International Journal of Drug Development and Research

Key words:

Flavonoids, Prophylactic, Thioacetamide.

INTRODUCTION

Liver is one of the major target organ in the human body and the main site for metabolism and excretion of the substance. It is involved in most of the biochemical pathways for growth, fight against disease, supply of the nutrients, energy provision, and reproduction [1]. Hepatic injury is associated with alteration of these metabolic functions [2] and sometimes, leads to severe health problems.

Human is exposed to a variety of foreign chemicals such as drugs, food additives and environmental pollutants in their daily life. Various drugs like acetaminophen, alcohol, ibuprofen, CCl4, lead and contraceptives are some of the drugs that cause liver injury. Most of these compounds are known to cause chemical alteration in the human body. The liver plays a significant role in this process [3].

Acute liver failure is a major problem for hepatologists to face. Multiorgan failure and death are associated with failure of liver. The most common cause of acute liver failure includes acute viral hepatitis and drug induced hepatic injury. Acute liver failure is associated with high rates of mortality inspite advances in intensive care and the progress of new treatment methods. Liver transplantation remains the only effective treatment [4].

Since past several years thioacetamide (TAA) has been used as a model to induce acute liver toxicity in rats [5]. TAA is a potent centrilobular liver toxicant which undergoes a two-step bioactivation mediated by microsomal CYP2E1 to thioacetamide sulphoxide, and lead to formation of a reactive metabolite thioacetamide-S, S-dioxide [6]. Toxic effects of TAA are due to the production of (thioacetamide-S-oxide which reacts with proteins resulting in their denaturation. It has been evaluated that the administration of TAA leads to the cell death by necrosis as well as apoptosis in experimental animals [7].

Disorders of Liver are a great problem in the world today. In spite of its recurrent incidence, high morbidity and high mortality, its medicinal management is inadequate at present; no such therapy has effectively prevented the development of liver disorders [8]. There is a long history that environment has provided a remarkable number of modern drugs obtained from natural sources and are used in traditional medicine. Since time various medicinal plants have been used in daily life to protect against several diseases and also to improve health and therefore have been used as a source of medicine. The extensive uses of herbal preparation which are described in ancient period have been traced to the incidence of herbal products with medicinal properties. Plants are the main source of the production of various chemical compounds which are used as a source of diverse type of medicines and provide protection against various diseases [9]. Although crude drugs are not accepted across the world but their assessment has revealed their efficacy in assured health problems [10, 11]. The edible plants contains various bioactive compounds acting together to prevent the necrotic changes [12].

Hibiscus rosa sinensis is a medicinal plant, belongs to family Malvaceae and used in Ayuvedic preparation for a long period of time described in Indian literatures. Quercetin, anthocyanins, cyclopeptide alkaloid and several vitamins e.g. thiamine, riboflavin, niacin, ascorbic acid are the main constituents of HRS [13]. Previous studies have shown that HRS possesses antioxidant, antispermatogenic, androgenic, antitumor and anticonvulsant activities [14]. Therefore, the main aim of our study was to investigate the hepatoprotective effect of HRS against TAA induced liver toxicity.

MATERIALS AND METHODS

Chemicals and reagents

Glutathione Reductase (GR), oxidized (GSSG) and reduced glutathione (GSH), 1,2-dithio-bisnitrobenzoic acid (DTNB), 1-chloro-2, 4- dinitrobenzene (CDNB), bovine serum albumin (BSA), oxidized and reduced nicotinamide adenine dinucleotide phosphate (NADP), (NADPH), flavine adenine dinucleotide (FAD), glucose-6-phosphate, 2,6- dichlorophenolindophenol (DCPIP), thiobarbituric acid (TBA), quercetin etc were obtained from Sigma-Aldrich, USA. Sodium hydroxide, ferric nitrate, trichloroacetic acid (TCA) and perchloric acid (PCA). All other reagents used are of highest purity and commercially available.

Animals

Male Wistar rats (150–200 g), 6–8 weeks old were obtained from the Central Animal house Facility of Hamdard University. Rats were housed in animal care facility under room temperature 25±1oC with 12- h light/dark cycle and have given free assess to standard pellet diet and tap water. Before the treatment rats were left for seven days to acclimatize. Research committee of Hamdard University ethically approved protocols undertaken.

Collection and preparation of extract

Hibiscus rosa sinensis were collected from the herbal garden of Hamdard University, New Delhi, India. Freshly collected plant material was shade-dried and coarsely powdered in a grinder. The extraction procedure was followed as described by Didry et al. Briefly, 100gm dried powdered parts of Hibiscus rosa sinensis were extracted with methanol in a soxhlet for 72hrs.Then by removing the solvent under reduced pressure in rotatory evaporator (Buchi Rotavapour, Switzerland), the concentrated methanolic fraction obtained was stored at 4°C and was dissolved in distilled water to make the required doses.

Treatment regimen

Group I served as a control group and was given saline. Group II was given distilled water by oral gavage for 14 consecutive days and was given a dose of TAA (300 mg/kg b.wt.i.p) on 14th day. Groups III was given HRS at a dose (D1) 100 (mg/kg b.wt.) dissolved in distilled water for 14 consecutive days followed by TAA (300 mg/kg b.wt.i.p) on 14th day. Groups IV was given HRS at a dose (D2) 200 (mg/kg body weight) dissolved in distilled water for 14 consecutive days followed by TAA (300 mg/kg b.wt.i.p) on 14th day. Group V was given HRS (D2) 200 (mg/kg body weight) only. All animals were sacrificed on 15th day. Liver tissue was processed for biochemical estimations. Blood was collected and serum was separated out and processed for serological parameters.

Post Mitochondrial Supernatant preparation (PMS)

Liver was removed and cleaned with ice-cold saline (0.85% sodium chloride). Liver tissues were homogenized in chilled phosphate buffer (0.1 M, pH 7.4) using a Potter Elvehjen homogenizer and were centrifuged at 3000 rpm for 10 min at 4oC by Eltek Refrigerated Centrifuge to separate the nuclear debris. The aliquot so obtained was centrifuged at 12000 rpm for 20 min at 4oC to obtain PMS, which was used as a source of enzymes [15].

Biochemical parameters in liver tissue of wistar rats

Estimation of lipid peroxidation (LPO)

Membrane lipid peroxidation LPO was done by the method of Wright et al [16] with minute modification. The reaction mixture in a total volume of 1.0 ml contained 0.60 ml phosphate buffer (0.1 m, pH 7.4), 0.2 ml microsomes and 0.2 ml ascorbic acid (100 mm). The reaction mixture was incubated at 37°C in a shaking water bath for 1 h. The reaction was stopped by adding 1.0 ml 10% trichloroacetic acid. Following the addition of 1.0 ml 0.67% thiobarbituric acid, all tubes were placed in a boiling water bath for 20 min and then transferred to a crushed ice-bath before centrifuging at 2500g for 10 min. The amount of malondialdehyde (MDA) formed in each of the samples was assessed by measuring the optical density of the supernatant at 535 nm against a reagent blank. The results were expressed as nmol of MDA formed per minutes per gram of tissue using molar extension coefficient 1.56 X 105M-1 cm−1.

Estimation of reduced glutathione (GSH)

Reduced glutathione was assessed by the method of Jollow et al [17]. 1.0-ml of 10% PMS mixed with 1.0 ml of 4% sulphosalicylic acid, Then incubated at 4oC for a minimum time period of 1 h and then centrifuged at 4oC at 1200×g for 15min.Briefly reaction mixture have 0.4 ml supernatant, 2.2 ml phosphate buffer (0.1M, pH 7.4) and 0.4 ml DTNB (4 mg/ml) in a total volume of 3.0 ml. The yellow color developed was read immediately at 412 nm on spectrophotometer (Perkin Elmer, lambda EZ201). The reduced glutathione concentration was calculated as nmol GSH conjugates/g tissue.

Assay for catalase activity

Catalase activity was done by the method of Claiborne [18]. In short the reaction mixture comprised of 0.05 ml PMS, 1.0ml hydrogen peroxide (0.019M), 1.95 ml phosphate buffer (0.1M, pH 7.4), in a total volume of 3 ml. Changes in absorbance were recorded at 240 nm and the change in absorbance was calculated as nmol H2O2 consumed /min/mg protein.

Assay for glutathione peroxidase (GPx) activity

The activity of glutathione peroxidase was calculated by the method of Mohandas et al [19]. A total of 2 ml volume consisted of 0.1ml EDTA (1 mM), 0.1ml sodium azide (1 mM), 1.44ml phosphate buffer (0.1M, pH 7.4), 0.05 ml glutathione reductase (1 IU/ml), 0.05 ml reduced glutathione (1 mM), 0.1 ml NADPH (0.2mM) and 0.01ml H2O2 (0.25mM) and 0.1ml 10% PMS. The depletion of NADPH at 340 nm was recorded at 25oC. Activity of the enzyme was calculated as nmol NADPH oxidized/min/mg protein with the molar extinction coefficient of 6.22 X 103M−1 cm.−1

Assay for glutathione reductase (GR) activity

The activity of glutathione reductase was measured by the method of Carlberg and Mannervik [20]. The reaction mixture consisted of 1.65 ml phosphate buffer (0.1 M, pH 7.6), 0.1 ml NADPH (0.1 mM), 0.05 ml oxidized glutathione (1 mM), 0.1 ml EDTA (0.5 mM) and 0.1 ml 10% PMS in a total volume of 2 ml. Enzyme activity was assessed at 25oC by measuring disappearance of NADPH at 340 nm and was calculated as nmol NADPH oxidized/min/ mg protein using molar extinction coefficient of 6.22 X103 M-1 cm.-1

Assay for quinone reductase (QR) activity

The activity of quinine reductase was measured by the method of Benson et al [21]. The 3-mL reaction mixture consisted of 2.13 ml Tris-HCl buffer (25 mM, pH 7.4), 0.7 mL BSA, 0.1 mL FAD, 0.02 mL NADPH (0.1 mM), and 50 μl (10%)PMS. The reduction of DCPIP was recorded calorimetrically at 600 nm and enzyme activity was calculated as nmol of DCPIP reduced min-1mg protein-1 using molar extinction coefficient of 2.1 × 104 M-1cm-1.

Assay for xanthine oxidase (XO) activity

The activity of xanthine oxidase was measured by the method of Stripe et al [22]. The reaction mixture consisted of 0.2 ml PMS that was incubated for 5 min at 370C with 0.8 ml phosphate buffer (0.1 M, pH 7.4). The reaction was started by adding 0.1 ml xanthine (9 mM) and kept at 370C for 20 min. The reaction was terminated by adding of 0.5 ml ice-cold perchloric acid (PCA) (10% v/v). After 10 min, 2.4 ml of distilled water was added and centrifuged at 4000 rpm. for 10 min and mg uric acid formed per minute per mg protein was recorded at 290 nm.

Measurement of liver toxicity markers serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT)

AST and ALT activity were determined by the method of Reitman and Frankel [23]. Each substrate, 0.5 ml (2mM α-ketoglutarate and either 200 mM. Lalanine or L-aspartate) was incubated for 5 min at 37oC in a water bath. 0.1 ml serum was then added and the volume was adjusted to 1.0 ml with 0.1 M, pH 7.4-phosphate buffer. The reaction mixture was incubated for exactly 30 and 60 min at 37oC for ALT and AST, respectively. Then to the reaction mixture, 0.5ml of 1mM DNPH was added, after another 30 min at room temperature, the colour was developed by addition of 5.0 ml of 0.4N NaOH and the product read at 505nm.

Assay for lactate dehydrogenase (LDH) activity

LDH activity has been estimated in serum by the method of Kornberg [24]. The assay mixture consisted of 0.2 ml serum, 0.1ml 0.02 M NADH, 0.1ml 0.01 M sodium pyruvate, 1.1ml 0.1 M phosphate buffer PH 7.4 and distilled water in a total volume of 3ml. Enzyme activity was recorded at 340 nm and activity was calculated as nmol NADH oxidized/min/mg protein.

Estimation of protein

The protein concentration in all samples was determined by the method of Lowry et al using bovine serum albumin (BSA) as standard [25].

Histopathological evaluation

After blood sampling, the rats were dissected and their livers were separated. The livers were fixed with 10% formalin solution. Histolologic sections were prepared from the livers, stained with haematoxylin and eosin. Analysis was done on 400x magnification.

Statistical analysis

Differences between groups were analyzed using analysis of variance (ANOVA) followed by Dunnet’s multiple comparisons test. All data points are presented as the treatment groups mean ±Standard error of the mean (S.E.).

RESULTS

Hibiscus rosa sinensis pretreatment attenuates the serum LDH, AST and ALT level

Protective effect of HRS extract on serum LDH, AST and ALT level was observed. Significant change in these parameters was found in TAA treated groups as compared to control group (p < 0.001). Pretreatment with HRS was found significantly effective at lower dose (100 mg/kg b.wt.) LDH (p < 0.01), AST (p < 0.01) and ALT (p < 0.001) and at higher dose (200 mg/kg b.wt.) LDH (p < 0.001), AST (p < 0.001), ALT (p < 0.001) in the normalization of these markers when compared to TAA treated group. HRS alone pretreated group did not show any significant difference when compared with control group.

Hibiscus rosa sinensis pretreatment decreased XO activity and MDA level

MDA formation was measured to reveal the oxidative damage on lipid peroxidation on TAA induced liver damage of wistar rats. A significant increase in the XO (p < 0.001) activity and MDA (p < 0.001) level was found in TAA treated group as compared to control group. It has been observed that pretreatment with HRS extract at lower dose (100 mg/kg b.wt.) leads to the significant restoration of XO activity (p < 0.05) and MDA level (p < 0.01) and at higher dose (200 mg/kg b.wt.) leads to the significant restoration of XO activity (p < 0.01) and MDA level (p < 0.001) when compared with TAA treated group. No significant difference was found in the XO activity and MDA level between control and only D2 group.

Hibiscus rosa sinensis pretreatment restored the hepatic GSH level

Protective effect of HRS extract on hepatic GSH level was marked. The level of GSH was depleted significantly (p < 0.001) in TAA treated group as compared to control group. HRS pretreatment increased its level significantly (p < 0.001) in both lower (100 mg/kg b.wt.) and higher (200 mg/kg b.wt.) doses as compared to TAA treated group. Only HRS pretreated group exhibited no significant changes in GSH level as compared to control group.

Effects of Hibiscus rosa sinensis pretreatment on the activities of hepatic antioxidant enzymes

TAA administration was found to deplete hepatic antioxidant enzymes GPx (p < 0.01), GR (p < 0.001), QR (p < 0.01) and catalase (p < 0.001) significantly as compared to control. Pretreatment with HRS extract before TAA administration has been found significantly effective in restoring the activities of these enzymes at lower dose (100 mg/kg b.wt.) GR (p < 0.05) and QR (p < 0.05) but non significant for GPx and at higher dose (200 mg/kg b.wt.) GPx (p < 0.05), GR (p < 0.001), QR (p < 0.001) and catalase (p < 0.001). It has been observed that there is no significant difference in the activity of these antioxidant enzymes between control and only HRS treated groups.

Histopathological analysis

Histological evaluation (Figure1) reveals that liver sections from control rats showed normal architecture, characterized by polyhedral shaped hepatocytes and cytoplasm granulated with small uniform nuclei. Hepatocytes were arranged in wellorganized hepatic cords and separated by narrow blood sinusoids. The liver sections of rats treated with TAA showed centrolobular necrosis and inflammatory cell infiltration. Pretreatment of rats receiving TAA with HRS extract at lower dose (100 mg/kg b.wt.) showed less disarrangement and degeneration of hepatocytes. In contrast, rats treated with higher dose (200 mg/kg b.wt.) showed restoration of the liver morphology.

DISCUSSION

Liver has multiple important functions in which one of the major functions is detoxification of drugs and toxic compounds [26]. It has been investigated that in many cases free radicals are produced during detoxification of the drugs [27]. Over dose of drugs or long time use of various drugs might produce large amounts of free radicals that cause oxidative stress and hepatic injury [28]. TAA is known to cause apoptosis as well as necrosis in the hepatocytes by the production of free radicals during TAA metabolism [29]. TAA is a potent hepatotoxicant and its toxic effects have been attributed to the results from its bioactivation which produces toxic reactive intermediate called thioacetamide S-oxide [30, 31]. Thioacetamide S-oxide causes oxidative stress in the hepatocytes [32]. It cause changes in permeability of the cell, increase in Ca++ concentration level in intracellular region, as a result nuclear volume increases, enlargement in the size of the nucleoli and inhibition of mitochondrial activity which causes cell death [33, 34].

In the recent times consideration has been given to herbal products for the prevention of human diseases. It has been reported that HRS possess multiple biological activities including antioxidant, antispermatogenic, androgenic, antitumor and anticonvulsant suggesting the hepatoprotective effect of HRS may be due to its antioxidant activity [14]. Lipid peroxidation in liver caused by TAA administration is responsible for oxidative stress which leads to liver damage in experimental animals and also through clinical trials in human studies [35]. MDA is produced as a result of lipid peroxidation during the conversion of polyunsaturated fatty acid or lipid peroxides [36]. In the present study, we found that pretreatment with HRS extract significantly decreased MDA formation and XO activity due to ROS in rats treated with TAA. Pretreatment with HRS extract restored the MDA level signifying that the extract might be successful in quenching the free radicals, thus inhibiting LPO and protect the membrane damage from oxidative damage in rats and also restored the XO activity. Various studies of rats and cultured cells have reported that oxidative stress is responsible for TAA-induced liver damage which leads to lipid peroxidation [37, 38, 39]. Amelioration of antioxidant enzymes activity was also observed. HRS restore the antioxidant enzymes activity of GR, GPx, QR, catalase and GSH possibly by quenching excessive ROS and free radicals generated by TAA. Catalase is an enzymatic antioxidant distributed extensively in all animal tissues, and its activity is found to be highest in liver and erythrocytes. It decomposes hydrogen peroxide and help in the protection of the tissues from highly reactive hydroxyl radicals [40]. Thus reduction in the catalase activity might result in lethal effects due to the assimilation of superoxide radical and hydrogen peroxide. HRS pretreatment restored the activity of catalase. Reduced glutathione is a first line of defense neutralizes the hydroxyl radical and plays a major role against inflammatory responses and oxidative stress [41]. A significant dose dependent restoration of glutathione and dependent enzymes, namely glutathione reductase and glutathione peroxidase, to normal levels in HRS pretreated groups was found. Simultaneously the HRS pretreatment was found to restore the depleted level of quinone reductase. Good correlation is found between cellular damage and leakage of enzymes as supported by the elevated levels of serum marker enzymes [42]. It has been previously reported that TAA caused elevations in the levels of serum marker enzymes AST, ALT and LDH as seen in this study [43]. The level of serum toxicity marker enzymes (AST, ALT and LDH) were increased in the TAA treated group and were restored in the HRS extract pretreated group. Histopathological findings also suggested that of HRS have ameliorated effect on TAA-induced hepatic necrosis. Hepatic injury induced by TAA administration in the experimental animals cause histopathological changes in the liver which shows similarities with the human diseases [44]. The present findings showed that liver section from control group showed radial arrangement of hepatocytes around the central vein, granulated cytoplasm having uniform nuclei. In contrast to this liver section from TAA treated group showed loss of hepatic architecture and necrosis in the centrilobular area of hepatocytes. These histological changes were further restored and showed normal architecture of hepatocytes in the experimental animals that received HRS pretreatment.

CONCLUSION

It can be concluded that the results we have obtained reveal that of HRS possess hepatoprotective and antioxidant action on TAA induced liver toxicity in rats. The hepatoprotective effect of may be due to its ability to block the bioactivation of thioacetamide and by scavenging the free radicals and inhibition of lipid peroxidation. TAA administration caused oxidative stress in the liver. Pretreatment of HRS stabilize that stress in the liver. Further work is however needed to define the exact mechanisms which will explain the hepatoprotective action of HRS. The biochemical and histopathological findings obtained from the present study showed that HRS can distinctly reduce hepatic injury induced by TAA administration in experimental rats.

ACKNOWLEDGEMENTS

The authors are thankful to the University Grants commission (UGC), New Delhi, India for providing the funds to carry out this work.

Conflict of interest declaration

The authors of the present research work do not have any conflict of interest to present this research work.

Tables at a glance

Table 1 Table 2 Table 3

Figures at a glance

Figure 1

References

1. F. M. Ward and M. J. Daly. “Hepatic disease,” in Clinical Pharmacy and Therapeutics, R. Walker and C. Edwards, Eds., pp. 195–212, Churchill Livingstone, New York, NY, USA, 1999.
2. P. L. Wolf. “Biochemical diagnosis of liver disease,” Indian Journal of Clinical Biochemistry, vol. 14, no. 1, pp. 59–90, 199.
3. Akilavalli N., Radhika J., Brindha P. Hepatoprotective activity of Ocimum sanctum linn. against lead induced toxicity in albino rats. Asian Journal of Pharmaceutical and Clinical Research. 2011; 4 (2), 84-87.
4. Tse-Min Chen,*Yi-Maun Subeq, et al. Single dose intravenous thioacetamide administration as a model of acute liver damage in rats. J Exp Pathol. 2008; 89(4), 223–231.
5. Shapiro H, Ashkenazi M, Weizman N, Shahmurov M, Aeed H, Bruck R. Curcumin ameliorates acute thioacetamide-induced hepatotoxicity. J. Gastroenterol. Hepatol. 2006; 21,358–366.
6. Chilakapati J, Shankar K, Korrapati MC, Hill RA, Mehendale HM. Saturation toxicokinetics of thioacetamide: role in initiation of liver injury. Drug Metab. Dispos. 2005; 33,1877–188.
7. Li-Hsuen Chen, Chia-Yu Hsu and Ching-Feng Weng. Involvement of P53 and Bax/Bad triggering apoptosis in thioacetamide-induced hepatic epithelial cells. World J Gastroenterol. 2006; 12 (32), 5175-5181.
8. Bruck R et al. Inhibition of experimentally-induced liver cirrhosis in rats by a nonpeptidic mimetic of the extracellular matrix associated Arg-Gly-Asp epitope. J. Hepatol. 1996; 24, 731-738.
9. Farombi and EO. African. Indigenous plants with chemotherapeutic potentials and biotechnological approach to the production of bioactive prophylactic agents. African J Biotech. 2003; 2, 662-671.
10. Hikino H. Traditional remedies and modern assessment: the case of ginseng. In: Wijesekera, R.O.B. (Ed). The medicinal plant chemistry.CRC press, Boca Raton, 1991. Pp149-166.
11. Lamartimere C et al. Genistein alters the ontogeny of mammary gland development and protects against chemically induced mammary cancer in rats. Proc.Soc.Exp.Biol.Med. 1998; 217, 358-364.
12. Koshimizu K et al. Screaning of edible plants against possible antitumor promoting activity. Cancer Lett. 1988; 39, 247-257.
13. Igomathi N and Malarvili T. Effect of Hibiscus Rosa-sinensis On Carbohydrate Metabolizing Enzymes In Monosodium Glutamate Induced Obesity In Female Rats Journal of Cell and Tissue Research. 2009; 9(3), 1969-1974.
14. Nidhi Mishra, Vijay Lakshmi Tandon and Ashok Munjal. Evaluation of Medicinal Properties of Hibiscus rosa sinensis in Male Swiss Albino Mice. International Journal of Pharmaceutical and Clinical Research. 2009; 1(3), 106-111.
15. Athar M and Iqbal M. Ferric nitrilotriacetate promotes N-diethylnitrosamine induced renal tumorigenesis in the rat: implications for the involvement of oxidative stress. Carcinogenesis. 1998; 19, 1133–9.
16. Wright JR, Colby HD and Miles PR. Cytosolic factors which affect microsomal lipid peroxidation in lung and liver. Arch Biochem Biophys. 1981; 206, 296–304.
17. Jollow DJ, Mitchell JR, and Zampaglione. N. Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,4- bromobenzene oxide as the hepatotoxic metabolite. Pharmacology. 1974; 11, 151–169.
18. Claiborne A. Catalase activity. In Greenwald RA ed. CRC handbook of methods in oxygen radical research.Boca Raton, FL: CRC Press, 1985: 283–84.