Thioacetamide-Induced Acute Liver Toxicity in Rats Treated with Balanites

JOAD141_proof ■ 20 August 2016 ■ 1/6

Journal of Acute Disease 2016; ▪(▪): 1–6 1

56 HOSTED BY Contents lists available at ScienceDirect 57 58 Journal of Acute Disease 59 60 journal homepage: www.jadweb.org 61 62 1 63 2 Original article http://dx.doi.org/10.1016/j.joad.2016.08.009 64 3 Thioacetamide-induced acute liver toxicity in rats treated with extracts 65 4 Balanites roxburghii 66 5 * 67 6 Q4 Mallikarjuna Rao Talluri, Rajananda Swamy Tadi , Ganga Rao Battu 68 7 Department of Pharmacognosy and Phytochemistry, Andhra University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, AP, 69 8 530003, India 70 9 71 10 ARTICLE INFO ABSTRACT 72 11 73 12 Article history: Objective: To evaluate the antioxidant and thioacetamide induced liver toxicity using 74 13 Received 8 Jun 2016 extracts of aerial parts of Balanites roxburghii (B. roxburghii). 75 14 Received in revised form 28 Jun 2016 Methods: The in-vitro antioxidant activity was estimated for different extracts on su- 76 15 Accepted 14 Jul 2016 peroxide, hydroxyl and 1,1-diphenyl-2-picrylhydrazyl free radicals and the hep- 77 16 Available online xxx atoprotective activity of the selected plant extracts was evaluated by using thioacetamide- 78 induced liver intoxication in rats model. 17 79 Results: There were no visible signs of toxicity, mortality and no behavioral changes 18 Keywords: 80 were observed for the selected plant extracts (hydroalcoholic, ethyl acetate and hexane) at 19 Balanites roxburghii 81 2000 mg/kg body weight. The plant extracts showed dose-dependent effectiveness in the 20 Thioacetamide 82 inhibition of free radicals generation and hepatoprotective nature. The hydroalcoholic 21 Antioxidant activity 83 extract showed more scavenging activity as compared to other extracts with IC50 values 22 Hepatoprotective activity m m m 84 23 of (332.50 ± 1.30) g, (302.00 ± 1.50) g and (230.40 ± 1.10) g on superoxide, hydroxyl and 1,1-diphenyl-2-picrylhydrazyl free radicals. Aerial parts of B. roxburghii at 85 24 three dose levels such as 125 mg/kg, 250 mg/kg and 500 mg/kg showed dose dependent 86 25 percentage protection as standard drugs and on enhancement activities of serum 87 26 biomarker enzymes (aspartate aminotransferase, alanine transaminase, alkaline phos- 88 27 phatase, total bilirubin and total protein) observed in thioacetamide-induced hepatotox- 89 28 icity. The hydroalcoholic extract showed significant results at 500 mg/kg body weight as 90 29 compared to other extracts (P < 0.05) with 65.65%, 62.39%, 59.89%, 55.90% and 91 30 54.61% on aspartate aminotransferase, alanine transaminase, alkaline phosphatase, total 92 31 bilirubin and total protein levels. 93 32 Conclusions: It was observed that the B. roxburghii plant extracts are non-toxic and 94 33 have capacity to restore the physiological changes caused by the hepatotoxic compounds. 95 34 96 35 97 36 98 37 99 38 and finally drugs toxicity[2]. All these liver diseases affect 100 39 1. Introduction persons of all ages, especially people with ages from 40 to 60 101 40 years, and cause the alterations in the normal physiological 102 41 Liver is a very important organ in the body playing a vital functions of the body[3]. The untreated liver diseases and long 103 42 role in the metabolic functions. Over the last few decades, the term usage of medication sometimes cause side effects and 104 43 morbidity and mortality of different types of liver diseases have [1] finally lead to death[4]. So, researches on liver diseases are 105 44 increased around the world . Liver diseases include alcoholic, critically important in these days to bring the major toll of 106 45 non-alcoholic, microorganisms, genetic variation (autoimmune) liver diseases under control for human health. 107 46 Medicinal plants are used in the natural medicines such as 108 47 *Corresponding author: Rajananda Swamy Tadi, Department of Pharmacognosy Chinese traditional medicine, Indian Ayurveda and Unani 109 48 and Phytochemistry, Andhra University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, AP, 530003, India. medicine in middle east countries[5]. Many of the currently 110 49 Tel: +91 9490052637 available drugs are derived either directly or indirectly from 111 50 E-mail: [email protected] medicinal plants. In recent times, many plants have been used 112 51 Peer review under responsibility of Hainan Medical College. The journal implements double-blind peer review practiced by specially invited international for a wide spectrum of liver diseases so far to recovery 113 52 editorial board members. processes of the intoxicated livers[6]. But there are many 114 53 All the experimental procedures involving animals were conducted according to medicinal plants available to provide scientific evidence for 115 54 OECD guidelines and approved by the Institutional Animal Ethics Committee, 116 Q1 Andhra University. their biological activities which have been used in traditional 55 117

2221-6189/Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Talluri MR, et al., Thioacetamide-induced acute liver toxicity in rats treated with Balanites roxburghii extracts, Journal of Acute Disease (2016), http://dx.doi.org/10.1016/ j.joad.2016.08.009 JOAD141_proof ■ 20 August 2016 ■ 2/6

2 Mallikarjuna Rao Talluri et al./Journal of Acute Disease 2016; ▪(▪): 1–6

1 or herbal medicine for treating diseases including liver was studied by the competition between deoxyribose and the 63 2 diseases[7]. One of those medicinal plants is Balanites extract's antioxidant molecules for hydroxyl radicals generated 64 [8] 2+ 3 roxburghii (B. roxburghii) . from the Fe /ethylene diamine tetraacetic acid/H2O2 system. 65 4 B. roxburghii which is commonly known as hingot belongs 66 5 to the family Zygophyllaceae. It is commonly found in sandy 2.3.3. DPPH radical scavenging activity 67 6 plain areas like Western Rajasthan, West Bengal, Maharashtra The DPPH radical scavenging activity was measured as per 68 7 and drier parts of India [9]. Different parts of the B. roxburghii methods[13,14]. This method is based on measure of color 69 8 have been used to treat different diseases in traditional absorbance of alcoholic DPPH solution (Blue color) after 70 9 medicine. For example, the fruits are used in the treatment of addition of antioxidant solution (extract/compound). If 71 10 whooping cough, skin diseases and antifertility; leaves are antioxidants present in the test compound blue color due to 72 11 used for the treatment of jaundice and the bark is used for DPPH changes to yellow color. 73 12 swelling in body organs and inﬂammation and used to treat 74 – 13 snake-bite and dog bite[9 12]. But there are few scientiﬁc 2.3.4. Calculation of percentage inhibition 75 14 evidences regarding its biological activities. So, the author The percentage inhibition of the superoxide production by 76 15 selected the aerial parts of B. roxburghii to evaluate its the extract was calculated using the formula: 77 16 antioxidant activity on superoxide, hydroxyl and 1,1-diphenyl- 78

17 2-picrylhydrazyl (DPPH) free radicals and the hepatoprotective Inhibitory ratio = (A0 − A1) 100/A0 79 18 activity against thioacetamide (TAA)-induced liver toxicity. 80 19 where A0 was the absorbance of control and A1 referred to the 81 20 2. Materials and methods absorbance of plant extract or/and ascorbic acid. 82 21 83 22 2.1. Drug and chemicals 2.3.5. IC50 calculation form percentage inhibition 84 23 The optical density obtained with each concentration of the 85 24 Chemicals used for the study were of analytical grade. extract/ascorbic acid was plotted taking concentration on X-axis 86 25 Silymarin, TAA and DPPH were purchased from Sigma and percentage inhibition on Y-axis. The graph was extrapolated 87 26 Chemicals, USA, Nitroblue tetrazolium was purchased from to find the 50% inhibition concentration of extract/ascorbic acid. 88 27 Sisco Research Laboratories Pvt Ltd., Mumbai. Riboflavin was 89 28 purchased from Loba Chemie Pvt Ltd., Mumbai. The kits for the 2.4. Selection of animals 90 29 assessment of different biochemical parameters like aspartate 91 30 aminotransferase (AST), alanine transaminase (ALT), alkaline Healthy albino rats of either sex weighing between 180 and 92 31 phosphatase (ALP), total bilirubin and total protein were pur- 250 g aging from 60 to 90 days were used for the study. The rats 93 32 chased from Span Diagnostics Ltd., Gujarat, India. were taken care of at standard light and humidity by supplying 94 33 proper food and water. 95 34 2.2. Plant material and preparation of extracts 96 35 2.5. Acute toxicity studies 97 36 The plant material was collected at Andhra Pradesh, India on 98 37 99 July, 2012. The authentication of the plant was done by Rtd. The acute toxicity study was conducted for B. roxburghii 38 100 Prof. M. Venkaih, Department of Botany, Andhra University, extracts as per Organisation for Economic Co-operation and 39 101 Visakhapatnam. The plant material was dried under shade and Development (OECD) guidelines 420 (OECD. 2001) and reg- 40 102 powdered for extraction separately using maceration process ulations of the Institutional Animal Ethics Committee (No. 516/ 41 103 with hexane, ethyl acetate and hydroalcoholic (ethanol 70%), 01/A/CPCSEA).The albino mice of single sex were selected into 42 104 and the extracted solvents were concentrated to dryness under three groups (n = 6). They were maintained for one week before 43 105 vacuum using rotavapour. the experiment under room temperature and allowed free access 44 106 to water and diet. The animals were subjected for acute toxicity 45 107 2.3. In vitro antioxidant activity study using each extract at a dose of 2000 mg/kg orally in 2 46 108 groups at regular intervals of time, i.e.,1,2,4,8,12and24h. 47 109 In-vitro antioxidant activity was assessed by using the pre- During this time, the animals were under observation to note 48 110 pared extract of B. roxburghii using dimethyl sulphoxide as the different conditions like skin changes, morbidity, aggressive- 49 111 vehicle on superoxide, hydroxyl and DPPH free radicals[13,14]. ness, oral secretions, sensitivity to sound and pain, respiratory 50 112 The percentage of inhibition and IC values were calculated. movements and finally their mortality. 51 50 113 52 114 2.3.1. Superoxide radical scavenging activity 2.6. Assessment of hepatoprotective activity of 53 115 B. roxburghii 54 The superoxide scavenging activity of the selected plant 116 [13] 55 extracts was evaluated as per standard methods . It was by the 117 The selected plant extracts were tested for their hep- 56 absorption of light at 560 nm induction of superoxide free 118 fl atoprotective nature using TAA-induced liver toxicity in rats[15]. 57 radical generation by ribo avin and corresponding reduction 119 For this experiment, animals were grouped into twelve groups 58 by nitroblue tetrazolium. 120 (n = 6). Animals in group I were treated with normal saline 59 121 (vehicle) for one week through oral administration at 2 mL/kg 60 2.3.2. Hydroxyl radical scavenging activity 122 body weight. Rats in group II and group III were treated with 61 The scavenging activity of selected plants extracts on hy- 123 [14] TAA as a 2% w/v solution in water on the first day at 50 mg/ 62 droxyl radical was measured as per established methods .It 124

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1 kg body weight subcutaneously, then group II was continuously Table 2 63

2 treated with saline and group III with silymarin at a dose of IC50 of different extracts of B. roxburghii against superoxide, hydroxyl 64 3 25 mg/kg body weight orally for three weeks. Groups IV to and DPPH radicals. 65 4 XII were treated with TAA as a 2% w/v solution in water on 66 Name of the IC (mg) 5 ﬁ 50 67 the rst day at 50 mg/kg body weight subcutaneously, then extracts 6 groups IV, V and VI were treated with the hydroalcoholic Superoxide Hydroxyl DPPH radical 68 7 extract, and groups VII, VIII, IX were treated with the ethyl radical radical 69 8 acetate extract and groups X, XI, XII were treated with the Hydroalcoholic 332.50 ± 1.30 302.00 ± 1.50 230.40 ± 1.10 70 9 hexane extract at a doses of 125, 250, 500 mg/kg body weight Ethyl acetate 356.00 ± 0.80 260.00 ± 1.20 359.50 ± 0.68 71 Hexane 486.00 ± 0.62 503.00 ± 0.76 507.80 ± 0.24 10 orally for three weeks. Animals of all groups were Ascorbic acid 54.40 ± 1.10 68.00 ± 1.30 22.00 ± 0.50 72 11 anaesthetized using chloroform after 48 h of ﬁnal dose 73 12 administration of extracts. The blood samples were collected 74 13 from animal groups by retro-orbital plexus, then samples were 75 14 centrifuged at 2400 r/min for quarters of an hour. After 76 15 centrifugation, clearly separated serum was used for measuring 77 700 16 the different biochemical parameters using autoanalyzer with 78 600 17 the help of reagent kits[16,17]. All the experimental procedures 79 500 18 involving animals were conducted according to OECD 80 g) 400 μ 19 guidelines and approved by the Institutional Animal Ethics ( 81

50 300

20 Committee, Andhra University. IC 82 200 21 83 100 22 84 3. Results 0 23 Superoxide radical Hydroxyl radical DPPH radical 85 24 Hydroalcoholic extract Ethyl acetate extract 86 3.1. Antioxidant activity Hexane extract Ascorbic acid 25 87 26 Figure 1. IC50 of different extracts of B. roxburghii against superoxide, 88 In the present study, the tested extracts of B. roxburghii were hydroxyl and DPPH radicals. 27 found to possess concentration dependent antioxidant activity on 89 28 superoxide, hydroxyl and DPPH free radicals. 90 29 91 Superoxide anion radical (O2) are formed by activated (Fenton reaction). The mean IC50 values on hydroxyl radical of 30 phagocytes, such as monocytes, macrophages, eosinophils and hydroalcoholic, ethyl acetate and hexane extracts of 92 31 − 93 neutrophils, and the production of O2 is an important factor in B. roxburghii were found to be (302.00 ± 1.50) mg, 32 the killing of bacteria by phagocytes. In the present method, (260.0 ± 1.20) mg and (503.0 ± 0.76) mg, respectively. The mean 94 33 ﬂ 95 superoxide anion was derived from ribo avin and reduced by IC50 value of ascorbic acid was found to be (68.00 ± 1.30) mg. 34 nitroblue tetrazolium. The decrease in absorbance at 560 nm The hydroalcoholic extract had better antioxidant activity on 96 35 with tested extracts/compounds indicated the utilization of su- hydroxyl radical at 640 mg as compared to other extracts 97 36 98 peroxide anion in the reaction mixtures. The mean IC50 values (Tables 2 and 3, Figure 1). 37 on superoxide radical for hydroalcoholic, ethyl acetate and DPPH is a stable free radical having the highest absorption at 99 38 hexane extracts of B. roxburghii were found to be 517 nm. DPPH is easily stabilized by combining with the 100 39 (332.50 ± 1.30) mg, (356.00 ± 0.80) mg and (486.00 ± 0.62) mg, electrons or hydrogen radicals. The antioxidants have the ability 101 40 102 respectively. The mean IC50 value of ascorbic acid was found to of donating hydrogen. This makes the reduction of absorbance 41 be (54.40 ± 1.10) mg. The hydroalcoholic extract at a concen- by DPPH, because of its stabilization with hydrogen ions (re- 103 42 tration of 640 mg showed the better scavenging activity on su- action mixtures' purple color changed into yellow color). So, 104 43 peroxide free radical (Table 1, Table 2 and Figure 1). DPPH free radical scavenging activity is commonly used for the 105 44 Hydroxyl radical is the main reactive oxygen species (ROS) identiﬁcation of antioxidant activity of different extracts/com- 106 45 with very short life span damaging every part of the living cell as pounds. The tested extracts (hydroalcoholic, ethyl acetate and 107 46 compared to other ROS. These molecules sometimes bind with hexane) of B. roxburghii were found to be dose dependent 108 47 109 the nucleotides in the genetic materials of the body causing percentage of inhibition on DPPH free radical with IC50 values 48 cancers, mutations and toxicity. Hydroxyl radical scavenging of (230.40 ± 1.10) mg, (359.50 ± 0.68) mg and 110 49 activity was measured by studying the competition between (507.80 ± 0.24) mg, respectively. The hydroalcoholic extract 111 50 deoxyribose and the extracts for hydroxyl radicals generated have more inhibition, i.e., (78.25 ± 1.80) mg, at 640 mg(Tables 2 112 51 2+ 113 from the Fe /ethylene diamine tetraacetic acid/H2O2 system and 4, Figure 1). 52 114 53 115 54 116 Table 1 55 117 Concentration dependent percentage inhibition of different extracts of B. roxburghii on superoxide radical. 56 118 57 Name of the extracts Concentration (mg/0.1 mL) 119 58 20 40 80 160 320 640 120 59 121 Hydroalcoholic 8.50 ± 0.30 18.50 ± 1.00 30.40 ± 1.10 42.51 ± 1.20 56.15 ± 1.20 69.30 ± 0.50 60 Ethyl acetate 9.20 ± 0.50 18.60 ± 0.23 27.45 ± 0.15 38.62 ± 0.34 48.18 ± 0.25 61.92 ± 0.35 122 61 Hexane 7.24 ± 0.80 15.20 ± 0.60 24.80 ± 1.00 32.90 ± 0.30 44.25 ± 1.40 54.80 ± 0.50 123 62 Ascorbic acid 28.15 ± 0.50 43.19 ± 1.50 56.87 ± 1.40 74.46 ± 0.70 80.72 ± 2.10 84.41 ± 1.20 124

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1 Table 3 63 2 Concentration dependent percentage inhibition of different extracts of B. roxburghii on hydroxyl radical. 64 3 65 Name of the extracts Concentration (mg/0.1 mL) 4 66 5 20 40 80 160 320 640 67 6 Hydroalcoholic 8.40 ± 0.50 16.50 ± 1.10 25.40 ± 0.50 36.10 ± 1.20 51.30 ± 1.00 62.80 ± 1.10 68 7 Ethyl acetate 10.20 ± 0.30 21.58 ± 0.50 32.24 ± 0.30 43.60 ± 0.50 52.98 ± 0.50 66.46 ± 0.30 69 8 Hexane 7.28 ± 0.20 15.86 ± 0.24 23.48 ± 0.46 34.52 ± 0.32 45.42 ± 0.84 53.88 ± 0.72 70 Ascorbic acid 24.32 ± 1.00 35.12 ± 0.40 55.61 ± 1.10 65.31 ± 1.20 76.25 ± 1.20 82.11 ± 0.70 9 71 10 72 11 73 12 Table 4 74 13 Concentration dependent percentage inhibition of different extracts of B. roxburghii on DPPH radical. 75 14 Name of the extracts Concentration (mg/0.1 mL) 76 15 77 20 40 80 160 320 640 16 78 17 Hydroalcoholic 9.70 ± 0.40 15.80 ± 1.10 20.60 ± 0.50 32.70 ± 1.10 56.80 ± 1.20 78.25 ± 1.80 79 Ethyl acetate 8.58 ± 0.38 17.50 ± 0.14 22.68 ± 0.28 31.88 ± 0.62 48.28 ± 0.14 62.46 ± 1.22 18 Hexane 7.54 ± 0.13 16.36 ± 0.34 21.56 ± 0.82 29.58 ± 0.36 41.78 ± 1.20 54.62 ± 0.80 80 19 Ascorbic acid 48.00 ± 0.50 88.08 ± 1.00 90.68 ± 0.30 93.63 ± 0.50 94.21 ± 0.30 94.74 ± 1.10 81 20 82 21 83 22 3.2. Acute toxicity studies Group XII 84 23 Group XI 85 24 Group X 86 There were no visible signs of toxicity, mortality and no Group IX 25 behavioral changes such as alertness, motor activity, breath- Group VIII 87 26 lessness, restlessness, diarrhea, tremor, convulsion and coma Group VII 88 27 Group VI 89 were observed at the administered doses. The animals were Group V 28 physically active and no death was recorded even at the dose of Group IV 90 29 up to 2000 mg/kg body weight. Hence, all the tested extracts Group III 91 30 were considered as safe and non-toxic. Group II 92 31 Group I 93 0 50 100 150 200 250 300 350 32 3.3. Hepatoprotective activity SGOT enzyme level 94 33 Figure 2. SGOT enzyme levels due to the effect of B. roxburghii extracts 95 34 We examined the aerial parts of B. roxburghii at three dose at different doses on TAA-induced liver toxicity in rats. 96 35 levels, 125 mg/kg, 250 mg/kg and 500 mg/kg, and assessed by 97 36 measuring liver-related biochemical parameters [serum 98 37 glutamic-oxaloacetic transaminase (SGOT), serum glutamic Group XII 99 38 Group XI 100 pyruvic transaminase (SGPT), ALP, total serum bilirubin and Group X 39 total protein] levels for their hepatoprotective nature using TAA- Group IX 101 40 Group VIII 102 induced hepatotoxicity in rats (Table 5 and Figures 2–6). Group VII 41 Group VI 103 42 Group V 104 43 Group IV 105 Group III 44 Group II 106 45 Table 5 Group I 107 Percentage protection (%) of enzymes levels due to the effect of 0 50 100 150 200 46 SGPT enzyme level 108 B. roxburghii extracts at different doses on TAA-induced liver toxicity in 47 Figure 3. SGPT enzyme levels due to the effect of B. roxburghii extracts at 109 rats. 48 different doses on TAA-induced liver toxicity in rats. 110 49 Extract of Quantity Percentage protection on enzymes levels 111 50 B. roxburghii of the of TAA-induced hepatotoxicity 112 extract 51 SGOT SGPT ALP Total Total 113 (mg/kg Group XII 52 (IU/L) (IU/L) (IU/ bilirubin protein 114 body Group XI L) (mg/dL) (g/dL) 53 weight) Group X 115 54 Group IX 116 Silymarin 50 96.24 95.25 93.90 97.83 96.14 Group VIII 55 Hydroalcoholic 125 37.70 35.33 37.24 23.42 24.97 Group VII 117 Group VI 56 250 51.85 49.43 47.86 36.24 37.48 Group V 118 57 500 65.65 62.39 59.89 55.90 54.61 Group IV 119 58 Ethyl acetate 125 36.13 31.20 30.20 28.55 28.92 Group III 120 250 47.08 43.30 41.72 40.51 41.44 Group II 59 Group I 121 500 59.17 58.55 55.01 57.61 56.59 60 0 50 100 150 200 250 300 350 400 450 500 550 600 122 Hexane 125 32.86 29.91 26.12 22.56 22.33 ALP enzyme level 61 123 250 44.10 40.88 38.30 35.38 34.85 Figure 4. ALP enzyme levels due to the effect of B. roxburghii extracts at 500 56.76 54.27 52.49 52.48 50.66 62 different doses on TAA-induced liver toxicity in rats. 124

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1 Group XII comparably similar to silymarin. Among all extracts, hydro- 63 2 Group XI alcoholic extract of the selected plants showed a better activity. 64 Group X 3 Group IX 65 4 Group VIII 4. Discussion 66 5 Group VII 67 Group VI 6 Group V The liver participates in different physiological metabolisms 68 7 Group IV of the body, such as the exclusion of body wastage. So, the 69 Group III 8 Group II protection of liver is very important for the human wealth. 70 9 Group I However, in recent years, liver diseases are one of the most 71 10 0.0 0.5 1.0 1.5 2.0 2.5 72 Total bilirubin enzyme level severe diseases causing deaths around the world. Hepatotoxic 11 73 Figure 5. Total bilirubin enzyme levels due to the effect of B. roxburghii agents can react with the basic cellular components and conse- 12 extracts at different doses on TAA-induced liver toxicity in rats. quently induce acute liver failure[18]. TAA is a thiono-sulfur 74 13 containing compounds and has been used as a fungicide, 75 14 organic solvent. At the same time, it is hepatotoxic. TAA causes 76 15 Group XII centrilobular necrosis with a subsequent regenerative response 77 16 Group XI of liver leading to liver cirrhosis and hepatocarcinoma[19,20]. 78 17 Group X 79 Group IX TAA induces the formation of reactive metabolites (free 18 Group VIII radicals) derived from thioacetamide-S-oxide and by ROS 80 19 Group VII generated as intermediates, which leads to the cell death from 81 20 Group VI apoptosis and necrosis[16]. The metabolites produce are 82 21 Group V 83 Group IV covalently attached to the cellular macromolecules induce the [17] 22 Group III oxidative stress . ROS production resulting from TAA 84 23 Group II administration was followed by lipid peroxidation, glutathion 85 24 Group I depletion and reduction in the SH-thiol groups and TAA also 86 0 3 6 9 [21] 25 Total protein enzyme level affects the calcium movement from intracellular parts . All 87 26 Figure 6. Total protein enzyme levels due to the effect of B. roxburghii these effect the cellular permeability like different cell 88 27 extracts at different doses on TAA-induced liver toxicity in rats. organelles, mainly effect the function of mitochondria, power 89 28 house of the cell, by inhibiting the respiration[22]. 90 29 Group I treated with vehicle showed no significant changes in The results of our experiment provide evidence for the hep- 91 30 the biomarkers of liver enzymes (AST (SGOT), ALT (SGPT), atoprotective and antioxidant activities of B. roxburghii.The 92 31 ALP, total bilirubin and total protein) levels. Group II treated elevated enzymes level after treating with the TAA were signifi- 93 32 TAA showed significant changes in levels of biomarker enzymes. cantly restored by treating with 125, 250 and 500 mg/kg body 94 33 The animals of group III were administered with TAA and then weight doses of B. roxburghii extracts in the different groups. The 95 34 silymarin. There were significant changes in biomarker enzymes results of percentage protection of the extracts are well comparable 96 35 levels as compared to group II and the percentage protection with the standard drug, silymarin, and the tested extracts also 97 36 offered by the silymarin against the changes in AST (SGOT), showed the significant antioxidant activity on different free radi- 98 37 ALT (SGPT), ALP, total bilirubin and total protein levels were cals[16,21,22]. It could be concluded by the above results that 99 38 96.24%, 95.25%, 93.90%, 97.83% and 96.14%, respectively. B. roxburghii plant extracts have the capacity in reduce the 100 39 The percentage protection produced by the hydroalcoholic formation of the ROS by TAA, cellular apoptosis, lipid 101 40 extract (Groups IV, V and VI) on the enhancement of AST peroxidation by reducing the bonding of the radicals to 102 41 (SGOT), ALT (SGPT), ALP, total bilirubin and total protein membrane proteins. All these conditions protect the liver cells 103 42 levels were 37.70%, 35.33%, 37.24%, 23.42% and 24.97%, from the loss of cellular metabolites and maintain the normal 104 43 51.85%, 49.43%, 47.86%and 36.24% and 37.48%, 65.65%, function of the mitochondria. The hydroalcoholic extract of 105 44 62.39%, 59.89%, 55.90% and 54.61% at doses of 125, 250 and B. roxburghii showed more hepatoprotective and antioxidant 106 45 500 mg/kg body weight, respectively. activities because of the lack of solubility of the bioactive 107 46 The percentage protection produced by the ethyl acetate ex- molecules in the other extracted solvents. The observed activities 108 47 tracts (Groups VII, VIII and IX) of the enhancement of AST may be due to the presence of different phytocompounds. 109 48 (SGOT), ALT (SGPT), ALP, total bilirubin and total protein Further studies are needed to isolate and characterize the active 110 49 levels were 36.13%, 31.20%, 30.20%, 28.55% and 28.92%, entities presenting in B. roxburghii extracts and complete the 111 50 47.08%, 43.30%, 41.72%, 40.51% and 41.44%, 59.17%, explanation of their mechanism of action. 112 51 58.55%, 55.01%, 57.61% and 56.59% at doses of 125, 250 and 113 52 500 mg/kg body weight, respectively. Conflict of interest statement 114 53 The percentage protection produced by the hexane extracts 115 54 116 (Groups X, XI and XII) on the enhancement of AST (SGOT), The authors report no conflict of interest. 55 ALT (SGPT), ALP, total bilirubin and total protein levels were 117 56 118 32.86%, 29.91%, 26.12%, 22.56% and 22.33%, 44.10%, Acknowledgments 57 40.88%, 38.30%, 35.38% and 34.85%, 56.76%, 54.27%, 119 58 120 52.49%, 52.48% and 50.66% at doses of 125, 250 and 500 mg/ The authors are grateful to the authorities of Andhra University 59 121 kg body weight, respectively. for providing the necessary facilities and also to the Rajiv Gandhi 60 122 The decrease in the SGPT, SGOT, ALP and total bilirubin National Fellowship (RGNF)-UGC, New Delhi, for the financial 61 levels, increase in levels of protein to normal and percentage 123 support, which enabled us to successfully complete the research. Q2 62 protection produced by the higher dose of the extracts were 124

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