Indian Journal of Traditional Knowledge Vol. 5(4), October 2006, pp. 459-462

Acute and subacute toxicity of an antidiabetic Siddha herbal formulation

C Ramesh1*, V Gopal1 & K Sembulingam2 1College of Pharmacy, Mother Theresa Institute of Health Sciences, Gorimedu, Government Pharmacy, Pondicherry 605 006

2Department of Physiology, Sri Ramachandra Medical College & Research Institute, Porur, Chennai 600 116, Tamil Nadu Email: rampharm23@yahoo.co.in

Received 19 April 2005; revised 10 July 2006

The management of diabetes is considered a global problem. FS002, a polyherbal Siddha formulation intended to be used for diabetic patients, has been screened for toxic effect. For sub-acute toxicity studies, different doses of FS002 were administered orally to rats once daily for 30 days. Animals were observed for physiological and behavioral responses, mortality, food, water intake and body weight changes. Hematological evaluation was performed weekly. Biochemical studies were done on liver and serum enzyme levels. No mortality was observed up to 4 gm/kg of FS002 in acute studies. Daily administration of as high as 2 gm/kg of FS002 did not result in mortality or changes in behaviour, body weight, organ weight, histology, serum and liver biochemistry. The dose required to produce significant antidiabetic activity in rats, 0.2-0.5 gm/Kg, is much lower than doses used in present study. For a chronic disease like diabetes such doses may be safe for daily administration without causing any serious side effects.

Keywords: Antidiabetic activity, Diabetes, Siddha drug IPC Int. Cl.8: A61K36/00, A61P5/00, A61P5/50

Diabetes is a heterogeneous metabolic disorder characterized by altered carbohydrate, lipid and protein metabolism1. The management of diabetes is considered a global problem. The modern drugs including insulin and oral hypoglycemic agents, control the blood sugar level as long as they are regularly administered, and also produce a number of undesirable side effects2,3. The treatment of diabetes has been attempted with different indigenous plants and polyherbal formulations2,4-7. Preliminary screening of this formulation showed significant hypoglycemic activity in fasting rabbits and antihyperglycaemic activity in streptozotocin induced diabetes in Swiss albino rats8,9. However pre-clinical toxicity studies are essential for determining a safe dose for human trials10. Therefore, an attempt was made to evaluate acute and sub-acute toxicity of a polyherbal Siddha formulation, FS002 in laboratory animals. Methodology Acute and sub-acute toxicity studies were carried out in Swiss albino mice and Wistar rats, respectively. Animals were obtained from animal house, JIPMER, Pondicherry. Adult mice (6-8 weeks old) of either sex weighing 25-30 gm were housed in polyproplene

cages, 4 animals per cage with free access to food and water. 6-8 weeks old Wistar rats of either sex, weighing 150-200gm were housed, two per cage in elevated wire mesh cages with free access to food and water. Formulation, FS002 was prepared using bark of Ficus racemosa Linn. collected from in and around Pondicherry and fresh sesame (Sesamum indicum Linn.) cake (marc left out after extraction of sesame oil). For acute toxicity studies, 60 mice were used for the study. Food and water was withdrawn 18hrs before administration of formulation FS002. The mice were divided into 6 groups containing 10 animals. Group I received 0.2 ml of gingelly oil (vehicle for formulation) orally and served as the control. Group 2-6 received 0.2 to 4G/kg of FS002 orally. The animals were observed continuously for the first 2 hrs, then occasionally up to 6hrs and then daily up to 14 days, post treatment to observe for any toxic symptoms and mortality. For sub-acute toxicity studies, rats were divided into 4 groups of 6 animals each. (3 males and 3 females). One group was given 0.5 ml of gingelly oil orally. Other 3 groups were administered 0.2,0.4,1.2 gm/kg of FS002 daily for 30 days. All the rats were observed for any physiological and behavioural changes and mortality9. Food and water consumption was checked daily. Body weight was recorded at the beginning and twice weekly

______________ *Corresponding author

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throughout the study. Haematological parameters, total RBC, WBC, differential leukocyte count and haemoglobin were estimated weekly in blood collected from the orbital sinus into sterilized heparinized tube 24hrs after the last (on the 31st day of the experiment) administration11,12. Blood samples were collected from each rat individually into non-heparinized tubes and were allowed to coagulate. Serum was separated by centrifugation, alkaline and acid phosphatases were analyzed by spectrophototmetric method. Alanine transaminase (ALT), aspartate transaminase (AST), urea and creatinine were analysed using reagent (Qualigens) kit13,14. Liver, heart, thymus, spleen, adrenal, uterus and kidneys were removed and weighed immediately. Acid and alkaline phosphatases were also estimated as above. Pieces of organs were fixed in Bouin’s fixative and processed routinely for histological examination. The slides were stained with haematoxylin and eosin and observed for pathological changes15. The Students t-test was employed to analyze the results. Difference

below the probability level 0.05 was considered statistically significant. The experimental protocols were approved by the Institutional Ethics Committee of Regional Research Institute (Siddha), Pondicherry. Results & discussion No acute mortality was observed on oral administration even at the dose of 2 gm /kg of FS002 and all the animals were found to be normal during and at the end of the observation period (14 days). In the sub acute toxicity study, there was no death in the treatment period either in the control or in the treated groups. Food and water consumption did not differ significantly. There was no change in the general behaviour or other physiological activities of the animals. None of the organs in the treated rats showed any significant change in weight. Haematological analysis showed no significant increase in RBC count, haemoglobin, serum alkaline phosphatase, acid phosphatase, ALT, AST, urea or creatinine levels as compared to control (Tables 1 & 2). Histological

Table 1—Effect of FS002 on haematological parameters (mean ± S.E of 6 animals)

Differential leukocyte count Time (week)

Treatment (gm/kg)

106/ m3 RBC count

Haemoglobin gm/dl

WBC 103/mm3 N L M

I Control (0.5ml gingelly oil) 5.92± 0.1 12.73± 0.30 6.40± 0.35 22.7± 0.35 74.0± 0.76 0.7±0.2 FS002 0.2 6.02± 0.1 12.93± 0.21 6.30± 0.15 23.9± 0.15 75.5± 0.35 0.7± 0.35 0.4 6.12± 0.2 12.84± 0.32 6.15± 0.18 24.1± 0.44 74.6± 0.51 0.8± 0.1 1 6.22± 0.3 13.01± 0.31 6.54± 0.21 22.5± 0.51 76.2± 0.44 0.7± 0.4 2 6.33± 0.2 13.08± 0.22 6.77± 0.17 24.7± 0.37 75.2± 0.51 0.8± 0.5

II Control (0.5ml gingelly oil) 6.05± 0.1 12.92± 0.30 6.40± 0.35 22.7± 0.35 79.2± 0.37 1.1±0.2 FS002 0.2 6.31± 0.1 13.18± 0.21 6.30± 0.15 23.9± 0.15 76.3± 0.32 0.8± 0.26 0.4 6.21± 0.2 13.60± 0.32 6.15± 0.18 24.1± 0.44 75.4± 0.79 1.0± 0.1 1 5.99± 0.3 13.10± 0.31 6.54± 0.21 22.5± 0.51 76.4± 1.11 0.8±0.23 2 6.30± 0.2 13.18± 0.22 6.77± 0.17 24.7± 0.37 76.5± 0.51 0.8±0.33

IV Control (0.5ml gingelly oil) 6.42± 0.1 12.73± 0.30 6.40± 0.35 22.7± 0.35 75.7± 0.37 0.7±0.12 FS002 0.2 6.23± 0.1 12.93± 0.21 6.30± 0.15 23.9± 0.15 74.4± 0.79 0.8± 0.42 0.4 6.32± 0.2 12.84± 0.32 6.15± 0.18 24.1± 0.44 75.6± 1.05 0.8± 0.36 1 6.23± 0.3 13.01± 0.31 6.54± 0.21 22.5± 0.51 76.6± 0.44 0.8± 0.4 2 6.35± 0.2 13.08± 0.22 6.77± 0.17 24.7± 0.37 74.4± 0.51 0.8± 0.5

P< 0.05 All values are NS when compared to normal values

Table 2—Effect of FS002 on serum enzymes (30 days treatment) (mean± S.E of 6 animals)

Treatment gm/Kg

Alkaline phoshpatase (U/L)

Acid phosphatase (U/L)

AST (U/L)

ALT (U/L)

Urea mg/dl

Creatinine mg/dl

Control (0.5ml gingelly oil) 63.71±3.29 27.43 ± 3.03 69.03± 3.74 57.06± 2.85 44.25± 2.02 0.51± 0.12 FS002 (0.2 g/Kg) 63.67± 4.26 32.95 ± 4.93 67.64± 4.48 54.23± 5.15 43.23± 2.25 0.53± 0.22 0.4 64.11± 4.21 30.39 ± 2.83 66.26± 2.48 55.12± 3.12 45.11± 2.23 0.48± 0.17 1 64.61± 3.51 31.19 ± 4.91 68.14± 3.41 55.14± 1.14 46.22± 2.35 0.50± 0.11 2 63.97± 3.42 30.18 ± 3.52 67.15± 2.18 54.32± 2.61 45.22± 2.34 0.45± 0.13

P< 0.05, All values are NS when compared to normal values

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examination of the organs did not reveal any pathological changes. These observations were similar in the male and female rats (Figs. 1-12). The results shows that a very high oral doses were tolerated by the mice without producing any toxicity symptoms. The ingredients present in the formulation are purely phytochemical in origin and contain sterols, triterpenoid saponins, aminoacids, triterpenes, and flavonoids. Since, a number of phytoconstitutents are present in the formulation, these experiments were designed to screen for any toxic effects. Since, no toxic effects were observed, it could be inferred that the basic principle in the use of crude plant products or polyherbal preparations in traditional medicine, is that the toxic effect of one component is nullified by the protective effect of the other components, without interfering with their therapeutic properties. Daily administration of FS002 at different doses of 0.2, 0.4, 1 and 2 gm/kg for 30 days was well tolerated and there was no cumulative toxicity of the drug, making it suitable for the treatment of chronic diseases like Diabetes Mellitus. In another study, FS002 has shown significant hypoglycemic activity at a dose of 0.2 gm/kg in rabbits. The study shows that such a dose of FS002 may be safe for daily administration, as required in controlling the elevated blood sugar levels in diabetic patients. Since there are no toxic effects produced by the drug, further clinical studies would be conducted to prove the efficacy of FS002 in the treatment of diabetes. Further, chronic toxicity and teratogenic studies have to be conducted before the preparation could be recommended for long-term treatment of diabetes.

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