Protective Effect of Ethanolic Extract of Fruit of Luffa aegyptiaca Mill. on Carbon Tetrachloride Induced Hepatotoxicity in Rats.

 

Sushama D. Patil*, Leena Patil and Vilasrao J. Kadam

Department of Pharmacology, Bharati Vidyapeeth’s College of Pharmacy, Sector 8 , C.B.D, Navi Mumbai 400614, Maharashtra, India.

*Corresponding Author E-mail: sushama1688@yahoo.in

ABSTRACT:

Oxidative damage is implicated in the pathogenesis of various liver injuries. The study was aimed to investigate the antioxidant activity of fruit of Luffa aegyptiaca Mill. on carbon tetrachloride (CCl₄) induced oxidative stress in female wistar rats. CCl₄ injection induced oxidative stress by a significant rise in serum marker enzymes and thiobarbituric acid reactive substances (TBARS) along with the reduction of antioxidant enzymes. In serum, the activities of enzymes like alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) were evaluated. Pre-treatment of rats with different doses of plant extract (250 and 500 mg/kg) significantly lowered ALT, AST and TBARS levels against CCl₄ treated rats. Hepatic enzymes like catalase and reduced glutathione were significantly increased by treatment with plant extract, against CCl₄ treated rats. Histopathological examinations showed extensive liver injuries, characterized by extensive hepatocellular degeneration/ necrosis, inflammatory cell infiltration, congestion, and sinusoidal dilatation. Oral administration of the fruit extract at a dose of 500mg/kg body weight significantly reduced the toxic effects of CCl₄. The activity of fruit extract at the dose of 500 mg/kg was comparable to the standard drug, silymarin. Based on these results, it was observed that Luffa aegyptiaca Mill. fruit extract protects liver from oxidative stress induced by CCl₄ and thus helps in evaluation of traditional claim on this plant.

 

KEYWORDS: CCl₄, oxidative damage, fruit of Luffa aegyptiaca Mill., serum enzyme markers, hepatoprotection

 

INTRODUCTION:

The liver is the key organ regulating homeostasis in the body. It is involved with almost all the biochemical pathways related to growth, fight against disease, nutrient supply, energy provision and reproduction.¹ The liver is expected not only to perform physiological functions but also to protect against the hazards of harmful drugs and chemicals. In spite of tremendous scientific advancement in the field of hepatology in recent years, liver problems are on the rise. Jaundice and hepatitis are two major hepatic disorders that account for a high death rate.^{2, 3}Conventional drugs used in the treatment of liver diseases are often inadequate. It is therefore necessary to search for alternative drugs for the treatment of liver diseases to replace the currently used drugs of doubtful efficacy and safety. India is well known for a plethora of medicinal plants. The medicinal use of many plants (as hepatoprotectants) like Andrographis paniculata, Azadirachta indica, Cassia fistula, Elephantopus scaber, Hibiscus rosasinensis, Phyllanthus debilis, Picrorrhiza kurroa has been reported in the literature.^4,5

 

Luffa aegyptiaca Mill. (Cucurbitaceae) is a climber with slender, slightly hairy, furrowed stem commonly known as Sponge Gourds, Vegetable Sponge, Wash Sponge, Gourd Towel, Dishcloth Gourd, Loofah Gourd and found throughout India, wild in wastelands especially along the coastal areas and also cultivated.^6,7 Luffa cylindrica is the synonym of Luffa aegyptiaca Mill. It is an annual vegetable upland crop which is used as a vegetable either prepared like squash or eaten raw like cucumbers.^{8, 9}

 

The plant is reputed to have anti tubercular and antiseptic properties. The extract of leaves has been used in snake bites, convulsions, cramps, tetanus and also in the treatment of syphilis.¹⁰ The fruit is used in dropsy, nephritis, chronic bronchitis.¹¹ The seeds are considered as emetic and cathartic. The seed oil is reported to be used for skin infections. In the form of tincture, the fruit used in the treatment of ascites, jaundice, biliary and intestinal colitis and also in enlarged spleen and fever.¹²

 

Literature reviews indicated that the hepatoprotective activity of fruit of L. aegyptiaca Mill. has not been clinically evaluated so far. An active and safe drug is needed for the treatment of hepatitis. In view of this, the present study was aimed at evaluating the hepatoprotective activity of the ethanolic extract of fruit of L. aegyptiaca Mill. against CCl₄ induced hepatotoxicity in rats.

 

MATERIAL AND METHOD:

Plant material:

The fruit was collected in the month of August from local market in Mumbai and authenticated by Dr.Ganesh Iyer, Department of life science, Ramnarayan Ruia College, Matunga, Mumbai.

 

Extraction:

The fruits were collected, thoroughly washed, sliced and shade dried for 4-5 days and powdered in an electric mixer grinder. The 70g of powdered drug was then extracted using ethanol as a solvent by using soxhlet apparatus. After extracting all colouring matter, the solvent was removed by heating on water bath which give rise to a semisolid mass of extract. The yield was 28.5 %w/w. The extract was then subjected to preliminary phytochemical tests.¹³

 

Chemicals:

Carbon tetrachloride was purchased from SD fine chemicals and silymarin was gift sample from Micro labs. The kits for biochemical estimation were purchased from Coral clinical systems, Goa, India. The solvents and other chemicals used were of analytical grade.

 

Animals:

Female wistar rats (150-200 g) were procured from National Toxicology Center, Pune. Rats, 6 in a group, were housed in clean polypropylene cages under standard conditions of humidity (50 ± 5%), temperature (25 ± 2°C) and light (12 hr light: 12 hr dark cycle) and free access to food and water ad libitum. Experiments were conducted after obtaining the approval from Institutional Animal Ethics Committee constituted as per CPCSEA guidelines.

 

Acute oral toxicity study:

Acute oral toxicity study was conducted for the extract by OECD guideline 425.¹⁴ The extract was found safe up to 2000 mg/kg dose. Thus, two doses of drug 250 mg/kg (Dose 1) and 500 mg/kg (Dose 2) were selected for the study.

 

Evaluation of Hepatoprotective activity:

Hepatoprotective study was carried out as described by Satyanarayana et al.¹⁵ Five groups of animal containing 6 rats each were used for the study. Animals from group I were served as vehicle  control and received the vehicle that is 5ml/kg of distilled water per oral (p.o.) for 8 days and olive oil (0.5 ml/kg) intraperitonially (i.p.) on day 7.Group II, III, IV and  V were served as CCl₄ control, test 1, test 2and standard control respectively. Group II, III, IV and V received 5ml/kg of water, 250 mg/kg of extract, 500mg/kg of extract and 25mg/kg of standard drug silymarin once a day p.o. for 8 days respectively. All the four groups received CCl₄ (1ml/kg) in olive oil (1:1) i.p. on day 7, 30 minutes after administration of water, extract or silymarin.

The blood samples were kept at room temperature for 1 hr for clotting. Serum was separated by centrifugation at 2500 rpm at 37⁰ C for 10 minutes and the biochemical parameters like ALT, AST, ALP, total protein and total and direct bilirubin were estimated.^16-20

 

Immediately after sacrificing the animals, livers were separated, washed with pH 7.4 buffer and used for estimation of tissue parameters like lipid peroxidation, reduced glutathione and catalase.^21-23

 

After draining the blood, liver samples were excised, washed and fixed in 10% buffered neutral formalin for 48 h and then with bovine solution for 6 h. Paraffin sections were taken at 5 mm thickness and were stained with hematoxylin and eosin. The sections were examined microscopically for histopathological changes.²⁴

 

RESULTS:

The administration of CCl₄ to the animals resulted in a marked increase in total and direct bilirubin, ALT, AST and ALP activities. However, the serum total protein level was decreased. The toxic effect of CCl₄ was controlled in the animals treated with the ethanolic extract of fruit of L. aegyptiaca Mill. by way of restoration of the levels of the liver function biochemistry similar to that of the standard drug silymarin .Table 1 and 2 shows effect of ethanolic extract of fruit of L. aegyptiaca Mill. and standard drug silymarin on serum parameters which were changed by CCl₄induced hepatotoxicity in rats.

 

Level of lipid peroxidation in tissue was measured in terms of malondialdehyde (MDA) levels. MDA levels in the CCl₄ treated group were significantly higher than that in the vehicle control group and MDA levels in the groups treated with extract of L. aegyptiaca Mill. (at dose of 250 and 500 mg/kg) were significantly lower than that in the CCl₄ treated group. Activity of catalase and reduced glutathione were measured as an index of antioxidant status of tissues. Significantly lower liver catalase and reduced glutathione activity were observed in rats from the CCl₄ treated group as compared to the vehicle control group. There was a significant increase of catalase and reduced glutathione activity in the groups treated with extract and standard drug silymarin, as compared to the CCl₄ treated group. Table 3 shows Effect of L.aegyptiaca Mill. extract and standard drug silymarin on the level of MDA and activities of catalase and reduced glutathione in CCl₄ induced hepatotoxicity in rats.

 

The histopathological study also provided supportive evidence for the biochemical estimations. Vehicle control group (figure-1) showed normal liver architecture, hepatocytes and triads of hepatic veins without any alternations. Liver section of the rats treated with CCL₄ (figure-2) showed intense centrilobular necrosis and vacuolization. The rats treated with two doses of extract of L. aegyptiaca Mill. (figure- 3 and 4) and silymarin (figure-5) along with toxicant showed sign of protection against this toxicant to considerable extent as evident from formation of normal hepatic cords and absence of necrosis and vacuoles.

Table 1- Effect of ethanolic extract of fruit of L.aegyptiaca Mill. on AST, ALT and ALP levels

Treatment group	ALT (IU/L)	AST (IU/L)	ALP (IU/L)
Vehicle control	39.00±1.065c	91±1.949c	273.167±3.719c
CCl4 control	146±2.708	203.667±1.706	604.333±3.190
Test 1 (250 mg/kg of extract)	89.167±1.167c	168.333±4.161b	472.167±3.371c
Test 2  (500 mg/kg of extract)	74.167±1.537c	146.167±1.851c	377±3.464c
Standard (25mg/kg of silymarin)	58.833±1.302c	119.5±1.478c	348.167±1.956c

Values are expressed as mean ± SEM, n= 6 animals per group. One way ANOVA followed by Dunnett’s test when compared with CCl₄ control ^ap< 0.05, ^bp< 0.01, ^cp< 0.001

 

Table 2- - Effect of ethanolic extract of fruit of L.aegyptiaca Mill. on total bilirubin, direct bilirubin and total protein

Treatment group	Total bilirubin (mg/dL)	Direct bilirubin (mg/dL)	Total protein (g/dL)
Vehicle control	0.166±0.002c	0.088±0.0009c	7.608±0.027c
CCl4 control	0.934±0.006	0.597±0.006	2.82±0.052
Test 1 (250 mg/kg of extract	0.630±0.003c	0.461±0.007c	5.663±0.08c
Test 2 (500 mg/kg of Extract	0.507±0.017c	0.329±0.018c	6.488±0.098c
Standard (25 mg/kg of Silymarin)	0.230±0.002c	0.169±0.007c	7.208±0.038c

Values are expressed as mean ± SEM, n= 6 animals per group. One way ANOVA followed by Dunnett’s test when compared with CCl₄ control ^ap< 0.05, ^bp< 0.01, ^cp< 0.001

 

Table 3- Effect of ethanolic extract of fruit of L.aegyptiaca Mill. on lipid peroxidation, reduced glutathione activity and catalase activity

Treatment group	MDA (nMol/g liver wt)	Reduced glutathione (nMol/g liver wt)	Catalase (mMol H2O2/min/g liver wt)
Vehicle control	15.082±0.373c	4.442±0.295c	80.25±2.095c
CCl4 control	35.663±1.261	1.397±0.137	36.89±2.736
Test 1 (250mg/kg of extract)	27.682±0.303c	2.663±0.061c	49.53±3.689b
Test 2 (500mg/kg of extract)	24.488±0.249c	3.180±0.039c	58.14±1.203c
Standard (25mg/kg of silymarin)	21.370±0.433c	3.862±0.045c	71.59±1.025c

Values are expressed as mean ± SEM, n= 6 animals per group. One way ANOVA followed by Dunnett’s test when compared with CCl₄ control ^ap< 0.05, ^bp< 0.01, ^cp< 0.001

 

 

Figure 1- Vehicle control

 

Figure 2- CCl₄ control

 

Figure 3- Test 1 (250 mg/kg of extract)            

 

Figure 4- Test 2 (500 mg/kg of extract)

 

Figure 5- standard (25 mg/kg silymarin)

 

DISCUSSION:

The present investigation indicated that the extract of fruit of L. aegyptiaca Mill. provide significant protection against CCl₄ induced hepatotoxicity in rats. CCl₄ is widely used as hepatotoxin in the experimental studies. The CCl₄ is biotransformed by the cytochrome P450 system to produce the trichloromethyl free radicals, which in turn covalently binds to cell membranes and organelles to elicit lipid peroxidation.²⁵ Several plants viz., Cassia aungustifolia²⁶, Wrightia tinctoria²⁷, Foeniculum vulgare²⁸and Panax notoginseng²⁹ have been tested for their efficacy in controlling the CCl₄ induced liver damage. Further it has been evident that several phytoconstituents have the ability to induce the excretion of CCl₄ or by inhibition of lipid peroxidation induced by CCl₄.³⁰ Phytoconstituents  like flavonoids,³¹ triterpenoids³², saponins³³ and alkaloids³⁴ are  known  to  possess hepatoprotective activity. Phytochemical investigations of ethanolic extract of fruit of L. aegyptiaca Mill. revealed the presence of alkaloids, phenols, saponins, flavonoids and tannins. The present study revealed that ethanolic extract of fruit of L. aegyptiaca Mill. possess significant protective effect against hepatotoxicity induced by carbon tetrachloride which may be attributed to the individual or combined action of phytoconstituents present in it. The component(s) of the extract responsible for this effect however was not investigated. Further investigations are needed for identification of the active compounds responsible for hepatoprotective activity. The present finding provides scientific evidence to the ethno medicinal use of this plant by traditional systems of medicine in treating jaundice.

 

Abbreviations:

TBARS- Thiobarbituric acid reactive substances

ALT-Alanine aminotransferase

AST- Aspartate aminotransferase

ALP- Alkaline phosphatase

MDA- Malondialdehyde

 

ACKNOWLEDGMENT:

Authors are thankful to Dr. Vilasrao Kadam, Principal Bharati Vidyapeeth’s College of Pharmacy for motivation and support and for providing necessary facilities.

 

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Received on 16.02.2011          Modified on 27.02.2011

Accepted on 03.03.2011         © RJPT All right reserved