MS and hepatoprotective activity of Cardiospermum halicacabum against CCl4 induced liver injury in Wistar rats

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Phenolic profiling by UPLC–MS/MS and hepatoprotective activity of Cardiospermum halicacabum against CCl4 induced liver injury in Wistar rats R. Jeyadevia, T. Sivasudhaa,*, A. Rameshkumara, James M. Harnlyb, Long-Ze Linb a

Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India Food Composition and Methods Development Laboratory, Beltsville Human Nutrition Research Center, US Department of Agriculture, Beltsville, United States

b

A R T I C L E I N F O

A B S T R A C T

Article history:

The hepatoprotective potential of ethanolic extract of Cardiospermum halicacabum (ECH) was

Received 3 July 2012

evaluated in Wistar rats having liver injury induced by CCl4. The free radical scavenging

Received in revised form

activity of ECH was evaluated by ABTS and DPPH assay. Phenolic compounds like luteolin

29 October 2012

7-o-glucoronide, apigenin 7-o-glucoronide and chrysoerisol 7-o-glucoronide were identified

Accepted 30 October 2012

in ECH by UPLC–MS/MSn analysis. On the other hand, the anti mutagenic effect of ECH was

Available online 26 November 2012

examined using tester strain Salmonella typhimurium TA100 with and without S9 extract.

Keywords:

which were significantly (P 6 0.05) restored toward normal level by the therapy of ECH.

Cardiospermum halicacabum

ECH significantly (P 6 0.05) reduced LPO level in the liver tissue. Treatment with ECH

UPLC–MS/MS analysis

showed curative effect on histopathological alterations and DNA damage in CCl4 adminis-

Phenolic profile Ames test Carbon tetrachloride

CCl4 administered Wistar rats exhibited alteration in various biochemical parameters

tered rats in a dose dependent manner. Silymarin, a routine hepatoprotective drug was used as reference standard.

Hepatoprotectivity

1.

Introduction

The liver plays a major role in regulating various physio– chemical functions of the body, including synthesis, secretion and metabolism of xenobiotics. Damage to the hepatic parenchyma will lead to deleterious effect on liver physiochemical functions. Many factors may induce such damage, including infectious agents and hepatotoxic chemicals. Also many drugs used for the treatment of various diseases mainly affect liver by generating reactive oxygen species (ROS). ROS such as the superoxide anion (O 2 ), hydrogen

 2012 Elsevier Ltd. All rights reserved.

peroxide (H2O2), peroxyl radicals (ROO), reactive hydroxyl (OH), and nitric oxide (NO) radicals, are continuously generated during normal cell metabolism. ROS can act as a physiological defense system against microbial infection and are involved in maintaining normal cellular functions, including proliferation, apoptosis, and intracellular signal transduction (Filippin, Vercelino, Marroni, & Xavier, 2008). However, excessive amounts of ROS can damage lipids, proteins, DNA, and the extracellular matrix eventually leading to many chronic diseases, such as cancer, diabetes, aging, and other degenerative diseases in humans (Droge, 2002). Antioxidants have

* Corresponding author. Tel.: +91 0431 2407088; fax: +91 0431 2407045. E-mail address: [email protected] (T. Sivasudha). Abbreviations: CCl4, carbon tetrachloride; ABTS, 2,2 0 -azinobis-3-ethylbenzothiazoline-6-sulphonic acid; DPPH, 2,2-diphenyl-1picrylhydrazyl; UPLC, ultra performance liquid chromatography; SGOT, serum glutamate oxaloacetate transaminase; SGPT, serum glutamate pyruvate transaminase; ALP, alkaline phosphatase; LPO, lipid peroxidation; SOD, superoxidedismutase; GSH, reduced glutathione; CAT, catalase 1756-4646/$ - see front matter  2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jff.2012.10.019

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been reported to prevent oxidative damage caused by free radicals and thereby reduce the risk of cancer, cardiovascular diseases and arthritis (Shahidi & Zhong, 2010). They can interfere with the oxidation process by reacting with free radicals, chelating, catalytic metals and also by quenching singlet oxygen and thus can be utilized to scavenge the excessive free radicals generated (Wanasundara & Shahidi, 2005). Recently, the ability of phenolic substances, including flavonoids and phenolic acid acting as antioxidants has been extensively investigated. So far, many researchers have shown much interest in plants because of their safety and their wide acceptance by consumers (Katalinic, Milos, Kulisic, & Jukic, 2006). Cardiospermum halicacabum Linn. belongs to the family Sapindaceae is an annual or perennial climber, widely distributed in tropical and subtropical Asia and Africa, and consumed as green leaf vegetable. C. halicacabum roots have been used traditionally for the treatment of epilepsy and anxiety disorders (Rajesh Kumar, Murugananthana, Nandakumar, & Sahil Talwar, 2011), the anti-inflammatory activity of ethanol extract of C. halicacabum leaves against carrageenan-induced rat paw edema has been established (Sadique, Chandra, Thenmozhi, & Elango, 1987). The analgesic and vasodepressant activities of C. halicacabum plant have been reported by Gopalakrishnan, Dhananjayan, and Kameswaran (1976). The ethanol extract of C. halicacabum has antipyretic activity against yeast-induced pyrexia in rats (Asha & Pushpangadan, 1999), anti-ulcer activity against ethanol induced gastric ulcer in rats (Sheeba & Asha, 2006) and antihyperglycemic effect against streptozotocin induced diabetic Wistar rats (Veeramani, Pushpavalli, & Pugalendi, 2008). The ethanol extract of the C. halicacabum suppressed the production of TNF-alpha and nitric oxide in human peripheral blood mononuclear cells (Venkteshbabu & Krishnakumari, 2006). Recently the antiarthritic effect of ethanolic extract of C. halicacabum against adjuvant induced arthritis in Wistar rats have been reported by the authors (Jeyadevi, Sivasudha, Rameshkumar, & Dineshkumar, 2012). To the best of our knowledge, there has so far been no report on phenolic profile, antioxidant activity as well as hepatoprotective activities of C. halicacabum. Hence, the aim of this study was to evaluate the antioxidant and hepatoprotective properties of ethanolic extract of C. halicacabum leaves against CCl4 induced oxidative damage in rats and profiling of phenolics by UPLC–MS/MSn.

2.

Materials and methods

2.1.

Chemicals

All chemical products used were of high purity and analytical grade. Carbon tetrachloride (CCl4), Folin–Ciocalteu phenol reagent, sodium carbonate, gallic acid, rutin, trichloroacetic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2 0 -azinobis-3ethylbenzothiazoline-6-sulphonic acid (ABTS), potassium ferricyanide, butylated hydroxytoluene (BHT), aluminium chloride, sodium azide, and ferric chloride were purchased from Sigma Chemicals Co. (St. Louis, MO, USA).

2.2.

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Plant material and extract preparation

C. halicacabum plant were collected from Tiruchirappalli district, Tamilnadu, India and taxonomically identified by Department of Plant Science, Bharathidasan University, Trichirappalli, Tamil Nadu, India. The leaves were picked, washed and cut into small pieces, frozen in liquid nitrogen, lyophilized using a vacuum freeze dryer (Christ alpha 1–2/LD plus, Osterode am Harz, Germany) and the lyophilized powder was stored at 20 C for a period of three months. Ten grams of powder was extracted with 200 ml of 99% ethanol using a soxhlet apparatus. The solvent was evaporated under reduced pressure at 45 C using rotary evaporator (Buchi R-210, Flawil, Switzerland) to produce a yield of 9% of dry extract. The dried extract obtained was stored at 20 C for a period of three months. Then the extract was reconstituted with distilled water to produce the desired concentrations and used for further analysis.

2.3.

Phytochemical screening

Phytochemical analysis of the different plant extracts was performed using the methods described by Trease and Evans (1989). Approximately 0.2 g of the extract was dissolved in 2 ml of methanol and heated in flame for a minute. A chip of magnesium metal was added to the mixture, followed by the addition of few drops of concentrated hydrochloric acid. The formation of red color was indicative of the presence of flavonoids. Approximately 0.5 g of the extract was dissolved in 3 ml of chloroform, and a few drops of filtered concentrated sulphuric acid were carefully added to the filtrate to form a lower layer. A reddish-brown color at the interface was a positive indicator for the presence of steroids. Approximately 1 ml of ethanolic extract was diluted separately with 20 ml of distilled water and shaken in a graduated cylinder for 15 min. A 1-cm layer of foam indicated the presence of saponins. 2 ml of trichloroacetic acid was added to 1 ml of extract and the formation of a yellow-to-red precipitate showed the presence of terpenoids. About 0.5 ml of the extract was boiled and few drops of 0.1% ferric chloride were added. The formation of brown green or blue black color indicates the presence of tannins. To the extract, few drops of 5% ferric chloride reagent were added. A dark green color indicates the presence of phenolics.

2.4.

Determination of total phenolics

The total phenolic content was determined using the Folin– Ciocalteu method, based on the reduction of phosphor wolframate to phosphomolybdate complex by phenolics to a blue reaction product (Wolfe, Wu, & Liu, 2003). To 100 ll of the extract, 1.5 ml of distilled water and 300 ll of Folin–Ciocalteu reagent were added and incubated for 3 min at room temperature. About 300 ll of sodium carbonate (2 g/10 ml) were then added and allowed to stand for 30 min at 40 C for color development. Absorbance was then read at 755 nm using the spectrophotometer (Shimadzu UV-1601, Columbia, MD, USA). The total phenolics were expressed as mg of gallic acid equivalents/g of extract. The estimation of total phenolics in the

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extracts was carried out in triplicates and the results were averaged.

extract/standard. All determinations were carried out in triplicates.

2.5.

2.8.

Determination of total flavonoids

Total flavonoids were estimated using the method of Ordon ez, Gomez, Vattuone, and Isla (2006). To 0.5 ml of sample, 0.5 ml of 2% aluminium chloride ethanol solution was added. After one hour at room temperature, the absorbance was read at 420 nm using a Shimadzu UV-1601 spectrophotometer, and expressed as mg of rutin equivalents/g of extract. The estimation of total flavonoids in the extracts was carried out in triplicates and the results were averaged.

2.6.

ABTS+ radical scavenging assay

For ABTS+ assay, the method of Re et al. (1999) was adopted. The stock solutions included 7 mM ABTS+ solution and 2.4 mM potassium persulfate solution. The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12 h at room temperature in the dark. The solution was then diluted by mixing 1 ml ABTS+ solution with 60 ml methanol to obtain an absorbance of 0.706 ± 0.001 units at 734 nm using the spectrophotometer. Fresh ABTS+ solution was prepared for each assay. Plant extracts (1 ml) of various concentration ranging from 20 to 100 mg were allowed to react with 1 ml of the ABTS+ solution and the absorbance was taken at 734 nm after 7 min using the spectrophotometer. The ABTS+ scavenging capacity of the extract was compared with that of BHT standard and percentage of inhibition calculated as ABTSþ radical scavenging activity ð%Þ ¼ ½ðAbs control  Abs sampleÞ=ðAbs controlÞ  100 where Abs control is the absorbance of ABTS radical + methanol; Abs sample is the absorbance of ABTS radical + sample extract/standard. The sample concentration providing 50% inhibition (IC50) was calculated from the graph of inhibition percentage against sample concentration. All determinations were carried out in triplicates. The ABTS radical-scavenging activity of BHT was also assayed for comparison.

2.7.

DPPH radical scavenging assay

The effect of extracts on DPPH radical was estimated using the method of Liyana-Pathirana and Shahidi (2005). A solution of 0.135 mM DPPH in methanol was prepared and 1 ml of this solution was mixed with 1 ml of extract in ethanol containing 20–100 mg of the extract. The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm. BHT was used as reference standard. The ability to scavenge DPPH radical was calculated by the following equation: DPPH radical scavenging activity ð%Þ ¼ ½ðAbs control  Abs sampleÞ=ðAbs controlÞ  100 where Abs control is the absorbance of DPPH radical + methanol; Abs sample is the absorbance of DPPH radical + sample

Determination of reducing power

The reducing power of the ECH was determined according to the method of Oyaizu (1986). The extract (10–50 lg) in 1 ml of distilled water was mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50 C for 20 min. 2.5 ml of 10% trichloroacetic acid was added to the mixture, and mixture was centrifuged at 3000 rpm for 10 min. The supernatant (2.5 ml) was mixed with 2.5 ml distilled water and 0.5 ml, 0.1% ferric chloride. Absorbance was measured at 700 nm using a spectrophotometer. Increased absorbance of the reaction mixture indicated increased reducing power.

2.9. ECH

UHPLC-PDA-ESI/HRMS/MSn analysis of phenolics in

The UHPLC–HRMS system used consisted of a LTQ Orbitrap XL mass spectrometer with an Accela 1250 binary Pump, a PAL HTC Accela TMO autosampler, a PDA detector (ThermoScientific, San Jose, CA, USA) and a G1316A column compartment (Agilent, Palo Alto, CA, USA). The separation was carried out on a UHPLC column (200 · 2.1 mm i.d., 1.9 lm, Hypersil Gold AQ RP-C18) (Thermo-Scientific) with an HPLC/UHPLC precolumn filter (Ultra Shield Analytical Scientific Instruments, Richmond, CA, USA) at a flow rate of 0.3 ml/min. The mobile phase consisted of a combination of A (0.1% formic acid in water, v/v) and B (0.1% formic acid in acetonitrile, v/v). The linear gradient was from 4% to 20% B (v/v) at 40 min, to 35% B at 60 min and to 100% B at 61 min, and held at 100% B to 65 min. The PDA was set at 520, 330 and 280 nm to record the peaks, and UV/Vis spectra were recorded from 200 to 700 nm. Negative ionization modes were used and the conditions were set as follows: sheath gas at 70 (arbitrary units), aux and sweep gas at 15 (arbitrary units), spray voltage at 4.8 kV, capillary temp at 300 C, capillary voltage at 15 V, and Tube lens at 70 V. The mass range was from 200 to 2000m/z with a resolution of 15,000, FTMS AGC target at 2e5, FT-MS/ MS AGC target at 1e5, isolation width of 1.5 amu, and max ion injection time of 500 ms. The most intense ion, including some second intense ions, was selected for the datadependent scan to offer their MS2, MS3 and MS4 product ions with the a normalization collision energy at 35%.

2.10.

Antimutagenicity assay

The antimutagenicity of ECH was studied using tester strain Salmonella typhimurium TA100 through the standard plate incorporation test as described by Maron and Ames (1983). To test the mutagenic potential of ECH, plant extract of various concentration ranging from 500 to 2000 mg/ml, 0.5 mM biotin and 0.1 ml of overnight grown culture of S. typhimurium TA100 were mixed with molten soft agar (2 ml) and plated on the bottom agar containing minimal media. Above procedure is repeated with 500 ll of S9 mix to know role of liver enzymes on the test sample. Sodium azide was used as a diagnostic

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mutagen (1.5 lg per plate) in positive control and plates without test sample were considered as negative control. His+ revertants were counted after incubation of the plates at 37 C for 48 h. All determinations were carried out in triplicates.

2.11.

Animals

Acute oral toxicity test

To evaluate the acute toxicity of ECH, a single oral dose of ECH of various concentrations ranging from 0.5 to 3.0 g/kg body weight was given to Wistar rats then allowed free access to food and water. The animals were observed for signs of toxicity such as, urination, salivation, asthenia and defecation for 72 h after the oral administration for the acute oral toxicity. The extract is safe up to the dose of 2.5 g/kg b.w. p.o. for Wistar rats and did not shown any acute toxicity signs (Table 1).

2.13.

possible. One part of the liver samples was immediately stored at 20 C until analysis, another part was excised and fixed in 10% formalin solution for histopathological analysis.

2.13.1. Measurement of biochemical parameters

Male albino Wistar rats (150–200 g) were obtained and housed in poly-acrylic cages and maintained under standard laboratory conditions (temperature 24–28 C, relative humidity 60–70% and 12 h dark-light cycles). Animals were fed with commercial rat feed (Sai Durga feeds and food stocks, Chennai, India) and water, ad libitum. All the animals were acclimatized to laboratory conditions for 7 days before commencement of the experiment. Experimental protocols were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Chennai, Tamil Nadu, India (BDU/IAEC/2012/26/28.03.2012).

2.12.

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Treatment

The animals were randomly divided into five groups of 6 rats per group. Group 1 served as normal control and was given olive oil (1 ml/kg) daily for a period of 8 weeks. For inducing hepatotoxicity (in vivo), animals of Groups 2–5 were administered orally 1 ml/kg b.w. of CCl4 (1:1, v/v, CCl4 in olive oil) twice a week for 8 weeks. Group 2 served as CCl4-treated model group. In addition to CCl4 administration, Groups 3 and 4 were administered orally ECH at 250 and 500 mg/kg daily for 8 weeks. Besides CCl4, Group 5 was given silymarin (50 mg/ kg) daily for a period of 8 weeks, which served as positive control. At the end of the experiment, all animals were sacrificed. Serum samples were collected into heparinized tubes (50 U/ ml). Liver samples were dissected out and washed immediately with ice cold saline to remove as much blood as

Table 1 – Acute oral toxicity test after oral administration of ECH in Wistar rats. S. no

Dose (g/kg b.w.)

Mortality (dead/treated)

Toxicity signs

1 2 3 4 5 6

0.5 1 1.5 2 2.5 3

0/6 0/6 0/6 0/6 0/6 0/6

None None None None None Urination

Serum biochemical parameters such as SGOT, SGPT (Reitman & Frankel, 1957), and ALP (Kind & King, 1954) and serum bilirubin (Malloy & Evelyn, 1937) were analyzed according to the reported methods. In addition, the levels of cholesterol, TG, LDL, HDL, creatinine and blood urea nitrogen (BUN) were estimated in the serum of experimental animals using commercial reagent kits and were performed according to the manufacturer’s (Nice Chemicals [P] Ltd., Cochin, Kerala, India) instructions.

2.13.2. DNA fragmentation assay DNA fragmentation assay was performed according to the protocol given in QIAamp DNA Mini Kit, Qiagen, Germany. To visualize the DNA damage, 10 lg of DNA was separately loaded in 1.5% agarose gel containing 0.5 lg/ml ethidium bromide. DNA fragmentation pattern was studied under gel doc system (Gel Doc XR+ System, Marnes la Coquette, France) and photographed.

2.13.3. Measurement of antioxidant enzyme activities The liver homogenates (10%, w/v) prepared in phosphatebuffered saline (PBS containing 137 mM NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4 and 1.76 mM KH2PO4 in 1000 ml distilled water pH 7.2) were used for antioxidant studies such as LPO (Buege & Aust, 1978), SOD (McCord & Fridovich, 1969), CAT (Aebi, 1984), and GSH activities (Moron, De Pierre, & Mannervik, 1979).

2.13.4. Histological observations Liver samples were fixed in Bouin’s fixative and processed to obtain 5 lm thick paraffin sections and stained with hematoxylin and eosine (H&E) for histological observations.

2.14.

Statistical analysis

The statistical analyses were performed using the statistical package SPSS (Statistical Package for Social Science, SPSS Inc., Chicago, IL, USA). Analyses of variance were performed by ANOVA procedures and significance of each group was verified with one-way analysis of variance followed by Duncan post hoc test (P 6 0.05). Values obtained are means of six replicate determinations ± standard deviation.

3.

Results and discussion

The phytochemical analysis conducted on C. halicacabum extract revealed the presence of phenolics, flavonoids, steroids, tannins, saponins and terpenoids. The total phenolic content of the ECH was 17.94 mg gallic acid equivalents/g of extract. The total flavonoid contents of the ECH were 14.97 mg rutin equivalents/g of extract. The percentage inhibition of ABTS and DPPH free radicals by ECH are shown in Fig. 1a and b, respectively. The extract significantly inhibited the activities of ABTS and DPPH

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Fig. 1 – (a) ABTS+ radical scavenging activity and (b) DPPH radical scavenging activity of ECH compared with Butylated hydroxytoluene (BHT). Each value is expressed as the mean ± standard deviation (n = 3).

radicals in a dose-dependent manner. The IC50 values of the extract to quench ABTS and DPPH radicals are 58 and 29 lg/ ml, respectively. Fig. 2 shows the reducing power potentials of the ethanol extract of the test plant in comparison with a standard BHT at 700 nm. In the reducing power assay, the presence of antioxidants in the sample would result in the reduction of Fe3+ to Fe2+ by donating an electron. The amount of Fe2+ complex can then be monitored by measuring the

formation of perl’s blue at 700 nm. Increase in absorbance indicates an increase in reductive ability. The reducing power of 50 lg/ml ECH had an optical density of 0.4074, which was comparable to that of the standard BHT with an optical density of 0.4702. The identified phenolic compounds of C. halicacabum using UHPLC-PDA-ESI/HRMS/MSn were listed in Table 2. These compounds are summarized along with their retention time,

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Fig. 2 – Reducing power of ECH compared with Butylated hydroxytoluene (BHT). Each value is expressed as the mean ± standard deviation (n = 3). ESI [M–H] and MS/MS4, m/z base ions. In the previous work, the authors demonstrated the MS/MS fragmentation of tentatively identified phenolic compounds in C. halicacabum (Jeyadevi et al., 2012). Interestingly in the present work, three specific bio active compounds namely luteolin 7-o-glucoronide (Lee, Jung, Choi, Kim, & Lee, 2011), apigenin 7-o-glucoronide (Zheng, Sun, Xu, Li, & Song, 2005) and chrysoerisol 7-o-glucoronide responsible for hepatoprotective activity have been identified in C. halicacabum. Flavonoids and phenolic acids have antibacterial, antifungal, antiviral, hepatoprotective, immunomodulating, and anti-inflammatory properties. The pharmacological effects of phenolics and flavonoids are mostly associated with their antioxidant activity (Havsteen, 1983). Fig. 3 shows the MS/MS fragmentation pattern of (a) apigenin 7-o-glucoronide, (b) luteolin 7-o-glucoronide and (c) chrysoerisol 7-o-glucoronide. Though the effective mechanism behind the pharmacological activities of phenolics and flavonoids is unclear, it may be mainly due to the free radical scavenging activity of these compounds. The antimutagenic potential of ECH was studied using tester strain S. typhimurium TA100. ECH of different doses did not show any significant mutagenic effect on TA100 strain with (+S9) and without S9 (S9) extract. The number of His+ revertants in test samples, positive control (sodium azide) and negative control (spontaneous revertants) were counted and given in Table 3. CCl4 is one of the most commonly used hepatotoxins for inducing liver injury in experimental animal studies (Johnston & Kroening, 1998). CCl4 is metabolized by cytochrome P4502E1 (CYP2E1) to the trichloromethyl radical

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(CCl3) and proxytrichloromethyl radical (OOCCl3), which are assumed to initiate free radical-mediated lipid peroxidation leading to the accumulation of lipid-derived oxidation products which cause liver injury (Poli, Albano, & Dianzani, 1987; Shyur et al., 2008). Many biological substances such as membrane lipids, proteins, and nucleic acids are known to be damaged by trichloromethyl radicals (Nomura & Yamaoka, 1999; Weber, Boll, & Stamtl, 2003). The hepatic damage induced by administration of CCL4 resulted in marked elevation in serum levels of ALP, SGPT, and SGOT. Normally, SGOT, SGPT and ALP are present in high concentration in the liver. Due to hepatocyte necrosis or abnormal membrane permeability, these enzymes are released from the cells and their levels in the blood increases. SGPT is a sensitive indicator of acute liver damage and elevation of this enzyme in non-hepatic diseases is unusual. The rise in the SGOT is usually accompanied by an elevation in the levels of SGPT, which play a vital role in the conversion of amino acids to keto acids (Sallie, Tredger, & William, 1999). Treatment with ECH decreased the ALP, SGOT, SGPT and serum bilirubin level indicates that ECH stabilized the normal functional status of the liver. The results of blood biochemical parameters are presented in Table 4. Administration of CCl4 induced significant increase in the enzymatic activities of ALP, SGOT, and SGPT as compared to the control group. 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, and GSH (Wang, Leeb, Chenc, Yud, & Duhb, 2012). It is known that SOD converts superoxide anion into H2O2 and O2, whereas CAT reduces H2O2 to H2O, resulting in the detoxification of free radicals (Paoletti & Mocali, 1990). CCl4 treatment also caused a significant decrease in the level of tissue antioxidant enzymes such as SOD, CAT, and the cellular antioxidant molecule GSH in liver (Yu, Chia, Wen, & Fung, 2009). Treatment with ECH up regulated the activities of these endogenous antioxidant enzymes. The effects of ECH treatment on the activities of SOD, CAT, and GSH in the liver are shown in Table 4. The activities of liver SOD, CAT, and GSH in the CCl4-treated group were significantly decreased compared with the normal control group. There was a dramatic increase (P 6 0.05) in SOD, CAT and GSH activity in the ECH treated groups at the high dose (500 mg/kg b.w.) compared to the CCl4-treated group. Moreover, the activity of ECH at the dose of 500 mg/kg was comparable to that of the reference drug, silymarin. Reduced glutathione is presumed to be an important endogenous defense against peroxidative destruction of cellular membranes. In the present study, significant decline was seen in the reduced glutathione level (P 6 0.05) in CCl4 administrated animals. Treatment with extract was

Table 2 – Tentative identification of phenolic and flavonoid compounds in ethanolic leaf extract of C. halicacabum through LC-MS/MS analysis. No

RT

[M]

MS2 ions

MS3 ions

MS4 ions

UV kmax

Compounds

1. 2. 3.

25.83 31.35 33.31

461 445 475

357, 285 269, 175 299

243, 241, 217, 199, 175 269, 225, 201, 149 284

197, 196, 183, 181 284, 256

232, 253, 265, 347 237, 266, 346 232, 252, 266, 346

Luteolin 7-o-glucuronide Apigenin 7-o-glucuronide Chrysoeriol 7-o-glucuronide

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Fig. 3 – The MS/MS fragmentation pattern of (a) Apigenin 7-o-glucoronide; (b) Luteolin 7-o-glucoronide; and (c) chrysoerisol 7o-glucoronide. very effective in restoring the glutathione content which had been substantially decreased by CCl4 (Table 4).

In biological systems, aldehydes, including malondialdehyde are the end product of LPO, which cause cellular damage

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Table 3 – Number of His+ revertants (Salmonella typhimurium TA100) induced by the ECH. Number of His+ revertants (Salmonella typhimurium TA100)

Test samples

+S9

S9 c

Negative control (spontaneous revertants) Positive control (sodium azide) ECH 500 mg/ml ECH 1000 mg/ml ECH 2000 mg/ml

3 ± 1c 1072 ± 46a 1 ± 1c 2 ± 1c 7 ± 3c

2±1 124 ± 6b – – 5 ± 3c

Data are expressed as mean ± SD (n = 3). Mean values with different superscripts are significantly different from each other as revealed by Duncan post hoc test (P 6 0.05).

Table 4 – Protective effect of ECH on CCL4 induced elevation in hepatic enzymes, TBARS, GSH and antioxidant enzymes in Wistar rats. Groups Group Group Group Group Group

1 2 3 4 5

(normal control) (disease control) (silymarin) (ECH 250 mg/kg b.w.) (ECH 500 mg/kg b.w.)

SGOT (IU/L)

SGPT (IU/L)

ALP (IU/L)

TBARS*

GSH**

SOD***

CAT****

50.46 ± 5.48d 159.07 ± 8.04a 58.10 ± 3.43c 65.73 ± 4.12b 57.46 ± 4.99c,d

37.31 ± 2.32c 118.2 ± 13.92a 53.30 ± 6.53b 54.90 ± 4.03b 55.10 ± 6.34b

92.06 ± 11.64d 277.7 ± 12.42a 183 ± 9.48b 110.07 ± 5.71c 92.53 ± 2.81d

6.40 ± 1.14c 14.78 ± 2.59a 9.14 ± 1.04b 11.00 ± 1.58b 8.84 ± 1.30b

24.63 ± 3.27a 9.79 ± 5.05c 19.80 ± 1.92b 19.13 ± 1.38b 24.19 ± 3.12a

22.15 ± 1.61a 8.80 ± 1.92c 19.40 ± 2.70a 16.55 ± 1.28b 21.58 ± 3.85a

59.95 ± 3.14a 38.32 ± 1.77c 52.60 ± 10.73a,b 50.91 ± 4.76b 58.76 ± 3.06a

b

Data are expressed as mean ± SD (n = 6). Mean values with different superscripts are significantly different from each other as revealed by Duncan post hoc test (P 6 0.05). * mmol/mg protein. ** lg of reduced glutathione/mg protein. *** U/mg of tissue. **** lmol of H2O2 utilized/min/mg of protein.

Table 5 – Protective effect of ECH on CCL4 induced elevation in serum biochemical parameters in Wistar rats. Groups Group Group Group Group Group

1 2 3 4 5

(normal control) (disease control) (silymarin) (ECH 250 mg/kg b.w.) (ECH 500 mg/kg b.w.)

TG (mg/dl)

TC (mg/dl)

LDL (mg/dl)

BUN (mg/dl)

Creatinine (mg/dl)

Bilirubin (mg/dl)

HDL (mg/dl)

115.20 ± 1.30c,d 146.60 ± 5.77a 138.80 ± 27.17a,b 126.40 ± 6.18b,c 104 ± 9.47d

56.40 ± 7.5a 106.20 ± 11.81a 51.20 ± 6.05a 215 ± 302.02a 44.06 ± 11.43a

33.58 ± 3.79c 83 ± 4.52a 37.6 ± 6.94c 59.8 ± 9.67b 41 ± 6.55c

17.60 ± 2.07c 35.20 ± 3.11a 17.11 ± 1.58c 23.45 ± 1.52b 17.47 ± 1.44c

0.38 ± 0.17b 2.48 ± 1.06a 0.72 ± 0.08b 1.86 ± 1.02a 0.42 ± 0.13b

0.73 ± 0.076d 1.40 ± 0.17a 0.94 ± 0.034b 0.87 ± 0.015b,c 0.76 ± 0.059c,d

80.80 ± 8.58a 40.40 ± 136.18a 47.60 ± 3.43a 53.60 ± 4.03a 65 ± 3.60a

Data are expressed as mean ± SD (n = 6). Mean values with different superscripts are significantly different from each other as revealed by Duncan post hoc test (P 6 0.05).

and disruption of cell membrane when endogenous antioxidants are depleted. The production of secondary oxidation products, measured by the thiobarbituric acid reactive substances (TBARS) is enhanced upon lipid peroxidation in hepatic tissue (Karthikesan, Pari, & Menon, 2010). A significant increase was observed in the level of LPO in liver after 48 h of CCl4 intoxication when compared with the control group (P 6 0.05). Treatment with different doses of crude extract reversed the oxidative stress significantly toward control by inhibiting LPO in a dose dependant manner (Table 4). A significant increase in serum level of bilirubin, TC, TG, creatinine, LDL, BUN while decrease in HDL were observed after CCl4 intoxication. The ECH extract significantly decreased the elevated levels toward control (Table 5). The 500 mg/kg dose of ECH extract revealed a more significant therapeutic effectiveness (P 6 0.05). DNA fragmentation assay showed that CCl4

intoxication induces severe DNA damage in the liver tissues of rats and fragmentation of DNA was observed as smear in the agarose gel. Treatment with ECH and the reference drug, silymarin had reduced the DNA damage to the greater extant (Fig. 4). The presence of injuries in livers of CCl4 treated rats was revealed by histopathological examinations. Fig. 5a, shows photomicrographs of hematoxylin–eosin stained liver tissues. In case of the control, hepatocytes had normal architecture. Severe hepatocyte necrosis, fatty degeneration and vacuolation were found in liver of rat administered with CCl4 (Fig. 5b). Treatment of ECH at 250 and 500 mg/kg b.w. reduced the severity of CCl4 induced liver intoxication (Fig. 5c and d). Silymarin treated group depicted symmetrically arranged well formed hepatocytes separated by sinusoids and maintained cord arrangement (Fig. 5e). These results clearly

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5 ( 20 1 3) 2 8 9–29 8

(a)

(b)

(c)

(d)

(e)

Fig. 4 – Antimutagenic potential of ECH through DNA fragmentation assay. Lanes from left (M) DNA marker; normal control group (1); disease control (CCl4) group (2); reference drug (Silymarin) (3); ECH 250 mg/kg b.w. (4) and ECH 500 mg/kg b.w. (5).

indicate the protection provided by ECH .The histopathological observations also support the results obtained from serum biochemical assays.

4.

Conclusion

The present work reports for the first time, the phenolic profile, antioxidant and hepatoprotective potential of ethanolic extract of C. halicacabum against CCl4 induced liver injury. The phenolic and flavonoids present in the ECH may attribute to its antioxidant and hepatoprotective potential. The results indicated the potential of the plant extracts to offer protection against the acute hepatotoxicity induced by CCl4 is mainly due to the phenolic compounds. Further studies are in progress to better understand the mechanism of action of bioactive compounds in ECH, responsible for its hepatoprotective and antioxidant effects.

Acknowledgements The financial assistance provided by University Grants Commission (UGC), New Delhi is gratefully acknowledged. We also thank Dr. J. Senthilkumar, Veterinary Surgeon and Dr. R. Banumathi for their assistance in interpretation of histological results. We thank Dr. D. Prabu and Mr.V. Thangapadiyan, Department of Microbiology, ANJA College, Sivakasi for providing S. typhimurium TA100 strain. Finally we sincerely thank

Fig. 5 – Histopathological analysis of liver tissue of Wistar rats administrated with CCl4 and treated with the ECH. (a) Control group shown normal lobular architecture with clear central vein (X-100); (b) CCl4 administered group shown severe hepatocyte necrosis, fatty degeneration, and vacuolation (X-100); (c) ECH treated group at 250 mg/kg shown mild improvement in chord arrangement but perinuclear vacuolation was visible (X-100); (d) ECH treated group at 500 mg/kg shown normal cellular architecture with distinct hepatic cells sinusoidal spaces (X-100); (e) silymarin (positive drug) treated group exhibited almost normal histology (X-100).

DST-FIST for providing instrumentation facility (HPLC and Lyophilizer).

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