Protective effect of ethyl acetate fraction of Rhododendron arboreum flowers against carbon tetrachloride-induced hepatotoxicity in experimental models.

OBJECTIVE: To evaluate the hepatoprotective potential of ethyl acetate fraction of Rhododendron arboreum (Family: Ericaceae) in Wistar rats against carbon tetrachloride (CCl(4))-induced liver damage in preventive and curative models.
MATERIALS AND METHODS: Fraction at a dose of 100, 200, and 400 mg/kg was administered orally once daily for 14 days in CCl(4)-treated groups (II, III, IV, V and VI). The serum levels of glutamic oxaloacetic transaminase (SGOT), glutamate pyruvate transaminase (SGPT), alkaline phosphatase (SALP), γ-glutamyltransferase (γ -GT), and bilirubin were estimated along with activities of glutathione S-transferase (GST), glutathione reductase, hepatic malondialdehyde formation, and glutathione content. RESULT AND DISCUSSION: The substantially elevated serum enzymatic activities of SGOT, SGPT, SALP, γ-GT, and bilirubin due to CCl(4) treatment were restored toward normal in a dose-dependent manner. Meanwhile, the decreased activities of GST and glutathione reductase were also restored toward normal. In addition, ethyl acetate fraction also significantly prevented the elevation of hepatic malondialdehyde formation and depletion of reduced glutathione content in the liver of CCl(4)-intoxicated rats in a dose-dependent manner. Silymarin used as standard reference also exhibited significant hepatoprotective activity on post-treatment against CCl(4)-induced hepatotoxicity in rats. The biochemical observations were supplemented with histopathological examination of rat liver sections. The results of this study strongly indicate that ethyl acetate fraction has a potent hepatoprotective action against CCl(4)-induced hepatic damage in rats.

Introduction

Herbal drugs play a major role in the treatment of hepatic disorders. In India, a number of medicinal plants and their formulations are used to cure hepatic disorders in traditional systems of medicine.[12] In Homeopathic Materia Medica, the tincture of dried leaves of Rhododendron arboreum (Family: Ericaceae, commonly known as Laligurans) has been used in gout.[3] The leaves of this plant contain the flavone glycoside and dimethyl ester of terephthalic acid and certain flavonoids.[4] Flavonoids isolated from the leaves of R. arboreum were found to have potent antioxidant property.[5] Ayurvedic preparation “Asoka Aristha,” containing R. arboreum possesses oxytocic, estrogenic, and prostaglandin synthetase-inhibiting activity.[6] However, the ethanol soluble portion of the flowers of R. arboreum shows α-glucosidase inhibitor and potent antidiabetic activity.[7] We have observed the ethyl acetate fraction of R. arboreum to possess significant anti-inflammatory and antinociceptive activities.[8] The flowers of the plant are used traditionally in west Himalaya as a remedy for liver complaints and in anemia, but have not been investigated for a hepatoprotective activity.[9] The main objective of the study was to evaluate this hepatoprotective activity of R. arboreum to verify tribal or folk claims in using it for liver disorders. In the present investigation, the antihepatotoxic activity of ethyl acetate fraction of R. arboreum was investigated in acute liver injury model against carbon tetrachloride (CCl4) in preventive and curative treatments.

Materials and Methods

Chemicals

All chemicals were of analytical grade. CCl4 was purchased from Merck India Ltd., Mumbai. Silymarin, corn oil, dithio-bis-2-nitrobenzoic acid, thiobarbituric acid, and assay kit for lactate dehydrogenase were purchased from Sigma Chemical Co., St. Louis, MO, USA. Assay kits for serum alanine aminotransferase and aspartate aminotransferase were purchased from Dialab, Austria.

Plant Material

The flowers of R. arboreum were collected from Rudraprayag, Uttaranchal and authenticated with the existed specimen (NBRI/CIF/83/2009) at National Botanical Research Institute [NBRI], Lucknow. The prepared herbarium was deposited in the laboratory of NBRI for future reference.

Animals

Wistar albino rats (150–200 g) of either sex were selected for the study and maintained at a controlled temperature of 25 to 28°C with a 12-hour light/dark cycle and fed a standard diet and water ad libitum. Animal studies were conducted according to the Institute Animal Ethics Committee regulations approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals.

Extraction and Fractionation

The air-dried powdered flowers (200 g) were extracted with ethanol (50%) using Soxhlet apparatus, which was then evaporated under Rotavapor to afford the ethanolic extract of flowers. The extract thus obtained was partitioned with organic solvents to afford the n-hexane, chloroform, n-butanol, and ethyl acetate fractions.

Carbon Tetrachloride-Induced Hepatotoxicity

Rats were divided into six groups of six animals each, Group I (control) was administered a single daily dose of liquid paraffin (1 ml/kg body weight, p.o.). Group II received CCl4 (1 ml/kg body weight, i.p.). Test groups III, IV, and V received 100, 200, and 400 mg/kg of the ethyl acetate fraction of R. arboreum orally. Group VI received silymarin, a known hepatoprotective compound at a dose of 100 mg/kg, p.o., along with CCl4. Treatment duration was 14 days. CCl4 was administered as 30% solution in liquid paraffin every 72 hours. Animals were sacrificed 48 hours after the last injection. Blood was collected, allowed to clot, and serum separated. Liver was dissected out and used for biochemical studies.

Biochemical Determinations

The Biochemical parameters like serum enzymes: serum glutamic oxaloacetic transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), serum alkaline phosphatase (SALP), and bilirubin were assayed according to standard methods[10-11] using an assay kit. γ-glutamyltransferase (γ-GT) was determined by the method of Szasz.[12] The contents of glutathione (GSH) and malondialdehyde (MDA) were determined by the methods of Ellman[13] and Okhawa et al.,[14] respectively. The activities of glutathione S-transferase (GST) and glutathione reductase (GR) were determined by the method of Habig et al.[15] and Mize and Langdon,[16] respectively. The protein content was measured by the method of Lowry et al.,[17] with bovine serum albumin as standard.

Liver Histopathological Assessment

Liver sections were immediately fixed in 10% formalin, dehydrated in gradual ethanol (50–100%), cleared in xylene, and embedded in paraffin. Sections (4–5 μm thick) were prepared and then stained with hematoxylin and eosin (H-E) dye for photomicroscopic observation, including cell necrosis, fatty change, hyaline degeneration, ballooning degeneration, infiltration of Kupffer cells, and lymphocytes.

Statistical Analysis

The data are expressed as mean ± S.E.M. The difference among means has been analyzed by student's t test, method of Woolson.[18] A value of P<0.05 was considered to be statistically significant.

Results

Effects of Ethyl Acetate Fraction on Serum Glutamic Oxaloacetic Transaminase, Serum Glutamate Pyruvate Transaminase, Alkaline Phosphatase, Bilirubin, and γ-Glutamyltransferase Activities

The results of hepatoprotective effects of ethyl acetate fraction of R. arboreum on CCl4-intoxicated rats are shown in Table 1. In the CCl4-treated control, serum GOT, GPT, ALP, γ-GT, and bilirubin were substantially (statistical significance vs. normal) increased. In contrast, the groups treated with 100, 200, and 400 mg/kg of fraction showed a significant decrease in the elevated levels of SGOT, SGPT, SALP, γ-GT, and bilirubin in a dose-dependent manner toward normal. Treatment with ethyl acetate fraction of R. arboreum at a dose of 400 mg/kg showed highly significant activity which is almost comparable with the group treated with silymarin, a potent hepatoprotective drug used as reference standard.

Table 1

Effects of ethyl acetate fraction of Rhododendron arboreum flowers on serum and liver biochemical indices in CCl4-intoxicated rats

Histopathological Observations

The histological observations 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 [Figure 1]. The liver sections of CCl4-intoxicated rats showed massive fatty changes, necrosis, ballooning degeneration, and broad infiltration of the lymphocytes and Kupffer cells around the central vein and the loss of cellular boundaries [Figure 2]. CCl4-induced group was more severe than the other groups. The histological architecture of liver sections of rats treated with ethyl acetate fraction 100, 200, and 400 mg/kg showed a more or less normal lobular pattern with a mild degree of fatty change, necrosis, and lymphocyte infiltration [Figures 3–6].

Figure 1

Liver section of normal rats (Group I) showing: normal hepatic cells with well-preserved cytoplasm; well brought out central vein; prominent nucleus and nucleolus

Figure 2

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

Figure 3

Liver section of rats treated with CCl4 (1 ml/kg, i.p.) + ethyl acetate fraction (100 mg/kg, p.o.) × 14 days (Group III) showing well brought out central vein, hepatic cell with well-preserved cytoplasm, prominent nucleus and nucleolus

Figure 6

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

Liver section of rats treated with CCl4 (1 ml/kg, i.p.) + ethyl acetate fraction (200 mg/kg, p.o.) × 14 days (Group IV) showing well brought out central vein, hepatic cell with well-preserved cytoplasm, prominent nucleus and nucleolus

Liver section of rats treated with CCl4 (1 ml/kg, i.p.) + ethyl acetate fraction (400 mg/kg, p.o.) × 14 days (Group V) showing well brought out central vein, hepatic cell with well-preserved cytoplasm, prominent nucleus and nucleolus

Effects of Ethyl Acetate Fraction on Hepatic Malondialdehyde and Glutathione Levels

The production of MDA in CCl4-induced group was increased drastically as compared with the normal group [Figure 7]. Treatment with 100, 200, and 400 mg/kg of ethyl acetate fraction reduced CCl4-induced MDA production in a dose-dependent manner significantly (P<0.001). Administration of CCl4 decreased the hepatic GSH level drastically. Treatment with 100, 200, and 400 mg/kg of ethyl acetate fraction elevated the hepatic GSH levels toward normal in a dose-dependent manner [Figure 8]. The results were comparable with silymarin.

Figure 7

Rats were administered with ethyl acetate fraction 100, 200, and 400 mg/kg orally once a day for 14 days, and then CCl4 (1 ml/kg, i.p.) was injected four times at every 72 hours after the administration of ethyl acetate fraction. The rats were decapitated 48 hours after the final administration of CCl4. The data are expressed as mean ± S.E.M (n = 6).aP<0.01, bP<0.001 as compared with the CCl4-treated group by Duncan's new multiple range test

Figure 8

Rats were administered with ethyl acetate fraction 100, 200, and 400 mg/kg orally once a day for 14 days, and then CCl4 (1 ml/kg, i.p.) was injected four times at every 72 hours after the administration of ethyl acetate fraction. The rats were decapitated 48 hours after the final administration of CCl4. The data are expressed as mean ± S.E.M (n = 6). aP<0.01, bP<0.001 as compared with the CCl4 treated group by Duncan's new multiple range test

Effects of Ethyl Acetate Fraction on Glutathione S-Transferase and Glutathione Reductase Activities

GST and GR activities in CCl4-intoxicated rats were decreased drastically as compared with the normal group. Treatment with ethyl acetate fraction caused a recovery toward normal in a dose-dependent manner. From the results, it is very clear that ethyl acetate fraction 100 and 200 mg/kg showed highly significant activity (P<0.001) which is almost similar to the silymarin [Table 1].

Discussion and Conclusion

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 biotransformed by the cytochrome P450 system in the endoplasmic reticulum to produce trichloromethyl free radical (•CCl3). Trichloromethyl free radical further combines 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, trichloromethyl peroxyl free radical leads to elicit lipid peroxidation, the destruction of Ca2+ homeostasis, and finally, results in cell death.[19-21] This results 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.[22-25] The present study showed that ethyl acetate fraction of R. arboreum possess hepatoprotective activity, as evidenced by the significant inhibition in the elevated levels of serum enzyme activities induced by CCl4. Ethyl acetate fraction of R. arboreum given orally (100–400 mg/kg), once daily for 14 days, showed dose-dependent hepatoprotective activity, and highly significant effect was seen with doses of 200 and 400 mg/kg bodyweight, against CCl4-induced hepatic damage. Hepatotoxic compounds like CCl4 are known to cause marked elevation in serum enzyme activity. In the present study, treatment with ethyl acetate fraction of R. arboreum attenuated the increases in the activities of SGOT, SGPT, SALP, γ-GT, and bilirubin produced by CCl4, indicating that ethyl acetate fraction of R. arboreum protects liver injury induced by CCl4. It has been hypothesized that one of the principal causes of CCl4-induced liver injury is lipid peroxidation by free radical derivatives of CCl4. 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. Ethyl acetate fraction of R. arboreum significantly inhibited lipid peroxidation and recovered the decreased hepatic GSH level induced by CCl4 toward normal. To prevent lipid peroxidation, it is very important to maintain the level of GSH. GSSG plays a role in maintaining adequate amounts of GSH. It is reduced to GSH by GFR which is NADPH dependent. Accordingly, the reduction of GR results in decreasing GSH.[26] In CCl4-intoxicated rats, the activity of GR is significantly decreased. However, ethyl acetate fraction of R. arboreum brought the activity of GR toward normal. The function of GST is divided into catalysis and binding. GST is a soluble protein which is located in cytosol, which plays an important role in the detoxification and excretion of xenobiotics.[2728] The cytosolic GST catalyzes the conjugation reaction of GSH and electrophilic substrates, and therefore, has an integral role in the detoxification of electrophilic toxicants.[29] In CCl4-intoxicated rats, the activity of GST decreased drastically compared with the normal group. The activity of GST recovered significantly (P<0.001) at 200 and 400 mg/kg of ethyl acetate fraction of R. arboreum compared with that of CCl4 group. In contrast, the GST activity at 400 mg/kg was almost similar to that shown by silymarin, a potent hepatoprotective agent. The above observations strongly indicate the hepatoprotective activity of ethyl acetate fraction of R. arboreum against CCl4-intoxicated rats. GR is involved in diverse cellular phenomena, including the defensive response against free radicals and reactive oxygen species (ROS) as well as protein and DNA biosynthesis, by maintaining a high ratio of GSH/GSSG.[30-32] Therefore, it is concluded that effects of ethyl acetate fraction of R. arboreum on liver protection are related to glutathione-mediated detoxification as well as free radical scavenging activity. Further studies are in progress for better understanding of the mechanism of action and to evaluate the efficacy of the ethyl acetate fraction of R. arboreum on liver organelle that are possibly damaged during experimental hepatitis.