HEPATOPROTECTIVE AND ANTIOXIDANT EFFECT OF BALANITES AEGYPTIACA (L.) DEL AGAINST CCL4 INDUCED HEPATOTOXICITY IN RATSHTML Full Text
HEPATOPROTECTIVE AND ANTIOXIDANT EFFECT OF BALANITES AEGYPTIACA (L.) DEL AGAINST CCL4 INDUCED HEPATOTOXICITY IN RATS
- Mayba Gnana Suky 1, B. Parthipan 2, C. Kingston 3, V.R. Mohan*4 and P. Tresina Soris 4
Department of Botany, Holy Cross College (Autonomous) 1, Tiruchirapalli, Tamil Nadu, India
PG & Research Department of Botany, S. T. Hindu College 2, Nagercoil, Tamil Nadu, India
PG & Research Department of Botany, Scott Christian College 3, Nagercoil, Tamil Nadu, India
Ethnopharmacology Unit, Research Department of Botany, V. O. Chidambaram College 4, Tuticorin, Tamil Nadu, India
The various parts of Balanites aegyptiaca (L.) Del (Balanitaceae) is used in Ayurvedic and other folk medicine for the treatment of various ailments such as syphilis, jaundice, liver and spleen problem, epilepsy and yellow fever. This study was designed to evaluate the hepatoprotective and antioxidant effect of ethanol extract of aerial part of Balanites aegyptiaca on CCl4 induced hepatotoxicity in rats. Activities of liver marker enzymes, SGOT, SGPT and ALP, total protein, albumin, globulin, total, conjugated and unconjugated bilirubins at an oral dose of ethanol extract of Balanites aegyptiaca (100 and 200mg/kg) showed a significant hepatoprotective effect. Regarding antioxidant activity, ethanol extract of Balanites aegyptiaca exhibited a significant effect showing increasing levels of SOD, CAT, GPX and GRD by reducing malondialdehyde (MDA) levels.
|Keywords:Hepatoprotective activity, Antioxidant,
INTRODUCTION: Liver diseases remain as one of the serious health problems. However, we do not have satisfactory liver protective drugs in allopathic medical practice for serious liver disorders. Herbal drugs play a role in the management of various liver disorders most of which speed up the natural healing processes of the liver. A number of plants have been shown to possess hepatoprotective properties by improving the antioxidant status 1. However, there is still, lack of scientific proofs to authenticate the hepatoprotective properties of some plants which are used traditionally to treat liver disorders.
Balanites aegyptiaca (L.) Del belongs to the family Balanitaceae. It is commonly known as nanjunda. It has been used in a variety of folk medicine in India and Asia. Various parts of the plant are used in Ayurvedic and other folk medicine for the treatment of various ailments such as syphilis, jaundice, liver and spleen problem, epilepsy and yellow fever and the plant also has insecticidal, antihelminthic, antifeedant, molluscicidal and contraceptive activities 2. In view of the above medicinal properties, the present study was designed to investigate the hepatoprotective activity of ethanol extract of aerial part of Balanites aegyptiaca in CCl4 intoxicated rats.
MATERIALS AND METHODS:
Plant material: The aerial part of Balanites aegyptiaca (L.) Del was collected from Vadavalli, Coimbatore, Tamil Nadu and identified by the Botanical Survey of India, Coimbatore. A voucher specimen was retained in Ethnopharmacology Unit, Research Department of Botany, V. O. Chidambaram College, Tuticorin for further reference.
Preparation of plant extract for phytochemical Screening and Hepatoprotective Studies: The aerial part of the plant was dried under shade and then powdered with a mechanical grinder to obtain a coarse powder, which was then subjected to successive extraction in a Soxhlet apparatus using ethanol. The extract was subjected to qualitative test for the identification of various phytochemical constituents as per standard procedures 3, 4, 5. The ethanol extracts were concentrated in a rotary evaporator. The concentrated ethanol extract were used for antidiabetic studies.
Animals: Normal healthy male Wistar albino rats (180-240g) were used for the present investigation. Animals were housed under standard environmental conditions at room temperature (25±2°C) and light and dark (12:12h). Rats were fed with standard pellet diet (Goldmohur brand, MS Hindustan lever Ltd., Mumbai, India) and water ad libitum.
Acute Toxicity Studies: Acute oral toxicity study was performed as per OECD-423 guidelines (acute toxic class method), albino rats (n=6) of either sex selected by random sampling were used for acute toxicity study 6. The animals were kept fasting for overnight and provided only with water, after which the extracts were administered orally at 5mg/kg body weight by gastric intubations and observed for 14 days. If mortality was observed in two out of three animals, then the dose administered was assigned as toxic dose. If mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. If mortality was not observed, the procedure was repeated for higher doses such as 50, 100 and 2000 mg/kg body weight.
Experimental Design: In the investigation, a total of 25 rats (20 CCl4 hepatic toxicity induced rats and 5 normal rats) were taken and divided into five groups of 5 rats each.
Group I: Rats received normal saline was served as a normal control.
Group II: CCl4 hepatic toxicity induced control: Rats received 2.5ml/kg body weight of CCl4 for 7 days.
Group III: Liver injured rats received ethanol extract of aerial part of Balanites aegyptiaca at the dose of 100mg/kg body weight for 7 days.
Group IV: Liver injured rats received ethanol extract of aerial part of Balanites aegyptiaca at the dose of 200mg/kg body weight for 7 days.
Group V: Liver injured rats received standard drug silymarin at the dose of 100mg/kg body weight for 7 days.
Biochemical Analysis: The animals were sacrificed at the end of experimental period of 7 days by decapitation. Blood was collected, sera separated by centrifugation at 3000g for 10 minutes. Serum protein 7 and serum albumins was determined quantitatively by colorimetric method using bromocresol green. The total protein minus the albumin gives the globulin. Serum glutamate pyruvate transaminase (SGPT) and serum glutamate oxaloacetate transaminase (SGOT) was measured spectrophotometrically by using the method of Reitman and Frankel 8. Serum alkaline phosphatase (ALP) was measured by the method of King and Armstrong 9. Total bilirubin and conjugated bilirubin were determined as described by Balistrei and Shaw 10. The unconjugated bilirubin concentrations were calculated as the difference between total and conjugated bilirubin concentrations. Liver homogenates (10%W/V) were prepared in ice cold 10mM tris buffer (pH7.4). Quantitative estimation of MDA formation was done by determining the concentration of thiobarbituric acid reactive substances (TBARS) in 10% liver homogenates by the method of Okhawa 11. Enzymatic antioxidants, superoxide dismutase (SOD) 12 catalase 13, 14 and non enzymatic antioxidant glutathione peroxidase (GPx) 15 and glutathione reductase (GRD) 16 were also assayed in liver homogenates.
Statistical Analysis: The data were expressed as the mean ± S.E.M. The difference among the means has been analyzed by one-way ANOVA. p<0.01 and p<0.01 were considered as statistical significance using SPSS Software.
RESULTS: The ethanol extract of aerial part of Balanites aegyptiaca subjected for phytochemical study showed the presence of alkaloids, coumarin, glycosides, flavonoids, saponins, steroids, phenols, tannins and xanthoproteins. The ethanol extract did not show any sign and symptoms of toxicity and mortality upto 2000mg/kg dose. The effect of ethanol extract of Balanites aegyptiaca on serum total protein, albumin, globulin, A/G ratio, serum transaminases, alkaline phosphatases in CCl4 intoxicated rats are summarized in Table 1.
There was a significant (p< 0.01) increase in serum GOT, GPT and ALP levels in CCl4 intoxicated group (Group II) compared to the normal control group (Group I). The total protein and albumin levels were significantly (p< 0.01) decreased to 4.31 g/dl and 2.61g/dl in CCl4 intoxicated rats from the levels of 9.38g/dl and 4.98g/dl respectively in normal group. Ethanol extract of Balanites aegyptiaca at the dose of 100 and 200mg/Kg orally significantly decreased the elevated serum marker enzymes and reversed the altered total protein and albumin to almost normal level.
The effect of ethanol extract of Balanites aegyptiaca on total, conjugated and unconjugated bilirubin is shown in Table 2. A significant elevation of total, conjugated and unconjugated bilirubin in the serum of CCl4 intoxicated group (Group II) when compared to normal control (Group I). The ethanol extract of Balanites aegyptiaca at the dose 100 and 200mg/Kg reduced the levels of total, conjugated and unconjugated bilirubin (Group III and Group IV). The decreases in the concentration of total bilirubin, conjugated bilirubin and unconjugated bilirubin were found to be greater in standard silymarin (Group V) followed by Group IV and Group III (Table 2).
TABLE: 1 EFFECT OF ETHANOL EXTRACT OF BALANITES AEGYPTIACA ON THE PROTEIN, ALBUMIN, GLOBULIN CONCENTRATION AND ENZYME ACTIVITY OF SERUM GOT, GPT AND ALP IN THE NORMAL, LIVER INJURED AND DRUG TREATED RATS
|T .Protein (mg/Dl)||Albumin(g/dl)||Globulin(g/dl)||A/G Ratio||SGOT(U/L)||SGPT(U/L)||ALP(U/L)|
|I||9.58± 0.2||4.98 ± 0.04||4.40± 0.02||1.1:1||54.13 ± 1.6||39.51±2.4||173.6±2.1|
|II||4.31± 0.6**||2.61± 0.02**||2.01± 0.01*||1.2:1||151.61 ± 5.35*||103..42±4.9**||269.3±4.0**|
|III||8.56± 0.2a||4.91± 0.03a||2.65± 0.01||1.8:1||59.32±3.15||41.31±1.3||178.51±4.11a|
|IV||9.11± 0.3a||4.86± 0.03a||3.25± 0.03||1.5:1||51.11±2.32a||33.56±1.05a||171.32±3.34a|
|V||7.99± 0.1a||4.16± 0.03a||3.83± 0.03a||1.0:1||46.23±1.98a||36.51±1.68a||161.14±2.89a|
Each Value is SEM ± 5 individual observations * P < 0.05; ** P<0.01 Compared normal control vs liver injured rats; a P < 0.05; aa P<0.01 Compared liver injured rats vs. drug treated
|Group||Total Bilirubin (µmol/L)||Conjugated (µmol/L)||Unconjugated (µmol/L)|
TABLE: 2 EFFECT OF ETHANOL EXTRACT OF BALANITES AEGYPTIACA ON THE SERUM TOTAL, CONJUGATED AND UNCONJUGATED BILIRUBIN LEVELS IN THE NORMAL, LIVER INJURED AND DRUG TREATED RATS
Each Value is SEM ± 5 individual observations * P < 0.05; ** P<0.01 Compared normal control vs. liver injured rats; a P < 0.05; aa P<0.01 Compared liver injured rats vs. drug treated
The effects of ethanol extract of Balanites aegyptiaca on lipid peroxidation (LPO), Glutathione peroxidase (GPx), Superoxide dismutase (SOD) and Catalase (CAT) activity is shown in Table 3. Lipid peroxidation level was significantly (p< 0.01) increased and glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase activity were significantly (p< 0.01) decreased in CCl4 intoxicated rats when compared with those of the animals in normal control group. Rats treated with ethanol extract of Balanites aegyptiaca at the doses of 100 and 200 mg/kg significantly decreased the elevated lipid peroxidation levels and restored the altered glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase levels towards the normal levels in a dose dependent manner. The results are well comparable with silymarin (standard drug) treated group.
TABLE 3: EFFECTS OF ETHANOL EXTRACTS OF BALANITES AEGYPTIACA ON LIVER LPO, GPX, GRD, SOD AND CAT IN THE NORMAL CONTROL, LIVER INJURED AND DRUG TREATED RATS
|Groups||LPO (n mole of MDA/mg protien)||GPX (μ/mg Protein)||GRD (μ/mg)||SOD (μ/mg)||CAT (μ/mg)|
Each Value is SEM ± 5 individual observations * P < 0.05; ** P<0.01 Compared normal control vs. liver injured rats a P < 0.05; aa P<0.01 Compared liver injured rats vs. drug treated
DISCUSSION: Liver diseases, especially viral hepatitis occurs predominantly in the developing world, an enormous impact on public health and economy. In the absence of reliable liver protective drugs in allopathic medical practices, herbs play an important role in the management of various liver disorders. A number of plants show hepatoprotective activity 17. Based on the promising results shown by the plant extracts in the in vitro studies, in vivo hepatoprotective studies were carried out in experimental rats using CCl4 induced hepatotoxicity. CCl4 is one of the most commonly used hepatotoxin. CCl4 produces an experimental damage that histological resembles viral hepatitis.
Toxicity begins with the change in endoplasmic reticulum, which results in the loss of metabolic enzymes located in the intracellular structures 18. The toxic metabolite, CCl3 radical, is produced and further reacts with oxygen to give trichloromethyl peroxy radical. Cytochrome P450 is the enzyme responsible for this conversion. Both the radicals can bind covalently to the macromolecules and induce peroxidative degradation of the membrane lipid of endoplasmic reticulum rich in polyunsaturated fatty acids 18. This leads to the formation of lipid peroxidases followed by pathological changes such as depression of protein synthesis, elevation levels of serum marker enzymes such as SGOT, SGPT and ALP, depletion of GPX, GRD, SOD and CAT and increase in lipid peroxidation.
In the present study, it was obtained that, the rats treated with CCl4 resulted in significant hepatic damage as shown by the elevated levels of serum markers. These changes in the marker levels will reflect in hepatic structural integrity. 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 19. Ethanol extract of Balanites aegyptiaca at the doses of 100mg/kg and 200mg/kg significantly attenuated the elevated levels of the serum markers. The normalization of serum markers by ethanol extract of Balanites aegyptiaca suggests that they are able to condition the hepatocytes so as to protect the membrane integrity against CCl4 induced leakage of marker enzymes into the circulation. The above changes can be considered as an expression of the functional improvement of hepatocytes.
The alkaline phosphatase is the prototype of these enzymes that reflects the pathological alteration in biliary flow 20. The CCl4 induced elevation of this enzymatic acivity in the serum is in line with high level of serum bilirubins content. The ethanol extract of Balanites aegyptiaca induced suppression of the increased ALP activity with the concurrent depletion of raised bilirubins suggests the possibility of the extract to have ability to stabilize biliary dysfunction in rat liver during hepatic injury by CCl4. Thus administration of ethanol extract of aerial part of Balanites aegyptiaca is against the toxic effect of CCl4.
Lipid peroxidation has been postulated to the destructive process of liver injury due to CCl4 administration. In the present study, the elevations in the levels of end products of lipid peroxidation in the liver of rat treated with CCl4 were observed. The increase in malondialdehyde (MDA) levels in liver suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanisms to prevent formation of excessive free radicals. Treatment with ethanol extract of Balanites aegyptiaca significantly reversed these changes. Hence, it may be possible that the mechanism of hepatoprotection by ethanol extraction of Balanites aegyptiaca is due to its antioxidant effect.
In the present investigations, CCl4 intoxicated rats decreased the content of GPX and GRD in liver, whereas, treatment with ethanol extract of Balanites aegyptiaca (100 and 200mg/kg) able to reverse such effects. Superoxide dismutase (SOD) is a key defense enzyme and catalyses the dismutation of superoxide anions. Catalase (CAT) is a haemeprotein that catalyses the reduction of H2O2 and able to prevent the tissue from reactive free oxygen and hydroxyl radicals. Decrease in SOD activity can result in the removal of superoxide anions that may inactivate SOD thereby causing an inactivation of H2O2 scavenging enzymes. Administration of ethanol extract of Balanites aegyptiaca treated rats able to prevent effectively the decrease in SOD and CAT activities, which may be directly correlated to scavenging or neutralizing of radicals by ethanol extract of Balanites aegyptiaca resulting in protection of these important defense enzymes.
The preliminary phytochemical analysis of the extracts has shown the presence of flavonoids and phenolics compounds which have been known for its antioxidant and hepatoprotective studies. It is suggested that, saponins in ethanol extract of Balanites aegyptiaca play an important role as antioxidant for prevention of oxidative hepatic damage. Furthermore, the flavonoids and saponins of ethanol extract of Balanites aegyptiaca may able to stabilize reactive oxygen species by reacting with them and oxidizes subsequently to more stable and less reactive radicals. Our findings support the reported therapeutic use of this herb in tribal medicine for liver ailments and jaundice. Further investigation is in progress to determine the exact phytoconstituent(s) responsible for hepatoprotective effect.
ACKNOWLEDGEMENT: Thanks to Dr. Sampathraj, Honorary Advisor, Samsun Clinical Research Laboratory, Tirupur for their assistance in animal studies.
- Gupta M, Mazumder UK, Kumar RS, Sivakumar T, Gomathi P and Rajeshwar Y: Antioxidant defense system induced by a methanol extract of Caesalpinia bonducella in rat liver. Pharmaceutical Biology. 2005; 43: 411-419.
- Yadhav JP and Panghal M: Balanites aegyptiaca (L.) (Hingot): A review of its traditional uses, phytochemistry and pharmacological properties. International Journal of Green Pharmacy. 2010; 4: 140-146.
- Brinda P, Sasikala P, Purushothaman KK: Pharmacognostic studies on Merugan kizhangu. Bulletin in Medical Ethnobotanical Research. 1981; 3:84-96.
- Anonymous: Phytochemical investigation of certain medicinal plants used in Ayurveda. Central Council for Research in Ayurveda and Siddha, New Delhi. 1990.
- Lala PK: Lab manuals of Pharmacognosy CSI Publishers and Distributers, Kolkata. 1993.
- OECD, (Organisation for Economic co-operation and Development). OECD guidelines for the testing of chemicals/Section 4: Health Effects Test No. 423; Acute oral Toxicity- Acute Toxic Class method. OECD. Paris. 2002.
- Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ: Protein measurement with the folin’s phenol reagent. Journal of Biological Chemistry. 1951; 193: 265-275.
- Reitman S, Frankel SA: Colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. American Journal of Clinical Pathology. 1957; 28: 56-63.
- King EJ. Armstrong AR: Determination of serum and bile phosphatase activity. Cannadian Medical Association Journal, 1934; 31: 56-63.
- Balistrei WR, Shaw LM. Liver function In: Fundamental of Clinical Chemistry, (Ed) Tietz N.W. 3rd W.B. Saunders Company, Philadelphia, 1987; pp 729-761.
- Okhawa H, Ohishi N and Yagi K: Assay of lipid peroxidases in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry, 1979; 95: 351-358.
- Mishra HP and Fridowich I: The role of superoxide dismutase anion in antioxidation of epinephrine and sample assay in superoxide dismutase. Journal of Biological Chemistry. 1972; 241: 3170.
- Aebi H: Catalase, in Methods of enzymatic analysis (Chemic Academic Press Inc. Verlag) 1974; 673.
- Colowick SP, Kaplan NO and Packer L: Methods in Enzymology (Academic press, London). 1984; 121.
- Pagila DE, Valentine WN: Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. Journal of Laboratory and Clinical medicine. 1967; 70: 158-169.
- Goldberg DM, Spooner RJ: Glutathione Reductase, In: Methods in Enzymatic Analysis, VCH Weinhem, Germany. 1983; 258-265.
- Malhotra S, Singh GA and Munja. Hepatotoxic potential of commonly used herbal products. Gastroenterology: 5: 110. 2001.
- Recknagel RO. A new direction in the study of carbon tetrachloride hepatoprotective. Life Science. 1983. 33:401-408.
- Sallie R, Tredger JN, William. Drugs and the liver. Biopharmacy Drug Dispos, 12:251-259.
- Ploa GC, Hewitt WR. Detection and evaluation of chemically induced liver injury. In: Principles and methods of Toxicology. Wallace Hayes.Ed.2ndpp 399-628, Raven press, New York, 1989.
T. Mayba Gnana Suky , B. Parthipan , C. Kingston , V.R. Mohan* and P. Tresina Soris
Ethnopharmacology Unit, Research Department of Botany, V. O. Chidambaram College, Tuticorin, Tamil Nadu, India
17 December, 2010
15 January, 2011
14 March, 2011
01 April, 2011