cholesterol values which are statistically insignificant suggest that Garbhpal Ras devoid of any metabolic adverse effects. The results are presented in Table 5. Effect of Garbhpal Ras on kidney: Garbhpal Ras treatment did not cause any adverse toxic effect on renal function. Biochemical parameter revealed that excretory function of kidney was well maintained, as there was no rise in the values of blood urea nitrogen (BUN), and serum creatinine levels in rats treated with Garbhpal Ras (Table 5). Histopathological study: Liver biopsy of Garbhpal Ras treated rats show normal lobular architecture of liver with normal central portal vein, radiating plates of hepatocytes and peripheral portal tracts composed of hepatic artery, bile ductule and distal portal vein. No focal or diffuse foci of necrosis of hepatocytes, and infiltration of chronic inflammatory cells were observed (Fig. 1). However, in samples at 80 mg/kg dose ballooning degeneration of hepatocytes was observed, which is reversible (Fig. 2). Kidney biopsy of Garbhpal Ras treated rats show normal renal architecture composed of normal renal glomerulii, collecting tubules, interstitial tissue and blood vascular channels. There were no foci of necrosis, degeneration or fibrosis in interstitium (Fig. 3 and ). In summary, there were no significant changes in hematological, biochemical and histopathological parameters that resulted from the long-term administaration of Garbhpal Ras in rodents indicating Garbhpal Ras did not have any toxic manifestations. Chronic toxicity study of Garbhpal Ras clearly demonstrate the non-toxic nature and safety profile of Garbhpal Ras in rodents. The combined history of human use of Garbhpal Ras and the data from the current study support the safe use of Garbhpal Ras. ACKNOWLEDGEMENTS Authors are thankful to Dr. P.N. Singh, Professor of Pharmacology, Department of Pharmaceutics, IT, BHU for providing constructive comments on this manuscript and to Mr. Gulam M. Husain, Research Scholar, for providing technical assistance in preparation of manuscript. Thanks are also due to Dabur India Ltd., Sahibabad, UP for providing gift sample of Garbhpal Ras. Deepa Mishra is thankful to the University Grants Commission, New Delhi for providing Junior Research Fellowship. REFERENCES [1] Sharma, H.: Rasyogasagar. Vol. I, Krishna Das Academy, Varanasi (1983). [2] Mishra, D., Sinha, M., Singh, P.N., Kumar, V.: Electronic J. Pharmacol. Therapy, 1: 31-3 (2008). [3] Kumar, V., Das, S.N., Rao, A.T., Agrawala, S.K.: Phytomedica, 6: (2005). [] Chavalittumrong, P., Chivapat, S., Attawish, A., Bansiddhi, J., Phadungpat, S., Chaorai, B., Butraporn, R.: J. Ethnopharmacol. 90: (200). [5] Schmidt, B.M., Ilic, N., Poulev, A., Raskin, I.: Food Chem. Toxicol., 5: (2007). 17