International Journal of Applied Research in Natural Products Vol. 2(2), pp. 19-32, June-July 2009 Available online http://www.healthy-synergies.

com 2009 Healthy Synergies Publications

Original Article

The Aqueous Seed Extract Of Carica papaya Linn. Prevents Carbon Tetrachloride Induced Hepatotoxicity In Rats
Adeneye AA1, *, Olagunju JA2, Banjo AAF3, Abdul SF4, Sanusi OA4, Sanni OO4, Osarodion BA4, Shonoiki OE4
Department of Pharmacology, 2Department of Medical Biochemistry, Faculty of Basic Medical Sciences, Lagos State University College of Medicine, Ikeja, Lagos State, Nigeria 3 Department of Morbid Anatomy, School of Basic Medical Sciences, College of Medicine, University of Lagos, Idi-Araba, Lagos State, Nigeria 4 Department of Biochemistry, Obafemi Awolowo College of Health Sciences, Olabisi Onabanjo University, Ikenne Campus, Ogun State, Nigeria
1

Summary: Carica papaya Linn. is known to have a versatile application in African folkloric medicine. In the current study, the dose-dependent (100 400 mg/kg/day/oral route) and time-course protective effects of the 400 mg/kg/oral route of the aqueous seed extract of unripe and mature Carica papaya fruit (CPE) were investigated in carbon tetrachloride (CCl4) hepatotoxic rats for 72 hours. Results showed the extract to cause significant (p<0.05, p<0.001), dose related attenuation in the elevation of serum liver enzyme markers of acute hepatocellular injury (ALT, AST), serum lipids (TG, TC, HDL-c, LDL-c and VLDL-c) and serum proteins (TP and ALB). Maximum hepatoprotection was offered at an oral dose of 400 mg/kg/day of the extract. The biochemical results obtained were corroborated by improvements in the CCl4-induced hepatic histological changes. In addition, maximum hepatoprotection was offered at the 400 mg/kg of CPE for up to 3 hours postCCl4 induction. In conclusion, the results obtained in the current study justify the folkloric application of CPE in the treatment of drug-related hepatic injury. Industrial relevance: Results of the current study provide some scientific information to develop safe and effective products such as food supplements, dietary supplements, etc. that could be useful in the clinical management of patients with drug related hepatic disorders. Keywords: Carica papaya Linn., hepatoprotection, CCl4 hepatotoxicity, Wistar rats

Introduction Carica papaya Linn. (family: Caricaceae) is a widely grown, perennial tropical tree, which grows up to 5 to 10 m tall with an erect and branchless trunk. Its leaves are large, 50-70 cm in diameter, deeply palmately lobed with 7 lobes (Duke, 1984). Its melon-like fruit (papaya) is known by different names in different parts of the world and these include fruta bomba (in Cuba), lechoza (in Venezuela, Puerto Rico, the Philippines and the Dominican Republic) and papaw (Sri Lankan) (Lohiya et al., 2002). In Nigeria, it is also known by different local names depending on the tribe. For example, among the Yoruba (South-West Nigeria) it is known as Ibepe and sigun, gwanda among the Hausa (Northern Nigeria), ojo and okwere among the Igbo (South-East Nigeria), etihi-mbakara among the Efik (South-South Nigeria).The ripe fruit is edible and is usually eaten raw, without the skin or seeds. The unripe green fruit (which is a rich source of vitamin A) can be eaten cooked, usually in curries, salads and stews as used in Thai cuisine (Lohiya et al., 2002). ____________________________ *Corresponding Author: Telephone (mobile): +234-802-0690-946 Fax: +234-702-800-2758 E-mail: adeneye2001@yahoo.com

Hepatoprotective Activity of the Aqueous Seed Extract of Carica papaya

Different parts of the plant are attributed with different medicinal values. For example, in African traditional medicine, the boiled green leaves of papaya combined with leaves of Azadirachta indica, Cymbopogon citratus, Psidium guajava and stem bark of Alstonia boonei boiled together and the hot infusion is drunk as one wine glass full thrice daily in the treatment of malaria (Gill, 1992). Its fresh leaves are also efficacious in the treatment of gonorrhea, syphilis and amoebic dysentery (Gill, 1992). The milky juice of the unripe fruit is a powerful abortificant, anti-helminthic for roundworms, stomach disorders and enlargement of liver and spleen (Gill, 1992). The seeds are also effective as a vermifuge and in the treatment of hypertension, diabetes mellitus and hypercholesterolemia (Gill, 1992). Results from studies on biological activities of Carica papaya parts, extracts and isolated compounds showed that the latex and root extracts inhibited Candida albicans while extracts of pulp and seeds showed bacteriostatic properties against Staphylococcus aureus, Escherichia coli, Salmonella typhi, Bacillus subtilis, and Entamoeba histolytica, in vitro (Emeruwa, 1982). Its root aqueous extract has equally been shown to have purgative effect (Akah et al., 1997). In an earlier study, the hypoglycemic and hypolipidemic effects of the aqueous seed extract of Carica papaya was reported and the LD50 of the oral toxicity was estimated to greater than 2000 mg/kg/oral route (Adeneye and Olagunju, 2009). Recent ethnobotanical survey conducted by us also revealed that hot infusion of Carica papaya seeds is used by Yoruba herbalists (Southwest Nigeria) in the treatment of poison related liver and renal diseases. In an earlier study, we reported the nephroprotective activity of the water extract of the plant seeds in acute model of CCl4 renal injured rats (Olagunju et al., 2009). Based on the folkloric use of plant seeds in the treatment of poison related liver disease and its oral safety on acute exposure, the present study was designed to investigate the effect of graded oral doses (100 - 400 mg/kg/day) and the time-course effect of 400 mg/kg of the aqueous seed extract of mature unripe Carica papaya (CPE) in CCl4 treated rats as a way of validating this folkloric use. The choice of the extract dose range employed in this study was based on the result obtained from the previous preliminary study. Materials and Methods Plant materials and the aqueous extraction procedure Six mature, unripe fruits of Carica papaya were collected from a cultivated Pawpaw Plantation at Oke-Afa, Isolo, Lagos State, Nigeria, in the first week of May, 2008. Plant identification and authentication had earlier been done by Olagunju et al. (1995). The pawpaw fruits were cut into pieces and the wet seeds were separated out. These were then gently but thoroughly rinsed in tap water two times and completely air-dried at room temperature for 4 weeks. The dried seeds were pulverized into fine powder using a new domestic mixer grinder (Kanchan Tycoon, Kanchan International Limited Unit III, Daman, India). 40 g of the powdered Carica papaya seeds was boiled in 500 mL of distilled water for 30 minutes after which it was filtered using a piece of clean white cotton gauze. The filtrate was evaporated to complete dryness at 40 C, producing a fine sweet smelling and chocolate color solid residue [yield: 22.5% (w/w)]. The extraction process was repeated 4 times and the solid residue was weighed after extraction and pooled together in an airand water-proof container kept in a refrigerator at 4 C. From this stock, fresh preparations were made whenever required. Experimental Animal and their management Young adult male Wistar rats, weighing between 150 and 180 g were used for this study. The rats were obtained from the Rat Colony of the Zoology Department of Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria, after an ethical approval was obtained from the existing Ethical Committee of Lagos State University College of Medicine, Ikeja, Lagos State, Nigeria. The rats were allowed two weeks of acclimatization under standard laboratory conditions. The rats were maintained on standard rat feed (Ladokun Feeds, Ibadan, Oyo State, Nigeria) and potable water ad libitum. The experimental rats were all handled in strict compliance with international guidelines as prescribed by the Canadian Council on the Care and use of Laboratory Animals in Biomedical Research (1984). Determination of the dose-dependent effect of CPE For the graded oral dose model, rats were randomly divided into five groups (A E) with six rats in each group. Group A served as the untreated control group while group B served as the model control. Rats in both groups were orally pretreated with 10 mL/kg/day of distilled water for 7 days. Rats in groups C E served as the treatment groups that were orally pre-treated with 100, 200 and 400 mg/kg/day of CPE, respectively, for 7 days. Determination of the time-course effect of CPE To determine the time needed for the extract to offer the maximum hepatoprotection, the rats were also randomly divided into seven groups (F L) consisting six rats per group. Group F rats served as the control, untreated group and were administered 10 mL/kg and 1 mL/kg of distilled water via the oral and intraperitoneal routes, respectively. Rats in groups G L were gastro-gavaged with 400 mg/kg of CPE at 3 hours pre-, 0 hour, 1 hour post-, 3 hours post- and 6 hours post-CCl4 induction, respectively.

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Experimental induction of CCl4 hepatotoxicity CCl4-induced acute hepatic injury was initiated by intraperitoneal injection of 1.5 mL/kg of 20% CCl4 (Merck, Darmstadt, Germany) dissolved in olive oil (Roberts Laboratories Limited, Belton, England) as previously described by Lu et al. (2002). For the oral graded dose model, CCl4 was intraperitoneally injected into groups B E fasted rats 24 hours after the last oral dose of CPE on the 7th day. Rat feed and potable water were further withheld for additional 4 hours before the rats were allowed free access to feed and water. Similarly, for the time course model, same dose of the hepatotoxicant was administered intraperitoneal except that the toxicant was injected at specified time intervals as earlier stated. Measurement of fasting blood glucose (FBG) in rats in pre- and post-CCl4 induction states in rats The rats were initially fasted overnight to obtain the baseline fasting blood glucose (FBG) value before the experiment began. The second and third FBG values were also determined on the termination of extract pretreatment and 72 hours after induction with CCl4. FBG concentration was determined by the tail tipping method and using glucose oxidase method of Trinder (1969) on a One Touch Basic Blood Glucose Monitoring System (LifeScan Inc., Milpitas, California, U.S.A.). Blood collection and biochemical assays Seventy-two hours post-induction with CCl4, the fasted rats were anesthetized with inhaled diethyl ether after which blood samples were collected through cardiac puncture into plain sample bottles. The collected samples were allowed to clot at the temperature of 4 C for 6 hours before they were centrifuged using Uniscope Laboratory Centrifuge (Model SM 902B, Surgifriend Medicals, England, U.K.) at 3000 rpm at same temperature for 20 minutes in order to separate the sera. The separated sera were then assayed for the liver enzymes (ALT and AST) by method of Reitman and Frankel (1957), lipids (triglycerides, total cholesterol, HDL-c, LDL-c, and VLDL-c), total protein and albumin using standard diagnostic test kits (Randox Laboratories, Crumlin, U.K.) on Automated Clinical System (Sychron Clinical System, model: CX5 PRO) (Beckman Coulter Inc., Galway, Ireland). Serum VLDL-c concentration was calculated by deduction of the sum of HDL-c and LDL-c concentrations from that of total cholesterol. Histopathological studies of rat livers After the animals were sacrificed, the rat livers were identified and carefully dissected out en bloc. The livers were rinsed in normal saline and sections were taken from them. The liver tissue sections obtained were fixed in 10% formo-saline, dehydrated with 100% ethanol solution and embedded in paraffin. They were then processed into 5 m thick sections stained with hematoxylin-eosin and observed under a photomicroscope (Model N 400ME, CEL-TECH Diagnostics, Hamburg, Germany). Statistical Analysis Data were expressed as mean SEM of six observations and statistically assessed by two ways analysis of variance and the group means were compared using Students t-test on statistical software program, SYSTAT 10.6. A probability of p<0.05, p<0.01 and p<0.001 were considered as significant. Results and Discussion Carbon tetrachloride is an industrial solvent known to induce various organ toxicities when exposed to it. This agent is actively metabolized in the body tissues to its highly reactive halogenated metabolites (.CCl3 and .Cl) and its metabolic activation is accompanied by the release of reactive oxygen species (ROS) (Slater, 1984). The reactive metabolites and the free radicals released subsequently result in the induction of lipid peroxidation leading to array of organ toxicities such as hepatotoxicity, nephrotoxicity, neurotoxicity, cardiotoxicity and hematotoxicity (Charbonneau et al., 1986; Ozturk et al., 2003). Several studies have shown that -tocopherol (Vitamin E), and silymarin are potent antioxidants that could protect the liver against CCl4 hepatotoxicity indicating that oxidative stress could play a pivotal role in CCl4 hepatic injury (Yoshikawa et al., 1982; Farghali et al., 2000). In Indian Ayurvedic medicine, several herbal extracts have been reported to protect the liver against CCl4-induced hepatic injury. Some of these herbal plants include Silybum marium (Faghali et al., 2000), Cajanus indicus (Ghosh and Biswas, 1973), Phyllanthus niruri (Syamasundar et al., 1985), Calotropis procera (Basu et al., 1992; Samvatsar and Diwanji, 2000; Padhy et al., 2007) and Terminalia arjuna (Manna et al., 2006). Biologically active principles isolated from these herbal plants have been reported to mediate their protective activities through intrinsic antioxidant and free radical scavenging activities (Manna et al., 2006). Also, in African Traditional Medicine, many medicinal plants have been documented to possess hepatoprotective properties and these include Garcinia kola (Akintonwa and Essien, 1990; Farombi, 2000; Ajani et al., 2007), Zingiber officinale (Yemitan and Izegbu, 2006), Harungana madagascariensis (Adeneye et al., 2008), Corydalis saxicola (Wang et al., 2008) and Lantana camara (Fagbounka et al., 2008) just to mention a few. Recent ethnobotanical survey conducted by us revealed that the hot infusion made from dry seeds of the mature, unripe fruit of Carica papaya is effectively employed in the traditional management of hepatic and renal diseases including drug-related liver and kidney diseases, among the Yoruba herbalists (South-West Nigeria). In order to validate this folkloric use, 100 400 mg/kg/day of CPE

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Hepatoprotective Activity of the Aqueous Seed Extract of Carica papaya

were investigated for their hepatoprotective activity against acute CCl4 hepatotoxicity in male rats. In addition, the kinetics of the hepatoprotection offered by the extract was also explored.
Table 1: Effect of graded oral doses (100 - 400 mg/kg/day) of the aqueous seed extract of Carica papaya (CPE) Linn. on serum ALT and AST in acute CCl4-induced hepatotoxic rats
_____________________________________________________________________ Group Treatment ALT AST (IU/L) (IU/L) _____________________________________________________________________ A 10 mL/kg DW/oral and IP, respectively 20.0 2.7 14.68 1.2 B 10 mL/kg DW/oral + 1.5 mL/kg/IP, CCl4 121.7 8.7c 163.67 11.8c e 71.5 3.6 85.87 22.3d C 100 mg/kg/day/oral + 1.5 mL/kg/IP, CCl4 48.3 5.2e 76.39 2.1e D 200 mg/kg/oral CPE + 1.5 mL/kg/IP, CCl4 E 400 mg/kg/oral CPE + 1.5 mL/kg/IP, CCl4 28.2 5.9f 36.6 14.9f _____________________________________________________________________

represents a significant increase at p<0.001 when compared to group A values while e and f represent significant decreases at p<0.01 and p<0.001 when compared to group B values.

In the current study, acute liver damage was chemically induced by CCl4 at a single intraperitoneal dose of 1.5 mL/kg of 20% CCl4 in olive oil. As shown in Table 1, induction with CCl4 was marked by a significant (p<0.001) rise in the serum levels of ALT and AST in the Group B rats when compared to untreated control (Group A). However, pretreatment with 100, 200, and 400 mg/kg/day of CPE significant (p<0.01, p<0.01, p<0.001, respectively) decreased the serum levels of these marker enzymes in dose-dependent manner when compared with Group B values. In addition, CPE ameliorated the earlier observed significant increase in the values of these enzymes for up to 3 hours post-exposure to CCl4 intoxication (Table 2).
Table 2: Time-course effect of 400 mg/kg of CPE on serum ALT and AST levels in CCl4 hepatotoxic rats
_____________________________________________________________________ Group Treatment ALT AST (IU/L) (IU/L) _____________________________________________________________________ F 10 mL/kg DW/oral and IP, respectively 20.0 2.7 14.7 1.2 121.7 8.7c 163.7 11.8c G 10 mL/kg DW/oral + 3 hr pre-1.5 mL /kg/IP, CCl4 H 400 mg/kg/day/oral + 3 hr pre-1.5 mL /kg/IP, CCl4 41.5 8.0e 61.7 13.2e 22.6 2.9f 13.9 1.0f I 400 mg/kg/oral CPE + 0 hr pre-1.5 mL/kg/IP, CCl4 31.5 5.7f 36.7 14.6f J 400 mg/kg/oral CPE + 1 hr post-1.5 mL/kg/IP, CCl4 35.4 5.7f 35.6 13.1f K 400 mg/kg/oral CPE + 3 hr post-1.5 mL/kg/IP, CCl4 139.7 2.7c 139.8 15.3c L 400 mg/kg/oral CPE + 6 hr post-1.5 mL/kg/IP, CCl4 _____________________________________________________________________

represents a significant increase at p<0.001 when compared to group F values while e and f represent significant decreases at p<0.01 and p<0.001 when compared to group G values, respectively

This is an indication that the extract protected against the deleterious effect of CCl4 on hepatic function and integrity for up to 3 hours post-CCl4 induction. As shown by the results, this dose reliably induced acute hepatic damage within 72 hours of its administration as evidenced by a marked elevation in the serum levels of the liver enzymes, ALT and AST, and a significant decrease in the circulating levels of total protein and albumin, which are in conformity with earlier independent reports of the deleterious biochemical effects of CCl4 on hepatic integrity (Fadhel and Amran, 2002; Weber et al., 2003; Rajesh and Latha, 2004; Nagano et al., 2007). It is well documented in the literature that CCl4 is metabolized by the mixed-function oxidase system in the endoplasmic reticulum of the liver to the highly reactive trichloromethyl radical and this reactive metabolite leads to autooxidation of the fatty acids present in the cytoplasmic membrane phospholipids and causes both functional and morphological distortion of the cell membrane (Recknagel and Glende, 1973). The hepatocyte membrane distortion is associated with membranal leakage of the hepatocyte cytosolic contents which is manifested by significant elevation of the serum marker enzymes of acute hepatocellular damage namely ALT and AST, and ALP as a marker for hepatobiliary damage (Bhattacharyya et al., 2003). However, of these marker enzymes, ALT is the most reliable. AST is known to be present in abundance in the cardiac muscles, skeletal muscles, kidneys and testes, and ALP abundant in the growing bone. Thus, any disease state affecting any of these extrahepatic tissues significantly elevates the serum levels of these enzymes (Friedman et al., 1996). In this study, extract pre-treatment significantly attenuated the acute elevation of these enzymes in dose-related manner, showing that CPE has hepatoprotective action. Carbon tetrachloride is a potent hepatotoxicant which has been documented to cause a wide range of metabolic derangements which include derangement in glucose, protein and lipid metabolism since these are

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synthesized in the liver (Bhattacharya et al., 2003). Thus, any condition affecting the metabolic function of the liver affects their hepatic syntheses as well as their circulating levels. The effect of graded oral doses of CPE pre-treatment and subsequent CCl4 treatment on the fasting blood glucose is shown in Table 3. As shown in Table 3, oral pretreatment of rats with 100, 200, and 400 mg/kg/day of CPE for 7 days caused significant (p<0.05, p<0.01, p<0.001, respectively) dose related lowering of FBG, with the most significant (p<0.001) hypoglycemic effect recorded for the 400 mg/kg/day dose. However, with CCl4 treatment, the hypoglycemic effect of the extract was potentiated also in dose-related fashion.
Table 3: Effect of graded oral doses of the aqueous seed extract of Carica papaya (CPE) on the fasting blood glucose (FBG) in pre- and post-CCl4 induced hepatotoxic rats
_____________________________________________________________________ 72 hr post-CCl4 treatment FBG Groups CCl4-pretreatment FBG (mg/dl) on (mg/dl) __________________________________ Day 1 Day 8 _____________________________________________________________________ A 64.3 3.3 73.8 2.1 69.0 3.4 B 76.2 2.6 72.5 5.3 48.0 2.8d d C 71.5 2.6 53.2 4.4 49.3 2.7d 50.0 3.5d 43.8 3.3e D 69.5 3.9 E 67.8 2.6 38.17 2.0e 32.2 1.7f _____________________________________________________________________

d, e, f

represent significant decreases at p<0.05, p<0.01 and p<0.001, respectively, when compared to negative control (group A) values A = 10 mL/kg DW/IP and oral routes, respectively B = 10 mL/kg DW/oral + 1.5 mL/kg/IP, CCl4 C = 100 mg/kg/oral CPE + 1.5 mL/kg/IP, CCl4 D = 200 mg/kg/oral CPE + 1.5 mL/kg/IP, CCl4 E = 400 mg/kg/oral CPE + 1.5 mL/kg/IP, CCl4

Table 4 shows the dose dependent preventive effect of graded oral doses of CPE against the deleterious effect of CCl4 intoxication on the serum lipids. To determine at which dose the extract exerts its maximum protective activity, different doses of the extract (100 mg/kg/day, 200 mg/kg/day and 400 mg/kg/day) were administered to 3 groups of rats for 7 days. As shown in Table 4, the protective effect was dose-related and the most significant (p<0.001) protective effect of the extract against the deleterious effect on the measured serum lipid parameters was at a dose of 400 mg/kg/day.
Table 4: Effect of graded oral doses of CPE on serum TG, TC, HDL-c, LDL-c and VLDL-c in CCl4-induced hepatotoxic rats
_____________________________________________________________________ Group TG TC HDL-c LDL-c VLDL-c (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) _____________________________________________________________________ A 108.0 29.6 61.5 5.5 21.5 5.2 18.7 4.2 21.3 5.8 B 114.7 18.7 88.3 9.7a 17.2 3.8d 40.2 4.8a 32.6 4.6a 12.0 3.3d 38.0 4.1a 24.9 4.0d C 111.8 16.8 75.7 10.4d 22.2 3.3a 38.3 3.0a 24.2 2.3d D 104.5 15.9 76.2 2.1d E 76.5 12.9d 77.2 1.7d 29.0 3.0a 26.2 3.7d 20.3 3.2d _____________________________________________________________________ a represents a significant increase at p<0.05 when compared to group A values while d represents a significant decrease at p<0.05 when compared to group B values. A = 10 mL/kg DW/IP and oral routes, respectively B = 10 mL/kg DW/oral + 1.5 mL /kg/IP, CCl4 C = 100 mg/kg/oral CPE + 1.5 mL/kg/IP, CCl4 D = 200 mg/kg/oral CPE + 1.5 mL/kg/IP, CCl4 E = 400 mg/kg/oral CPE + 1.5 mL/kg/IP, CCl4

Table 5 shows the time-course protective effect of the extract on the measured lipid parameters. As indicated in Table 5, 400 mg/kg of the extract neither offered prophylactic nor post-exposure hepatoprotection to the toxicant.

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Table 5: Time-course effect of 400 mg/kg of CPE on serum TG, TC, HDL-c, LDL-c and VLDL-c in CCl4-induced hepatotoxic rats
_____________________________________________________________________ Group TG TC HDL-c LDL-c VLDL-c (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) _____________________________________________________________________ F 108.0 29.6 61.5 5.5 21.5 5.2 18.7 4.2 21.3 5.8 G 114.7 18.7 88.3 9.7a 17.2 3.8 40.2 4.8b 32.6 4.6a H 130.7 10.3b 77.8 7.0a 10.7 3.3d 37.7 4.1b 29.5 4.8a I 149.5 11.6b 88.0 4.4a 17.8 2.1 31.9 4.4b 38.2 6.2a 81.5 10.3a 11.2 1.3d 34.3 7.4b 36.0 3.9a J 180.7 19.7c 89.5 11.1a 7.7 6.4e 46.9 8.3c 24.9 3.7d K 125.5 18.5a 88.7 4.6a 8.0 1.4e 46.8 2.8c 23.9 4.1d L 120.2 20.1a _____________________________________________________________________

and c represent significant increases at p<0.05, p<0.01, p<0.001 when compared to group A values while d and e represent significant decreases at p<0.05 and p<0.01, respectively when compared to group B values. F = 10 mL/kg DW/oral and IP, respectively G = 10 mL/kg DW/oral + 3 hr pre-1.5 mL/kg/IP, CCl4 H = 400 mg/kg/day/oral + 3 hr pre-1.5 mL/kg/IP, CCl4 I = 400 mg/kg/oral CPE + 0 hr pre-1.5 mL/kg/IP, CCl4 J = 400 mg/kg/oral CPE + 1 hr post-1.5 mL/kg/IP, CCl4 K = 400 mg/kg/oral CPE + 3 hr post-1.5 mL/kg/IP, CCl4 L = 400 mg/kg/oral CPE + 6 hr post-1.5 mL/kg/IP, CCl4

a, b

The liver is known to be involved in the syntheses of triglyceride and cholesterol which are synthesized from the substrate, acetyl CoA (produced through fatty acid oxidation) (West et al., 1966). In the present in vivo study, the graded oral doses and the time-course effect of the hepatoprotective activities of 400 mg/kg/day of CPE were evaluated using serum triglyceride, total cholesterol and cholesterol fractions as measuring parameters of liver functions since they are synthesized de novo in this organ. Results showed that induction with CCl4 reliably and significantly altered hepatic function in the treated rats by inducing significant increase in the serum triglyceride and cholesterol levels. However, oral pre-treatment with the extract caused nonsignificant (p>0.05), dose-related decrease in the serum levels of these lipids, an indication that the extract preserves the hepatic lipid syntheses dose-dependently.
Table 6: Effect of graded oral doses (100 - 400 mg/kg/day) of the aqueous seed extract of Carica papaya (CPE) Linn. on serum albumin (ALB) and total protein (TP) in acute CCl4-induced hepatotoxic rats
_____________________________________________________________________ Group Treatment ALB TP (mg/dl) (mg/dl) _____________________________________________________________________ A 10 mL/kg DW/oral and IP, respectively 2.6 0.8 4.7 0.3 1.1 0.1e 3.2 0.4d B 10 mL/kg DW/oral + 1.5 mL /kg/IP, CCl4 e C 100 mg/kg/day/oral + 1.5 mL/kg/IP, CCl4 1.5 0.1 3.9 0.2d D 200 mg/kg/oral CPE + 1.5 mL/kg/IP, CCl4 2.6 0.4 6.4 0.9b c E 400 mg/kg/oral CPE + 1.5 mL/kg/IP, CCl4 4.4 0.2 7.9 0.4c _____________________________________________________________________

and c represent significant increases at p<0.01 and p<0.001 when compared to group B values, respectively while d and e represent significant decreases at p<0.05 and p<0.01 when compared to group A values.

CCl4 induction was also associated with significant (p<0. 05, p<0.01) decreases in the serum levels of albumin and total protein (Table 6). The decrease could have resulted from the deleterious effect of the toxin on the hepatic syntheses of these proteins. However, pre-treatment with 100-400 mg/kg/day of CPE protected the liver from the deleterious effect of the toxin by ameliorating the decrease in the circulating levels of albumin and total protein in dose-related fashion. Again, the extract protected the protein synthetic function of the liver for up to 6 hours post-exposure (Table 7).

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Table 7: Time-dependent protective effect of 400 mg/kg of CPE on serum albumin (ALB) and total protein (TP) in CCl4 treated rats
_____________________________________________________________________ Group Treatment ALB TP (mg/dl) (mg/dl) _____________________________________________________________________ F = 10 mL/kg DW/oral and IP, respectively 2.6 0.8 4.7 0.3 1.1 0.1e 3.2 0.4d G = 10 mL/kg DW/oral + 3 hr pre-1.5 mL/kg/IP, CCl4 2.1 0.6a 6.1 0.2b H = 400 mg/kg/day/oral + 3 hr pre-1.5 mL/kg/IP, CCl4 3.8 0.6b 8.6 0.4c I = 400 mg/kg/oral CPE + 0 hr pre-1.5 mL/kg/IP, CCl4 J = 400 mg/kg/oral CPE + 1 hr post-1.5 mL/kg/IP, CCl4 3.2 0.8b 7.8 0.6c K = 400 mg/kg/oral CPE + 3 hr post-1.5 mL/kg/IP, CCl4 2.7 0.7a 6.6 0.5b 4.7 0.4a L = 400 mg/kg/oral CPE + 6 hr post-1.5 mL/kg/IP, CCl4 1.0 0.1e _____________________________________________________________________

a, b and c represent significant increases at p<0.05, p<0.01 and p<0.001, respectively, when compared to G values while d and e represent significant decreases at p<0.05 and p<0.01, respectively, when compared to group F values.

Figure 1. A representative section of an untreated control rat liver administered 10 mL/kg and 1 mL/kg of distilled water orally and intraperitoneally, respectively showing the central hepatic vein (CV) and normal hepatocytes (N) distribution (x400 magnification, hematoxylin and eosin stain)

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Figure 2. A sectional view of 1.5 mL/kg CCl4 in 20% in olive oil treated rat liver showing the severely congested hepatic central vein (CCV) and diffuse fat globules deposits (FG) in hepatocytes and bile pigment staining (BS), indicating diffuse hepatic steatosis (fatty hepatic degeneration) and destructive cholangiostasis (x400 magnification, hematoxylin and eosin stain)

Figure 3. A sectional view of rat liver pretreated with 400 mg/kg/day CPE for 7 days before induction with 1.5 mL/kg CCl4 in 20% in olive oil showing mildly congested hepatic central vein (CCV), mild bile ductal proliferation (BDP), bile stains (BS) and normal hepatocytes (N) indicating mild destructive cholangitis (x400 magnification, hematoxylin and eosin stain)

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Figure 4. A sectional view of rat liver pretreated with 400 mg/kg/day CPE 3 hr before induction with 1.5 mL/kg CCl4 in 20% in olive oil showing congested hepatic central vein (CCV), diffuse fat globule deposits (FG), bile stains (BS), and diffuse hydropic degeneration of the hepatocytes (HD) indicating diffuse hepatic necrosis and destructive cholangitis (x400 magnification, hematoxylin and eosin stain)

Figure 5. A sectional view of 400 mg/kg/day CPE co-treated with 1.5 mL/kg CCl4 in 20% in olive oil treated rat liver showing mildly congested hepatic central vein (CCV), mild bile ductal proliferation, bile stains and normal hepatocytes (N) (x400 magnification, hematoxylin and eosin stain)

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Hepatoprotective Activity of the Aqueous Seed Extract of Carica papaya

Figure 6. A sectional view of a rat liver post-treated with 400 mg/kg/day CPE 1 hr after induction with 1.5 mL/kg CCl4 in 20% in olive oil showing mildly congested central hepatic vein (CCV) with inflammatory cell infiltration (IC), perivenular bile stains (BS) and cloudy swelling of hepatocytes (CN) (x400 magnification, hematoxylin and eosin stain).

Figure 7. A sectional view of a rat liver post-treated with 400 mg/kg/day CPE 3 hrs after induction with 1.5 mL/kg CCl4 in 20% in olive oil showing mildly congested central hepatic vein (CCV) with mild inflammatory cell infiltration (IC), bile stain (BS) and hydropic degeneration of hepatocytes (HD) (x400 magnification, hematoxylin and eosin stain).

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Figure 8. A cross-sectional view of a rat liver post-treated with 400 mg/kg/day CPE 6 hrs after induction with 1.5 mL/kg CCl4 in 20% in olive oil showing moderate-to-severe central hepatic vein congestion (CCV), perivenular bile stains (BS) and fat globule deposits (FG) indicating fatty hepatic necrosis with destructive cholangitis (x400 magnification, hematoxylin and eosin stain).

It has been earlier reported in the literature that decreasing the metabolic activation of carbon tetrachloride, the antioxidant activity, prevention of generation of reactive oxygen species and scavenging of generated free radicals or by combination of these are important mechanisms in the protection against CCl4-induced hepatic lesion (Yutin et al., 1990; Bhattacharyya et al., 2003). Although the antioxidant status of the extract in the liver was not investigated in the current study, the biochemical and histological findings thus obtained suggest that the extract may be mediating its protective effects either by decreasing the metabolic activation of carbon tetrachloride, or by acting as a chain-breaking antioxidant for scavenging free radicals or by a combination of these effects. In an earlier study, the presence of alkaloids, flavonoids, saponin, tannin, anthraquinones, and anthacyanosides in CPE was reported (Adeneye and Olagunju, 2009). Also, previous independent studies have reported that the protective actions of hepatoprotective medicinal plants are mediated by their flavonoids or alkaloids components or by their combination via antioxidant and free radicals scavenging activities (Lanhers et al., 1991; Adeneye et al., 2008). The presence of these active biological principles may thus be accounting for the biological effect of CPE and could be via antioxidant and/or free radicals scavenging activities. However, further studies will still be required to substantiate this. The overall results obtained in this study suggest that the extract possesses protective effect on the deleterious effect of CCl4 in both dose- and time-related fashion. Maximum protection was offered by the extract when it was administered at the single, daily oral dose of 400 mg/kg body weight for 7 days before CCl4 induction. References Adeneye AA, Olagunju JA, Elias SO, Olatunbosun DO, Mustafa AO, Adeshile OI, Ashaolu AO, Laoye TA, Bamigboye AO, Adeoye AO. 2008. Protective activities of the aqueous root extract of Harungana madagascariensis in acute and repeated acetaminophen hepatotoxic rats. International Journal of Applied Research in Natural Products 3: 29-42. Adeneye AA, Olagunju JA. 2009. Preliminary hypoglycemic and hypolipidemic activities of the aqueous seed extract of Carica papaya Linn. in Wistar rats. Biology and Medicine 1(1): 1-10. Ajani EO, Shallie PD, Adegbesan BO, Salau BA, Akinpelu A. 2007. A study of the hepatoprotective effect of Garcinia kola water extract in amodiaquine and artesunate treated rats. Nigerian Journal of Health and Biomedical Sciences 6(2): 9-15. Akah PA, Oli AN, Enwerem NM, Gamaniel K. 1997. Preliminary studies on purgative effect of Carica papaya root extract. Fitoterapia 68(4): 327-331. Akintonwa A, Essien AR. 1990. Protective effects of Garcinia kola seed extract

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