Physiological Studies of the Effect of Moringa olifera and Vitamin (C) on Hepatotoxicity and Oxidative Stress Induced by Lead Acetate in Male Albino Rats

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Lead acetate, Moringa Olifera, Vitamin C, hepatotoxicity, Albino rats Lead is highly toxic naturally occurring element that affects numerous organ systems in humans, the purpose of this study was to investigate the role of Moringa olifera leaves extract and vitamin C in male albino rats against lead acetate toxicity.forty (40) male albino rats were used for the experiment.Group I was administered distilled water, Group II was administered lead acetate only (20mg/kg b.w.), Group III was administered lead acetate (20mg/kg b w) and Moringa olifera leaves extract (400mg/kg b.w.) and Group IV was administered lead acetate (20mg/kg b.w.) and Vitamin C (50 mg/kg b.w.).Animals were exposed to treatment once daily for 8 weeks orally.After the last day of treated animals were slaughtered and blood samples collected and serum separated for analyzing serum liver enzymes.Results obtained in this present study revealed a significant increased at (p<0.05) in aspartate aminotransferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP),bilirubin and Malondialdehyde (MAD), while showed a significant decreased at (p<0.05) in Total protein, Albumin, Glutathione (GSH), Superoxide dismutase (SOD) and Catalase (CAT) in group treated with lead acetate only when compared with normal group.However, Effects of Moringa oleifera (MO) and Vitamin C on hepatic injury due to lead induced oxidative stress revealed a significant decreased at (p<0.05) in aspartate aminotransferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP), bilirubin and Malondialdehyde (MAD), while showed a significant increased at (p<0.05) in Total protein, Albumin, Glutathione (GSH), Superoxide dismutase (SOD) and Catalase (CAT) when compared with the control group.It is concluded that Moringa olifera leaves extract and vitamin C have antioxidant properties.
Plants have played a substantial role in the maintenance of human health, from time immemorial.Moringa oleifera is one such multipurpose tree, commonly used in spices and cosmetic oils, and has various medicinal and therapeutic applications.
Moringa oleifera belongs to the Moringacaea family, a fast -growing, drought -resistant tree now distributed throughout the world in the tropics and sub -tropics, with extensive cultivation in Central and South America, Africa, Indonesia, Mexico, Malaysia, the Philippines and India.
Moringa oleifera is considered one of the most useful trees in the world, as nearly every part of the tree can be used for food or has other beneficial properties., Extracts from all parts of the plant also show pharmacological properties, which are recognized by popular use and confirmed by the scientific community.(Oliveira et al., 1999).
In addition, Moringa oleifera's active antioxidant properties, known to include specific plant pigments, include carotenoids such as lutein, chlorophyll, xanthins, alpha-carotene and beta carotene.Quercetin, rutin, kaempferol and caffeoylqunic acids are other isolated phytochemicals of Moringa oleifera with antioxidant potential.
Ascorbic acid is well known for its antioxidant activity, which acts as a reduction in liquid oxidation reversal agent.When there are more free radicals (reactive oxygen species, ROS) in the human body than antioxidants the condition is called oxidative stress.(McGregor and Biesalski, 2006) .
The aim of this work was to assess the ameliorative effect of Moringa oleifera leaf extract (MO) and vitamin C in order to reduce the toxicity of lead acetate in the liver.

Materials : a) Chemicals
1-Lead(II) acetate (Pb(CH3COO)2), is a white crystalline chemical compound with a sweetish taste.It was obtained from El-Nasr Pharmaceutical Chemical Co.,A.R.E.Laboratory chemical division.
2-Vitamin C , ascorbic acid powdered pure was obtained from the local market b) Plant Material (Moringa oleifera): Moringa oleifera leaves were purchased from local market.

c) Preparation of Extract of
Moringa oleifera Leaves: Moringa oleifera leaves were powdered and 5g of the powder was mixed with 50 ml of 80% ethanol.The mixture was stirred using magnetic stirrer in an air-tight container for about 1 hour and filtered afterward.Resulting Filtrate was evaporated to remove alcohol in a rotary evaporator free residue of sample was weight (500mg) and dissolved in 100ml distilled water to constitute the final extract solution (5mg /ml) (Babu et al., 2003).

d)
Animals: 40 adult male Albino rats weighing about (150-200g) were divided into four groups (10 rats/cage) in room temperature, for 2 weeks before starting the experiment, under natural day and night periods and supplied with a balanced stable commercial diet and water.

Methods 1.Experimental Design:
The experimental animals were divided into 4 groups, 10 rats for each group.Group 1 (Normal group): Normal received orally 1 ml distilled water, daily for 8 weeks.Group 2 (control group): rats were received Lead acetate (20mg/kg body weight) (Nehru and Kanwar, 2004), daily for 8 weeks.
At the end of the experiment, all animals were sacrificed and the blood from each animal was transported into clean tubes., then serum was separated by centrifugation and stored at -80 o c for biochemical analysis, liver tissue was collected from animals for biochemical analysis.Liver Tissue Was Homogenized as Following: a) Prior to dissection, perfuse tissue with phosphate buffer saline (PBS) solution pH=7.4 containing 0.16 mg/ml heparin to remove any red blood cells and clots.b) Homogenize the tissue in 5-10 ml cold buffer (i.e., 50 mM potassium phosphate, pH7.5 1mM EDTA and 1gm∕100ml of polyphenyl pyridine) per gram tissue in pastel homogenizer.C) Centrifuge at 4000 rpm for 15min at 4ºc. d) Remove for assay the supernatant and store it on ice.Freeze the supernatant at -80oc if not tested on the same day, the sample will be stable for at least one month.

Assessment of Biochemical Parameters and Antioxidants:
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined according to Reitman and Frankel, (1957), The level of Serum Alkaline Phosphates (ALP) was determined by the colorimetric described by (Bolfield and Goldberg, 1971).while albumin was determined according to Doumas et al., (1971), but total protein according to Gornal et al., (1949).

Statistical Analysis:
All quantitative measurements were expressed as means ± S.D. of control and experimental animals.The data were analyzed using oneway analysis of variance (ANOVA) on SPSS (statistical package for social sciences).Statistical significance was set up P < (0.05).As recorded in table 2, there were a significant increase at (p<0.05) in glutathione (GSH) and superoxide dismutase (SOD) activities but Catalase (CAT) activities showed a not significant increase at (p<0.05) in liver tissue of rats while there were a significant decrease at (p<0.05) in Malondialdehyde (MDA) activities in liver tissue of same rats compared with control animal but it not reach to normal animals.As recorded in table 2, there were a significant increase at (p<0.05) in glutathione (GSH) and superoxide dismutase (SOD) activities but Catalase (CAT) activities showed a not significant change at (p<0.05) in liver tissue of rats while there was a significant decrease at (p<0.05) in Malondialdehyde (MDA) activities in liver tissue of same rats compared with control animal but it did not reach to normal animals.

DISCUSSION
In the current results, the elevation in the levels of ALT, AST, ALP, Total bilirubin and Direct bilirubin was noted, while the level of serum albumin and Total protein decreased in rat serum oral administration of lead acetate (20mg / kg b.w.daily).These results agree with the recorded data of Abdel Aal and Abeer, (2008) they reported that lead administration (30 mg/kg body weight of 1/20 th of LD50) orally resulted in increased serum ALP, ALT and AST.This is because lead is known to cause oxidative injury in the liver tissue by producing membrane lipid peroxidation and causing derangement of several biochemical hepatic pathways and energy metabolism (Taki et al., 1985).
Increasing AST and ALT serum activity was most likely a result of the hepatotoxic effect of lead i.e. toxic hepatitis.By ingestion, the lead entering the body is delivered to the liver through the blood circulation portal and smaller portions of the lead "break the liver barrier" and enter the circulation of the body.The accumulated lead in the liver can act by directly damaging the hepatocytes primarily by destroying the cell membrane's permeability, resulting in the release of cellular enzymes increasing their serum values.(Todorvic, et al., 2005).Durgut, et al., (2008) found that, the increased serum activities of AST and ALT are associated with liver damage and or cardiac or skeletal muscle damage.Scholz et al., (1989) explained the increasing of ALP level in plasma.The increasing of ALP activity in plasma by toxins could be due to mechanical obstruction of bile ducts, failure to excrete the enzyme through relatively narrower bile passages leading to its accumulation and enzyme -level increase in plasma.
From obtained results, there was the elevation of plasma bilirubin, with the administration of lead acetate (20mg/kg body weight).This might be due to the induction of hemeoxygenase, the catabolism of heme from all heme proteins appears to be carried out in the microsomal fraction of cells by a complex enzyme system, hemeoxygenase, which converted heme to bilirubin.This suggestion agrees with (Seddik et al., 2010).
From obtained results, there was a reduction in plasma total soluble protein and albumin levels with administration of lead acetate (20mg/kg body weight).This might be due to the ingestion of lead acetate.These results show that the variation in total protein of plasma was correlated with the changes in albumin value.Murrey et al., (2006) explained the reduction in plasma total soluble protein and albumin levels May be caused by protein biosynthesis inhibition through the specific enzymes in cell processes and low significant excretion of hormones such as triiodothyronine (T3) and (T4) which regulated protein biosynthesis.The significant decrease in serum total protein and albumin can also be recognized to injury of hepatocyte functions resulting in decreased cytochrome P-450 activity and protein metabolism in the liver (Asagba, 2010 andIbiam et al., 2013).
In the present study, A significant decrease in ALT, AST, ALP, direct bilirubin and total bilirubin was observed during oral administration of daily doses of Moringa olifera (400 mg / kg body weight) while total protein and albumin increased significantly, these results agreed with Verma et al., (2009) Who reported the strong antioxidant properties of Moringa oleifera extracts due to the different bioactive components found in different solvent extracts.These bioactive antioxidant compounds effectively prevent toxicity induced by hepatotoxin.The reversal of high serum enzyme levels by Moringa oleifera leaf extract after administration of lead can be attributed to the Moringa oleifera's stabilizing ability on the cell membrane, preventing leakage of enzymes (Saalu et al., 2012).The stabilization of the serum total bilirubin level by the administration of Moringa oleifera extract is also a clear sign that the functional status of the hepatic cells is improved (Shah et al., 2013).
Moringa oleifera leaf has to repair effect on the liver due to their nutritional properties such as the presence of essential amino acid like methionine and cysteine and thus boosting the total proteins and albumin level Ekam et al., (2012).In this study, oral administration of daily doses of vitamin C (50 mg / kg body weight) revealed a significant decrease in ALT, AST, ALP, direct bilirubin and total bilirubin, whereas total protein and albumin increased significantly.These results showed an improvement in the rats treated with vitamin C compared to the control group.The reduction of AST and ALT by vitamin C is in accordance with Rekka et al., (1992), Who found that serum transaminases returned to normal activity with parenchyma tissue healing and hepatocyte regeneration Vitamin C significantly reduced serum transaminases, alkaline phosphatase, bilirubin and hepatic weight, and increased overall protein, albumin and body weight (Santhrani et al., 2012).
The present investigation revealed decrease of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) levels while there were a significant elevate in Malondialdehyde (MDA) levels in the liver with the administration of lead acetate orally (20mg/kg body weight).
A number of recent studies have confirmed that reactive oxygen species (ROS) may be involved in lead -induced toxicity (Gurer et al., 2000).Several antioxidant enzymes and molecules were used in animal and human studies to assess leadinduced oxidative damage.The most commonly used markers in tissues or blood are reduced glutathione (GSH) and catalase (CAT) and modifications in superoxide dismutase (SOD) activity (Khaki et al., 2010).Based on the observation that free radical was induced by lead exposure during pathogenesis processes.Sivaprasad et al., (2004) suggested that the decrease in (glutathione) GSH concentration May be due to the binding capacity of the lead with the SH group which decreases the GSH levels, thereby interfering with the antioxidant activity.Catalase is another major antioxidant enzyme with heme as the group of prostheses.Lead is known to reduce iron absorption and inhibit heme biosynthesis in the gastrointestinal tract.(Dresel et al., 1954).Decreased catalase activity observed in lead-exposed animals was attributed to the interference of lead by both processes (Sandhir et al., 1995).
Inhibition of SOD activity by lead was also demonstrated in an in vitro study in which the authors indicated that this effect of lead can lead to reduced ROS scavenging and oxidative stress.(Adler et al., 1993).Gurer et al., (2000) (Chumark et al., 2008).A possible explanation for the protective or therapeutic effect revealed in the present investigation of its antoxidant and scavenging properties.
The decreased levels of MDA in rats treated with Moringa oleifera may be due to the high levels of vitamin C and/or iron in the leaves (Aattar., 2006 ).Moringa leaves extracts have a powerful antioxidant action against free radicals, prevent oxidative damage to major biomolecules and provide substantial protection against oxidative damage (Sreelatha and Padma, 2009).Bajpai et al., (2005) reported antioxidant activity from the leaves of Moringa oleifera because of the founding of kaempferol in the leaves of Moringa The present study showed a significant increase in glutathione (GSH) in daily doses of vitamin C (50 mg / kg body weight) by oral administration.Superoxide dismutase (SOD) and catalase (CAT) activity in rat liver tissue and reduction of malondialdehyde (MDA) activity in same rat liver tissue.vitamin C has been reported to efficiently scavenge free radicals and contribute to the stability of cell and basal membranes, before initiating lipid peroxidation, (Sies et al., 1992).The result of increased SOD and CAT activities suggests that vitamin C contains a free radical scavenging activity that may have a beneficial effect on pathological changes caused by O2•− and OH•.The increased activity of SOD accelerates dismutation of O 2•− to hydrogen peroxide (H2O2) which is removed by CAT (Aebi, 1984).Vitamin C increased the glutathione content, reduced lipid peroxidation in liver samples (Santhrani et al., 2012).
Another mechanism of vitamin C mentioned by (Kadrabova et al.,1992) who reported that the vitamin C has been used to support cellular level of GSH because it acts as reducing agent preventing the oxidation of SH-group to nonfunctional disulphide groups, so the administration of vitamin C would enhance the antioxidant defense system.

Conclusion and Recommendation:
The results demonstrated that Moringa olifera leaves extract and Vitamin C showed a good ameliorating role against hepatotoxicity induced by lead exposure, this protection is mediated either by preventing the lead exposure -induced decline of liver antioxidant defense system or by their direct free radical scavenging activity.So the supplementation of Moringa olifera leaves and vitamin C containing several antioxidants may be important in occupationally exhibited workers to low doses of lead in undertakings, for instance, in paints, glass, aesthetic, batteries, shades, and welding.It is urged to be given with treatment traditions for lead lethality as it may have positive therapeutic repercussions.

Table ( 1
): Effect of lead acetate (20mg/kg b.w., orally daily), Moringa olifera (400 mg/kg b.w., orally daily) and vitamin C (50mg/kg b.w., orally daily) on serum level of ALT, AST, ALP, Total protein, Albumin, Direct bilirubin and Total bilirubin activities in blood of males albino rats post administration of lead acetate for 8 weeks.The result presented the mean  S.D. of 10 rats + significant increase at(p0.05).a → significantly different from normal rats.significant decrease at (p0.05).b → significantly different from a control animal.