Modulatory potentials of aqueous leaf and unripe fruit extracts of Carica papaya Linn. (Caricaceae) against carbon tetrachloride and acetaminophen-induced hepatotoxicity in rats.

INTRODUCTION: Carica papaya Linn is used in a traditional medicine for hepatobiliary disorders. This study investigated the hepatomodulatory effects of aqueous extracts of C. papaya leaf (CPL) and unripe fruit (CPF) at doses of 100 and 300 mg/kg on carbon tetrachloride (CCl4) and acetaminophen (ACM)-induced liver toxicities in rats.
MATERIALS AND METHODS: Rats were administered CCl4 (3 ml/kg in olive oil, i.p.) followed by oral administration of CPL and CPF at 2, 6 and 10 h intervals. The ACM model proceeded with the same method but inclusive of animals treated with N-acetyl cysteine (3 ml/kg i.p). At the end of the study, serum levels of liver biomarkers and antioxidant enzymes were assessed and histology of the liver tissues determined.
RESULTS: There was a significant (P < 0.05) reduction in CCl4 and ACM-induced increases in serum levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and direct bilirubin at 100 and 300 mg/kg, respectively. The levels of catalase (CAT), superoxide dismutase and reduced GSH were decreased in both models with corresponding significantly (P < 0.05) elevated level of malondialdehyde. However, these antioxidant enzymes were significantly (P < 0.05) increased in CPL and CPF-treated rats. Histopathological assessment of the liver confirmed the protective effects of CPL and CPF on CCl4 and ACM-induced hepatic damage evidenced by the normal presentation of liver tissue architecture.
CONCLUSION: These results indicate that aqueous extracts of C. papaya may be useful in preventing CCl4 and ACM-induced liver toxicities.

INTRODUCTION

Hepato-biliary disorders especially those induced by drugs are a worldwide problem [1]. More than a thousand drugs of the modern pharmacopoeia can induce liver injury with different clinical presentations [2,3]. In the most severe cases, drug-induced liver injury (DILI) can require liver transplantation or lead to the death of the patient [4] and can lead to the withdrawal of drugs from the market or earlier during clinical trials, thus causing huge financial losses [5].

In Nigeria, there is presently very little data about the etiology of DILI, but popular over-the-counter medications (OTC) especially acetaminophen (ACM), is a frequent culprit. Thus, the study of hepatotoxicants as well as the search for hepatoprotectants is on the rise.

Carbon tetrachloride (CCL4) has been proven to induce toxic effects in various organs on exposure to it. It 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 [6]. The reactive species 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 [7,8].

Carica papaya Linn (Pawpaw plant) is a perennial, herbaceous plant with copious milky latex, broad leaves and grows to heights reaching 6-10 m with large succulent fruits and a complicated means of reproduction [9]. The anti-sickling [10], antihelmintic [11], anti-diabetic [12], and anti-cancer [13] properties of the Pawpaw plant have been demonstrated in previous works.

The boiled leaves of C. papaya are used in African traditional medicine in combination with leaves of Azadirachta indica, Cymbopogon citratus, Psidium guajava, and stem bark of Alstonia boonei boiled together and the hot infusion drunk as one wine glass full thrice daily in the treatment of malaria [11]. Its fresh leaves are also efficacious in the treatment of gonorrhea, syphilis and amoebic dysentery [11]. The milky juice of the unripe fruit is a powerful abortifacient, antihelmintic for roundworms, stomach disorders and enlargement of liver and spleen [11]. The seeds are also used as a vermifuge [11].

The present study was carried out to investigate the hepatoprotective effects of aqueous extracts of unripe fruit pulp and leaves of C. papaya as well as elucidate its possible mechanism of hepatoprotection in rats.

MATERIALS AND METHODS

Plant Material

Collection and identification of plant parts

Freshly harvested leaves and mature, but unripe fruits of C. papaya were purchased from the Mushin Market in Lagos, Nigeria. Plant identification was carried out by Oyebanji and authenticated by Odewo, both of the Department of Botany, University of Lagos, Lagos, Nigeria and stored at the University Herbarium with LUH 5748 assigned as the reference number.

Drugs and Chemicals

CCl4 (JHD chemicals, China), KCl (GPR, Germany), phosphate buffer (BDH, Germany), Sodium carbonate buffer, hydrogen peroxide (Sigma-Aldrich, Germany), Dichromate-acetic reagent, polyethylene glycol (PEG) (Sigma-Aldrich, Germany), Normal saline (Unique pharmaceuticals, Lagos), ACM (May and Baker pharmaceuticals, Nigeria), N-acetyl cysteine (Zhangjiagang Huachang pharmaceuticals, China). All other reagents used were of analytical grade.

Extract Preparation

Leaves of C. papaya were air-dried at room temperature (24±2°C) for 3 weeks to remove moisture. The dried leaves were weighed, ground into powder and macerated in distilled water in the ratio: 1:2 (200 g of leaf:400 ml of water). The mixture was then boiled in a beaker placed on a hot plate for 1 h, forming a brownish solution in the process. Percentage yield was found to be 12.5%.

Yield = weight of dry crude extract obtained (g)/weight of initial dry leaves (g) × 100%

Also fresh, mature but unripe fruits of C. papaya were washed, peeled, cut to small pieces and soaked in 1 L of distilled water at a ratio of 1:1 (100 g fruit:100 ml water) at room temperature of 25°C, covered with a glass lid according to the method of Oduola et al. [10].

After 72 h, the solution was filtered using a clean piece of muslin cloth and filter paper. For the leaf and fruit extracts, the filtrate was oven-dried at 40°C in pre-weighed beakers and the concentrate refrigerated at 4°C until needed.

Experimental Animals

Sprague-Dawley rats of both sexes weighing between 150 g and 180 g were purchased from the Laboratory Animal Centre of the Lagos State University College of Medicine and were acclimatized to the laboratory conditions for 2 weeks. They were housed in clean propylene cages and fed with standard rodent pellets (Nigeria Institute of Medical Research, Yaba) and water was provided ad libitum. The experimental protocol adopted in this study was approved by the Experimentation Ethics Committee on Animal Use of the College of Medicine, University of Lagos, Nigeria and was in accordance with the United States National Institutes of Health Guidelines for Care and Use of Laboratory Animals in Biomedical Research [14].

Experimental Design

CCl4 hepatotoxicity

Rats (6 groups, 6 per group) were randomly allotted to several groups: Groups I and II served as controls. Group I was administered 10 ml/kg olive oil (vehicle i.p.) plus 1 ml/kg normal saline orally at 2, 6 and 10 h. Rats in Group II were administered 3 ml/kg CCl4 (20% v/v in olive oil i.p.) plus oral administration of normal saline at 2, 6 and 10 h. Group III were administered 3 ml/kg CCl4 (20% v/v in olive oil i.p.) plus an oral dose of 100 mg/kg C. papaya leaf (CPL) extract at 2, 6 and 10 h. Group IV; 3 ml/kg CCl4 (20% v/v in olive oil i.p.) plus oral administration of 300 mg/kg CPL extract at 2, 6 and 10 h. Group V; 3 ml/kg CCl4 (20% v/v in olive oil i.p.) plus oral administration of fruit extract at 100 mg/kg in 2, 6 and 10 h. Group VI; 3 ml/kg CCl4 (20% v/v in olive oil i.p.) plus oral administration of the fruit extract at 300 mg/kg in 2, 6 and 10 h.

ACM-induced Hepatotoxicity

ACM was dissolved in an equal volume of PEG 400 and normal saline solution. Rats (7 groups, 6 per group) were allotted randomly as follows: Groups I and II served as control. Group I rats were administered 10 ml/kg PEG (400/NS solution, per oral) plus 1 ml/kg NS at 2, 6 and 10 h. Group II; 600 mg/kg ACM per oral, plus 3 ml/kg NS at 2, 6 and 10 h. Groups III and IV were administered 600 mg/kg ACM per oral plus oral administration of 100 mg/kg and 300 mg/kg leaf extract of C. papaya at 2, 6 and 10 h, respectively. Groups V and VI were administered 600 mg/kg ACM a oral plus oral administration of 100 mg/kg and 300 mg/kg fruit extract of C. papaya at 2, 6 and 10 h, respectively. Rats in Group VII were administered 10 ml/kg of PEG (400/NS solution, per oral) plus 3 ml/kg N-acetyl cysteine intraperitoneally at 2, 6, and 10 h.

All animals were restricted from feeding for 24 h after oral administration of CCl4 and ACM solution before being sacrificed for assessment of liver histology, as well as serum biochemical and oxidative stress markers.

Collection of Liver for Hepatic Tissue Antioxidant Enzyme Assay

After blood collection through the cardiac puncture, a deep longitudinal incision was made into the ventral surface of the rat abdomen. The livers were identified and carefully dissected out from each rat. The right lobe of the liver was rinsed in ice-cold 1.15% KCl solution and stored in a clean sample bottle in ice.

Homogenization of Liver Tissue

The liver was washed in an ice cold 1. 15% KCl solution blotted and weighed. It was then homogenized with 0.1 M phosphate buffer (pH 7.2) and blended with laboratory sand (acid washed sand) in a mortar. The resulting homogenate was centrifuged at 2500 rpm for 15 min and the supernatant decanted and stored at −20°C until used.

Assessment of Serum Hepatic Biomarkers

At 24 h post-CCl4 or ACM administration, experimental rats were anaesthetized by putting each one in a glass jar containing ether-soaked cotton wool for about the 30s. Blood was obtained retroorbitally, through a heparinized capillary tube into a sample bottle. Each sample was centrifuged at 2500 G for 20 min to separate sera.

Samples were analyzed for alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DB), and uric acid level. The activity of serum ALP was determined at 405 nm using a standard method [15], serum ALT at 340 nm [16]. Uric acid levels were measured using the urease cleavage Berthlot’s reaction [17]. The colometric method of Malloy and Evelyn [18], (Sulfanilic acid method) was used for the estimation of serum bilirubin.

Serum AST and ALT Determination

The method of Reitman and Frankel [16] was used. Into a test tube, 0.1 ml substrate (D, L-aspartate, 0.2 mol/L and α-ketoglutaric acid, 1.8 mmol/L in phosphate buffer, pH 7.5) solution was pipetted and placed in a 37°C water bath to warm 0.2 ml plasma was added and shaken gently to mix. Exactly 1 h after adding plasma, 1.0 ml color reagent (2, 4-dinitrophenylhydrazine approximately 20 mg/100 ml, in 10% HCl solution) was added and mixed gently and left at room temperature (18-26°C). 20 min after adding color reagent, 10 ml 0.40 N sodium hydroxide solution was added and mixed by inversion. 5 min after, absorbance was read at 340 nm using water as a reference. AST activity in Sigma-Frankel units/ml was determined from the calibration curve. The same procedure was carried out for ALT except that procedures were started 30 min after starting AST. Substrate for ALT was L-alanine (0.2 mol/L) and α-ketoglutaric acid (1.8 mmol/L) in phosphate buffer, pH 7.5.

Serum ALP Determination

ALP was determined using the colorimetric endpoint method of Tietz et al. [15] and adapted by Teco Diagnostic Kits. The principle is based on the fact that ALP acts on the AMP-buffered sodium thymolphthalein monophosphate. The addition of an alkaline reagent stops enzyme activity and simultaneously develops a blue chorogem, which is measured photometrically. For each sample 0.5 ml of ALP, substrate was dispensed into labeled test tubes and equilibrated to 37°C for 3 min. At timed intervals 0.5 ml of standard, control and sample were added to their respective test-tube and mixed gently deionized water was used as blank. The samples were incubated for exactly 10 min at 37°C following the same sequences, 2.5 ml of ALP color developer at timed intervals were added. The wavelength of the spectrometer was set at 590 nm.

Assessment of Serum and Tissue Antioxidant Enzymes

The reduced glutathione (GSH) content of liver tissue as non-protein sulphydryls was estimated according to the method described by Sedlak and Lindsay [19]. Superoxide dismutase (SOD) activity was determined using the methods of Sun and Zigma [20]. Serum catalase (CAT) activity was determined according to the method of Beers and Sizer as described by Kakker et al. [21] and Usoh et al. [22]. CAT activity was determined by adopting the methods of Aksenes and Njaa [23]. Malondialdehyde (MDA) production was estimated using the methods as described by Buege and Aust [24].

Determination of Liver Tissue SOD Activity

SOD activity was determined by its ability to inhibit the auto-oxidation of epinephrine by the increase in absorbance at 480 nm as described by Sun and Zigma [20]. The reaction mixture (3 ml) contained 2.95 ml 0.05 M sodium carbonate buffer pH 10.2, 0.02 ml of liver homogenates, and 0.03 ml of epinephrine in 0.005 N HCl was used to initiate the reaction. The reference curette contained 2.95 ml buffer, 0.03 ml of substrate (epinephrine), and 0.02 ml of water. Enzyme activity was calculated by measuring the change in absorbance at 480 nm for 5 min.

Determination of Liver Tissue CAT Activity

Hepatic tissue CAT activity was determined according to Kakkar et al. [21] by measuring the decrease in absorbance at 240 nm due to the decomposition of H2O2 in UV recording spectrophotometer. The reaction mixture (3 ml) contained 0.1 ml of serum in phosphate buffer (50 mM, pH 7.0) and 2.9 ml of 30 mM of H2O2 in the phosphate buffer pH 7.0. An extinction coefficient for H2O2 at 240 nm of 40.0 M 1 cm 1 according to Aebi [25] was used for calculation. The specific activity of CAT was expressed as moles of H2O2 reduced per minute per mg protein.

Determination of Liver Tissue Reduced GSH Activity

The reduced GSH content in the liver tissue was estimated according to the method described by Sedlak and Lindsay [19]. To the homogenate 10% TCA was added and centrifuged. About 1 ml of the supernatant was treated with 0.5 ml of Ellman’s reagent (19.8 mg of 5,5-dithiobisnitro benzoic acid in 100 ml of 0.1% sodium nitrate) and 3.0 ml of phosphate buffer (0.2 M, pH 8.0). The absorbance was read at 412 nm.

Determination of Liver Tissue MDA Activity

MDA an index of lipid peroxidation was determined using the method of Buege and Aust [25]. 1 ml of supernatant was added to 2 ml of (1:1:1 ratio) tricarboxylic acid (TCA)-thiobarbituric acid (TBA)-HCl reagent (TBA 0.37%, 0.24 N HCl and 15% TCA) TCA, TBA, reagent boiled at 100°C for 15 min, and allowed to cool. The flocculent material was removed by centrifuging at 3000 rpm for 10 min. The supernatant was removed, and the absorbance was read at 532 nm against a blank. MDA was calculated using the molar extinction for MDA TBA complex of 1.56 × 105/m/cm.

Histopathological Assessment

The method of Baker and Silverston [26] was employed for the preparation of liver tissue before examination under the light microscope at a magnification of ×40.

Statistical Data Analysis

Data were calculated as mean ± standard error of mean. The mean of each treated group was compared for significance at P < 0.05, using analysis of variance, followed by Dunnet’s post hoc multiple comparison tests. The Graph pad prism 6.0 was used for all statistical analysis.

RESULTS

Effects of Leaf and Fruit Extracts of C. papaya L. on Serum Hepatic Enzymes in CCl4-Induced Hepatotoxicity in Rats

The results obtained with the intraperitoneal administration of 3 ml/kg, 20% v/v CCl4 and aqueous preparations of plant extracts CPL and CPF showed significant (P < 0.05) decrease in all the serum enzymes in comparison with the control (CCl4 alone). There was a decrease in ALT (126±11.92), AST (55.6±1.7), ALP (54.0±10.80), uric acid (138.7±21.80), and DB (1.5±0.20) at 100 mg/kg CPL compared with the control, indicative of attenuation in hepatocyte destruction. However, at 300 mg/kg CPL, serum liver markers did not change significantly for ALT (128.3±1.80), AST (61.6±7.20), ALP (94.8±3.30), uric acid (115.9±10.20), and DB (1.3±0.08). There was no significant difference in serum enzyme levels between aqueous leaf extract (CPL) and unripe fruit extract (CPF) of C. papaya at all the doses (100 mg/kg and 300 mg/kg) however, at each of the doses, serum enzymes were slightly lower with the leaf extract (CPL) for serum liver markers ALT, AST, ALP, and DB when compared with the fruit extract (CPF) [Figures 1-5].

Figure 5

Effects of leaf and fruit extracts of Carica papaya L. on levels of direct bilirubin in carbon tetrachloride-induced hepatotoxicity in rats. Values are mean±SEM (n=5). ap < 0.05 vs CCl4, bp < 0.05 vs olive oil (One Way ANOVA followed by Dunnett’s posthoc multiple comparison test)

Effects of leaf and fruit extracts of Carica papaya L. on levels of alanine aminotransferase in carbon tetrachloride-induced hepatotoxicity in rats. Values are mean±SEM (n=5). ap < 0.05 vs CCl4, bp < 0.05 vs olive oil (One Way ANOVA followed by Dunnett’s posthoc multiple comparison test

Effects of leaf and fruit extracts of Carica papaya L. on levels of aspartate aminotransferase in carbon tetrachloride-induced hepatotoxicity in rats. Values are mean±SEM (n=5). ap < 0.05 vs CCl4, bp < 0.05 vs olive oil (One Way ANOVA followed by Dunnett’s posthoc multiple comparison test)

Effects of leaf and fruit extracts of Carica papaya L. on levels of alkaline phosphatase in carbon tetrachloride-induced hepatotoxicity in rats. Values are mean±SEM (n=5). ap < 0.05 vs CCl4, bp < 0.05 vs olive oil (One Way ANOVA followed by Dunnett’s posthoc multiple comparison test).

Effects of leaf and fruit extracts of Carica papaya L. on levels of uric acid in carbon tetrachloride-induced hepatotoxicity in rats. Values are mean±SEM (n=5). ap < 0.05 vs CCl4, bp < 0.05 vs olive oil (One Way ANOVA followed by Dunnett’s posthoc multiple comparison test)

Effects of Leaf and Fruit Extracts of C. papaya L. on Serum Antioxidant Enzymes in CCl4-Induced Hepatotoxicity in Rats

The results obtained with CCl4 administration at 3 ml/kg intraperitoneally showed a significant (P < 0.05) reduction in serum levels of GSH, SOD and CAT but increased levels of MDA when compared with olive oil treated rats. At 100 mg/kg, CPL showed significant (P < 0.05) increase in GSH (1.52 ± 0.20) compared with CCl4 treated (0.59 ± 0.10), SOD (3.87 ± 1.26) compared with CCl4 treated (2.53 ± 0.40) as well as lower levels of MDA (0.04 ± 1.50) also compared with CCl4 treated (0.07 ± 0.00) rats. However, there were no significant (P < 0.05) changes in serum antioxidant enzymes in rats treated with leaf (CPL) and fruit extracts (CPF) at 100 mg/kg and 300 mg/kg respectively compared to those that were given CCl4 alone.

Effects of Leaf and Fruit Extracts of C. papaya L. on Liver Antioxidant Enzymes in CCl4-Induced Hepatotoxicity in Rats

The rats treated with 3 ml/kg CCl4 in olive oil alone showed significant reductions (P < 0.05) in GSH, SOD, CAT but an increase in levels of MDA when compared with olive oil treated rats. At 300 mg/kg, CPL showed significant (P < 0.05) increase in GSH (4.7 ± 0.6) compared with CCl4 treated (1.52 ± 0.17), SOD (3.03 ± 3.17) compared with CCl4 treated (1.65 ± 0.33) and CAT (16.05 ± 1.65) also compared with CCl4 treated rats (3.40 ± 0.81). The same trend was also observed for the fruit extract CPF with GSH compared with CCl4 treated, SOD compared with CCl4 treated and CAT compared with CCl4 treated rats.

Effects of Leaf and Fruit Extracts of C. papaya L. on Serum Hepatic Enzymes in ACM-Induced Hepatotoxicity in Rats

The rats administered with 600 mg/kg, p.o. ACM showed significant (P < 0.05) increases in serum enzymes ALT, ALP, AST as well as DB when compared to rats who received no extracts. At 300 mg/kg CPL, serum liver markers were less expressed with ALT (53 ± 3.53), AST (15.25 ± 2.62), ALP (79.83 ± 26.40) and DB (1.60 ± 0.07). There was also significant (P < 0.05) reduction in serum enzymes in animals treated with the highest dose (300 mg/kg) of CPF with ALT (52.25 ± 5.26), AST (18.0 ± 2.34), ALP (50.41 ± 11.42) and DB (1.47 ± 0.16) compared to the control. There was no significant difference (P > 0.05) between rats treated with N-acetyl cysteine and the rats who received no extracts [Table 1].

Table 1

Effects of leaf and fruit extracts of C. papaya on serum hepatic enzymes in ACM-induced hepatotoxicity in rats

Effects of Leaf and Fruit Extracts of C. papaya L. on Serum Antioxidant Enzymes in ACM-Induced Hepatotoxicity in Rats

ACM at 600 mg/kg, caused significant (P < 0.05) reduction in the levels of GSH, CAT, and SOD, but increases in MDA when compared with the rats who received no extracts. However, there was no significant difference (P > 0.05) in serum oxidative markers in groups treated with plant extracts of C. papaya CPL and CPF. However, at 300 mg/kg CPL, there was a slight increase in SOD (3.33 ± 0.13) and CAT (10.78 ± 0.57). These slight elevations were, however, lower compared with the N-acetyl cysteine-treated group with GSH (0.52 ± 0.05), SOD (2.32 ± 0.13) and CAT (10.06 ± 0.56) [Table 2].

Table 2

Effects of leaf and fruit extracts of C. papaya on serum antioxidant enzymes in ACM-induced hepatotoxicity in rats

Effects of Leaf and Fruit Extracts of C. papaya L. on Liver Antioxidant Enzymes in ACM-Induced Hepatotoxicity in Rats

ACM at 600 mg/kg, p.o., significantly (P < 0.05) decreased organ levels of GSH, SOD, CAT as well as increased MDA in treated rats compared with the rats who received no extracts. N-acetylcysteine and CPL and CPF, both at 100 mg/kg and 300 mg/kg reversed this effect significantly (P < 0.05). There was, however, no significant difference (P > 0.05) between N-acetylcysteine-treated rats and the rats who received no extracts. However, the N-acetyl cysteine-treated group showed slightly higher values of both GSH (9.71 ± 1.12) and CAT (14.70 ± 4.07) compared with those treated with plant extracts CPL and CPF at all doses [Table 3].

Table 3

Effects of leaf and fruit extract of C. papaya on liver antioxidant enzymes in ACM-induced hepatotoxicity in rats

Histology

The histology results revealed the hepatoprotective effect of the liver architecture at all doses in comparison with control (CCl4) [Figure 6 and Table 4].

Figure 6

(a). Olive oil with no cellular abnormalities (b). CCl4 showing congested vascular channels, fatty lesions and necrotic formation (c). 300 mg/kg CPL and 20% CCl4 with no cellular abnormalities (d). 300 mg/kg CPF + CCl4 with no cellular abnormalities (e). 600 mg/kg ACM showing fatty lesions and necrotic formation (f). NAC with no cellular abnormalities (g). 600 mg/kg ACM + 300 mg/kg CPL showing mild necrosis (h). 600 mg/kg ACM + 300mg/kg CPF with no cellular abnormalities

Table 4

Histological presentations of the liver

DISCUSSION

ACM, a common OTC drug, widely used for its analgesic and antipyretic purposes is a common cause of acute hepatocellular damage which could be lethal if not treated. It is one of the common causes of poisoning worldwide [27]. Oxidative necrosis and depletion of liver stores of GSH have been implicated in the mechanism of action of ACM poisoning [27-29].

It is important, therefore, to evaluate plant extracts that can be used for the improved treatment of hepatic failure caused by severe oxidative stress and necrosis [30,31]. A estimation of serum enzymes is a useful quantitative marker of the extent and type of hepatocellular damage. Increases in serum AST, ALT, ALP, and lactate dehydrogenase (LDH) levels have been attributed to damage of the structural integrity of the liver because these enzymes are cytoplasmic in location and are released into the circulation after autolytic breakdown or cellular necrosis. Marked release of AST, ALT, ALP, and LDH into the circulation indicates severe damage to hepatic tissue membranes during CCl4 intoxication [32].

Tarkang et al. [33] reported the oral LD50 of C. papaya aqueous leaf extract as >5 g/kg. Oduola et al. [34] had also reported the oral LD50 of the unripe fruit extract of C. papaya to be 2.52 g/kg. This suggests that the extracts are safe on acute exposure. In this study, extracts of C. papaya (leaf and unripe fruits) decreased the serum level of hepatic biomarkers in rats pre-treated with CCl4 compared to control animals administered CCl4 alone, indicating the hepato-protective potential of the extracts. This is in agreement with the findings by Adeneye et al. [35].

In ACM toxicity, the metabolite NAPQI is known to deplete GSH levels, thereby inducing hepatic necrosis [36]. More so, oxidative stress and lipid peroxidation which are early events of free radical generation have been implicated in ACM metabolism [37]. Extracts of C. papaya (CPL and CPF) reversed the depletion of GSH, SOD, CAT induced by ACM toxicity and significantly decreased CCl4-induced elevation in levels of MDA compared to the control. It has been stated that SOD and CAT are the most important enzymes involved in ameliorating the effects of oxygen metabolism [38,39].

Treatment with the extracts led to significant increases in GSH, SOD and CAT compared to ACM treated rats. Current evidence suggests that intracellular GSH plays an essential role in ACM detoxification and in the prevention of ACM-induced toxicity in the liver and the kidney [40]. The effect of the raised antioxidant enzymes may be responsible for the prevention of tissue damage seen with the presentation of normal architecture in the liver of ACM treated rats. Treatment with N-acetyl cysteine, which is the standard for treating ACM toxicity [41,42] showed no significant difference compared to the extracts of C. papaya indicating the potential of the extracts (CPL and CPF) in conferring hepatoprotection against ACM toxicity.

Several medicinal plants have been found to inhibit xenobiotic-induced hepatotoxicity as well as nephrotoxicity due to their potent anti-oxidant or free radical scavenging effects [43,44]. The leaves of the pawpaw plant have been shown to contain many active components such as papain, chymopapain, cystatin, α-tocopherol, ascorbic acid, flavonoids, cyanogenic glucosides, and glucosinolates that can increase the total antioxidant power in blood and reduce lipid peroxidation level [45]. Furthermore, alkaloids have been reported to strongly inhibit lipid peroxidation induced in isolated tissues in-vitro via their antioxidant activity [46]. The leaves and unripe fruits of C. papaya have been found to contain saponins, tannins, flavonoids, cardiac glycosides, alkaloids and anthraquinones [47,48]. The protection offered by the extract may also be due to its high content of flavonoids, ascorbic acid and alkaloids contained in the extract which may account for the reducing and free radical scavenging properties of the extract. The liver protective activity elicited by the extract might be due to its ability to activate antioxidant enzymes suppressed by ACM-induced toxicity [49,50]. The results from this study indicate that extracts of C. papaya have hepatoprotective properties which could be harnessed in the treatment of the acute liver injury.

In CCl4 toxicity, there was marked increase of hepatic enzymes; ALT, AST ALP and uric acid concentration as well as DB [51]. There is a link between hyperuricemia and some hepatic disorders. Serum uric acid level is significantly associated with non-alcoholic fatty liver disease and elevated serum uric acid level has been shown to be an independent risk factor for non-alcoholic fatty liver disease [52-54]. Rats treated with CCl4 showed significant increases in ALT, ALP and AST compared to control. The increased serum levels of hepatic biomarkers have been attributed to liver injury because these enzymes are found in the cytoplasmic area of the cell and are released into circulation in the event of cellular damage [55,56]. Zimmerman et al. [57] stated that CCl4 induced the increase of serum ALT and AST levels as a result of cell membrane and mitochondrial damage in liver cells. Khan et al. [58], Wang et al. [59], Mehmetcik et al. [60], Arici and Cetin, [61] had all reported that these enzyme activities were significantly elevated after CCl4 treatment.

Histological examination of liver tissue in rats treated with CCl4 alone expressed hepatocellular necrosis, fatty cell accumulation, inflammatory cell infiltration and other manifestations that are consistent with acute liver damage. This is in agreement with the findings of Khan and Al-Zohairy [62]. Extracts of C. papaya significantly reversed the increased levels of liver biomarkers at 100 mg/kg and 300 mg/kg compared to animals treated with CCl4 alone. This hepatoprotective effect, however, was not dose-dependent.

SOD, GSH, CAT were significantly decreased with MDA significantly increased in CCl4 treated rats when compared with untreated animals. However, in rats treated with aqueous plant extracts of C. papaya, there was a significant increase in the levels of these antioxidant enzymes, especially in the liver. This effect is also in coherence with the findings by Wang et al. [59] and Slater [63]. The depletion of antioxidant enzymes is an indicator of liver damage in CCl4 toxicity. Further examination of histological slides prepared from rats treated with CCl4 confirmed hepatic damage with hepatocellular necrosis and inflammatory lesions compared to animals treated with extracts of C. papaya which were seen to be of remarkably less severity with regards to hepatocellular necrosis.

CONCLUSION

Decreased concentration of liver serum biomarkers such as ALP, ALT and AST and increased activities of various antioxidant enzymes indicates that the aqueous extracts of C. papaya Linn may be able to protect against drug and chemical induced acute liver pathologies through improvement of antioxidant indices. This may explain its use in traditional medicine for the treatment of hepato-billiary disorders. A further study is required to elucidate the fraction and constituents of the extract that produces this hepatoprotective effect.