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:



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




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, 3Conventional 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.



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.



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



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.



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



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




Vehicle control




CCl4 control




Test 1 (250 mg/kg of extract)




Test 2  (500 mg/kg of extract)




Standard (25mg/kg of silymarin)




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




CCl4 control




Test 1 (250 mg/kg of extract




Test 2 (500 mg/kg of Extract




Standard (25 mg/kg of Silymarin)




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


(nMol/g liver wt)

Reduced glutathione

(nMol/g liver wt)


(mMol H2O2/min/g liver wt)

Vehicle control




CCl4 control




Test 1 (250mg/kg of extract)




Test 2 (500mg/kg of extract)




Standard (25mg/kg of silymarin)




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)



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.



TBARS- Thiobarbituric acid reactive substances

ALT-Alanine aminotransferase

AST- Aspartate aminotransferase

ALP- Alkaline phosphatase

MDA- Malondialdehyde



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



1.         FM, Daly MJ. Hepatic disease. In: Walker R, Edwards C, editors. Clinical Pharmacy and Therapeutics. Churchill Livingstone: New York; 1999. p. 195-212.

2.         Pang S, Xin X, Stpierre MV. Determinants of Metabolic Disposition. Ann Rev Pharmacol Toxicol 1992; 32:625-6.

3.         Ross MH, Romrell LJ, Kaye GI. Histology a text and atlas. William and Wilkin: Baltimore; 1996.

4.         Rajesh MG, Latha MS. Hepatoprotection by Elephantopus scaber Linn in CCl 4- induced liver injury. Indian J Physiol Pharmacol 2001; 45:481-6.

5.         Anandan R, Deepa Rekha R, Devaki T. Protective effect of Picrorrhiza kurroa on mitochondrial glutathione antioxidant system in D-galactosamine-induced hepatitis in rats. Curr Sci 1999; 76:1543-5.

6.         Satyavati G.V., Raina M.K., Sharma M. Indian Medicinal Plants, Vol-II, ICMR, New Delhi, 1976.p. 178.

7.         Nair N.C., Hemg A.N. Flora of Tamil nadu, India, Part-I, 1983.p. 173.

8.         Yang, Y., X. Ma, W. Wu and P. Guo. Biological characters of the different varieties for Luffa cylindrica. Zhong Yao Cai, 1999; 22: 165–167.

9.         Oboh, I.O. and E.O. Aluyor. Luffa cylindrica-an emerging cash crop. African J. Agric. Res., 2009 4: 684–688.

10.      Jain S.K., Tarafder C.R. Econ, Bot, 1970; 24:241.

11.      Chopra R.N. Glossary of Indian Medicinal Plant, CSIR, New Delhi, 1956.p.279.

12.      Anonymors, the Wealth of India, Vol-VI, CSIR, New Delhi.1962.p.231.

13.      Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy, Pune, India: Nirali Prakashan; 1990.

14.      OECD, Guidelines for testing of chemicals, Acute oral toxicity, Environmental Health and Safety Monograph Series on Testing and Adjustment No. 425, 2001.

15.      Bhattacharyya D, Pandit S, Mukharjee R, Das N and Sur TK. Hepatoprotective effect of Himoliv®, a polyherbal formulation in rats, Indian J Physiol Pharmacol 2003; 47: 435-440.

16.      IFCC Methods for the measurement of catalytic concentrations of enzymes, J. Clin. Chem. Clin. Biochem, 1986; 24: 481.

17.      IFCC Methods for the measurement of catalytic concentrations of enzymes, J. Clin. Chem. Clin. Biochem, 1986; 24: 497.

18.      Bowers GN, McCommb RB. Clin. Chem.; 1972: 18: 97.

19.      Doumas BT. Clin Chem. 1975; 21: 1159.

20.      Jendrassik L, Grof P. Biochem.1938; 297: 81.

21.      Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978; 52: 302-310.

22.      Anderson ME. Determination of Glutathione and Glutathione disulfide in biological sample. Methods Enzymol 1985; 113: 548-555.

23.      Aebi H. Catalase in Vitro. Methods Enzymol 1984; 105: 121- 126.

24.      Galighter AE, Koyloff EN. Essential of practical micro technique. Philadelphia: Lea and Febiger; 1971.

25.      Recknagel RO, Glende EA, Dolak JA Jr, Waller RLC. Mechanism of carbon tetrachloride toxicity. Pharmacol Ther 1989; 43:139-54.

26.      Ilavarasan R, Mohideen S, Vijayalakshmi M, Manonmani G. Hepatoprotective effect of Cassia angustifolia Vahl. Indian J Pharm Sci 2001; 63:504-7.

27.      Chandrashekhar VM, Abdul Haseeb TS, Habbu PV, Nagappa AN. Hepatoprotective activity of Wrightia tinctoria (Roxb) in rats. Indian Drugs 2004; 41:366-70.

28.      Ozbek H, Ugras S, Bayram I, Tuncer I, Ozturk G, Ozturk A. Hepatoprotective effect of Foeniculum vulgare essential oil. Fitoter 2003; 74:317-9.

29.      Yoshikawa M, Morikawa T, Kashima Y, Ninomiya K, Matsuda H. Structure of new dammarane-type triterpene saponins from the flower buds of Panax notoginseng and hepatoprotective effects of principal ginseng saponins. J Nat Prod 2003; 66:922-4.

30.      Mehta RS, Shankar MB, Geetha M, Saluja AK. Hepatoprotective activity of Trianthema portulacastrum. Indian Drugs 1999; 36:241-4.

31.      Baek NL, Kim YS, Kyung JS, Park KH. Isolation of anti-hepatotoxic agent from the roots of Astragalus membranaceous. Korean J Pharmacog 1996; 27:111-6.

32.      Xiong X, Chen W, Cui J, Yi S, Zhang Z, Li K. Effects of ursolic acid on liver protection and bile secretion. Zhong Yao Cai 2003; 26:578-81.

33.      Tran QI, Adnyana IK, Tezuka Y, Nagaoka T, Tran QK, Kadota S. Triterpene saponins from Vietnamese ginseng (Panax vietnamensis) and their hepatocyteprotective activity. J Nat Prod 2001; 64:456-61.

34.     Vijyan P, Prashanth HC, Dhanaraj SA, Badami S, Suresh B. Hepatoprotective effect of total alkaloid fraction of Solanum pseudocapsicu leaves. Pharmaceut Biol 2003; 41:443-8.







Received on 16.02.2011          Modified on 27.02.2011

Accepted on 03.03.2011         © RJPT All right reserved

Research J. Pharm. and Tech. 4(6): June 2011; Page 938-941