Hepatoprotective effect of root bark of Oroxylum indicum on carbon tetrachloride (CCl4) - induced hepatotoxicity in experimental

Abstract

Hepatoprotective effect of root bark of Oroxylum indicum on carbon tetrachloride (CCl4) - induced hepatotoxicity in experimental animals

Maitreyi Zaveri^{a, c} and Sunita Jain^b

 

ABSTRACT

The present study was designed to investigate the effect of Oroxylum indicum against carbon tetrachloride (CCl4)-induced hepatotoxicity in mice and rats. The hepatotoxicity was induced with the administration of 1:1(v/v) mixture of CCl4 in arachis oil at the dose of 1ml/kg intraperitoneally on day 3rd and 6th. Pretreatment with different extracts and standard silymarin (25mg/kg) was done orally from day 1 to 7. Alcoholic (300 mg/kg), petroleum ether (300 mg/kg) and n-butanol (300–100 mg/kg) extracts produced significant (p<0.05) lowering of the elevated Serum glutamic oxaloacetic transaminase (SGOT), Serum glutamic pyruvate transaminase (SGPT), alkaline phosphatase (ALP) and total bilirubin (TB) when compared with the toxic control. The increased lipid peroxide (LPO) formation in the tissues of CCl4 –treated animals was significantly inhibited by Oroxylum indicum. The observed decreased antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH), and antioxidant concentration of glutathione were nearly normalized by Oroxylum indicum treatment. Carbon tetrachloride (CCl4)-induced damage pro+duced alteration in the antioxidant status of the tissues, which was also manifested by abnormal histopathology. Pretreatment with Oroxylum indicum restored all these changes. Hence, it is suggested that root bark of Oroxylum indicum showed significant antioxidant activity, which might be in turn responsible for its hepatoprotective activity.

 

Key words: Oroxylum indicum; hepatoprotective; antioxidant; carbon tetrachloride; silymarin.

Introduction

Indian medicinal plants and many herbal formulations belonging to the traditional systems of medicines have been investigated as liver protective drugs (Jose and Kuttan, 2000). Oroxylum indicum, Vent. (Syonakh), is an indigenous plant, found in India, Ceylon, Malaya, Cochin, China, Philippines and Indonesia. Oroxylum indicum is used in traditional Ayurvedic medicine to alleviate thirst, rheumatism, dysentery, anorexia, bronchitis, eruptive fevers and dropsy. It is also regarded as tonic, aphrodisiac, carminative, bitter and anthelmintic. Roots of Oroxylum indicum is one of the ingredients of traditional Ayurvedic formulation viz. Dasamula, Chyavanaprasa, Brahma Rasayana, Dhanawantara Ghrita, Dantyadyarista, Awalwha and Narayana Taila. It is reported that Oroxylum indicum plant possesses anti-inflammatory, diuretic, anti-arthritic, antifungal and antibacterial activity (Warrier et al., 1997). The stem bark of this plant is reported to contain flavonoids namely, baicalein, chrysin, oroxylin-A, scutellarin etc (Sankara et al., 1972-a; Sankara et al., 1972-b). Seeds of this plant are reported to contain ellagic acid (Vasantha et al., 1991). Baicalein is reported to possess an anti-inflammatory (Tie et al., 2002), anti-ulcer (Kennouf et al., 2003), antioxidant (Ng et al., 2000), hepatoprotective (Niedworok et al., 1999) and immunomodulatory activity (Lien et al., 2003), while chrysin and baicalein both are reported to have antibacterial, antifungal and antiviral activity (Tahara et al., 1987; Kujumgier et al., 1999). Furthermore, biochanin-A possesses anti-fungal action and tumor necrosis factor-α (Knight et al., 1996). Ellagic acid is an important polyphenolic compound (Jadhav et al., 2004). As a result of these findings, the present study was undertaken to evaluate, the effects of different fractions of Oroxylum indicum against liver damage in experimental animals model and further to establish the relationship between the hepatoprotective and antioxidant enzyme activity. 

Materials and Methods

Procurement of Plant material and extraction procedure

The fresh root bark of Syonakh was collected from Vanaushadhi Ektrikaran Udyan, Ahwa, Dang forest, Gujarat, India. The authentification of this plant was established by the taxonomist of Gujarat Ayurved University, Jamnagar, India. The voucher specimen (#404) was deposited in the Department of Pharmacognosy and Phytochemistry at L. M. College of Pharmacy, Ahmedabad. The root bark was sun dried and powdered to 60 mesh (approximately 250 µm diameter; by grinding with a porcelain pestle a shifting through appropriate mesh screens). The powder of root bark first defatted using petroleum ether (remaining post-extraction material constituted 0.32% [w/w] of the original solute [i.e., root bark powder] that underwent the extraction). The residual unextracted material was then air-dried; this defatted powder was then moistened with an ammonia (NH₃) solution and then extracted with chloroform (post-extract remaining solute =0.78% [w/w] of pre-extracted material). After, drying the remaining unextracted material was then extracted with ethyl acetate (post-extract remaining solute =1.52% w/w). Finally, the thrice-extracted powder was then extracted with n-butanol (post-extract remaining solute=1.68% w/w). The solvent-specific eluents recovered in each extraction regimen were then air-dried and their corresponding powdered fractions stored in airtight container until usage.  

 

Phytochemical test

Phytochemical analysis of the extract was performed using standard methods. Specifically, the extract was analyzed for the presence of alkaloids (Sim, 1969), flavanoids (Geissman, 1955), saponins (Fischer, 1952), tannins (Robinson, 1964), anthraquinone, and carbohydrates (Freudenberg et al., 1962). Thin layer chromatography was employed to check for the presence of a baicalein. Quantification of the aglucone was performed using reverse phase HPLC (Khandhar et al., 2006, Zaveri et al., 2008) and extrapolation against a standard curve generated using purified baicalein.

 

Drugs and chemicals

All different organic solvents used for extraction were obtained from the S.D. Chemicals Private Limited (Mumbai, India), and were analytical grade. Fresh drug solutions were prepared in 1% carboxymethylcellulose (CMC) for oral administration. Hydrogen peroxide and ciocalteau phenol reagent were obtained from S.D. Fine Chemicals Limited (Mumbai). Trichloroacetic acid, thiobarbituric acid, phosphate buffer, Tris buffer, 5, 5'- dithibis-2-nitrobenzoic acid (DTNB), bovine serum albumin, epinephrine and silymarin were all obtained from Sigma-Aldrich (St Louis, MO). The kits for the estimation of Serum glutamic oxaloacetic transaminase (SGOT), Serum glutamic pyruvate transaminase (SGPT), alkaline phosphatase (ALP) and total bilirubin (TB) were purchased from Span Diagnostics Ltd.

 

Animals 

Wistar albino rats (Zydus Cadila Limited, Ahmedabad, India) of either sex weighing 170-225 g as well as male albino mice weighing 25-30 g were selected for the present study. Animals were provided a standard chow diet (certified Amrut brand rodent feed, Pranav Agro Industries, Pune, India) and filtered tap water was given ad libitum. The animalswere maintained under standard that was freely available under standard condition of a 12 h dark-light cycle, 60±10% humidity and a temperature of 21.5 ±1⁰c. Coprophagy (and thus re-ingestion of any drug) was prevented by keeping the animals in cages with gratings on the floors. The distribution of animals in the groups, the sequence of trials and treatment allotted to each group was randomized. Freshly prepared solutions of drugs or chemicals were used throughout the study. After completion of the experiments, animals were sacrificed by over-anesthetization with ether. All experiments complied with University guidelines for animal experimentation. Throughout the entire study period, the rats were monitored for growth, health status, and food intake capacity to be certain that they were healthy.

 

Methodology 

The animals were randomly divided (to assure equal distribution of weights) into the following groups containing six animals each.

Group 1: (control) animals received only aqueous suspension of 1% CMC as vehicle   without any treatment once daily for seven days.

Group 2: (CCl₄   treated) animals were intoxicated with CCl₄ in arachis oil (1:1 v/v) (1 ml/kg of b.w., i.p., and twice a week, on 3^rd and 6^th day). The animals were sacrificed 24 h after the last CCl₄   treatment.

Group 3: (Standard) CCl₄ treated animals were received silymarin (25 mg/kg of b.w. suspended in 1% CMC solution, p.o.) once daily for seven days.

Group 4: (Drug treated) CCl₄ treated animals were received 50% alcoholic extract and petroleum ether, chloroform, ethyl acetate, and n-butanol fractions (100 & 300 mg/kg of b.w., p.o.) once daily for seven days.  

 

Carbon tetrachloride (CCl₄) - induced hepatotoxicity in experimental animals (Sarmistha et al., 1998)

Carbon tetrachloride (CCl₄) (1 ml/kg body weight, twice a weak on 3^rd and 6^th day, i.p.) - induced acute hepatic necrosis in animals. Blood samples were collected separately under ether anesthesia through retro-orbital plexus 24 h after the last CCl₄ injection. Blood was centrifuged at 2000 rpm for 30 min to separate the serum and analyzed for the assay of following marker enzymes and total protein. All the tests were carried out using serum diagnostic kits supplied by Span Diagnostic Ltd. Animals were sacrificed to collect the liver tissue for estimation of protein content, antioxidant enzyme activity, lipid peroxidation and for histopathological observations.

 

Assessment of liver function:

Effect of drug administration on assay of serum enzymes 

Measurement of Serum glutamic oxaloacetic transaminase (SGOT), (Reitman and Frankels methods, 1957), Serum glutamic pyruvate transaminase (SGPT), Reitman and Frankels methods, 1957), alkaline phosphatase (ALP)(Kind and Kings Method, 1954) and total bilirubin (TB) levels (Malloy and Evelyn Method, 1937) was carried out. Protein content (PR) was estimated in liver homogenate by the method of Lowry et al., (1951). 

 

Effect of drug administration on liver antioxidant enzymes and on lipid peroxide levels

Liver in each case was dissected out quickly, blotted off blood, washed with ice-cold saline and a 10% homogenate was prepared in phosphate buffer (PH 7). The homogenate was centrifuged at 3000 rpm for 15 min at 4 ⁰C and the supernatant was used for the estimation of the following parameters. The total protein concentration in each sample was determined (Lowry et. al., 1952). The effects of theroot bark extract on the activity of the antioxidant enzymes superoxide dismutase (SOD; in terms of mU/mg protein) (Misra and Frodvich, 1973), catalase (CAT; as U/min/mg protein) (Aebi, 1974) and on the levels of reduced glutathione (GSH; as µmole/mg protein) (Beutler et. al., 1963) in the homogenate were assayed. The levels of malondialdehyde (MDA) in each sample were estimated (expressed as µmole thiobarbituric acid reactive substances [TBARS]/mg protein) at 535 nm in a Shimandzu UV Spectrophotometer (Shimadzu, Japan) (Kiso et. al., 1984).

SOD activity in the samples was determined by mixing 0.1 ml of sample with 0.1 ml of EDTA (1 x 10^-4 M), 0.5 ml of carbonate buffer (pH 9.7), and 1 ml of epinephrine (3 x 10³ M) (Sigma). The optical density of the adrenochrome was assessed at 480 nm at 30 sec intervals for a total of 3 min. SOD activity was expressed as mU/mg of protein. One unit of activity was defined as the enzyme concentration required to inhibit the chromogen produced, by 50%, in one minute under the defined assay condition.

Catalase activity in each sample was measured by assessing the decomposition of hydrogen peroxide (H₂O₂) at 240 nm. In a cuvette, 50µl sample was mixed with 2.95 ml of reaction buffer (0.05 M phosphate buffer [pH 7.0] containing 30 mM H₂O₂) and the absorbance was measured at 15 sec intervals for 3 min. As the optical density measured reflects the peroxide concentration in the cuvette, the activity of catalase in the 3 min period was deduced and expressed as mM H₂O₂ consumed/mg tissue/min.

Reduced glutathione (GSH) content in each sample was measured after initial precipitation of proteins with 10% chilled trichloroacetic acid. After 30 min incubation, the samples were then centrifuged at 1000 g for 10 min at 4⁰C. The GSH levels in the supernatant were then determined by mixing 0.5 ml of the material with 2.0 ml 0.3 M phosphate buffer (pH 7.0) and 0.25 ml DTNB reagent (40 mg/l00 ml in 1% sodium citrate buffer), and then measuring the absorbance at 412 nm. Standard solutions containing different concentrations of GSH were prepared in parallel to generate a standard curve. Results are expressed as µmoles of GSH/mg of protein.

The levels of malondialdehyde (MDA, representative of peroxidative damage to cell membranes) were measured by mixing 2 ml of 5% suspension of recovered, samples (in 0.1 M phosphate-buffered saline [pH 7.4]) with 2 ml of a 28% trichloroacetic acid solution. After thorough mixing, the mixture was then centrifuged at 10,000g at 4°C for 5 minutes and the supernatant was separated for estimation of MDA. For this, 4 ml supernatant was mixed with 1 ml of 1% thiobarbituric acid solution (TBA), and heated at 100°C for 60 min. The mixture was then cooled to room temperature and the absorbance was measured spectrophotometrically at 532 nm. After accounting for background absorbance using buffer blanks, the total TBARS (TBA-reactive substrate) concentration in each sample was derived from the TBA extinction coefficient Є = 1.56 x 10⁵ M^-1 cm^-1. The level of MDA in each sample was calculated and data was expressed in terms of nmoles of MDA/mg of protein in each sample.

 

Histopathological studies

On day 7, the final day of the treatments, all the animals were sacrificed by overdose of ether anesthesia and several tissues recovered during necropsy for use in histopathological analyses. The liver tissues from both control and treated (standard and fractions) were preserved in 10 % formalin solution. Thereafter, after embedding in paraffin, 6 µm thick sections were cut and then stained with haematoxylin to permit histological examination. All tissues were then assessed for any morphological changes like necrosis, ballooning degeneration, fatty changes or inflammation of lymphocytes under a photomicroscope. Photomicrographs of representative sections were taken at 10X magnification using a Trinocular Research Zeisss Microscope (Gottingen, Germany) for histopathological observation.    

 

Statistical analysis  

All the results were expressed as Mean ± SEM. The significance of difference between mean values for the various treatments was tested using one way analysis of variance (ANOVA) followed by Tukey's multiple range test (Bolton, 1997). Differences between treatment groups were considered as statistically significant at p < 0.05. 

Results

Phytochemical Analyses

On preliminary phytochemical screening, the root bark of plant showed presence of alkaloids, flavonoids, tannins, and anthraquinones. The preliminary screening using thin layer chromatography was employed to specifically check for the presence of a flavonoid, baicalein. Using reverse phase-HPLC analysis to quantify the baicalein present in the extract, the results show that n-butanol fraction used in these particular studies contained 11.56 % (w/w) baicalein (Khandhar et. al., 2006). Therefore, in the present study, we used Oroxylum indicum root bark for the screening of hepatoprotective activity.

 

Carbon tetrachloride (CCl₄) - induced hepatotoxicity

CCl₄ administration resulted in significant rise in enzymes activity of serum transaminases, alkaline phosphatase alongwith decreased total protein content of the liver (Table-1, 3). Besides, CCl_4- treatment also resulted into significant rise in LPO alongwith significant fall in SOD, CAT, and reduced GSH levels as compared to control (Table-2, 4).

Alcoholic extract (50%) of Oroxylum indicum and its fractions petroleum ether and n-butanol (300 mg/kg b.w., p.o.) significantly (p<0.05) reduced serum enzymes SGOT, SGPT, ALT, and TB levels (Table-1). In addition to above, these fractions, also showed significant reduction (p<0.05) in LPO alongwith significant rise (p<0.05) in SOD, CAT, and reduced GSH levels (Table-2). Maximum protection was observed with the use of n-butanol fraction. The study was therefore, further extended on another species, rats using lower dose of n-butanol fraction (100 mg/kg, b.w.). Pretreatment with n-butanol fraction showed significant protection in both serum enzyme levels and lipid peroxidation alongwith antioxidant enzyme activity as shown in Table-3, 4. 

 

Histopathological studies

The histologic analyses of the rats indicated in the liver of the normal control group showed no sign of necrosis or degeneration (Fig-1-a). Liver tissue obtained from the CCl₄-treated mice revealed severe cell necrosis around central veins, fatty changes, wide spread hepatocellular necrosis, kupffer cells, hyperplasia, centro-lobular necrosis and steatosis (Fig-1-b). Examination of the isolated liver tissues of animals indicated that the pretreated with different fractions (300 mg/kg of b.w., p.o.) showed microfatty changes with dense collection of lymphoid cells suggesting evidence of very little necrosis or degeneration (Fig-1-d). There was no significant hepatocellular damage. Only small areas of focal degeneration and sinusoidal dilation were observed. Similar observations were found with silymarin treated group of animals (Fig-1-c). Further, use of the n-butanol fraction (100 mg/kg, b.w., p.o.) in rats also showed protection in liver tissue that was evident from the normalcy of hepatic cells and central vein (Fig-2).

Discussion

Liver injury induced by CCl₄ is commonly used as model for the screening of hepatoprotective drugs (Janbaz et al., 1995). Liver dysfunction begins soon after injection of CCl₄ and maximum malfunction occurs within 48-72 hr.  Induction of CCl₄ eventually leads to hepatocellular necrosis and is reflected in our experiment by marked change in various enzymatic and non-enzymatic parameters of CCl₄ treated mice and rats. The most serious delayed toxic effects of CCl₄ results from its hepatotoxic and nephrotoxic action (Deichmann and Gerade, 1969).

Raised action of serum transaminases (Slater, 1984) in CCl₄-intoxicated animals is reported to be due to the damaged structural integrity of the liver because these are cytoplasmic in location and are released into circulation after cellular damage (Sallie et al., 1991). In the present study, many fold increase of enzyme leakage as demonstrated by an increased level of serum enzymes ALT, AST, ALP & TB have been noted indicating liver cell damage by CCl₄ (Tesehke et al., 1983). Use of different extracts of Oroxylum indicum prevented leakage of these enzymes and restored the activity of enzymatic variables.

Further, significant increase in hepatic LPO and decreased level of SOD, CAT and reduced GSH were observed after CCl₄ exposure. CCl₄ toxicity requires cleavage of the carbon-chloride bond and cleavage takes place after binding of CCl₄ to cytochrome P-450 apoprotein in the mixed function system located in the hepatocellular endoplasmic reticulum (Recknagel and Glande, 1973). CCl₄ is metabolized into trichloromethyl radical (CCl₃°), which further reacts with molecular oxygen, resulting in the formation of trichloromethyl peroxy radical (CCl₃O₂°). Trichloromethyl radical and trichloromehtyl peroxy radical combine with cellular lipid and protein to induce lipid peroxidation by hydrogen abstraction (Kadhska et al., 2000; Lim et al., 2000).

Free radical induced oxidative stress has been implicated in disorders, resulting from deficient antioxidant defences. Potential hepatoprotective agents therefore, include either free radical scavenging property or agents, which are capable of augmenting the activity of antioxidant enzymes (Bhattacharya et al., 1997). In the present study, increased malondialdehyde content of liver indicated enhanced lipid peroxidation due to tissue injury in CCl₄ treated control animals. Significant restoration of LPO was observed in animals pretreated with alcoholic, petroleum ether, and n-butanol fraction of root bark of Oroxylum indicum to mice and rats as compared to CCl₄ control. Pretreatment with extracts of Oroxylum indicum also restored the depleted SOD and CAT levels, demonstrating that the root bark supports the antioxidant defense mechanisms by increasing the activity of enzymes like SOD and CAT.

There is a direct co-relation between the enhanced SOD and CAT levels and reduced LPO levels and vice-versa (Gyamfi et al., 1999). In states of oxidative stress, reduced glutathione (GSH) is converted to oxidize glutathione (GSSG) and depletion in it leads to lipid peroxidation. Therefore, the role of GSH as a reasonable marker for evaluation of oxidative stress is important as it acts as an antioxidant, both extracellularly and intracellularly and is produced in the liver (Recknagel et al., 1982). Alcoholic, petroleum ether and n-butanol fractions of root bark of Oroxylum indicum inhibited lipid peroxidation significantly and recovered the decreased hepatic GSH level induced by CCl₄ towards normal. Generation of malondialdehyde like substances were inhibited and delayed by the presence of different extracts of the root bark of Oroxylum indicum during initiation and propagation phases of lipid peroxidation in serum (Sharma et al., 1994).

Phytochemical analysis of the root bark of Oroxylum indicum has previously revealed the presence of alkaloids, flavonoids, tannins, and anthraquinones.  In the present study, it is suggested that root bark of Oroxylum indicum possesses significant hepatoprotective activity. The mechanism of this activity can be attributed to its antioxidant enzyme activity. It is reported that, flavonoids are known to be antioxidants, free radical scavengers and antilipoperoxidants leading to hepatoprotection. Baicalein, one of the flavonoid is reported to be present in the stem bark of Oroxylum indicum. Baicalein has been shown to possess hepatoprotective activity (Niedworok et al., 1999). HPLC of our earlier study (Data not presented here) has confirmed the presence of baicalein in our plant (Khandhar et. al., 2006 and Zaveri et. al., 2008). Therefore, this hepatoprotective activity of our plant might be attributed to the presence of baicalein in O. indicum root bark. 

Conclusion

It is concluded from our study that alcoholic, petroleum ether and n-butanol fractions of root bark of Oroxylum indicum showed significant hepatoprotective activity. The hepatoprotective activity of this plant can be correlated with its antioxidant enzyme activity. Further, with the aid of HPLC based profiling techniques, the hepatoprotective activity could also be linked to a significant extent, to the presence of baicalein, a major flavonoid in n-butanol fraction of this plant. Thus, this plant may serve as a useful adjuvant in several clinical conditions associated with liver damage, which is evident from the results of biochemical assays and histopathological study.  

Acknowledgement:

The authors are thankful to GUJCOST (Gujarat Council on Science and Technology) for financial assistance by providing minor research project scheme

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Figures

	Figure 1   (Click image to enlarge)
	Figure 2   (Click image to enlarge)

 

Tables

Table 1  Effect of different extracts of root bark of Oroxylum indicum (300 mg/ kg; p.o.) on different biochemical parameters in serum of mice in CCl4-induced hepatotoxicity.   All values represent mean ± SEM, n=6 in each group. * P < 0.05, when compared with the CCl4 control group (ANOVA, followed by Tukey's multiple range test), F tab = 2.249; F cal(7,40) = 938.44 (SGPT), 634.90 (SGOT), 106.56 (ALP), 589.07 (TB). (Click image to enlarge)

Table 2  Effect of 50% alcoholic extract and different extracts of root bark of Oroxylum indicum (300 mg/kg; p.o.) on lipid peroxidation and anti-oxidant enzymes on CCl4- induced hepatotoxicity in mice. All values represent mean ± SEM, n=6 in each group. * P < 0.05, when compared with the CCl4 control group. (ANOVA, followed by Tukey's multiple range test), F tab = 2.249; F cal(7, 40) = 495.63(LPO), 402.96(SOD), 130.84(CAT), 273.33(Reduced GSH) (Click image to enlarge)

 

http://www.biology-online.org/articles/hepatoprotective-effect-root-bark-oroxylum.html