Toxicological Assessment of Herbal Medicine (Tragia involucrate L.) in Rats: Biochemical Analysis of Proteins from Various Organs

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1 Toxicological Assessment of Herbal Medicine (Tragia involucrate L.) in Rats: Biochemical Analysis of Proteins from Various Organs R. Perumal Samy*,1,2, S. Ignacimuthu 3, P. Gopalakrishnakone 2, Vincent TK Chow 1 1 Infectious Diseases Programme, Department of Microbiology, 2 Venom and Toxin Research Programme, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore , 3 Entomology Research Institute, Loyola College, Chennai , India. Abstract Protein profiles analysis of liver, kidney and spleen by SDS-PAGE in aqueous leaf extract of Tragia involucrata L. (Euphorbiaceae) was injected by intraperitoneally in albino rats for 30 days. The variation of protein changes was recorded. In liver, aqueous leaf extracts treated (1500 mg/kg body/weight) group did not exhibit a marked increase in all the polypeptides over control. But in the case of other treatments (250, 500, 1000 and 2000 mg/kg body weight doses) the profile showed significant increases in the accumulation of all polypeptides were quantified using densitometry. It concluded that the aqueous leaf extract, which influenced the accumulation of polypeptide in higher concentration. Extra polypeptide may be responsible for the protective action. Key words: Poly acrylamide gel electrophoresis; Protein profile; albino rat; Tragia involucrata. *Corresponding author. Tel..: ; fax: address: micramar@nus.edu.sg (R.Perumal Samy).

2 1. Introduction Tragia involucrata L. is a commonly available as a weed plant belongs to the family Euphorbiaceae. The tribals (Malaiali) in Kolli hills and Kalrayan, Western Ghats of Tamil Nadu are using different parts of this plant for scabies, cut wounds, inflammation and skin infection (Perumal Samy et al., 1998). The leaves are used for headache and the roots are said to possess diaphoretic property to treat fever. A paste prepared from the root is given to inactivate and kill guinea worms. It is also mixed with the leaf juice of Ocimum sanctum and the paste is employed for the treatment of skin and venereal complaints. The fruit extracts are rubbed on the head to cure baldness. The efficacy of this plant is well known in Indian traditional medicine systems in which it is used to cure diseases (Chopra, 1956; Nadkarni, 1982). When the plant is alive the stinging hairs have sharp siliceous points that break off when touched and penetrate the skin. The acid present in the hair causes burning pain and inflammation. Interesting features of this plant is dried or the powdered plant material is not produced any harmful effects to human and animals. This part we have proved in the field as well as in our laboratory experiment (Perumal Samy and Ignacimuthu, 1997). The active substances present in the extract at higher concentration interact with target cells and cause changes, which lead to the symptoms of physiological and behavioral changes of the rat. The continuous administration of the extract cause functional deficiency in the tissue. Particularly in liver it causes necrosis, conjunction and sinnosidal effects were observed in the histopathology at higher concentration. There is no literature is available on the molecular level assessment of toxicity. So the present investigation has been designed to examine the effect of T. involucrate on the exact protein profile patterns of animal tissues such as liver, kidney, spleen and blood serum before and after the treatment of crude herbal medicine assessed by gel electrophoresis and densitometer scanning. 2. Materials and methods 2.1. Collection of plant materials Plant was collected from Kalrayan and Chinnakalrayan hills of Salem district, Tamil Nadu, India, based on the medicinal information obtained from the tribals (natives), during an ethnobotanical survey in Voucher specimen was kept at the department herbarium.

3 2.2. Preparation of plant extracts Leaf was separated and shade dried at 31 C and ground into powder by using an electric blender. 100 gm of dry powdered plant material was soaked in 600 ml of double distilled water at room temperature for over night days. The extracts were filtered and the filtrates were evaporated using water bath at 60 C (Kandil et al., 1994). Crude extracts were collected and stored at - 20 C Animals 4-6 weeks old albino rats (wistar strain) were divided into seven groups and each group consisted of 6 animals of either sex. Animal body weight-ranging form gm was kept in each cage (45 x 30 cm). The cages were maintained under standard environmental condition (12 h light/12 h dark cycle, room temperature 26±1 C; relative humidity 70 ± 2 C) in a clean and hygienic condition. To prevent infection, husk was placed in the bottom of the cages and cleaned regularly. The animals were fed with a standard laboratory diet and tap water (Fujiwara et al., 1994) Toxic assessment Group I Normal control animals were fed with standard diet and water Group II Vehicle control rats treated with methylcellulose Group III rats treated with 250 mg/kg, body weight of extracts Group IV rats treated with 500 mg/kg, body weight of extracts Group V rats treated with 1000 mg/kg, body weight of extracts Group VI rats treated with 1500 mg/kg, body weight of extracts Group VII rats treated with 2000 mg/kg, body weight of extracts The rats were administered with aqueous leaf extract at different doses such as 250, , 1500 and 2000 mg/kg body weights by intra peritoneal (i.p). One group of rats fed with standard diet and tap water was used as control. The toxic symptoms and behavioral changes were monitored daily up to 30th day. After 30th day the animals were sacrificed using diethyl ether anesthesia. Blood samples were collected in eppendorf tubes and then liver, kidney and spleen were quickly removed, weighed and stored at -20 C for further analysis (Twaij et al., 1983; Al- Deen et al., 1987 and Stanely et al., 1993).

4 2.5. Extraction of animal tissues 10 mg tissue of liver, kidney and spleen was homogenized using 500 µl of 2N Tris-HCl buffer (ph 7.2), and centrifuged (4 C at 10,000 rpm) for 15 min. The supernatant such as kidney, liver, spleen and blood serum protein samples were collected and stored at 4 C for SDS-PAGE Preparation of Linear Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (Linear SDS-PAGE) The denature protein from kidney, liver and spleen (100µg), blood serum (70µg) quantified using Bradford, (1976) method. The slab gel with discontinuous SDS-PAGE (17.5 cm (L) x 15 cm (B) x 1.5 cm (T) prepared by the method of Laemmli (1970). The separating gel (resolving) of 10% was prepared by adding 3.30 ml of 30% acrylamide, 2.5 ml of lower Tris-HCl, (ph 8.8) and 4 ml of distilled water and 100 µl of 10% SDS. After adding 75 µl of ammonium per sulphate and 10µl of TEMED, the solution was mixed well and glass plate with polymerized gel was fixed to the electrophoresis apparatus. Samples were mixed with 20 µl of sample buffer (bromophenol blue as a tracking dye) and kept in a boiling water bath for 2 min. The above samples were loaded into the well of the gel Standard protein markers run along with tissues samples. Electrophoresis was carried with tank buffer ph8.3 (3 g of Tris, 14.4 g of glycine and 1 g of SDS was dissolved in 1000 ml double distilled water and store at 4 C) for about 6 h at constant voltage of 60v in stacking gel and 120v in separating gel. The gel was stained using 200 mg Coomassie brilliant blue R-250, 7ml acetic acid, 50 ml methanol and 43 ml double distilled water, filtered and stain for 6 h. It was later de-stained using 7 ml of acetic acid, 30 ml of methanol and 63 ml of double distilled water and stored in 7% acetic acid. The visualized bands were photographed. The polypeptides were scanned using Laser Densitometer Scanner (Agrawal and Bedwal, 2000). 3. Results and Discussion The results are revealed in table 1. Alterations in the protein profiles patterns in the liver, spleen and kidney were as a result of aqueous leaf extract treated rats. SDS-PAGE protein pattern analysis of rat organs revealed the accumulation proteins in aqueous leaf extract treated animals and absent in their respective control animals. The electrophoretic profile of total soluble proteins of liver, kidney and spleen showed various polypeptides of different molecular weights 97.4 kda polypeptide, 68 kda polypeptide, 43 kda polypeptide and 29 kda polypeptide. In case

5 of liver samples all the treated groups showed a marked increase in all the polypeptides over control except the group treated with 1500 dose of extract. During the treatment of the crude medicine that influenced the bulk of stained material at the region of 68 kda and 43 kda. Besides, some other polypeptides also present but the concentration of the protein were very low in quantity. In blood serum there was no change in all proteins. In spleen, the rats treated with aqueous extract 200 mg/kg body weight did not affect the protein profile of treated rats and were almost equal to the profile of control rats. But in the case of other treatments (250, 500, 1000 and 1500 mg/kg body weight respectively) the profile showed a significant increase in the accumulation of all polypeptides over control. Similar results were observed in densitometer scanning of all the profiles. In kidney samples, treated as well as control groups did not show any significant variation. However there was a slight increase in the accumulation of all proteins in treated groups when compared to control. The electrophoretic profile of total soluble proteins of kidney samples treated with aqueous leaf extracts did not show any significant variation. In case of liver sample, except 1500 mg/kg dose, all other dose treated groups showed a marked increase in all the polypeptides over control. Goel et al., (1992) reported that 80 kd protein was increased in tirchloroethylene treated kidney and liver samples. In spleen, the rats treated with aqueous leaf extracts of 250, 500, 1000 and 1500 mg/kg doses, showed significant increase in the accumulation of all polypeptides in comparison to the control. Our present findings corroborate with Chithra et al., (1998) who reported increased level of protein and DNA contents of Aloe vera treated wound compared to untreated controls (Malini and Vanithakumari, 1990). In blood sample all the proteins slightly reduced than the control these results corporate with Sunderman, (1995) reported that the reduced concentration and complete loss of some of the proteins may be on account of protein degradation. 4. Conclusions The aqueous leaf extract of T. involucrata, which influenced the accumulation of polypeptide at higher concentration. The extra polypeptide may be responsible for the protective action of organs damage. This need further study of the exact mechanism action on molecular level. The

6 results proved the traditional claims of this plant; moreover the plant extract did not show any external poison or danger in using this plant species. Acknowledgements We are grateful to the Council of Scientific Industrial Research (CSIR), New Delhi, Government of India for the financial support to carry out this project. References Agrawal, R. and Bedwal, R.S SDS-PAGE analysis of caput-epididymis protein in rats receiving a zinc deficient diet. Indian Journal of Experimental Biology 38, Al-Deen, I.H.S., Twaij, H.A.A., Al-Badr, A.A. and Istai-Abadi, T.A.W Toxicological and histopathological studies of Pleurotus ostreatus mushroom in mice. Journal of Ethnopharmacology 21, Chithra, D., Sajithlal, G.B., Chandrakasan, G Influence of Alove vera on the healing of dermal wounds in diabetic rats. Journal of Ethnophamacology 59, Fradford, M.M. (19976) Annual Bicohemistry 72. Fujiwara, T., Yuasa, H., Ogiku, N., and Kawi, Y Histopathological investigation on saltloaded stroke-prone spontaneously hypertensive rats whose biochemical parameters of renal dysfunction were ameliorated by administration of imidapril. Japanese Journal Pharmacology 66, Goel, S.K., Rao, G.S., Pandya, K.P. and Shanker, R Trichloroethylene toxicity in mice: A biochemical, hematological and pathological assessment. Indian Journal Experimental Biology 30, Kandil, O., Radwan, N.M., Hassan, A.B., Amer, A.M.M.and El-Banna, H.A Extracts and fractions of Thymus capitatus exhibit antimicrobial activities. Journal Ethnopharmacology 44, Laemmli, U.K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, Malini, T. and Vanithakumari, G Rat toxicity studies with β-sitosterol. Journal Ethnopharmacology 28, Nadkarni, K.M Indian Materia Medica. Vol. I and II, Popular Prakashan Private Limited, Popular Press, Bombay, pp Stanely, A., Averal, H.I. and Akbarsha, M.A Reproductive toxicity of vincristine in male rats. Indian Journal of Experimental Biology 31, Sunderman, F.W.Jr., (1995) The influence of zinc apoptosis. Annual Clinical Laboratory Science 25, 134. Twaij, H.A.A., A. Kery and N.K. Al-Khazarji, Some pharmacological, toxicological and phytochemical investigations on Centaurea phyllocephala. Journal of Ethnopharmacology 9,

7 Table 1. Densitometer quantification of various polypeptides of rat kidney, liver and spleen showing the percentage area occupied by individual polypeptides. Kidney K Liver L Spleen S Kidney K1 Liver L1 Spleen S RT- Retention Time % - occupied by individual polypeptide of gene