Hepatoprotective potential of Indigofera tirunelvelica Sanjappa: in vitro and in vivo studies on CCl4 induced wistar albino rats

The hepatoprotective efficiency of Indigofera tirunelvelica Sanjappa whole plant against CCl4 induced hepatotoxicity was examined. Rat hepatocyte monolayer culture and wistar albino rats were exercised as in vitro and in vivo screening models of protective agent for liver. In in vitro analyses, the whole plant ethanolic extract of Indigofera tirunelvelica Sanjappa were inspected. Silymarin was chosen as a standard treatement drug. In vitro, free radical scavenging property was also evaluated. In animal studies, hepatotoxicity was produced in Wistar albino rats by dispensing CCl4. The degree of hepatotoxicity was examined by determining the ranges of serum enzyme. The antioxidant parameters such as superoxide dismutase, catalase, reduced glutathione, and malondialdehyde of the hepatocytes were also evaluated. In in vitro studies, ethanol extract of I. tirunelvelica whole plant was identified to be the most active than other assessed extracts. Besides, whole plant ethanol extract of I. tirunelvelica was noticed to be rich in phenolic and flavonoids. It exhibited expressive free radical scavenging property versus diphenylpicryl hydrazyl (DPPH) and superoxide ion radicals. In the animals studies, whole plant ethanolic extract of I. tirunelvelica at a ranges of doses (100, 200 and 400 mg/kg body weight) revealed considerable amount of protection against CCl4 induced hepatotoxicity as evident by the protection of CCl4 induced changes biochemical parameters. The results of the present study suggested that the significant hepatoprotective property of whole plant ethanol extract of I. tirunelvelica against CCl4 induced hepatotoxicity and intimates its use as a potential medicinal drug for liver diseases.

Indigofera tirunelvelica, Antioxidants, Hepatotoxicity-CCl4, Hepatoprotective activity ABSTRACT The hepatoprotective ef iciency of Indigofera tirunelvelica Sanjappa whole plant against CCl 4 induced hepatotoxicity was examined. Rat hepatocyte monolayer culture and wistar albino rats were exercised as in vitro and in vivo screening models of protective agent for liver. In in vitro analyses, the whole plant ethanolic extract of Indigofera tirunelvelica Sanjappa were inspected. Silymarin was chosen as a standard treatement drug. In vitro, free radical scavenging property was also evaluated. In animal studies, hepatotoxicity was produced in Wistar albino rats by dispensing CCl 4 . The degree of hepatotoxicity was examined by determining the ranges of serum enzyme. The antioxidant parameters such as superoxide dismutase, catalase, reduced glutathione, and malondialdehyde of the hepatocytes were also evaluated. In in vitro studies, ethanol extract of I. tirunelvelica whole plant was identi ied to be the most active than other assessed extracts. Besides, whole plant ethanol extract of I. tirunelvelica was noticed to be rich in phenolic and lavonoids. It exhibited expressive free radical scavenging property versus diphenylpicryl hydrazyl (DPPH) and superoxide ion radicals. In the animals studies, whole plant ethanolic extract of I. tirunelvelica at a ranges of doses (100, 200 and 400 mg/kg body weight) revealed considerable amount of protection against CCl 4 induced hepatotoxicity as evident by the protection of CCl 4 induced changes biochemical parameters. The results of the present study suggested that the signi icant hepatoprotective property of whole plant ethanol extract of I. tirunelvelica against CCl 4 induced hepatotoxicity and intimates its use as a potential medicinal drug for liver diseases.

INTRODUCTION
Oxidative stress has been linked in the acute and chronic development of diseases in liver injury in a variety of pathophysiological conditions such as alcoholic liver injury,intrahepatic cholestasis, hepatotoxin exposure, viral liver disease and also liver ischemia (Stehbens, 2003) ; (Jaeschke, 2003) ; (Sera ini et al., 1998) . Excessive synthesis of reactive oxygen species (ROS) and reactive nitrogen species (RNS), together with a substantial decline of antioxidant defence in these diseased conditions, hinders numerous cell performances throughout the courses of lipid peroxidation (LPO), nucleic base oxidation as well as healthy protein oxidation. Lipid peroxidation causes vicissitudes in the chemical abd physical properties of cell membrane layers, thus modifying their luidness and also permeability, resulting in impairment in membrane layer signal transduction and ion exchange, causing swelling, cytolysis, and also inally, cell death. The oxidation of healthy proteins and DNA also connects openly to cell dysfunction and fatality (Fang et al., 2002). As necessary, results of free radical scavengers or antioxidants have been extensively evaluated for the avoidance as well as healing of immediate and persistent liver damage. In recent research studies, antioxidants have indicated bene icial impacts, especially for protection as well as therapy of persistent liver damage (Shakya et al., 2012) ; (Rajeshkumar and Kayalvizhi, 2015) ; (Dogan and Celik, 2012) .
Indigofera tirunelvelica Sanjappa (Fabaceae) is an annual erect herbs, about 60 cm high, branches woody, angular, light brown pubescent when young terete, striate and glabrous at maturity. Leaves 3,5.4 cm long, pinnately trifoliolate, alternate; petioles 1-.3 cm long, slender, canaliculated above., Flowers pink, 5mm long; pedicels short, pubescent, glandular; bracts 1-1.5 mm long, lanceolate, acute, pubescent without, caducousl cayx 2mm long, 5lobes, lobes, 1-1.5 mm long. Flowering from November to December months and fruiting is from December to march. Indigofera tirunelvelica distributed in and around Tirunelveli Hills, Tamil Nadu. The literature review revealed that the pharmacognostic standardization, physicochemical analysis. Preliminary phytochemical studies and antibacterial activity of the plant were reported (Subburayalu and Asha, 2017) . Our present study was destined to scrutinize the shielding potential of whole plant ethanolic extract of Indigofera tirunelvelica against CCl 4 induced hepatotoxicity and oxidative stress in Wistar albino rats.

Plant material
The fresh plant material of Indigofera tirunelvelica ItW-He=Hexzne extract; ItW-Ch=Chloroform extract; ItW-Ea=Ethylacetate extract; ItW-Et= Ethanol extract; ItW-Aq= Water extract; +=present; -= absent was gathered from Thirunelveli district of Tamilnadu, India in December month. The whole plant was identi ied and veri ied by Dr. V. Chelladurai, Research of icer, Botany C. C. R. A. S. Govt. of India, (Retired). The whole plant was parched in shadows, created into a abrasive powder with a mechanical grinder, distributed through 40 mesh sieves and stowed in covered vessels for further utilization.

Determination of total phenolic and lavonoid content
The total phenolic and lavonoid substances of various plant extracts were determined (Madinah et al., 2015) . Both the total lavonoid and total phenolic content was expressed in mg/g. Ascorbic acid, a well-known antioxidant, was utilized as positive control.

In vitro antioxidant activity
Superoxide ion radical scavenging assay: the superoxide ion radical scavenging activity was determined conferring to the procedure of Robak and Gryglewski (Zhang and Lu, 2006) . The reaction mixture, comprising 3 ml of plant extract (10-500 µg/ml), 10µl of phenazine methosulphate (60 µM) and 1 ml of NADH (468 µM) was incubated at 25ºC for 5 min, and the absorbance was taken at 560 nm.

In vitro hepatoprotective activity
Hepatocytes were isolated from rat liver as per the reported method by Jain and Singhai (Chu et al., 2016) . The isolated liver cells were suspended in William's E medium (pH 7.4) and seeded in collagen pre-coated culture plates at a density of 2 to 3 x 10 3 cells/well at 37ºC in a humidi ied atmosphere of 5% CO 2 in a CO 2 incubator. After 24h of culturing, cells were exposed to CCl 4 (2.5 mM) with or without selected plant extracts (100 µg/ml) or silymarin (10 µM) and incubated for another 24h at 37ºC in CO 2 incubator. After 24h incubation, the leakage of alanine transaminase (ALT) (Reitman and Frankel, 1957) and lactate dehydrogenase (LDH) Alpini (Alpini et al., 1994) .

Chronic oral toxicity studies
The chronic oral toxicity studies were performed following OECD guidelines (OECD, 2000). Based on these studies, oral doses of 0.5, 1, 2, and 4g/kg b.w. were selected for in vivo studies.

In vivo hepatoprotective activity
The experiment was directed concurring to the method described of (Hu et al., 2014) . Experimental animals were separated into 6 groups, each group have 6 animals and given doses of drug as follows: Group I (normal control), Group II administrated orally with CCl 4 (0.5 ml/150g of bw-v/v in olive oil) on 1 st , 8 th and 16 th days, Group III-V, administrated orally with CCl 4 (0.5 ml/150 g of bw-v/v in olive oil on 1 st , 8 th and 16 th days) and treated with ItW-Et (100, 200. 400mg/ Kg BW) orally for 21 days respectively, Groups VI administrated orally with CCl 4 (0.5 ml/150 g of bw-v/v in olive oil on 1 st , 8 th and 16 th days) and treated with Silymarin (20mg/ Kg BW) orally for 21 days. On the 21 st day, animals were sacri iced by cervical decapitation, liver and blood samples were gathered and processed for biochemical estimations.

Statistical Analysis
Experimental results were presented as mean ± SEM, and the statistical signi icance between the groups was studied through one way ANOVA followed by Tukey's multiple comparison test. P ≤ 0.05 was considered as statistically signi icant.

Preliminary Secondary Metabolites screening
In the preliminary Secondary Metabolites analysis, various extracts (Hexane, Chloroform, Ethylacetate, Ethanol and Water) of I. tirunelvelica indicated the existence of different secondary metabolites Table 1 .

Total phenolic and lavonoid contents
Our phytochemical analysis showed the presence of moderate to high concentration of phenolics (4.56±0.28 to 21.65±0.82mg/g) and lavonoids (2.62±0.15 to 13.65±0.70mg/g) in different extracts of I. tirunelvelica. The rich amount of phenolic and lavonoid contents were found in ItW-Et Table 2 .

In vitro antioxidant activity
In the in vitro evaluation, the ItW-Et had more powerful antioxidant capacity when we compare with other tested extracts. The IC 50 values of ItW-Et against DPPH and superoxide ion radicals were found to be 40.36 and 55.62µg/ml, respectively. Meanwhile, the ascorbic acid showed potent antioxidant activity with IC 50 values of 54.29 and 57.02µg/ml, respectively Figure .

In vitro hepatoprotective activity
Incubation of hepatocytes with CCl 4 (2.5 mM) resulted in a signi icant (p < 0.05) elevation of ALT and LDH (3 and 1.5 fold, respectively) in CCl 4 control hepatocytes Table 3 . Treatment with different extracts of I. tirunelvelica (100 µg/ml) or silymarin (10 µM) exhibited a medium to high hepatoprotective result as evidenced by the recompense of ALT and LDH levels. The high amount of restitution versus enzyme out low was noticed with ItW-Et (69.5% and 71.4%, respectively, for ALT and LDH). In comparison, the silymarin, standard drug, exposed a shielding effect (73.1% and 79.0% regained, respectively, for ALT and LDH). The highest effective extract, ItW-Et, was chosen for in vivo hepatoprotective studies.

Chronic toxicity studies
In chronic toxicity studies, the ItW-Et did indicate no sign and symptoms of toxicity and mortality up to 4g/kg dose, measured comparatively harmless.

In vivo hepatoprotective activity
The result of ItW-Et on hepatic enzymes throughout CCl 4 caused hepatotoxicity is received Table 4 . The increased degrees of ALP, AST, ALT and LDH due to CCl 4 drunkenness were substantially (p < 0.05) protected against ItW-Et therapy when evaluated with CCl 4 control rats. The optimum function was noticed with the high dose. Silymarin additionally indicated considerable safety impact versus CCl 4 caused alterations. The raised LPO and also minimized level of chemical and non-enzymatic antioxidants as perceived in CCl4 control rats Table 5 , were substantially (p < 0.05) avoided in ItW-Et treated groups, showing the exceptional antioxidant result.
CCl4, a well-known hepatotoxin, is traditionally used to monitor new hepatoprotective agents (Al-Sna i et al., 2019) ; (Ali et al., 2016) . This xenobiotic is swiftly altered by cytochrome P450 2E1 to a CCl 3 extreme which is changed into a peroxyl major in the inclusion of oxygen. These radicals may relate with macromolecules of cell and instigate the peroxidative deprivation of lipid membranes (Saravanan and Malarvannan, 2016) . Stimulated fabrication of ROS and proin lammatory cytokines by triggered Kupffer cells are also associated in liver injury started by CCl 4 derivative (El-Bialy et al., 2019) .
In the present study, experimental rats treated with CCl 4 alone, developed signi icant hepatic injury and oxidative damage, as evident by the high increase activities of AST, ALT, ALP and LDH when compared with normal rats. This is an manifestation of cellular leakage and damage of functional reliability of liver cell membrane (Hu et al., 2014) . Lessening enzyme functions to the respective Group I animals by ItW-Et at various dose levels (100, 200 and 400 mg/kg) is an symptoms of stadyiness of plasma membranes and healing of liver injury.
Elimination of free radicals and prevention of LPO is essential in the treatment of CCl 4 mediated liver damage (Narwal et al., 2011) . Inactivation, detoxi ication, elimination of ROS and other free radicals be igured out by enzymatic as well as non-enzymatic antioxidants. The crucial chemical antioxidants in the tissue are SOD, CAT and also glutathione peroxidase (GPx). These antioxidants along with GSH performace to prevent the advancement of toxic substances as well as therefore prevent oxidative anxiety (Khan and Sultana, 2011) .
In our experimental conditions, CCl 4 induced a severe depletion in hepatic GSH, SOD and CAT concerning normal control group. Moreover, this effect was accompanied by a high level of LPO. This would initmaet that LPO is a signi icant factor in the pathogenesis of CCl 4 induced liver damage. Rats administered with ItW-Et revealed a considerable rise in the levels of GSH, SOD and CAT along with a marked reduction in MDA when compared with CCl 4 control rats, indicating remarkable antioxidant effects.
Previous phytochemical investigations on I. tirunelvelica reported the existence of lavonoids and phenolics (Jaeschke, 2003) ; (Sera ini et al., 1998). Consistent with previous results, we also found the abundant amount of phenolics and lavonoids in I. tirunelvelica. Taking into account the fact that such constituents have demonstrated signi icant hepatoprotective and antioxidant activity  ;  ; (Patel et al., 2014) , their presence in the ethanol extract could explain the biological effects here reported. Our experimental indings also revealed a positive association among phenolic content and antioxidant activity, signifying that phenolics and lavonoids might be the active secondary metabolites in I. tirunelvelica.

CONCLUSIONS
In conclusion, the hepatoprotective activity of Indigofera tirunelvelica Sanjappa whole plant extract against CCl 4 induced hepatic toxicity was evaluated. Our results demonstrated that whole plant ethanol extract of Indigofera tirunelvelica possessed signi icant (p < 0.05) protection against CCl 4 induced hepatotoxicity, which might be associated with its antioxidant activities through scavenging free radicals to reorganize oxidative stress and protect lipid peroxidation. The secondary metabolites analysis exposed the high amount of phenolics and lavonoids in ItW-Et, which might be accountable for its more potent biological activities. These preliminary results on antioxidant and hepatoprotective activities here reported lend support to the use of I. tirunelvelica as a hepatoprotective agent. Further studies on identify and characterize the active principle(s) and the working mechanism will through more light on its possible use of whole plant extract of I. tirunelvelica in hepatoprotective drug formulation.