INTRODUCTION:

Liver is a vivacious organ that functions as metabolic centre for various nutrients such as carbohydrates, proteins and lipids [1]. It also takes part in metabolism of drugs, xenobiotics and excretion of their waste metabolites from the body and protects the organs against various toxicants [2].

It is well established that liver injury is caused by various toxicants [3] such as certain chemotherapeutic agents (anti-tubercular drugs, anti-HIV drugs and some antibiotics), carbon tetrachloride, thioacetamide, chronic alcohol consumption (e.g., liver cirrhosis) and microbes.

As herbal based therapeutic drugs has been popularized worldwide for the treatment of liver disorders by leading pharmaceutical industries and is worthwhile to search safe hepatoprotective agents [4]. Most of the liver protective plants may contain various biologically active phytochemicals in it. Recently, investigators have reported about hepatoprotective activity of alkaloids [5], polyphenols [6], glycosides [7,8]), carotenoids [9,10], coumarins [11] and flavonoids [12].

Paracetamol (Acetaminophen) is one of the most widely used pharmaceutical analgesic and antipyretic which causes liver toxicity and damage due to excessive use or overdose [13]. Paracetamol induced-hepatotoxicty in experimental animals as well as human subjects is widely recognized and reported [14-16].

Studies have shown that several plants have anti-hepatotoxic properties that can protect animals from paracetamol toxicity [17-18]. Due to its high tolerance and its availability, over-the-counter misuse and overdose of paracetamol is common and well recognized all over the world [19]. However, the widespread consumption of plant based diets such as Hylocereus polyrhizus may mask the apparent hepatotoxicity from the misuse of paracetamol.

Selected species of the genus Hylocereus, which consists of climbing three-ribbed stems and mostly white, fragrant, nightblooming flowers, have been recently developed as fruit crops [20, 21]. According to the color of the skin and pulp of the fruit(Fig.1), these species were named as white pitaya (Hylocereus undatus Britt & Rose, red skin, white pulp), red pitaya (Hylocereus polyrhizus, red skin and red pulp), yellow pitaya (Hylocereus megalanthus, yellow peel and white pulp) [22, 23].

Fig.1

The red pitaya otherwise called as Red Pitahaya, Dragonfruit, Night blooming Cereus, Strawberry Pear, Belle of the Night, Conderella Plant, was reported to offer many health benefits including cancer chemoprevention, anti-inflammatory, anti-diabetic and cardiovascular mortality risk reducing properties [24-26]

The fruits of red pitaya [Hylocereus polyrhizus (Weber) Britton & Rose] have recently drawn much attention both for their economic value and potential health benefits. Previous studies reported on the content of betacyanins in both flesh and peels [27]. These deep red–purple pigments, with their stable appearance in a broad pH range, have a great potential as natural coloring agents for a wide array of food [28]. In addition, recent studies have focused attention on their antioxidant activity, suggesting that these pigments may provide protection against certain oxidative stress-related disorders [29].

Hence, the aim of this present study was to investigate the preventive potentials of consumption of fruit of red pitaya against PAM – induced liver poison through biochemical, and haematological effects, as well as histological changes.

MATERIALS AND METHODS:

Sample preparation and processing

Red pitaya, Hylocereus polyrhizus, used in this study was obtained locally from four lots of fruits, which were washed and stored at -20 ⁰C before analysis. The fruit was peeled prior to analysis.

Formulation of experimental diets: Three isocaloric and isonitrogenous experimental diets namely control diet, 5 and 10% Buah naga supplemented diets were formulated. The diets were formulated from commercially available feed grade feedstuffs including maize, corn flour, fishmeal, groundnut meal, bone meal and vitamin premix. The control diet was formulated without the inclusion of H. polyrhizus while the Buah naga supplemented diets were incorporated with 5 and 10% of H. polyrhizus pulp.

Experimental design and paracetamol-induced hepatotoxicity: The experimental design involved random distribution of six rats each into six experimental groups namely non-hepatotoxic control (Group I), PAM induced hepatotoxic control (Group II), non-hepatotoxic fed 5% Buah naga supplemented diet (Group III), non-hepatotoxic fed 10% Buah naga supplemented diet (Group IV), hepatotoxic fed 5% Buah naga supplemented diet (Group V), and hepatotoxic fed 10% Buah naga supplemented diet (Group VI). The animals in non-hepatotoxic control and hepatotoxic control groups were fed control diet while those in non-hepatotoxic fed 5% Buah naga supplemented diet and hepatotoxic fed 5% Buah naga supplemented diet. The animals in non-hepatotoxic fed 10% Buah naga supplemented diet and hepatotoxic fed 10% Buah naga supplemented diet. All the animals were given the various feed and water ad libitum for 56 days. Prior to the end of feeding, animals in hepatotoxic control, hepatotoxic fed 5% Buah naga supplemented diet and hepatotoxic fed 10% Buah naga supplemented diet groups were orally administered daily with 3 g kg-1 b.wt. of PAM for seven days while those in non-hepatotoxic control, non-hepatotoxic fed 5% Buah naga supplemented diet and non-hepatotoxic fed 10% Buah naga supplemented diet groups were administered with the vehicle (distilled water) only. Weight changes of animals in all the groups were recorded throughout the experiment. All the experiments were performed under standard animal husbandry conditions and after the protocols had been approved by the animal ethics committee of the department of Bio-sciences.

Collection of blood and tissue samples: At the end of the feeding and administration of PAM, the animals from each group were anesthetized and blood samples were collected in labeled sample bottles with drops of Ethylene diamine tetra acetic acid (EDTA). Serum samples were collected in sample bottles without EDTA and allowed to clot before being centrifuged at 5000 rpm for 10 min. The livers of the animals in all the groups were promptly excised soon after sacrifice and stored in 10% formyl saline.

Determination of biochemical parameters: Glucose, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined using test kits. Total triglyceride and cholesterol were determined using test kits produced by Linear chemicals SL, Spain. Total protein was determined by the method described by [30] Lowry. Reduced glutathione was determined using the method of [31] Ellman. Lipid peroxidation was determined by the thiobarbituric acid reactive substances (TBARS) method [32].

Determination of haematological parameters: Full blood count and white blood cells (WBC) differentials were determined according to methods described by [33]Dacie and Lewis. The haemoglobin and haematocrit estimations were also carried out [34]

Histological study: Histological examinations were done on sections of liver tissues from animals in the different groups according to procedures described by [35]Disbrey and Rack.

Expression of results and statistical analysis: Results were expressed as Mean±Standard Error of Mean (SEM) for triplicate determinations. A One-way Analysis of Variance (ANOVA) for a completely randomized design was used to analyse experimental data. Values were considered significant at p<0.05.

RESULTS:

Effect on body weight: The effects of feeding Red pitaya-supplemented diet on weight prior to and after induction of hepatotoxicity with paracetamol are shown in Table 1. Prior to the induction of hepatotoxicity, Red pitaya-supplemented diet caused varying percentage weight gains in all the groups which were not statistically significant when compared to the corresponding controls. In contrast, significant weight loss were recorded in group II after induction of hepatotoxicity in groups except the non-hepatotoxic group fed 5% Buah naga supplemented diet.

The effect on biochemical parameters: The effect of Red pitaya-supplemented diet on the biochemical parameters in non-hepatotoxic and hepatotoxic rats are presented in Table 2, the levels of the liver enzymes were not significantly altered except AST that was significantly increased (p<0.05) in the non-hepatotoxic group fed 5% Red pitaya-supplemented diet. Serum ALT levels were markedly elevated in group II and slight increase in group III while the group VI favored steep fall and group V with moderate decline in their levels w.ref.t. group I. Blood glucose concentration was significantly reduced (p<0.05) in hepatotoxic groups fed 5 and 10% Red pitaya-supplemented diet w. ref. t. group II. There were slight fall in the protein and increase in cholesterol levels in all the hepatotoxic groups. Triglyceride concentration was significantly increased (p<0.05) in the hepatotoxic group and reduced in the hepatotoxic extract treated groups. Reduced glutathione was significantly increased (p<0.05) only in the hepatotoxic group fed 10% Red pitaya supplemented diet. There was moderate to marked fall in levels in 5 and 10 % extract fed hepatotoxic group animals (Group V & VI).

Effect on haematological parameters: The result of feeding Red pitaya-supplemented diet on haematological parameters in control and paracetamol-induced hepatotoxic rats are presented in Table 3.The PCV was significantly reduced (p<0.05) only in the hepatotoxic group fed Red pitaya supplemented diet while neutrophils were also reduced only in the hepatotoxic control.

Effect on liver tissues: The histological features of the liver of the animals in all the groups are shown in Plates 1-6. The liver sections of the rats in the control and non-hepatotoxic groups fed with Red pitaya-supplemented diet (Plates 1-3) showed less disarrangement and degeneration of hepatocytes, indicating marked preservation of hepatic architecture. The liver sections of the rats in the control hepatotoxic group (Plate 4) showed disarrangement and degeneration of normal hepatic cells with intense centrilobular necrosis, sinusoidal hemorrhages and dilatation. There was also inflammatory cell infiltrate in the portal tracts. However, Plates 5 and 6 shows that the intensity of centrilobular necrosis was less in the liver sections of the hepatotoxic groups fed extract supplemented diet indicating marked regeneration. In histopathological examination of the liver tissues also revealed the dose-dependent protection effect of extract in treatment groups from liver damage, when compared with PAM toxicated group. Change in liver histology, such as fatty liver change, degeneration of central hepatic vein, hepatocyte proliferation, necrosis, inflammation, lymphatic infiltration and in sinusoidal irregularities were reduced by the treatment of extract. Maximum protection from liver damage in animals was observed at higher dose. All biochemical findings of liver functions were supported by the positive results of histopathological study

DISCUSSION:

Paracetamol (Acetaminophen or n-acetyl-p-aminophenol), a commonly used analgesic drug has the potential to cause centrilobular hepatic necrosis in experimental animals and in humans [36-38]. Damage to the liver or hepatotoxicity, does not result from paracetamol itself, but from one of its metabolites, N-acetyl-p-benzoquinoneimine (NAPQI) [39]. NAPQI is a highly reactive toxic and cytotoxic intermediate metabolite, which is damaging to cell components if not detoxified by conjugation with glutathione (GSH). NAPQI can rapidly react with reduced glutathione (GSH) and lead to a 90% total hepatic GSH depletion in the cells and mitochondria, which can result in hepatocellular death and mitochondrial dysfunction [40].

Phytochemical products including plant herbs and extracts have been used for centuries to promote liver health. Although the exact mechanisms behind this protection are uncertain, many theories have been proposed. Paracetamol is being used extensively to investigate hepatoprotective activity of different treatments on various experimental animals [41]. It is selected as hepatotoxicant in inducing injury to the liver as it is known to cause hepatotoxicity in man and experimental animals when taken overdose [42].

Red pitaya is highly valued as a nutritious, medicinal and therapeutic fruit across Malaysia. The major purpose of this investigation was to study the effect of Red pitaya on paracetamol- induced hepatotoxicity and some associated parameters in rats. Aspartate transaminase (AST) and alanine transaminase (ALT) were used as parameters for assessing of liver toxicity, while total protein, triglycerides, cholesterol and glucose were used as supplementary tests for hepatic synthetic and other allied functions. Liver histopathology served as the most important tool for identifying and characterizing liver injury. The feeding of Red pitaya-supplemented diet of different concentration (5 and 10%) showed a general non-significant (p<0.05) increase in weight of animals. The increase in weight indicates that the fruit of Red pitaya was not toxic to the animals and could be attributed to their content of nutrients such as proteins, carbohydrates, lipids, minerals and vitamins which are needed for growth, body repair and maintenance [43,44]. Thus, the fruit could be a valuable and viable source of bioactive nutrients and non-nutrient substances with potential hepatoprotective properties.

Increases in activities of liver enzymes such as alanine transaminase and aspartate transaminase are roughly proportional to the extent of liver tissue damage [45]. Generally the consumption of Red pitaya-supplemented diet did not significantly (p<0.05) change any of the liver enzymes in the animals which shows that Red pitaya does not have any noticeable or apparent toxic effect on the liver.

Total protein levels are rough measures of protein status but reflect major functional changes in liver functions [46]. In this research there was a non-significant change (p<0.05) in the protein level of hepatotoxic animals, which could be due to stabilization in protein synthesis secondary to a decreased amount and availability of mRNA in the liver and this could indicate liver dysfunction [47].

Oxidative stress caused by Reactive Oxygen Species (ROS) plays a central role in hepatotoxicity [48]. GSH is an important antioxidant and free radical scavenger that has the ability to combat Reactive Oxygen Species (ROS) in the liver [49]. However, during paracetamol poisoning, NAPQI depletes markedly hepatocellular levels of reduced glutathione making the hepatocytes susceptible to it’s the toxic effects [50]. The result of this study showed that there was a significant increase (p<0.05) in the level of reduced glutathione in the hepatotoxic animals fed with the 10% Red pitaya-supplemented diet. This suggests that increase in quantity of Red pitaya consumed may improve hepatoprotection during paracetamol-induced toxicity [51, 52, and 29].

Excess reduction of hepatic glutathione concentration follows paracetamol challenge and is associated with heightened lipid peroxidation via free radical damage and directly damages cells in the liver [36, 39]. There was a stabilization in the level of lipid peroxidation of hepatotoxic control animals and those fed Red pitaya-supplemented diet which could be attributed to the presence of antioxidant phytochemicals including phenolic substances, flavonoids and anthocyanidins in the Red pitaya whose phenolic structure favor their reaction with free radicals and Reactive Oxygen Species (ROS)[53-55].

The reduction (p<0.05) in the levels of glucose in hepatotoxic rats fed Red pitaya-supplemented diet suggests that consumption of Red pitaya could reduce the blood concentration of glucose in the case of hyperglycemia seen in disease conditions such as diabetes with associated liver damage.

There were also non-significant changes (p<0.05) in the levels cholesterol in the groups. This supports research suggesting that Red pitaya may have cholesterol reducing properties that is important in preventing atherosclerosis [56]. The increase in triglycerides of hepatotoxic animals that consumed 10% Red pitaya-supplemented diet may be due to liver dysfunction that causes their excessive production while their reduction in the non-hepatotoxic group is attributed to the hypolipidemic properties of fruit diets.

Haematological parameters namely PCV, WBC and differentials were monitored in this study because of their diagnostic significance and role in providing information concerning haematological changes caused by paracetamol-induced toxicity [57]. Most phytochemical constituents of plant foods affect the immune system and other haematological parameters [58]. The increase in Hb and PCV (p<0.05) in hepatotoxic rats fed 10% Red pitaya-supplemented diet reflects the results obtained from the consumption of noni [59] and avocado [60]. This may be due to the presence of Hematinic factors in Red pitaya such as iron which plays a role in iron metabolism that increases the level of PCV and synthesis of hemoglobin [61-63]. The non-significant negative change in the level of white blood cells of the animals could be attributed to a rare case of hematologic side effects called thrombocytopenia often associated with paracetamol overdose. The cell membrane damage associated with inflammation results in leucocyte release of lysosomal enzymes that can be injurious to nearby cells [64]. Stimulation of neutrophils can lead to the production of oxygen – derived free radicals that produce further cellular damage. The increase in the levels of lymphocytes and neutrophils level of the hepatotoxic rats and those fed Red pitaya-supplemented diet supports the study done by [65] Duthie et al., which stated that antioxidant phytochemicals that can be found in Red pitaya are known to protect them. The phytochemical constituents of Red pitaya which include flavonoids and phytosterol are possible candidates that increase white blood cells.

The liver of non- hepatotoxic animals fed with Red pitaya-supplemented diet showed normal histological features. This result shows that the consumption of Red pitaya-supplemented diet does not have any apparent toxicity on the liver of rats. The necrotic effects of paracetamol seen in the abnormal histological changes in the liver of the animals is similar to that gotten by Hewawasam et al.[66] where Epaltes divaricata plant extract against carbon tetrachloride induced hepatotoxicity. However the consumption Buah naga-supplemented diet reduced this necrosis indicating some level of hepatoprotective and regenerative properties.

CONCLUSION:

The results of this study suggest that

1. Buah naga may be used in the treatment or prevention of paracetamol-induced hepatotoxicity probably due to its ability to preserve the natural integrity of hepatocytes when challenged with hepatotoxicants.

2. It is also revealed the dose-dependent protection effect of Buah naga fruit extract in treatment groups from liver damage. i.e. the 10% extract exhibits better therapeutic efficacy than the other one.

3. Further study is suggested to be carried out to help unravel the precise mechanism(s) for paracetamol-induced toxicity and the specific constituents of Buah naga involved in hepatoprotection against liver poison.

4. It could also be speculated that the observed hepatoprotective effects of Buah naga fruit extract might be related to the rich phytochemicals such as flavonoids, polyphenols, alkaloids, steroids, amino acids, and vitamins with strong antioxidant properties.

5. The process of extraction and identification of active principles responsible for the observed pharmacological actions of Buah naga fruit through bioactivity guided fraction is under progress to understand the possible mechanism of action of Buah naga fruits.

Table 1: Weight Change profile in Hepatotoxic and Non-Hepatotoxic Rat Administrated with ‘Buah naga’ supplemented diet

Weight change	Group I	Group II	Group III	Group IV	Group V	Group VI
Prior to hepatotoxicity (%)	40.1	51.2	41.5	41.5	43.4	43.7
After hepatotoxicity (%)	40.1	-10.3	6.4	8	-6.1	-2.5

Table 2 : Biochemical Profile in Control and Experimental Group Rats Fed ‘Buah naga’ Fruit Extract Supplemented Diet

Blood/serum parameter		Group I	Group II	Group III	Group IV	Group V	Group VI
Glucose ( mg/dL)	120.1±8.22		196.54±6.47	141.06±7.31	121.91±10.52	152.01±8.96	129.56±13.6
Protein (mg/mL)	6.96±0.14		4.72±0.90	6.86±0.30	6.99±0.01	5.01±0.31	5.91±0.76
GSH (mmol/mL)	0.0108±0.001		0.0096±0.006	0.0114±0.0052	0.0138±0.0016	0.0101±0.004	0.0147±0.092
Cholesterol (mg/dL)	180.16±1.21		292.89±5.47	176.53±8.01	164.64±4.77	274.01±3.77	214.89±9.83
TG (mg/dL)	165.72±6.01		243.89±6.99	169.88±1.001	153.86±8.01	222.49±3.68	196.87±6.77
LPO (mmol/mL)	13.51±0.93		24.11±1.01	13.01±1.96	12.95±0.001	20.01±0.86	17.36±1.52
AST (IU/L)	13.77±1.01		26.06±2.71	13.71±0.05	13.01±1.68	24.37±1.68	60.22±0.98
ALT (IU/L)	15.88±2.71		36.11±3.78	16.01±1.07	15.32±0.01	31.84±4.06	20.14±3.33

Table 3: Hematological Profile Status in Control and Experimental Groups of Rat Fed ‘Buah naga’ Supplemented Diet

Parameter	Group I	Group II	Group III	Group IV	Group V	Group VI
Hemoglobin (g/dL)	13.11±0.67	10.88±1.11	13.89±0.01	13.96±1.63	11.37±1.79	12.83±0.11
Hematocrit (%)	39.33±1.84	32.06±2.04	39.12±0.05	39.56±0.50	35.01±1.69	38.07±0.13
Leukocyte (10³/mm³)	6500±88.61	5000±10.87	6500±128.65	6750±60.17	5400±121.06	6100±82.64
Lymphocyte (%)	24.26±1.76	31.00±1.76	24.15±0.79	23.99±1.67	29.01±0.19	26.79±2.86
Polymorphs (%)	62.73±1.95	50.98±1.66	62.11±0.78	64.97±1.44	54.72±3.01	59.87±2.01

Plate 1. Non-hepatotoxic rat fed with control diet showing normal architecture of liver tissues (MAG. X 100)

Plate 2. Section of liver of non-hepatotoxic rat fed with 5%Buah naga extract supplemented diet (MAG. X 100)

Plate 3. Section of liver of non-hepatotoxic rat fed with 10%Buah naga extract supplemented diet (MAG. X 100)

late 4. Section of hepatotoxic rat liver fed with control diet showing congested central vien and vaculation of hepatocytic nuclei. (MAG. X 100)

Plate 5. Section of liver tissue from 5% Buah naga extract treated hepatotoxic rat showing more or less normal architecture with concentric arrangement of the hepatocytes around the central vein (MAG. X 100)

Plate 6. Section of liver tissue from 10% Buah naga extract treated hepatotoxic rat showing marked recvery towards normal architecture with concentric arrangement of the hepatocytes around the central vein (MAG. X 100)

Histological observation of hepatocytes of control and experimental group of rats

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Received on 05.01.2012 Accepted on 08.02.2012

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