Trichosanthes dioica Roxb. prevents hepatic inflammation and fibrosis in CCl4-induced ovariectomized rats

Abstract

•HPLC analysis revealed the presence of phenolic antioxidants in TD peel extract.•TD peel inhibited the AST, ALT and ALP levels in CCl4-induced ovariectomized rats.•TD peel attenuated the MDA, NO and APOP levels in both plasma and liver tissues.•TD peel improved the endogenous antioxidant enzymes (CAT and SOD) levels in rats.•TD peel protected the liver by diminishing inflammation and fibrosis in rats. Plant-derived natural products have shown beneficial effects in many diseases associated with liver. The present study aimed to appraise the effects of Trichosanthes dioica (TD) peel extract on oxidative stress, inflammation as well as fibrosis exhibited by the ovariectomized rats in which hepatic damage had been induced by the administration of carbon tetrachloride (CCl4). The CCl4 administration in the ovariectomized rats elevated the plasma ALT, AST, and ALP level in comparison with the control while TD peel extract showed significant reduction of those activities. Besides, oxidative stress and lipid peroxidation also increased in CCl4-treated rats. TD peel extract significantly reduced the oxidative stress marker's concentration and improved the catalase and SOD activities in CCl4-administered ovariectomized rats. These results suggest that TD peel extract is capable of protecting the liver from CCl4induced damage through preventing oxidative stress, lipid peroxidation, inflammation, fibrosis and boosting the diminished cellular antioxidant defense. Plant-derived natural products have shown beneficial effects in many diseases associated with liver. The present study aimed to appraise the effects of Trichosanthes dioica (TD) peel extract on oxidative stress, inflammation as well as fibrosis exhibited by the ovariectomized rats in which hepatic damage had been induced by the administration of carbon tetrachloride (CCl4). The CCl4 administration in the ovariectomized rats elevated the plasma ALT, AST, and ALP level in comparison with the control while TD peel extract showed significant reduction of those activities. Besides, oxidative stress and lipid peroxidation also increased in CCl4-treated rats. TD peel extract significantly reduced the oxidative stress marker's concentration and improved the catalase and SOD activities in CCl4-administered ovariectomized rats. These results suggest that TD peel extract is capable of protecting the liver from CCl4induced damage through preventing oxidative stress, lipid peroxidation, inflammation, fibrosis and boosting the diminished cellular antioxidant defense. Liver diseases i.e., hepatitis, fatty liver, liver cirrhosis, hepatocellular carcinoma etc. in which structure or functions of the liver are mostly affected and has been ranked as fifth position in terms of common cause of death after the heart disease, stroke, chest disease and cancer worldwide. Fibrosis in liver can be considered as a vigorous and highly integrated response of cells to long-term hepatic damage [[1]Friedman S.L. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury.J Biol Chem. 2000; 275: 2247-2250Crossref PubMed Scopus (1847) Google Scholar,[2]Williams R. Global challenges in liver disease.Hepatology. 2006; 44: 521-526Crossref PubMed Scopus (618) Google Scholar]. Various internal or external factors may trigger the malfunctioning of a liver such as oxidative stress, biotoxins or xenobiotics [[3]Fernandez-Checa J.C. Kaplowitz N. Hepatic mitochondrial glutathione: transport and role in disease and toxicity.Toxicol Appl Pharmacol. 2005; 204: 263-273Crossref PubMed Scopus (225) Google Scholar] which can additionally stimulate different pathological conditions like subclinical icteric (jaundice) hepatitis to necro-inflammatory carcinoma, cirrhosis, and hepatitis [[4]Vrba J. Modriansky M. Oxidative burst of Kupffer cells: target for liver injury treatment.Biomedical Papers-Palacky University in Olomouc. 2002; 146: 15-20Crossref PubMed Scopus (55) Google Scholar]. Carbon tetrachloride (CCl4) is used to induce hepatic fibrosis in different types of animal models [[5]Recknagel R.O. Glende Jr E.A. Dolak J.A. Waller R.L. Mechanisms of carbon tetrachloride toxicity.Pharmacol Therapeut. 1989; 43: 139-154Crossref PubMed Scopus (1156) Google Scholar]. CCl4 injures the liver by producing reactive oxygen species (ROS) [[6]Tada S. Nakamoto N. Kameyama K. Tsunematsu S. Kumagai N. Saito H. et al.Clinical usefulness of edaravone for acute liver injury.J Gastroenterol Hepatol. 2003; 18: 851-857Crossref PubMed Scopus (33) Google Scholar]. Hepatotoxicity of CCl4 involves its biotransformation into free radicals for example trichloromethyl free radical (CCl3) and trichloroperoxyl radical (CCl3O2−)which are highly reactive and increase lipid peroxidation [[7]Feng Y. Siu K.Y. Ye X. Wang N. Yuen M.F. Leung C.H. et al.Hepatoprotective effects of berberine on carbon tetrachloride-induced acute hepatotoxicity in rats.Chin Med. 2010; 5: 33Crossref PubMed Scopus (59) Google Scholar]. Previous studies showed that natural antioxidants, particularly polyphenols and flavonoids are capable of inhibiting lipid peroxidation, suppressing liver enzyme activities, and may also increase antioxidant enzyme activities against hepatotoxicity which is induced by CCl4 [[8]Kumaravelu P. Dakshinamoorthy D.P. Subramaniam S. Devaraj H. Devaraj N.S. Effect of eugenol on drug-metabolizing enzymes of carbon tetrachloride-intoxicated rat liver.Biochem Pharmacol. 1995; 49: 1703-1707Crossref PubMed Scopus (67) Google Scholar]. An ovariectomized rat is a well established model for many clinical studies to circumvent the beneficial role of estrogen which is a female hormone involved in the different physiological processes. Within the liver, estrogen inhibits proliferation of stellate cells and fibrogenesis by inhibiting the deposition of collagen, increasing innate immunity, acting as antioxidants, and also by suppressing the pro-inflammatory mediators [[9]Brady C.W. Liver disease in menopause.World J Gastroenterol: WJG. 2015; 21: 7613Crossref PubMed Scopus (61) Google Scholar]. Trichosanthes dioica Roxb. is an annual or perennial medicinal herb distributed in tropical Asia and Australia. The fruit is the main part of the herb. It has been reported to be used by the traditional healers for curing jaundice and hepatic disorders [[10]Khandaker M. Akter S. Imam M.Z. Trichosanthes dioica Roxb.: a vegetable with diverse pharmacological properties.Food Sci Hum Wellnessnd Human Wellness. 2018; 7: 34-48Crossref Scopus (8) Google Scholar,[11]Shah B.N. Seth A. Pharmacological potential of Trichosanthes dioica-an edible plant.Hygeia J Drug Med. 2010; 2 (Short Rev): 1-7Google Scholar]. Also, it has been used traditionally to treat enlarged liver and spleen [[12]Ghani A. Medicinal plants of Bangladesh with chemical constituents and uses.2nd ed. Asiatic Society of Bangladesh, Dhaka, Bangladesh2003Google Scholar]. However, it has been found that different parts of T. dioica are effective as an antipyretic, diuretic, cardiotonic, laxative and antiulcer agent [[12]Ghani A. Medicinal plants of Bangladesh with chemical constituents and uses.2nd ed. Asiatic Society of Bangladesh, Dhaka, Bangladesh2003Google Scholar]. Experimental studies demonstrated the effectiveness of this plant to treat different inflammatory diseases for instance gastritis, arthritis, ischemia, diabetes, alopecia, febrifuge, and in edema [[13]Kumar N. Singh S. Manvi R.G. Trichosanthes dioica Roxb.: an overview.Phcog Rev. 2012; 6: 61-67Crossref PubMed Scopus (13) Google Scholar], and as an anticoagulant. However, the anti-inflammatory action of T. dioica is not clearly understood. It has been mainly used as an antioxidant in many previous studies [[13]Kumar N. Singh S. Manvi R.G. Trichosanthes dioica Roxb.: an overview.Phcog Rev. 2012; 6: 61-67Crossref PubMed Scopus (13) Google Scholar]. So, in case of an inflammatory response, it can be assumed that T. dioica activates the antioxidant properties either by a mechanism called chain-breaking in which an electron is donated by primary oxidants to the free radicals or by the removal of ROS/reactive nitrogen species initiator through quenching chain-initiating catalyst [[14]Shivhare Y. Singour P.K. Patil U.K. Pawar R.S. Wound healing potential of methanolic extract of Trichosanthes dioica Roxb (fruits) in rats.J Ethnopharmacol. 2010; 127: 614-619Crossref PubMed Scopus (50) Google Scholar]. However, the effect of this plant on liver fibrosis has not been explored yet and it is unclear whether T. dioica could improve liver fibrosis by modulating oxidative stress in liver. Therefore, this study was conducted to assess hepatoprotective potential of T. dioica where CCl4 was used to induce oxidative stress and inflammation in the liver of ovariectomized rats and T. dioica was used as a hepatoprotective agent. CCl4 obtained from Merck (Germany) and silymarin was purchased from the Hamdard Laboratories Bangladesh. From J.I. Baker (USA) and Sigma Chemical Company (USA), trichloroacetic acid (TCA) and thiobarbituric acid (TBA) were obtained respectively whereas ALT, AST, and ALP assay kits were procured from DCI Diatec diagnostics (Budapest, Hungary). In addition, 50, 50-Dithiobis-2-nitrobenzoate, also known as Ellman's reagent was purchased from Sigma, USA. Other chemicals and reagents of analytical grade were also utilized in this investigation. All the chemicals and reagents including biologicals and synthetics used in this study were endotoxin free. Green T. dioica fruits were collected from a local market in October 2016. Through verification of the colored pictures of the plant species followed by consideration of their description and distinguishing characteristics, the fruits were identified. Then, the fruits were properly cleaned with water to make them free from dust or any other unwanted particulates followed by peeling their skin off. The clean fruit peels were shade-dried in the Phytochemistry Laboratory and after drying, the dried peels were blended into powder which was sieved through a kitchen strainer to finally obtain a fine powder which was used for extraction. This finely grounded plant material (200 g) was kept in a 1000 mL conical flask and 600 mL of ethanol was added to it. The conical flask's mouth was then covered using aluminum foil and for several days (6 h per day), the flask was placed in a reciprocating shaker for continuous agitation at 150 revs/min in order to ensure thorough mixing. Afterward, the well-mixed extract was filtered initially through a clean muslin cloth and then by Whatman no. 1 filter paper. Finally, using a rotary vacuum evaporator, solvent of the filtered extract was evaporated and the residue obtained was used for further experimentation. The phenolic composition of the ethanol extract of T. dioica peel extract was determined by Dionex UltiMate 3000 HPLC system (Thermo Scientific, equipped with PDA (DAD-3000RS)), as described previously [[15]Rahman M.M. Ferdous K.U. Roy S. Nitul I.A. Mamun F. Hossain M.H. et al.Polyphenolic compounds of amla prevent oxidative stress and fibrosis in the kidney and heart of 2K1C rats.Food Sci Nutr. 2020; 8: 3578-3589https://doi.org/10.1002/fsn3.1640Crossref PubMed Scopus (6) Google Scholar,[16]Hossain H. Rahman S.E. Akbar P.N. Khan T.A. Rahman M.M. Jahan I.A. HPLC profiling, antioxidant and in vivo anti-inflammatory activity of the ethanol extract of Syzygium jambos available in Bangladesh.BMC Res Notes. 2016; 9: 191Crossref PubMed Scopus (17) Google Scholar]. Three to four-months-old, 36 female rats of Long-Evans species (180–200 g) were taken from Animal House of the Department of Pharmaceutical Sciences, North South University. They were kept in individual cages at room temperature of 25 ± 2 °C. Those rats were provided with coarse powder of standard laboratory pellet and drinking water ad libitum. The whole animal care and experiment were conducted by following the experiment protocol approved from the Animal Ethical Committee of North South University. Ovariectomy survival surgical procedure was conducted on all animals followed by an aseptic technique. Animals were anesthetized by intraperitoneally injecting ketamine at a dose of 100 mg/kg. A 2-cm dorsal flank incision was made in the experimental rats through which every ovarian fatty tissue was removed following identification. The ovarian arteries were then ligated and the ovaries were removed from the body by a cut. Using a 4-O sterile suture, the cut was finally closed and each rat was injected with Penicillin-G procaine (0.2 mL, 20,000 IU, IM). To study the effects of T. dioica in these ovariectomized rats, 36 rats were divided into six groups with six rats in each group as follows: Group I (Control), Group II (CCl4), Group III (CCl4 +100 mg/kg TD), Group IV (CCl4+ 200 mg/kg TD), Group V (CCl4 +400 mg/kg), and Group VI (CCl4+ silymarin) (standard). Group I rats was given chow diet (5% w/w of diet) and water throughout the study every day for 14 days. Rats of groups II, III, IV, V, and VI were treated with CCl4 (1: 3 in olive oil) at a dose of 0.5 mL/kg orally twice a week for two weeks. However, animals of groups III, IV, and V received T. dioica extracts orally every day for two weeks. All the animals were checked regularly for their body weight, food and water intake. After two weeks, all the experimental rats were killed to collect their various important organs such as liver, heart, kidney etc. and blood. All the collected organs were immediately weighed and were then kept in a refrigerator (−20 °C) for subsequent biochemical assays and also in neutral buffered formalin (NBF) (pH 7.4) for the histological test. The blood sample was centrifuged for 15 min at 8000 rpm at 4 °C. Finally, the plasma obtained was separated and transferred into microcentrifuge tubes which were kept at 4 °C for further analysis. Diatec diagnostic kits made in Hungary were utilized to estimate liver marker enzymes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels in rat plasma by following the manufacturer's protocol. The stored rat liver tissue was homogenized in ten times concentration of phosphate buffer of pH 7.4 followed by centrifugation for 12 min at 10,000 rpm by keeping the temperature at 4 °C. The upper portion was then separated to use in measuring protein and enzymatic activities as described below. TBARS concentrations in a solution indicate the level of lipid peroxidation. By using TBARS according to a method demonstrated earlier, lipid peroxidation in liver was estimated calorimetrically by measuring MDA level [[17]Niehaus W. Samuelsson B. Formation of malonaldehyde from phospholipid arachidonate during microsomal lipid peroxidation.FEBS J. 1968; 6: 126-130Google Scholar,[18]Mamun F. Rahman M.M. Zamila M. Subhan N. Hossain H. Hasan S.M.R. Polyphenolic compounds of litchi leaf augment kidney and heart functions in 2K1C rats.J Funct Foods. 2020; 64: 103662Crossref Scopus (7) Google Scholar]. A previously described method was used to determine the NO as nitrate and nitrite [[18]Mamun F. Rahman M.M. Zamila M. Subhan N. Hossain H. Hasan S.M.R. Polyphenolic compounds of litchi leaf augment kidney and heart functions in 2K1C rats.J Funct Foods. 2020; 64: 103662Crossref Scopus (7) Google Scholar,[19]Sagor A.T. Chowdhury M.R.H. Tabassum N. Hossain H. Rahman M.M. Alam M.A. Supplementation of fresh ucche (Momordica charantia L. var. muricata Willd) prevented oxidative stress, fibrosis and hepatic damage in CCl4 treated rats.BMC Compl Alternative Med. 2015; 15: 115Crossref PubMed Scopus (38) Google Scholar]. Naphthyl ethylenediamine dihydrochloride (0.1% w/v) was used in place of 1-naphthylamine (5%) to modify Griess-Illosvoy reagent. With the help of a standard curve, NO level was measured and indicated as nmol/g of tissue. According to a previously described method, APOP concentrations were determined [[20]Sagor M.A.T. Taher A. Tabassum N. Potol M. Alam M. Xanthine oxidase inhibitor, allopurinol, prevented oxidative stress, fibrosis, and myocardial damage in isoproterenol induced aged rats.Oxidative Med cell Longev. 2015; 2015Crossref PubMed Scopus (53) Google Scholar] and stated as mmol/L chloramine-T equivalents. Catalase levels were measured using a former method [[21]Chance B. Maehly A. [136] Assay of catalases and peroxidases.Methods Enzymol. 1955; 2: 764-775Crossref Scopus (3185) Google Scholar]. Changes in absorbance were recorded at 240 nm after 1 min of the reaction with each change of 0.01 as units/min considered to be one unit of catalase activity. SOD level in the plasma and homogenized liver sample was measured following a method described earlier [[15]Rahman M.M. Ferdous K.U. Roy S. Nitul I.A. Mamun F. Hossain M.H. et al.Polyphenolic compounds of amla prevent oxidative stress and fibrosis in the kidney and heart of 2K1C rats.Food Sci Nutr. 2020; 8: 3578-3589https://doi.org/10.1002/fsn3.1640Crossref PubMed Scopus (6) Google Scholar,[22]Misra H.P. Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase.J Biol Chem. 1972; 247: 3170-3175Abstract Full Text PDF PubMed Google Scholar]. An aliquot of enzyme preparation and PBS comprised 2.94 mL of the total volume of 3 mL of the reaction mixture. The reaction initiated following the incorporation of 0.06 mL of epinephrine (15 mM) and at an interval of 15 s, the absorbance was noted for 1 min at 480 nm. A control with a composition similar to that of the reaction mixture but only lacking the enzyme preparation was run simultaneously. To determine the myeloperoxidase activity, the dianisidine-H2O2 method was modified for 96-well plates [[18]Mamun F. Rahman M.M. Zamila M. Subhan N. Hossain H. Hasan S.M.R. Polyphenolic compounds of litchi leaf augment kidney and heart functions in 2K1C rats.J Funct Foods. 2020; 64: 103662Crossref Scopus (7) Google Scholar,[23]Bradley P.P. Priebat D.A. Christensen R.D. Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker.J Invest Dermatol. 1982; 78: 206-209Abstract Full Text PDF PubMed Scopus (2923) Google Scholar]. Briefly, plasma samples (10 mg protein) were added in triplicate to the mixture of 0.53 mM o-dianisidine hydrochloride (Sigma) and H2O2 (0.15 mM) in 50 mM potassium phosphate buffer (pH 6.0). The difference in absorbance was measured at 460 nm. Results were expressed as units of MPO/mg protein. For microscopic evaluation, liver tissues were preserved in 10% neutral buffered formalin and were then treated with ethanol and then with xylene. Following that, the liver tissues were fixed in small blocks of paraffin and cut into 5 mm slices. These liver tissue sections were then subjected to Hematoxylin and Eosin staining and Sirius red staining with a view to notice the presence of any hepatic inflammation and to evaluate collagen deposition and fibrosis in the liver respectively. Apart from these, Prussian blue staining was also done to detect iron deposition in the liver tissue sections. All the slides with stained tissue sections were photographed and analyzed under a light microscope at 40× magnifications (Zeiss Axioscope) after completion of the routine staining. All the values obtained in this study were expressed as mean ± standard error of mean (SEM). By using Graph Pad Prism Software, version 6.01, the results were evaluated according to One-way ANOVA followed by Newman–Keuls post hoc test. In all cases of this study, statistical significance was considered p < 0.05. The HPLC chromatogram of the T. dioica peel extract is presented in Fig. 1. It is revealed that caffeic acid (CA), (−)-epicatechin (EC), vanillic acid (VA), p-coumaric acid (PCA), quercetin hydrate (QU), and trans-ferulic acid (FA) are present in the extract. Among these six phenolic antioxidants, trans-ferulic acid was found in high amount in the extract (Table 1).Table 1Contents of polyphenolic compounds in the ethanol extract of Trichosanthes dioica peel (n = 5).Polyphenolic compoundEthanol extract of PotolContent (mg/100 g of dry extract)% RSDVA12.070.05CA39.610.18EC7.280.02PCA15.740.08FA51.020.21QU30.170.16RSD: Relative Standard Deviation. Open table in a new tab RSD: Relative Standard Deviation. Body weight, food and water consumption of each rat were recorded daily during the experiment and % change was calculated for all groups. The body weight decreased remarkably (p < 0.05) in CCl4-induced rat groups in comparison with the sham rats. On the other hand, CCl4-administered rats group showed significant (p < 0.05) increase of body weight who received the treatment of T. dioica peel extract (100 mg/kg, 200 mg/kg & 400 mg/kg). In addition, the body weight of CCl4+silymarin (SM) rats group constantly increased during the experiment compared to CCl4-administered rats (data not shown here). In case of the CCl4 induced ovariectomized group, food intake was constantly lower in comparison to the control group. However, food intake of the treatment group (100 mg/kg, 200 mg/kg & 400 mg/kg) was significantly higher (p < 0.05) compared to the CCl4-administered rats. Furthermore, the rats who received the treatment of 200 mg/kg and 400 mg/kg of T. dioica peel extract had a similar pattern of food intake compared to the group treated with 100 mg/kg T. dioica peel extract, which showed lower food intake than other two treatment groups. In addition, the food intake of CCl4+Silymarin (SM) rats group was higher than the CCl4-administered rats (data not shown here). Additionally, the water intake was significantly (p < 0.05) decreased in the disease group compared to the sham group. Nevertheless, water intake significantly (p < 0.05) increased in treatment group (100 mg/kg, 200 mg/kg & 400 mg/kg) than the CCl4-administered rats group. Besides, in between the treatment groups, the water intake was similarly higher in the 200 mg/kg and 400 mg/kg T. dioica peel extract treated group compared to the 100 mg/kg treatment group and among the three treatment groups, we have observed a dose–response relationship. Moreover, the water intake of CCl4+Silymarin (SM) rats group was higher than the CCl4-administered rats (Data not shown here). Significant (p < 0.05) increase in the liver wet weight of the CCl4 induced rats was noticed compared to the control rats. On the other hand, three groups treated with T. dioica peel extract (100 mg/kg, 200 mg/kg, & 400 mg/kg) displayed no remarkable difference in terms of wet liver weight compared to the disease group or even among themselves and dose–response relationship was also not found among these treatment groups. Additionally, a significant difference in the liver wet weight was not found in the treatment groups as compared to the CCl4+silymarin (SM) group (Fig. 2A). A highly significant (p < 0.05) elevation was found in the activities of ALT in plasma of CCl4-treated rats compared to the rats of control group. However, the treatment groups did not show any significant decrease in the ALT activity in plasma compared to the CCl4 group or to the group that received CCl4 with silymarin as a standard. Here, no dose–response relationship was found. However, the CCl4+silymarin group significantly lowered the ALT activity in plasma compared to the CCl4-administered rats (Fig. 2B). In addition, AST activities in the plasma of the CCl4-administered rats were significantly (p < 0.05) increased compared to the control rats. T. dioica peel extract treatment (100 mg/kg, 200 mg/kg & 400 mg/kg) in CCl4-administered rats significantly prevented the rise of AST activities in their plasma. Among the treatment (100 mg/kg, 200 mg/kg & 400 mg/kg) groups, AST activity in plasma was significantly (p < 0.05) decreased in a dose-dependent manner and clearly showed a dose–response relationship. Moreover, the CCl4+silymarin group also showed significantly (p < 0.05) decreased activity of AST in plasma in contrast to the CCl4-administered rats and also in comparison to the treatment group that received 100 mg/kg TD (Fig. 2C). Furthermore, in this study, CCl4-administered rats displayed remarkably (p < 0.05) elevated ALP activities compared to the control rats. T. dioica peel extract (100 mg/kg, 200 mg/kg & 400 mg/kg) in CCl4-administered rats significantly reduced ALP activity in plasma than in the disease group. Besides, both 200 mg/kg and 400 mg/kg treatment groups almost similarly reduced the ALP activity in plasma compared to the 100 mg/kg treatment group. On the other hand, in CCl4+silymarin (SM) group, the ALP enzyme activity was also substantially (p < 0.05) decreased than in the CCl4-administered rats. Besides, treatment with TD (100 mg/kg) significantly lessened the ALP activities in comparison to the CCl4+silymarin (SM) group (Fig. 2D). In this experiment, the MDA level in plasma and the liver of CCl4-administered rats was found to be significantly greater (p < 0.05) than in the control rats whereas 100 mg/kg treatment group displayed significantly lower plasma level of MDA in contrast to the disease group. However, no significant changes were found for 200 mg/kg and 400 mg/kg treatment groups in terms of decreasing MDA level in plasma compared to the diseased group. Besides, dose–response relationship could not be observed in this case (Fig. 3A). On the other hand, after administration of T. dioica peel extract (100 mg/kg, 200 mg/kg & 400 mg/kg), the MDA level in the liver tissue significantly (p < 0.05) decreased in CCl4+TD peel treated groups compared to the rats administered with CCl4 alone. However, there was no dose–response relationship found among the treatment groups. In CCl4+Silymarin (SM) rats, the concentration of MDA significantly (p < 0.05) reduced both in their plasma and liver compared to the CCl4-administered rats (Fig. 3A and B) as well as in comparison to the MDA level in the plasma of 100 mg/kg TD treatment group (Fig. 3A). However, there was no significant change of MDA level in liver was noted between CCl4+silymarin (SM) group and TD treatment group (Fig. 3B). Moreover, there was a significantly (p < 0.05) increased level of NO in both plasma and liver of CCl4 induced rats in contrast to the control ones. T. dioica peel extract significantly decreased the level of NO in the plasma and liver of CCl4 treated rat group compared to the disease group. However, dose-dependent relationship was not noted among the treatment groups (100 mg/kg, 200 mg/kg & 400 mg/kg) in terms of reducing the NO level in plasma and liver tissue. In addition, in case of the CCl4+silymarin (SM) rat group, the concentration of NO was significantly (p < 0.05) lowered both in the plasma and the liver compared to CCl4-administered rats (Fig. 3C and D) as well as compared to the plasma NO level in treatment group that received 100 mg/kg TD (Fig. 3C). On the other hand, there was no significant change of liver NO level was revealed between CCl4+silymarin (SM) group and TD treatment group (Fig. 3D). Furthermore, this study indicated that APOP level elevated significantly (p < 0.05) in the plasma and liver tissue of the CCl4-administered rats when it was compared with the sham rats (Fig. 3C and F). T. dioica peel extract (100 mg/kg, 200 mg/kg & 400 mg/kg) supplementation significantly (p < 0.05) decreased the APOP level in the plasma of CCl4-treated rats compared to the disease group. However, there was no dose–response relationship found between the treatment groups (Fig. 3C). Similarly, a significant increase of APOP level was also observed in the liver tissue homogenates of the CCl4-administered rats compared to the control group whereas T. dioica peel extract decreased the elevated APOP level significantly (p < 0.05) in the liver of CCl4-treated rats at the dose of 100 mg/kg and 200 mg/kg. However, there were no significant differences found in the APOP level in liver tissue of the 400 mg/kg treatment group compared to the disease group (Fig. 3F). On the other hand, the CCl4+silymarin (SM) group of rats significantly decreased the level of APOP in both plasma and the liver compared to the CCl4-administered rats (Fig. 3C and F). Nevertheless, there were no significant differences of plasma and liver APOP level noted in between the CCl4+silymarin (SM) group and TD treatment group (Fig. 3E and F). A significant (p < 0.05) decline in the SOD level in plasma and liver of the CCl4 induced rats (Fig. 4A and B) was observed in comparison with the control rats in this study. SOD activity was restored significantly (p < 0.05) in the plasma of the CCl4-administered rats compared to the diseased rats by administration of T. dioica peel extract (100 mg/kg, 200 mg/kg & 400 mg/kg). Besides, among the treatment groups (100 mg/kg, 200 mg/kg & 400 mg/kg), a dose–response relationship was also found in the plasma indicating an increasing SOD activity. In case of the CCl4+silymarine group, the SOD activity in plasma was significantly (p < 0.05) increased in comparison with the CCl4-administered rats (Fig. 4A). In addition, in case of the SOD activity in the liver tissue, the changes were significant among the treatment (100 mg/kg, 200 mg/kg & 400 mg/kg) groups compared to the CCl4-administered rats. Likewise, a significant difference was also observed in the SOD activity in liver of the CCl4+silymarin group rats compared to the CCl4-administered rats (Fig. 4B). However, in comparison to the 100 mg/kg TD treated group, CCl4+silymarin group did not show any significant effects in case of SOD level in liver and plasma (Fig. 4C and D). Moreover, the catalase level was also measured in both liver homogenates and plasma of all experimental groups. The plasma level of catalase was attenuated substantially (p < 0.05) in the CCl4-administered rats than in the control rats. However, T. dioica peel extract elevated the decreased catalase ac