Journal of Ethnopharmacology 103 (2006) 484490

Ethnopharmacological communication

Hepatoprotective effects of rubiadin, a major constituent of Rubia cordifolia Linn.

Guntupalli M. Mohana Rao a, , Chandana V. Rao a , Palpu Pushpangadan a , Annie Shirwaikar b
a

Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute, Lucknow 226 001, Uttarpradesh, India b Department of Pharmacognosy, College of Pharmaceutical Sciences, Manipal 576 119, Karnataka, India Received 16 April 2005; received in revised form 4 August 2005; accepted 16 August 2005 Available online 5 October 2005

Abstract The hepatoprotective effects of rubiadin, a major constituent isolated from Rubia cordifolia Linn., were evaluated against carbon tetrachloride (CCl4 )-induced hepatic damage in rats. Rubiadin at a dose of 50, 100 and 200 mg/kg was administered orally once daily for 14 days. The substantially elevated serum enzymatic activities of serum glutamic oxaloacetic transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), serum alkaline phosphatase (SALP) and -glutmyltransferase (-GT) due to carbontetrachloride treatment were dose dependently restored towards normalization. Meanwhile, the decreased activities of glutathione S-transferase and glutathione ruductase were also restored towards normalization. In addition, rubiadin also signicantly prevented the elevation of hepatic melondialdehyde formation and depletion of reduced glutathione content in the liver of CCl4 intoxicated rats in a dose dependent manner. Silymarin used as standard reference also exhibited signicant hepatopretective activity on post treatment against carbon tetrachloride induced hepatotoxicity in rats. The biochemical observations were supplemented with histopathological examination of rat liver sections. The results of this study strongly indicate that rubiadin has a potent hepatoprotective action against carbon tetrachloride induced hepatic damage in rats. 2005 Published by Elsevier Ireland Ltd.
Keywords: Rubia cordifolia; Rubiadin; Carbon tetrachloride; Hepatoprotective effect; Silymarin; Histopathology

1. Introduction Liver regulates various important metabolic functions. Hepatic damage is associated with distortion of these metabolic functions (Wolf, 1999). Liver disease is still a world wide health problem. Unfortunately, conventional or synthetic drugs used in the treatment of liver diseases are inadequate and sometimes can have serious side effects. This is one of the reasons for many people in the world over including those in developed countries turning complimentary and alternative medicine (CAM). Many traditional remedies employ herbal drugs for the treatment of liver ailments (Dhuley and Naik, 1997; Venkateswaran et al., 1997; Latha et al., 1999; Mitra et al., 2000). Rubiadin, 1,3dihydroxy-2-methyl anthraquinone has been isolated from the 50% aqueous EtOH extract of roots of Rubia cordifolia Linn.

Corresponding author. Fax: +91 522 2205836. E-mail address: mmraos@rediffmail.com (G.M.M. Rao).

(Rubiaceae). Rubia cordifolia is an important medicinal plant which is used for treatment of various ailments in Ayurvedic system of medicine (George, 1967; Pandey and Chunnekar, 1967; Sertoli et al., 1994; Adwankar et al., 1980; Singh et al., 1983; Tripathi et al., 1993; Pandey et al., 1994). Rubiadin, isolated from the roots of Rubia cordifolia was found to have potent antioxidant property (Tripathi et al., 1993), In addition, rubiadin also have been found to inhibit lipid peroxidation (Tripathi and Sharma, 1998) and the plant Rubia cordifolia have been reported for anti-inammatory (Antarkar et al., 1983), immunomodulatory (Joharapurkar et al., 2003), anticonvulsant and anxiolytic (Kasture et al., 2000) and anti-tumor activities (Manohar et al., 1982). In the ethnobotanical claims, it is mentioned as, the roots are used for the treatment of jaundice by the folk tribes of west Bengal and Uttaranchal (Jain, 1991), but to the best of our knowledge there is no scientic report on the hepatoprotective activity of Rubia cordifolia. Therefore, to justify the traditional claims we have assessed the hepatoprotective effects of rubiadin using CCl4 -intoxicated rats as experimental model.

0378-8741/$ see front matter 2005 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2005.08.073

G.M.M. Rao et al. / Journal of Ethnopharmacology 103 (2006) 484490

485

2. Materials and methods 2.1. Animals SpragueDawley rats weighing (140 20 g) of either sex were purchased from the animal house of the Central Drug Research Institute, Lucknow. They were kept in departmental animal house in well cross-ventilated room at 27 2 C, and relative humidity 4456%, light and dark cycles of 10 and 14 h, respectively, for 1 week before and during the experiments. Animals were provided with standard rodent pellet diet (Amrut, India) and the food was withdrawn 1824 h before the experiment thought, water was allowed ad libitum. All the experiments were performed in the morning according to current guidelines for the care of the laboratory animals and the ethical guidelines for the investigation of experimental pain in conscious animals (Zimmerman, 1983). All the chemicals used were of the analytical grade from standard companies and the water represents the double distilled water. The standard orogastric cannula was used for oral drug administration. 2.2. Plant material Roots of Rubia cordifolia were collected from campus garden of National Botanical Research Institute, Lucknow, India in January 2004. The plant material was identied and authenticated taxonomically at National Botanical Research Institute, Lucknow, India. A voucher specimen of the collected sample was deposited in the institutional herbarium and departmental museum for future reference. 2.3. Isolation and quantication of rubiadin The powdered roots of Rubia cordifolia (1000 g) were extracted with 50% aqueous EtOH by cold percolation at room temperature and the combined extracts from three successive extractions, were concentrated to a sticky gum in a rotary vacuum evaporator under reduced pressure to yield 95 g (9.5% (w/w)). Solvent free extract was portioned between 90% aqueous methanol and hexane in a separating funnel. Non-polar hexane layer was separated and dried over Na2 SO4 and concentrated (fraction 1). Methanol layer was dried and suspended in water for further extraction successively chloroform (fraction 2) and n-butanol (fraction 3). Fraction 2 was adsorbed on a bed of silicagel and eluted initially with hexane, toluene, ethylacetate and butanol by using different ratios of these solvents in the order of increasing polarity. The fractions were checked on TLC plate and similar fractions were pooled. Elution of the column with toluenehexane (1:1) mixture furnished a yellow solid. Later, the yellow solid was puried by several recrystallizations from toluene and conrmed (Tripathi et al., 1997) by TLC using toluene:ethyl acetate (85:15) as the mobile phase (Rf = 0.58) and the spots were detected under UV irradiation. Further rubiadin was quantied with the help of high-performance thin layer chromatography (HPTLC) studies, were carried out on pre coated silica gel plate (Merck 60 F 254).

The yellow solid (rubiadin) obtained from chloroform fraction was spotted along with test solution (50% aqueous EtOH extract of Rubia cordifolia) using a Camag Linomat IV spotter. These plates were observed at UV 254 and 366 nm, and were scanned on TLC scanner III using CAT software at 300 nm, record the peak area and prepare the calibration curve by plotting peak area against concentration of the rubiadin applied. The amount of rubiadin present in the 50% aqueous EtOH extract of Rubia cordifolia was calculated from the calibration curve of rubiadin. 2.4. CCl4 induced hepatotoxicity Rats were divided into six groups of six animals in each group. Group I (control) animals were administered a single daily dose of liquid parafn (1 ml/kg body weight, p.o.). Group II received carbon tetrachloride (1 ml/kg body weight, i.p.). Test groups (Groups IIIV) were administered orally 50, 100 and 200 mg/kg body weight rubiadin, respectively, in the form of aqueous suspension daily once a day. Group VI received silymarin, the known hepatoprotective compound (Sigma Chemicals Company, USA), at a dose of 100 mg/kg, p.o., along with carbon tetrachloride. The rubiadin was given simultaneously with carbon tetrachloride. Treatment duration was 14 days. Dosage of carbon tetrachloride was administered as 30% solution in liquid parafn for every 72 h. Animals were sacriced 48 h after the last injection. Blood was collected, allowed to clot and serum separated. Liver was dissected out and used for biochemical studies. 2.5. Biochemical determinations The Biochemical parameters like serum enzymes: serum glutamic oxaloacetic transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), serum alkaline phosphatase (SALP) were assayed according to standard methods of Malloy and Jollow Evelyn (1937), Reitman and Frankel (1957), respectively, using an assay kit. -Glutmyltransferase (-GT) was determined by the methods of Szasz (1969). The contents of glutathione (GSH) and malondialdehyde (MDA) were determined by the methods of Ellman (1959) and Okhawa et al. (1979), respectively. The activities of glutathione S-transferase (GST) and glutathione reductase (GR) were determined by the method of Habig et al. (1974) and Mize and Langdon (1962), respectively. The protein content was measured by the methods of Lowry et al. (1951) with bovine serum albumin as standard. 2.6. Liver histopathological assessment Liver sections taken immediately from the liver, xed in 10% formalin, dehydrated in gradual ethanol (50100%), cleared in xylene, and embedded in parafn. Sections (45 m thick) were prepared and then stained with hematoxylin and eosin (HE) dye for photomicroscopic observation, including cell necrosis, fatty change, hyaline degeneration, ballooning degeneration, inltration of kupfer cells and lymphocytes.

486

G.M.M. Rao et al. / Journal of Ethnopharmacology 103 (2006) 484490

2.7. Statistical analysis The data are expressed as mean S.E.M. The difference among means has been analyzed by students t-test, method of Woolson (1987). A value of P < 0.05 was considered statistically signicant. 3. Results 3.1. HPTLC analysis of rubiadin In view of the identication of the rubiadin as the constituent responsible for signicant hepatoprotective activity of Rubia cordifolia, the active marker has been isolated and quantied by HPTLC analysis. The marker compound isolated from the fresh plant material collected locally was thoroughly characterized by spectroscopic analysis. Its identity was also conrmed by comparing the data with reported values (Tripathi et al., 1997). HPTLC studies revealed that the toluene:ethylaacetate (85:15) was ideal mobile phase for rubiadin and gave single spot at Rf 0.58 (Plate 1). The spot on the chromatogram was visualized under UV light at 254 and 366 nm (Plate 1), and then densitometric scanning was done for rubiadin at 300 nm along with test sample (Plate 2). The percentage of the active marker was calculated by using peak area, and the rubiadin content was found to be not less than 0.3%. 3.2. Effects of rubiadin on SGOT, SGPT, ALP and -GT activities The results of hepatoprotective effects of rubiadin on CCl4 intoxicated rats are shown in Table 1. In the CCl4 -treated control, serum GOT, GPT, ALP and -GT were increased 103.12, 148.23 kA unit/ml, 98.12 unit/ml and 205.2 mU/ml, mean while these values showed 48.02, 63.12 kA unit/ml, 71.29 unit/ml and 28.6 mU/ml in normal group, respectively. In contrast, the groups treated with 50, 100 and 200 mg/kg of rubiadin decreased signicantly (50: P < 0.01; 100, 200: P < 0.001) the elevated levels of SGOT, SGPT, SALP, and GT in a dose dependent manner towards normalization. Treatment with rubiadin 200 mg/kg, showed highly signicant activTable 1 Effects of rubiadin on serum and liver biochemical indices in CCl4 -intoxicated rats Group I II III IV V IV Treatment Normal (liquid parafn) CCl4 1 ml/kg Rubiadin 50 (mg/kg) + CCl4 Rubiadin 100 (mg/kg) + CCl4 Rubiadin 200 (mg/kg) + CCl4 Silymarin 100 (mg/kg) + CCl4 SGOT (U/L) 48.02 103.12 84.32 78.26 69.23 50.43 6.32 11.12 11.61* 8.73** 3.12** 3.80** SGPT (U/L) 63.12 148.23 95.10 80.26 73.12 81.23 4.39 10.13 3.86* 5.07** 1.21** 4.20** SAKP (U/L) 71.29 98.12 102.11 88.12 84.23 81.11 12.16 4.13 4.24* 3.79** 3.12** 3.26** -GT (mU/ml) 28.6 205.2 97.35 75.12 68.12 108.12 3.35 18.15 2.23* 11.85** 10.73** 7.94* GSTa 258.1 18.56 144.6 9.10 189.1 6.88* 204.1 3.53** 212.5 6.72** 214.3 5.66** GRb 29.1 3.42 10.6 1.50 20.2 1.87* 22.5 1.29** 24.5 1.66** 25.6 2.0**

Plate 1. TLC prole of rubiadin along with test solution of Rubia cordifolia root. Track 1, rubiadin (isolated); Track 2, test solution of 50% aqueous EtOH extract Rubia cordifolia.

Plate 2. HPTLC densitometric chromatogram scan at 300 nm of rubiadin along with test solution of Rubia cordifolia root. A, rubiadin (isolated); B, test solution (50% aqueous EtOH extract of Rubia cordifolia).

Rats were administered with rubiadin 50, 100 and 200 mg/kg orally once for 14 days, and then CCl4 1 ml/kg (i.p.) was injected for every 72 h once during the 14 days. The rats were decapitated 48 h after the nal administration of CCl4 . Data are expressed as mean S.E.M (n = 6). a Units: 1,2-dichloro-4-nitrobenzene nmol/mg protein/min. b Units: GSH formed nmol/mg protein/min. * P < 0.01 as compared with Group II. ** P < 0.001 as compared with CCl treated group. 4

G.M.M. Rao et al. / Journal of Ethnopharmacology 103 (2006) 484490

487

Fig. 1. Group I: Liver sections of normal control rats showing: normal hepatic cells with well preserved cytoplasm; well brought out central vein; prominent nucleus and nucleolus.

Fig. 3. Group VI: Liver section of rats treated with CCl4 (1 ml kg, i.p.) + silymarin (100 mg/kg, p.o.) 14 days, showing: well brought out central vein, hepatic cell with well preserved cytoplasm, prominent nucleus and nucleolus.

ity which is almost comparable to the group treated with silymarin, a potent hepatoprotective drug used as reference standard. 3.3. Histopathological observations The histologcal observations basically support the results obtained from serum enzyme assays. Histology of the liver sections of normal control animals (Group I) showed normal hepatic cells with well preserved cytoplasm, prominent nucleus and nucleolus and well brought out central vein (Fig. 1). The liver sections of CCl4 -intoxicated rats showed massive fatty changes, necrosis, ballooning degeneration, and broad inltration of the lymphocytes and kupffer cells around the central vein and the loss of cellular boundaries (Fig. 2). CCl4 -induced group was more severe than the other groups. The histological architecture of liver sections of rats treated with rubiadin 50, 100 and 200 mg/kg showed a more or less normal lobular pattern with a mild degree of fatty change, necrosis and lymphocyte inltration almost comparable to the normal control and silymarin treated groups (Figs. 36).

Fig. 4. Group III: Liver section of rats treated with CCl4 (1 ml kg, i.p.) + rubiadin (50 mg/kg, p.o.) 14 days, showing: well brought out central vein, hepatic cell with well preserved cytoplasm, prominent nucleus and nucleolus.

3.4. Effects of rubiadin on hepatic MDA and GSH levels The production of MDA in CCl4 -induced group was increased drastically when compared with the normal group (Fig. 7). Treatment with 50, 100 and 200 mg/kg of rubiadin reduced CCl4 -induced MDA production in a dose depen-

Fig. 2. Group II: Liver section of CCl4 (1 ml/kg, i.p.) treated rats showing: massive fatty changes, necrosis, ballooning degeneration, and broad inltration of the lymphocytes and kupffer cells around the central vein and the loss of cellular boundaries.

Fig. 5. Group IV: Liver section of rats treated with CCl4 (1 ml kg, i.p.) + rubiadin (100 mg/kg, p.o.) 14 days, showing: well brought out central vein, hepatic cell with well preserved cytoplasm, prominent nucleus and nucleolus.

488

G.M.M. Rao et al. / Journal of Ethnopharmacology 103 (2006) 484490

Fig. 6. Group V: Liver section of rats treated with CCl4 (1 ml kg, i.p.) + rubiadin (200 mg/kg, p.o.) 14 days, showing: well brought out central vein, hepatic cell with well preserved cytoplasm, prominent nucleus and nucleolus.

dent manner, when compared with the CCl4 control group (P < 0.001). Administration CCl4 decreased the hepatic GSH level drastically. Treatment with 50, 100, and 200 mg/kg of rubiadin elevated the decreased hepatic GSH levels towards normalization in a dose dependent manner (Fig. 8). In contrast 100 and 200 mg/kg shown highly signicant (P < 0.001) activity. The results are comparable with silymarin, a potent hepatoprotective drug available in the market. 3.5. Effects of rubiadin on GST and GR activities GST and GR activities in CCl4 -intoxicated rats were decreased drastically when compared with normal group. Treatment with rubiadin recovered these decreased enzyme activities produced by CCl4 towards normalization in a dose dependent manner. From the results, it is very clear that rubiadin 100 and 200 mg/kg showed highly signicant activity (P < 0.001) which is almost similar to the silymarin produced results (Table 1).

Fig. 8. Effects of rubiadin on hepatic GSH levels in CCl4 -intoxicated rats. Rats were administered with rubiadin 50, 100 and 200 mg/kg orally once a day for 14 days, and then CCl4 was 1 ml/kg (i.p.) was injected four times at every 72 h after the administration of rubiadin. The rats were decapitated 48 h after the nal administration of CCl4 . The results are compared with silymarin (S). The data are expressed as mean S.E.M (n = 6). * P < 0.01, ** P < 0.001 as compared with the CCl4 control by Duncans new multiple range test.

4. Discussion and conclusion The present study showed for the rst time, rubiadin isolated from Rubia cordifolia possess hepatoprotective activity as evidenced by the signicant inhibition in the elevated levels of serum enzyme activities induced by CCl4 . Earlier studies have shown that rubiadin, a major component of Rubia cordifolia possesses potent antioxidant property, inhibited the lipid peroxidation induced by FeSO4 and t-butylhydroperoxide (t-BHP) in a dose dependent manner (Tripathi et al., 1997; Tripathi and Sharma, 1998). In the present study, rubiadin was given orally (50200 mg/kg), once daily for 14 days showed dose dependent hepatoprotective activity, and highly signicant effect was seen with of 100 and 200 mg/kg bodyweight, against CCl4 induced hepatic damage. It is well established that CCl4 induces hepatotoxicity by metabolic activation, therefore it selectively causes toxicity in liver cells maintaining semi-normal metabolic function. CCl4 is bio-transformed by the cytochrome P450 system in the endoplasmic reticulum to produce trichloromethyl free radical ( CCl3 ). Trichloromethyl free radical then combined with cellular lipids and proteins in the presence of oxygen to form a trichloromethyl peroxyl radical, which may attack lipids on the membrane of endoplasmic reticulum faster than trichloromethyl free radical. Thus, trichloromethylperoxyl free radical leads to elicit lipid peroxidation, the destruction of Ca2+ homeostasis, and nally, results in cell death (De Groot and Noll, 1986; Clawson, 1989; Reckengel et al., 1989). These result in changes of structures of the endoplasmic reticulum and other membrane, loss of enzyme metabolic enzyme activation, reduction of protein synthesis and loss of glucose-6-phosphatase activation, leading to liver damage (Recknagel and Glende, 1973; Gravela et al., 1979; Wolf et al., 1980; Azri et al., 1992). Hepatotoxic compounds like CCl4 are known to cause marked elevation in serum enzyme activities. In the present study, treatment with rubiadin attenuated the increases in the activities of SGOT, SGPT, SALP

Fig. 7. Effects of rubiadin in CCl4 -intoxicated rats. Rats were administered with rubiadin 50, 100 and 200 mg/kg orally once a day for 14 days, and then CCl4 was 1 ml/kg (i.p.) was injected four times at every 72 h after the administration of rubiadin. The rats were decapitated 48 h after the nal administration of CCl4 . The results are compared with silymarin (S). The data are expressed as mean S.E.M. (n = 6). * P < 0.01, ** P < 0.001 as compared with the CCl4 control by Duncans new multiple range test.

G.M.M. Rao et al. / Journal of Ethnopharmacology 103 (2006) 484490

489

and -GT produced by CCl4 indicating that rubiadin protects liver injury induced by CCl4 towards normalization. It has been hypothesized that one of the principal causes of CCl4 -induced liver injury is lipid peroxidation by free radical derivatives of CCl4 . Earlier studies have shown that rubiadin might be acting by metal chelation and thereby inhibiting lipid peroxidation, possibly due to the simultaneous presence of both quinine skeleton and phenolic groups in this molecule. The number and position of these groups in rubiadin also favour its role in an electorn trapping capacity (Shiang et al., 1995). In states of oxidative stress, GSH is converted into GSSG and depleted leading to lipid peroxidation. Therefore, the role of GSH as a reasonable marker for the evaluation of oxidative stress is important (Reckengel et al., 1991). Rubiadin inhibited significantly lipid peroxidation and revovered the decreased hepatic GSH level induced by CCl4 towards normalization. To prevent lipid peroxidation, it is very important to maintain the level of GSH, GSSG is reduced to GSH by GR, which is NADPHdependent. It plays a role in maintaining adequate amounts of GSH. Accordingly, the reduction of GR results in decreasing GSH (Reckengel et al., 1991). In CCl4 -intoxicated rats, the activity of GR is signicantly decreased. However, rubiadin with 100 and 200 mg, brought the activity of GR towards normalization. The function of GST is divided into catalysis and binding. GST is a soluble protein which is located in cytosol, plays an important role in the detoxication and excretion of xenobiotics (Boyer et al., 1984; Masukawa and Iwata, 1986). GST catalyzes the conjugation of the thiol functional groups of glutathione to electrophylic xenobiotics and results in increasing solubility. The xenobioticGSH conjugate is then either eliminated or converted to mercapturic acid. Another function of GST is the binding between GST and endogenous and exogenous substances. Since GST increases solubility of hydrophobic substances, it plays an important role in storage and excretion of xenobiotics. Compounds which increase the activity of GST, which metabolizes toxic compounds to non-toxic compounds, means they have an increasing protective activity of the liver. In CCl4 intoxicated rats, the activity of GST decreased drastically compared to that of normal group. The activity of GST recovered signicantly (P < 0.001) at 100 and 200 mg/kg of rubiadin compared to that of CCl4 group. In contrast, the GST activity at 200 mg/kg is almost similar to the activity shown by silymarin, a potent hepatoprotective agent. The above observations strongly indicate the hepatoprotective activity of rubiadin against CCl4 -intoxicated rats. Also, the activity of GST related to detoxication was increased towards normalization. In addition, the activity of GR, suppressing free radical and the content of GSH reduced by free radical were affected by rubiadin. Therefore, it is concluded that effects of rubiadin on liver protection are related to glutathione mediated detoxication as well free radical scavenging activity. Further studies are in progress for better understanding of the mechanism of action and to evaluate the efcacy of the rubiadin on liver organelle that are possibly damaged during experimental hepatitis.

Acknowledgement The authors are thankful to CSIR, New Delhi, India, for providing the nancial assistance to the rst author in the form of SRF. References
Adwankar, M.K., Chitnis, M.P., Khandalekar, D.D., Bhadsavale, C.G., 1980. Anti-cancer activity of the extracts of Rubia cordifolia Linn. Indian Journal of Experimental Biology 18, 102106. Antarkar, D.S., Chinwala, T., Bhatt, N., 1983. Anti-inammatory activity of Rubia cordifolia Linn. in rats. Indian Journal of Pharmacology 15, 185188. Azri, S., Mata, H.P., Reid, L.L., Gandlo, A.J., Brendel, K., 1992. Further examination of selective toxicity of CCl4 rat liver slices. Toxicology and Applied Pharmacology 112, 8186. Boyer, T.D., Vessey, D.A., Holcomb, C., Saley, N., 1984. Studies of the relationship between the catalytic activity and binding of non-substrate ligands by the glutathione S-transferase. Biochemical Journal 217, 179185. Clawson, G.A., 1989. Mechanism of carbon tetrachloride hepatotoxicity. Pathology and Immunology Research 8, 104112. De Groot, H., Noll, T., 1986. The crucial role of low steady state oxygen partial pressures in haloalkane free radical mediated lipid peroxidation. Biochemical Pharmacology 35, 1519. Dhuley, G.P., Naik, S.R., 1997. Protective effect of Rhinax, a herbal formulation against CCl4 induced liver injury and survival in rats. Journal of Ethnopharmacology 56, 159164. Ellman, G.L., 1959. Tissue sulfadryl group. Archives of Biochemistry and Biophysics 82, 7077. George, W., 1967. Dictionary of the Economic Product of India, vol. VI. Cosmo Publication, Library Road, Delhi, p. 570. Gravela, E., Albano, E., Dianzani, M.U., Poli, G., Slater, T.F., 1979. Effects of carbon tetrachloride on isolated rat hepatocytes: inhibition of protein and lipoprotein secreation. Biochemical Journal 178, 509512. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathone S-transferase: the rst enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 249, 71307139. Jain, S.K., 1991. Dictionary of Indian folk medicine and Ethnobotany. Deep Publication, New Delhi, p. 156. Joharapurkar, A.A., Deode, N.M., Zambad, S.P., Umathe, S.N., 2003. Immunomodulatory activity of alcoholic extract of Rubia cordifolia Linn. Indian Drugs 40, 179181. Kasture, V.S., Desmukh, V.K., Chopde, C.T., 2000. Anticonvulsant and behavioral actions of triterpine isolated from Rubia cordifolia Linn. Indian Journal of Experimetal Biology 38, 675680. Latha, U., Rajesh, M.G., Latha, M.S., 1999. Hepatoprotective effect of an ayurvedic medicine. Indian Drugs 36, 470473. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193, 265275. Malloy, J.R., Jollow Evelyn, K.A., 1937. Journal of Biological Chemistry 119, 481490. Manohar, K., Adwankar, M.K., Chitnis, M.P., 1982. In vivo anti-cancer activity of RC-80, a plant isolate from Rubia cordifolia Linn. against a spectrum of experimental tumour models. Chemotherapy 28, 291293. Masukawa, T., Iwata, H., 1986. Possible regulation mechanism of microsomal glutathione S-transferase activity in rat liver. Biochemical Pharmacology 35, 435438. Mitra, S.K., Seshadri, S.J., Venkataranganna, M.V., Gopumadhavan, S., Venkatesh Udupa, U., Sarma, D.N.K., 2000. Effect of HD-03-a herbal formulation in galactosamine-induced hepatopathy in rats. Indian Journal of Physiology and Pharmacology 44, 8286. Mize, C.E., Langdon, R.G., 1962. Hepatic glutathione reductase. I. Purication and general kinetic properties. Journal of Biological Chemistry 237, 15891595.

490

G.M.M. Rao et al. / Journal of Ethnopharmacology 103 (2006) 484490 Szasz, G., 1969. A kinetic photometric method for serum glutamyltranspeptidase. Clinical Chemistry 15, 124136. Tripathi, Y.B., Pandey, S., Tripathi, P., Shukla, S.D., 1993. Indian Journal of Experimetal Biology 31, 533535. Tripathi, Y.B., Sharma, M., 1998. Comparison of the antioxidant action of the alcoholic extract of Rubia cordifolia with rubiadin. Indian Journal of Biochemistry and Biophysics 35, 313316. Tripathi, Y.B., Sharma, M.K., Manickam, M., 1997. Rubiadin, a new antioxidant from Rubia cordifolia. Indian Journal of Biochemistry and Biophysics 34, 302306. Venkateswaran, S., Pari, L., Viswanathan, P., Menon, V.P., 1997. Protective effex, a herbal formulation against erythromycin estolate-induced hepatotoxicity in rats. Journal of Ethnopharmacology 57, 161167. Wolf, C.R., Harrelson Jr., W.G., Nastainczyk, W.M., Philpot, R.M., Kalyanaraman, B., Mason, R.P., 1980. Metabolism of carbon tetrachloride in hepatic microsomes and reconstituted monooxygenase systems and its relationship to lipid peroxidation. Molecular Pharmacology 18, 553558. Wolf, P.L., 1999. Biochemical diagnosis of liver diseases. Indian Journal of Clinical Biochemistry 14, 5990. Woolson, R.F., 1987. Statistical Methods for the Analysis of Biomedical Data. Wiley, New York. Zimmerman, M., 1983. Ethical guidelines for investigation of experimental pain in conscious animal. Pain 16, 109110.

Okhawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351358. Pandey, G.S., Chunnekar, K.C., 1967. Bhav Prakash Nighantu. Chaukhambha Vidya Bhavan, Varanasi, pp. 570576. Pandey, S., Sharma, M., Chaturvedi, P., Tripathi, Y.B., 1994. Indian Journal of Experimetal Biology 32, 180184. Reckengel, R.O., Glende Jr., E.A., Britton, R.S., 1991. Free radical damage and lipid peroxidation. In: meeks, R.G., Harrison, S.D., Bull, R.J. (Eds.), Hepatotoxicology. CRC Press, Florida, pp. 401436. Reckengel, R.O., Glende, E.A., Dolak, J.A., Waller, R.L., 1989. Mechanism of carbon tetrachloride toxicity. Pharmacological Therapy 43, 139154. Recknagel, R.O., Glende Jr., E.A., 1973. Carbon tetrachloride hepatotoxicity: an example of lethal cleavage. CRC Critical Review Toxicology 2, 263297. Reitman, S., Frankel, S., 1957. American Journal of Clinical Pathology 28, 5356. Sertoli, A., Francalanci, S., Giorgini, S., 1994. Contact Dermatitis 31, 322323. Singh, P.P., Pande, A.P., Goyal, A., Ghosa, R., Srivastva, A.K., 1983. Indian Drugs 20, 264280. Shiang, S.H., Sheau, F.Y., Chaunge, Y.H., 1995. Journal of Natural Products 58, 13651371.