Antioxidant and Protective Effect of Ethyl Acetate Extract of Podophyllum Hexandrum Rhizome on Carbon Tetrachloride Induced Rat Liver Injury

The antioxidant and hepatoprotective activities of ethyl acetate extract was carefully investigated by the methods of DPPH radical scavenging activity, Hydroxyl radical scavenging activity, Superoxide radical scavenging activity, Hydrogen peroxide radical scavenging activity and its Reducing power ability. All these in vitro antioxidant activities were concentration dependent which were compared with standard antioxidants such as BHT, α-tocopherol. The hepatoprotective potential of Podophyllum hexandrum extract was also evaluated in male Wistar rats against carbon tetrachloride (CCl4)-induced liver damage. Pre-treated rats were given ethyl acetate extract at 20, 30 and 50 mg/kg dose prior to CCl4 administration (1 ml/kg, 1:1 in olive oil). Rats pre-treated with Podophyllum hexandrum extract remarkably prevented the elevation of serum AST, ALT, LDH and liver lipid peroxides in CCl4-treated rats. Hepatic glutathione levels were significantly increased by the treatment with the extract in all the experimental groups. The extract at the tested doses also restored the levels of liver homogenate enzymes (glutathione peroxidase, glutathione reductase, superoxide dismutase and glutathione-S- transferase) significantly. This study suggests that ethyl acetate extract of P. hexandrum has a liver protective effect against CCl4-induced hepatotoxicity and possess in vitro antioxidant activities.


Introduction
The partially reduced metabolites of oxygen and nitrogen, commonly referred to as free radicals are highly toxic and reactive. They have been postulated to be increased in majority of diseases like aging, atherosclerosis, cancer, diabetes, liver cirrhosis, cardiovascular disorders, and so forth, [1,2]. The most common reactive oxygen species are superoxide anion (O 2 ·-), hydrogen peroxide (H 2 O 2 ), peroxyl radical (ROO • ), and highly reactive hydroxyl radical (OH • ). Oxidative processes are the most important routes for producing free radicals in living systems. The liver, because of its strategic anatomical action and its large capacity for metabolic conversions, is exposed to many kinds of xenobiotics and therapeutic agents. Due to these facts, efforts to find suitable curatative agents for treatment of liver diseases in natural products of plant and mineral origin are being made [2]. Liver injury induced by CCl 4 is the best characterized system of xenobiotics-induced hepatotoxicity in living system. It is a volatile organic chemical and causes liver, kidney and lung damage through free radical mediated process.
Antioxidants are the substances that when present in low concentration significantly delay or reduce the oxidation of the substrate [3]. Antioxidants protect the body from damaging oxidation reactions by reacting with free radicals and other reactive oxygen species; therefore, diseases linked with free radicals can be prevented by antioxidant therapy. The subject has gained an immense importance and current research trends are directed towards finding naturally occurring antioxidants particularly of plant origin [4][5][6]. Currently, available synthetic antioxidants like butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), tertiary butylated hydroquinone, and gallic acid esters have 2 Evidence-Based Complementary and Alternative Medicine been suspected to cause negative health effects. Hence, strong restrictions have been placed on their application, and there is a trend to substitute them with naturally occurring antioxidants. Moreover, these synthetic antioxidants also show low solubility and moderate antioxidant activity [7]. BHA and BHT are suspected of being responsible for liver toxicity and carcinogenesis [8,9]. Traditionally used natural antioxidants from tea, wine, fruits, vegetables, spices, and medicine (e.g., rosemary and sage) are already exploited commercially either as antioxidant additives or a nutritional supplements [10]. Also, many other plant species have been investigated in search of novel antioxidants [11][12][13][14], but generally, there is still a demand to find more information concerning the antioxidant potential of plant species. It has been mentioned that the antioxidant activity of plants might be due to their phenolic compounds [15]. Flavonoids are a group of polyphenolic compounds with known properties which include free-radical scavenging, inhibition of hydrolytic and oxidative enzymes, and anti-inflammatory actions [16][17][18]. The use of traditional medicine is widespread, and plants still present a large source of natural antioxidants that might serve as leads for the development of novel drugs.
The evaluation of plant extracts antioxidant capacity is not an easy task, since many different methods can be used to determine this activity. Substrates, conditions, methods, and concentrations can also affect the estimated activity [19]. Rhizome powder of P. hexandrum is locally used as a laxative, to treat intestinal parasites, worms, warts, and tumourous growth on skin [5]. The current study was carried out to examine the antioxidant and protective effects of ethyl acetate rhizome extract of P. hexandrum in the rat model of CCl 4induced liver injury. The plant material (rhizome) was dried in the shade at 30 ± 2 • C. The dried rhizome material was ground into a powder using mortar and pestle and passed through a sieve of 0.3 mm mesh size. The powder obtained was extracted with ethyl acetate using a Soxhlet extractor (60-80 • C). The extract was then concentrated with the help of rotary evaporator under reduced pressure and the solid extract was stored in refrigerator for further use.

Animals.
Adult male albino rats of Wistar strain weighing 200-250 g used throughout this study were purchased from the Indian Institute of Integrative and Medicine Jammu (IIIM). The animals had access to food and water ad libitum. The animals were maintained in a controlled environment under standard conditions of temperature and humidity with an alternating 12 hr light and dark cycle. The animals were maintained in accordance with the guidelines prescribed by the National Institute of Nutrition, Indian Council of Medical Research, and the study was approved by the Ethical Committee of the University of Kashmir.

DPPH Radical Scavenging
Activity. The 1, 1-diphenyl-2picryl-hydrazyl (DPPH) assay was performed by using the method of [4,20]. Various concentrations of plant extract (100-1000 μg/mL were added to 1ml of the 0.004% methanol solution of DPPH, and the mixture was vortexed vigorously. The tubes were then incubated at room temperature for 30 minutes in dark, and the absorbance was taken at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. Alpha tocopherol and BHT was taken as known free-radical scavengers. Percentage inhibition activity was calculated by.
where A 0 was the absorbance of the control and A 1 was absorbance in the presence of P. hexandrum extract/known antioxidant.

Assessment of Hydroxyl Radical Scavenging Property.
Hydroxyl radical, generated from the Fe 3+ -Ascorbate-H 2 O 2 (Fenton reaction), was evaluated by degradation of deoxyribose that produced thiobarbituric acid reactive species (TBARS) [21]. The reaction mixture contained 25 mM deoxyribose, 10 mM Ferric chloride, 100 mM ascorbic acid, 2.8 mM H 2 O 2 in 10 mM KH 2 PO4 (pH 7.4), and various concentrations of P. hexandrum rhizome ethyl acetate extract. The reaction mixture was incubated at 37 • C for 1 h. Then, 1 mL of 1% thiobarbituric acid and 1 mL of 3% trichloroacetic acid was added and mixture heated at 100 • C for 20 min. The TBARS was measured spectrophotometrically by taking absorbance at 532 nm. The results were expressed as percentage inhibition of deoxyribose oxidation, as determined by.
where A was the malondialdehyde produced by Fenton reaction treated alone, and B was the malondialdehyde produced in the presence of P. hexandrum extract/known antioxidant.

Assessment of Superoxide Anion Radical Scavenging
Property. Superoxide anion radical generated by the Xanthine/Xanthine oxidase system was spectrophotometrically determined by monitoring the product of nitroblue tetrazolium (NBT) using the method of [22]. A reaction mixture Evidence-Based Complementary and Alternative Medicine 3 containing 1.0 mL of 0.05 M phosphate buffer (pH 7.4), 0.04 mL of 0.15% BSA, 0.04 mL of 15.0 mM NBT, and various concentrations of (plant extract and known antioxidant) was incubated at 25 • C for 10 min, and the reaction was then started by adding 0.04 mL of 1.5 U/mL Xanthine oxidase and again incubated at 25 • C for 20 min. The absorbance of the reaction mixture was measured at 560 nm. Decreased absorbance of the reaction mixture indicates increased superoxide anion radical scavenging activity. The scavenging activity of the plant extract on superoxide anion radical was expressed as where A 0 was the absorbance of the control and A 1 was absorbance in the presence of P. hexandrum extract/known antioxidant.

Assessment of Hydrogen
where A 0 is the absorbance of the control and A 1 is absorbance in the presence of plant extract and known standard.

Reducing
Power. The reducing power of P. hexandrum rhizome extract was evaluated according to [25]. Different concentrations of the plant extract were mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium hexacyanoferrate II. The mixture was incubated at 50 • C for 20 minutes, 2.5 mL of 10% TCA was added to the mixture and centrifuged at 3000 rpm for 10 minutes. The upper layer of the solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl 3 (0.5 mL, 0.1%), and the absorbance was measured at 700 nm. BHT (butylated hydroxyl toluene) was taken as the known standard. Increased absorbance of the reaction mixture indicates stronger reducing power.
3.6. Dosage and Treatment. Rats were divided into six groups each containing seven rats. The plant extract was employed at oral doses of 20, 30, and 50 mg/kg-day. The extract was suspended in normal saline such that the final volume of extract at each dose was 1 mL which was fed to rats by gavage.
Group I: received olive oil vehicle only at 5 mL/kgday.
Group II: received CCl 4 in olive oil only.
Group III: were administered with vitamin E (50 mg/kg-day).
Group IV: received 20 mg/kg-day extract orally for fifteen days.
Group V: received 30 mg/kg-day extract orally for fifteen days.
Group VI: received 50 mg/kg-day orally for fifteen days.
On the thirteenth day, animals from groups II-VI were injected intraperitoneally with CCl 4 in olive oil vehicle at a dosage of 1 mL/kg bw. The rats were sacrificed, 48 hr after CCl 4 administration, and livers were collected; post mitochondrial supernatant of the liver tissue was prepared as written under preparation of PMS in Section 3.8.

Blood Collection for Estimation of AST, ALT, and LDH.
Before sacrificing the experimental animals, blood was collected from retro-orbital plexus without the use of anticoagulant. The blood was allowed to stand for 10 min before being centrifuged at 5,000 g for 10 min to obtain serum for analysis of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and serum lactate dehydrogenase (LDH).

Serum Alanine Aminotransaminase (ALT).
ALT was estimated by the method of Reitman and Frankel [26]. Briefly, 0.5 mL of substrate (2 mM α-ketoglutarate, 0.2 M DL-alanine in phosphate buffer 0.1 M pH 7.4) was incubated at 37 • C for 5 minutes. 0.1 mL of freshly prepared serum will be added to the aliquot and again incubated at 37 • C for 30 minutes. At the end of incubation 0.5 mL of 2, 4-dinitrophenylhydrazine was added, and the aliquot left for 30 minutes at room temperature. 0.5 mL of 0.4 N NaOH was added, and the aliquot is again left for 30 minutes. Absorbance was then recorded at 505 nm against water blank. [26]. The substrate, however, was 2 mM α-ketoglutarate, 0.2 M DL-aspartate, and the rest of the procedure was same.

Serum Lactate Dehydrogenase (LDH)
. LDH was assayed according to the method of King [27]. To 1.0 mL of buffered substrate (sodium pyruvate 37.5 mM in phosphate buffer 100 mM, pH 7.4), 0.1 mL of freshly prepared serum was added, and the tubes were incubated at 37 • C for 15 minutes. After adding 0.2 mL of NAD + solution (10 mg/mL in phosphate buffer), the incubation was continued for another 15 minutes. The reaction was arrested by adding 0.1 mL of 2, 4-dinitrophenylhydrazine (0.02% in concentrated HCl), and the tubes were incubated again at 37 • C for a further period of 15 min, after which 7.0 mL of 0.4 N NaOH was added, and the color developed was measured spectrophotometrically at 420 nm against a blank containing phosphate buffer only.

Preparation of Post Mitochondrial Supernatant (PMS).
Liver tissue was washed in ice-cold 1.15% KCl and homogenized in a homogenizing buffer (50 mM Tris-HCl, 1.15% KCl pH 7.4) using a Teflon homogenizer. The homogenate was centrifuged at 9,000 g for 20 minutes to remove debris. The supernatant was further centrifuged at 15,000 g for 20 minutes at 4 • C to get PMS, subsequently used for various biochemical assays. Protein concentration was estimated by the method of Lowry et al. [28].

Estimation of Lipid Peroxidation (PMS).
Lipid peroxidation in liver tissue homogenate was estimated by the formation of thiobarbituric acid reactive substances (TBARS) by the method of [29]. In brief, 0.1 mL of tissue homogenate (PMS; Tris-HCl buffer, pH 7.5) was treated with 2 mL of (1 : 1 : 1 ratio) TBA-TCA-HCl reagent (0.37% thiobarbituric acid, 0.25 N HCl, and 15% TCA), placed in boiling water bath for 15 min, cooled, and centrifuged at room temperature for 10 min. The absorbance of the clear supernatant was measured against reference blank at 535 nm.

Determination of Total Sulphydryl
Groups. The acid soluble sulphydryl groups (nonprotein thiols of which more than 93% is reduced glutathione (GSH) forms a yellow colored complex with DTNB that shows the absorption maximum at 412 nm. The assay procedure followed was that of [30]. 500 μL of homogenate precipitated with 100 μL of 25% TCA was subjected to centrifugation at 300 xg for 10 minutes to settle the precipitate. 100 μL of the supernatant was taken in a test tube containing the 2 mL of 0.6 mM DTNB and 0.9 mL of 0.2 mM sodium phosphate buffer (pH 7.4). The yellow color obtained was measured at 412 nm against the reagent blank which contained 100 μL of 25% TCA in place of the supernatant. Sulphydryl content was calculated using the DTNB molar extension coefficient of 13,100.

Glutathione Peroxidase (GPx).
GPx activity was assayed using the method of [31]. The assay mixture consisted of 1.49 mL of sodium phosphate buffer (0.1 M pH 7.4), 0.1 mL EDTA (1 mM), 0.1 mL sodium azide (1 mM), 0.1 mL 1 mM GSH, 0.1 mL of NADPH (0.02 mM), 0.01 mL of 1 mM H 2 O 2 , and 0.1 mL PMS in a total volume of 2 mL. Oxidation of NADPH was recorded spectrophotometrically at 340 nm and the enzyme activity was calculated as nmoles NADPH oxidized/min/mg of protein, using extinction coefficient of 6.22 × 10 3 M −1 cm −1 .
3.12. Glutathione Reductase Activity (GR). GR activity was assayed by the method of [31,32]. The assay mixture consisted of 1. The enzyme activity measured as absorbance at 340 nm was calculated as nmoles of NADPH oxidized/min/mg of protein using extinction coefficient of 6.22 × 10 3 M −1 cm −1 .

Glutathione-S-Transferase (GST) Activity.
GST activity was assayed using the method of [33]. The reaction mixture consisted of 1.67 mL sodium phosphate buffer (0.1 M pH 6.5), 0.2 mL of 1 mM GSH, 0.025 mL of 1 mM CDNB, and 0.1 mL of post mitochondrial supernatant in a total volume of 2 mL. The change in absorbance was recorded at 340 nm and the enzyme activity was calculated as nmoles of CDNB conjugates formed/min/mg protein using extinction coefficient of 9.6 × 10 3 M −1 cm −1 .
3.14. Superoxide Dismutase Activity (SOD). SOD activity was estimated by Beauchamp and Fridovich [34]. The reaction mixture consisted of 0.5 mL of hepatic PMS, 1 mL of 50 mM sodium carbonate, 0.4 mL of 25 μM NBT, and 0.2 mL of 0.1 mM EDTA. The reaction was initiated by addition of 0.4 mL of 1 mM hydroxylamine-hydrochloride. The change in absorbance was recorded at 560 nm. The control was simultaneously run without tissue homogenate. Units of SOD activity were expressed as the amount of enzyme required to inhibit the reduction of NBT by 50%.

Statistical Analysis.
The values are expressed as mean ± standard deviation (SD). The results were evaluated by using the SPSS (version 12.0) and Origen 6 software and evaluated by one-way ANOVA followed by Bonferroni t-test. Statistical significance was considered when value of P was <.5.

Results
In this present study, the antioxidant activity of the ethyl acetate extract of the rhizome of P. hexandrum was investigated by using DPPH scavenging assay, hydroxyl assay, H 2 O 2 assay, superoxide assay, and reducing power. Under in vivo situations, the extract was examined by determining the antioxidant enzymes activities and glutathione levels in the liver tissue homogenate. All the methods have proven the effectiveness of the ethyl acetate extract compared to known antioxidants BHT and α-tocopherol.

DPPH Radical Scavenging Activity.
Comparison of the antioxidant activity of the extract and standard antioxidants by DPPH method is shown in Figure 1. The ethyl acetate extract of P. hexandrum exhibited dose-dependent inhibition of DPPH activity, and the scavenging activities of the extract and known antioxidants increased with increasing concentration. At higher concentration (1000 μg/mL), the extract and known antioxidants gave the highest percentage activities, that is, (85.77%), (89.77) and (87.7%), respectively. The percentage inhibition observed is in the following descending order α-tocopherol > BHT > ethyl acetate extract. These results suggest that ethyl acetate extract of P. hexandrum has a noticeable effect on scavenging of DPPH radical.

Hydroxyl Radical Scavenging
Activity. The effect of ethyl acetate extract of P. hexandrum on the inhibition of hydroxyl radical production was assessed by the iron (II)-dependent deoxyribose damage assay. Figure 2 presents the results of the effects of examined ethyl acetate extract as well as known antioxidants on OH • radical production. The percentage of hydroxyl radical scavenging activity increased with the increasing concentration of extract and known antioxidants. At a concentration of 300 μg/mL, the extract shows the maximum inhibitory effect of about 83.44% which was comparable to that of α-tocopherol (88 and 86%). The scavenging activity was found in the following order αtocopherol > ethyl acetate extract.

Superoxide Anion Scavenging Activity.
Results of superoxide anion scavenging activity of ethyl acetate extract of rhizome of P. hexandrum and known antioxidant BHT are shown in Figure 3. The extract and the standard demonstrate a dose response inhibition on superoxide anion radical. The % inhibition of superoxide generation by ethyl acetate extract and BHT was found to be 70.5% and 78.4% at a higher concentration of 400 μg/mL. the results suggest that ethyl acetate extract has a potent superoxide scavenging effects. Figure 4, the ethyl acetate extract of P. hexandrum also demonstrated hydrogen peroxide decomposition activity in a concentration-dependent manner. At the higher concentration (300 μg/mL) of extract and BHT the H 2 O 2 scavenging activity was found as 65% and 70%, respectively. These results showed that ethyl acetate extract had effective H 2 O 2 scavenging activity. Figure 5 shows the reductive capabilities of ethyl acetate extract of P. hexandrum when compared to the standard, BHT. Like the antioxidant activity, the reducing power increased with increasing amount of  the extract. For the measurement of the reductive ability, the Fe 3+ -Fe 2+ transformation was investigated in presence of the extract. Presence of reductants causes the reduction of the Fe 3+ /ferricyanide complex to the Fe 2+ form. This Fe 2+ can be monitored by measuring the formation of Perl's Prussian blue at 700 nm [35]. At higher concentration of plant extract (300 μg/mL), reducing power value was around 0.53,

6
Evidence-Based Complementary and Alternative Medicine   Table 1, administration of CCl 4 induced a marked increase in the levels of AST, ALT, and LDH levels as compared to the control group. Group I: (this group was given neither Group comparison between Group III, Group V and Group VI shows no significant variation in these parameters indicating that ethyl acetate extract has got the same effect as that of the vitamin E which was used as the positive control in this study.

Effect of Extract on GSH and Antioxidant Enzyme
Activities. The findings of present investigation indicate the ethyl acetate extract of P. hexandrum and modulate the liver against oxidative stress in a dose-dependent manner without any adverse effect on the animals at the dose levels (20, 30, and 50 mg/kg body weight/day for 15 days, respectively). Table 2 shows oxidative stress by CCl 4 caused significant alterations in hepatic antioxidant defense system like GSH, GR, GPx, SOD, and GST contents in comparison to the normal controls.

Effect of Extract on Glutathione Reductase (GR).
Glutathione reductase (GR) activities in the liver homogenate of rats for all experimental groups are shown in the   Figure 6. From our results, it was found that P. hexandrum ethyl acetate extract could significantly decrease the formation of Malondialdehyde (MDA) in CCl 4treated rats. After CCl 4 administration, the liver MDA level significantly increased from 2.87 ± 0.36 nmol/mg protein (control group) to 10.16 ± 0.95 nmol/mg protein. However, oral administration of P. hexandrum extract at the concentration of 20, 30, and 50 mg/kg bw/day, the liver MDA level decreased to 7.27 ± 0.26, 4.80 ± 0.32, and 3.73 ± 0.38, respectively. At the same time, the effect of vitamin E (50 mg/kg/day) on MDA levels in CCl 4treated rats was found to be reduced to 3.81 nmol/mg protein.

Discussion
Free radical oxidative stress has been implicated in the pathogenesis of a wide variety of clinical disorders, resulting usually from deficient natural antioxidant defenses. Potential antioxidant therapy, therefore, should include either natural free-radical scavenging antioxidant enzymes or agents which are capable of augmenting the activity of these enzymes. Reactive oxygen species (ROS) has received considerable attention in the recent past because of its role in several pathological conditions including cancer, diabetes, arthritis, aging, and atherosclerosis. ROS produced in vivo O 2 − , hydrogen peroxide (H 2 O 2 ), and hypochlorous acid (HOCl), and H 2 O 2 can interact in the presence of transition metal ions to yield a highly reactive oxidizing species, the hydroxy radical [36]. If human disease is believed to be due to the imbalance between oxidative stress and antioxidative defense, it is possible to limit oxidative tissue damage and hence prevent disease progression by antioxidant defense supplements.
In our study, antioxidant effects of rhizome ethyl acetate extract of P. hexandrum were measured through a variety of biological parameters and compared with some of the known antioxidants. Because of the complex nature of phytochemicals, the antioxidant activities of plant extracts cannot be evaluated by a single method. Therefore, commonly accepted assays, including enzymatic and nonenzymatic methods under in vitro and in vivo conditions were employed to evaluate the total antioxidant potential of plant extracts.
DPPH is characterized as stable free radical by virtue of the delocalization of the spare electron, where the molecule as a whole, so that the molecule do not dimerise, as would be the case with most other free radicals. The delocalization gives rise to the deep violet colour, characterized by an absorption band (517 nm). When a solution of DPPH is mixed with a substance of H donor, it gets reduced into nonradical state [37]. Hence, the ethyl acetate extract of P. hexandrum exhibited a significant dose-dependant inhibition of DPPH activity. Similar results were reported by [38], while investigating the DPPH radical scavenging activity of wormwood (Artemisia absinthium L).
Similarly, results were also reported by [37] that extracts of Pterocarpus santalinus exhibited significant DPPH radical scavenging activity. The observed scavenging effect of P. hexandrum ethyl acetate extract and standards on the DPPH radical decreases in the following order: αtocopherol > BHT > Ethyl acetate and it was 89.77%, 87.77%, and 85.77% at concentration of 1000 μg/mL, respectively. Our results are in tune with earlier investigations that the methanol extracts of exhibited significant DPPH radical inhibition The reducing ability of a compound generally depends on the presence of reductants [39] which have been exhibited antioxidative potential by breaking the free-radical chain, donating a hydrogen atom [40]. Presence of reducers causes the conversion of the Fe 3+ /ferricyanide complex used in this method to the ferrous form. By measuring the formation of Perl's Prussian blue at 700 nm, it is possible to determine the Fe 2+ concentration. Our results suggest that polyphenolic components within the ethyl acetate extract of P. hexandrum play an important role in scavenging of free radicals, and the scavenging activity is increased with the increasing concentration of the plantextract. At the higher concentration (300 μg/mL), the reducing power of ethyl acetate extract of P. hexandrum and the known antioxidant BHT was 0.53 ± 0.005 and 0.675 ± 0.05, respectively. Our results showed that P. hexandrum rhizome ethyl acetate extract is an electron donor and could react with free radicals, convert them to more stable products, and terminate radical chain reaction. Similar results were reported by Noriham et al. [41], who demonstrated antioxidative activity on Pimpinella anisum seed extracts and different types of Malaysian plants, and [42] showed that P. fascisepala leaf extract is an electron donor and could react with free radicals and terminate radical chain reaction.
Highly reactive OH • radicals are responsible for the oxidative damage of DNA, lipids, and proteins [43]. Formation of a highly reactive tissue-damaging species like hydroxyl radical is caused by the interaction of iron ions with hydrogen peroxide in biological system [44]. The rhizome of P. hexandrum ethyl acetate extract exhibited hydroxyl radical scavenging activity in a dose-dependent manner. At a concentration of 300 μg/mL, the extract shows a maximum inhibitory effect of about 83%, which was comparable to the known antioxidant BHT (86%). These results show that ethyl acetate extract of P. hexandrum rhizome can act as effective antioxidants by reacting with free radicals.
Superoxide anion, which is a reduced form of molecular oxygen, has been implicated in the initiating oxidation reactions associated with aging [45]. Also, it has been implicated in several pathophysiological processes, due to its transformation into more reactive species such as hydroxyl radical that initiate lipid peroxidation. Superoxide has also been observed to directly initiate lipid peroxidation [46]. In the Xanthine/Xanthine oxidase system, superoxide anion derived from dissolved oxygen reduces NBT. Antioxidants are able to inhibit the blue NBT formation [47]. The superoxide radical scavenging activity of P. hexandrum ethyl acetate extract, and the standard BHT increased in a dosedependent manner. At the concentration of 300 μg/mL, the percentage inhibition on superoxide radical observed was 69.05 and 78.70%, respectively. Similar results reported by [48] that different extracts from Croton celtidifolius Baill (Euphorbiaceae) showed in vitro antioxidant properties through the superoxide scavenger capacity method by the nitro blue tetrazolium (NBT) reduction assay.
The measurement of H 2 O 2 -scavenging activity is one of the useful methods of determining the ability of antioxidants to decrease the level of pro-oxidants such as H 2 O 2 [49]. It can cross membranes and may slowly oxidize a number of compounds. Hydrogen peroxide itself is not very reactive, but sometimes, it can be toxic to cells because of rise in the hydroxyl radicals in the cells [50]. In our study, the H 2 O 2scavenging activity of P. hexandrum ethyl acetate extract and the standard BHT increased in a dose-dependent manner. At the concentration of 300 μg/mL, the percentage inhibition on H 2 O 2 was observed 65.54% and 70.91%, respectively. This ability to scavenge hydrogen peroxide could be an efficient assessment method to evaluate antioxidant property of extract of P. hexandrum.
In accordance with the results of the antioxidant effects of the extract of P. hexandrum rhizome obtained in in vitro assays, examinations of the in vivo activity of ethyl acetate extract of P. hexandrum was also conducted.
CCl 4 -induced hepatic injuries are commonly used animal models for the screening of hepatoprotective plant extracts and the magnitude of hepatic damage is assessed by measuring the level of released cytosolic transaminases including ALT and AST in circulation [51]. It is generally thought that CCl 4 toxicity is due to reactive free radical (CCl 3 • ), which is generated by its reductive metabolism by hepatic cytochrome P450. The reactive intermediate is believed to cause lipid peroxidation and breakdown of cellular membranes [52]. This present study evaluated the hepatoprotective effects of P. hexandrum ethyl acetate extract on CCl 4 -induced liver toxicity. Acute administration of CCl 4 produced a marked elevation of the serum levels of AST, ALT, and LDH in (Group II) treated animals when compared with that of the control group (Group I). Treatment with P. hexandrum ethyl acetate extract at a dose of 20, 30, and 50 mg/kg/day significantly reduced the elevated levels of the enzymes (Table 1). Similarly reported by [53] that the ethyl acetate extract of Cadaba farinosa exerts protective action by decreasing CCl 4 -derived free radicals and significantly inhibit the elevated level of serum enzyme activities.
Decreased serum levels of AST, ALT and LDH by the plant extract is an indication of stabilization of plasma membrane as well as repair of hepatic tissue damage caused by CCl 4 . The above changes can be considered as an expression of the functional improvement of hepatocytes, which may be caused by an accelerated regeneration of parenchyma cells.
The body has an effective mechanism to prevent and neutralize the free radical induced damage. This is accomplished by a set of endogenous antioxidant enzymes, such as SOD, CAT, GPX, GST, and GRD. When the balance between ROS production and antioxidant defenses is lost, "oxidative stress" results, which through a series of events deregulates the cellular functions leading to various pathological conditions [54] Any compound, natural or synthetic, with antioxidant properties might contribute towards the partial or total alleviation of this type of damage. In the present study, elevated level of TBARS in CCl 4 -treated rats indicates excessive formation of free radicals and activation of LP system resulting in hepatic damage. TBARS produced as byproducts of LP that occurs in hydrophobic core of biomembranes [55]. The significant decline in the concentration of these constituents in the liver tissue of CCl 4 and ethyl extract administered rats indicates antilipid peroxidative effect of P. hexandrum.
GSH is a major nonprotein thiol in living organisms which plays a central role in coordinating the body's antioxidant defense processes. Perturbation of GSH status of a biological system has been reported to lead to serious consequences [54]. Decline in GSH content in the liver of CCl 4 -intoxicated rats, and its dose dependent increased level with the administration of extract reveal antioxidant effect of P. hexandrum. SOD, GR, GST, and GP X constitute a mutually supportive team of defense against ROS. SOD is a metalloprotein and is the first enzyme involved in the antioxidant defense by lowering the steady-state level of O 2 − . GP X is a selenoenzyme two third of which (in liver) is present in the cytosol and one third in the mitochondria. It catalyses the reaction of hydroperoxides with reduced glutathione to form glutathione disulphide (GSSG) and the reduction product of the hydroperoxide. In our study, decline in the activities of these enzymes in CCl 4 -administered rats revealed that LP and oxidative stress elicited by CCl 4intoxication have been nullified due to the effect of P. hexandrum ethyl acetate extract. Similar results were reported by [56] who investigated hepatoprotective and antioxidant activity of Boehmeria nivea. GTS plays an essential role in liver by eliminating toxic compounds by conjugating them with glutathione. GR is concerned with the maintenance of cellular level of GSH (especially in the reduced state) by effecting fast reduction of oxidized glutathione to reduced form. In our study, the liver tissues of CCl 4 -administered rats, activities of GTS and GR were significantly decreased compared with control group. However, pretreatment with ethyl acetate extract of P. hexandrum, the activity of these enzymes significantly increased, thus unearthing the antioxidant effect of P. hexandrum. Similar results were reported by [57] showed that Nigella sativa L and Urtica dioica L increase the antioxidant defense system activity in experimentally CCl 4 -treated rats.

Conclusion
In conclusion, it is well known that the hepatoprotective effect has a good correlation with the antioxidant activities [58,59]. In our previous studies, methanolic and 70% ethanolic rhizome extracts of P. hexandrum have been demonstrated to possess excellent antioxidant activities by various in vitro and in vivo assays [5,6,60]. In the current study, the encouraging results of the ethyl acetate extract of P. hexandrum with the various in vitro antioxidant tests proved that the plant possess components with reducing activity has hydrogen-donating ability as well as effectiveness as scavengers of hydroxyl radical, hydrogen peroxide radical, and superoxide free radicals. Current study also demonstrates that ethyl acetate extract could reduce CCl 4induced toxicity, particularly hepatotoxicity, by inhibiting lipid peroxidation, suppressing alanine aminotransferase (ALT), aspartate aminotransferase (AST) activities, and lactate dehydrogenase activity (LDH), and increasing antioxidant enzyme activity. Therefore, ethyl acetate extract of P. hexandrum can be proposed to protect the liver against CCl 4induced oxidative damage in rats, and the hepatoprotective effect might be correlated with its antioxidant and free radical scavenger effects. The studies are in progress to isolate and purify the active principle involved in antioxidant activities of this plant. Further research is needed to study the mechanisms of activity of the active compounds responsible for antihepatotoxic activity.