Potential in vitro antioxidant and protective effects

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The present study was undertaken to evaluate the antioxidant capacities and the protective effects of aqueous extract of Opuntia vulgaris fruit extract (OE) against methanol-induced oxidative damage in experimental rats. The animals exposed to methanol at a dose of 2.37 g/kg body weight i.p for 30 days. OE was found to contain large amounts of polyphenols and carotenoids and significant antioxidant capacities highlighted by scavenging activities for DPPH. The treatment with methanol exhibited a significant increase of some serum hepatic and renal biochemical parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin, urea and creatinine). Methanol intoxication significantly increased hepatic and renal lipid peroxidation evaluated by thiobarbituric acid reactive substances (TBARS) in treated rats as compared to controls. However, hepatic and renal antioxidant enzymes namely superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were significantly decreased in methanol treated animals as compared to controls. In addition, treatment with OE for 15 days prior to methanol intoxication significantly improved hepatic and renal antioxidant markers. Histological studies on the ultrastructural changes of liver and kidney supported the protective activity of the OE. The results concluded that the treatment with OE prior to methanol intoxication has significant role in protecting animals from methanol-induced hepatic and renal toxicity.

Key-words: Antioxidant, Opuntia vulgaris, Methanol, Oxidative damage

Introduction

Methanol, first in the alcohol series, is normally used as an industrial solvent and cleanser. Also known as wood alcohol, methanol is a component of washing fluids, antifreeze formulations, photocopying fluids, perfumes, and paint removers. People handling products that contain methanol may inhale the toxic vapor during its evaporation from the product surface. Methanol is metabolized by the enzyme alcohol dehydrogenase (ADH) and catalase, mainly in the liver, via formaldehyde to formic acid (Bryson, 1989). In rats, a catalase-peroxidase system is considered to be responsible for oxidizing methanol to formaldehyde. The accumulation of formic acid is pointed out as the cause for the metabolic acidosis resulting in the clinical manifestations of methanol toxicity and characterized by optic nerve involvement. The toxicity evolves from a combination of the metabolic acidosis and an intrinsic toxicity from the formate anion itself (Barceloux et al., 2002). Methanol poisonings occur as isolated episodes caused by accidental or intentional ingestion, or epidemics. In the latter situation, a large number of victims are often reported (Paasma et al., 2007; Hovda et al., 2005). Methanol is also increasingly recognized as a hepatotoxin in that hepatocytes oxidize it first to formaldehyde and then to formate (Tephly, 1991). These processes are accompanied by elevation of NADH level and the formation of superoxide anion, which may be involved in lipid peroxidation (Poli, 1993). The mechanism of toxicity of methanol is characterized by mitochondrial damage and increased microsomal proliferation resulting in increased production of oxygen radicals. Methanol could initiate radical oxygen species (ROS) formation directly via a free radical intermediate, or possibly indirectly through mechanisms like the activation and/or enhancement of ROS-producing NADPH oxidases, which has been reported for ethanol (Dong et al., 2010). The generation of oxygen free radicals might be enhanced, leading to membrane damage, lipid peroxidation, and mitochondrial damage (Bralet et al., 1991; Chaco & Acosta, 1991). Further, Dikalova et al. (2001) detected free radical metabolites generation in bile and urine of rats from acute formate intoxication by the electron spin resonance spectroscopy spin-trapping technique. These generated free radicals cause extensive damage to cellular membranes leading to cellular dysfunction and death. Consequently, the cells' ability to survive the increased ROS is diminished as antioxidants become depleted (Monthe et al., 1992). However, cells also have a scavenging system to counteract the free radicals generated so that the integrity of the cell membrane may be maintained. Previously, we reported that methanol affects immune functions in albino rats (Parthasarathy et al., 2005). Data on the antioxidant defense potential in the liver and kidney after methanol intoxication in rats are lacking. It is, thus, interesting to evaluate the activities of methanol metabolizing enzymes in the liver cells which are targets of this alcohol. In this report, particular attention has been drawn to the antioxidant status expressed in enzyme activities in liver and kidney of rats after one dose of methanol, which were observed for 30 consecutive days. During the past few years, estimation of free radical generation and antioxidant defense has become an important aspect of investigation in mammals (Zini et al., 1993 R. Zini, E. Lamirande and G. Gagnon, Reactive oxygen species in semen of infertile patients. Levels of superoxide dismutase and catalase like activities in animal plasma and spermatozoa, J Androl 16 (1993), pp. 183-188.Zini et al., 1993). A positive correlation has been established between dietary supplementation with certain vegetables and plant products and the reduction of toxic effects of various toxicants and environmental contaminants (Nandi et al., 1997). Plant products are known to exert their protective effects by scavenging free radicals and modulating antioxidant defense system.

Fruits are beneficial to human health and contribute to the prevention of degenerative process (Vinson et al., 2001). Opuntia vulgaris, a native species to Tunisia, is widely distributed on the center and the south of Tunisia, and its fruits known as prickly pears or cactus pears are an excellent source of betalain natural colorants and functional compounds. Opuntia spp. fruits represents lower risk for microbiological contamination, have no nitrate content, are highly flavoured, show adequate nutritional properties (e.g. high levels of calcium, magnesium and vitamin C), and contains interesting functional compounds like quercetin (Stintzing et al., 2005). The aqueous extract of O. vulgaris on preliminary chemical analysis is found to contain saponin and alkaloid (Jiang et al., 2003). On the other hand, Opuntia spp. extracts have shown analgesic, anti-inflammatory, hypoglycemic, physiological antioxidant, cancer chemoprevention, and neuroprotective effects (Kim et al., 2006). It is also as a scavenger for reactive oxygen species and nitrogen (Gentile et al., 2004). Recent studies have shown that some phenolic compounds can prevent some chemical solvents-induced oxidative damage, and the ability of phenolic compounds might be related to their antioxidant properties. For that reason, it can be implicated in different pharmacological fields. The traditional use of this plant is based on the oral administration of a decoction prepared from fruits in water. This study was designed to determine the possible protective effects of O. vulgaris extract against biochemical disorders and oxidative damages induced in rats by intraperitoneal administration of methanol.

In the present study, to evaluate liver and kidney functions, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lacatate dehydrogenase (LDH), bilirubin, creatinine and urea were assayed. Oxidative stress was estimated by the lipid peroxidation increase (TBARS) and changes in the activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in liver and kidney tissues. To our knowledge, the use of OE to alleviate the oxidative damage induced by methanol was not previously examined.

Materials and Methods

Chemicals

2.2-diphenyl-l-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), thiobarbituric acid (TBA), glutathione (GSH), oxidised glutathione (GSSG), glutathione reductase (GR), bovine serum albumin (BSA) and 2,4-dinitrophenyl hydrazine (DNPH) were purchased from Sigma Chemical France. Trichloroacetic acid (TCA), hydrogen peroxide (H2O2), 5,5' dithiobis(2-nitrobenzoic acid) (DTNB), Folin-Ciocalteu reagent, sodium carbonate (Na2CO3) and other solvents, were of analytical grade and were freshly prepared in distilled water.

Preparation of O. vulgaris extracts (OE)

The O. vulgaris fruits (Figure 1) were collected from a culture area located in Kasserine region, Tunisia. Fruit samples were ground, put in water and shaken (10g/l, v/w) for 15-20 min, and then filtered using Whatman filter paper. The aqueous extract was given as beverage instead of tap water.

In vitro antioxidant properties

Total phenolic content

Total phenolics content was determined using the Folin-Ciocalteu method (Waterman and Mole, 1994) adapted to a microscale. Briefly, 10 µl diluted extract solution was shaken for 5 min with 50 µl of Folin-Ciocalteau reagent. Then 150 µl of 20% Na2CO3 was added and the mixture was shaken once again for 1 min. Finally, the solution was brought up to 790 µl by adding distilled water. After 90 min, the absorbance at 760 nm was evaluated using a spectrophotometer. Gallic acid was used as a standard for the calibration curve. The phenolic content was expressed as mg gallic acid equivalent/gram of dry extract using the linear equation based on the calibration curve.

Determination of total flavonoids content

The flavonoids content in extracts was determined spectrophotometrically according to (Quettier-Deleu et al., 2000), using a method based on the formation of a complex flavonoid-aluminium, having the maximum absorption at 430 nm. The flavonoids content was expressed in mg of quercetin equivalent per g of dry plant extract (mg QE/g).

Antioxidant testing assays

DPPH radical scavenging activity

Radical scavenging activity of O. vulgaris extracts was determined using DPPH as a reagent according to the method of Kirby and Schmidt (1997) with slight modifications. Briefly, 1 ml of a 4 % (w/v) solution of DPPH radical in methanol was mixed with 500 µl of sample solutions in ethanol at different concentrations. The mixture was incubated for 20 min in the dark at room temperature. The scavenging capacity was determined spectrophotometrically by monitoring the decrease in absorbance at 517 nm against a blank using a spectrophotometer (Bio-Rad SmartSpecTM plus). Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. Ascorbic acid was used as positive control. The percent DPPH scavenging effect was calculated using the following equation: DPPH scavenging effect (%) = (A control- A sample / A control) Ã-100 Where Acontrol is the absorbance of the control reaction containing all reagents except the tested compound. Asample is the absorbance of the test compound. Extract concentration providing 50% inhibition (IC50) was calculated from the graph plotting inhibition percentage against extract concentration. Tests were carried out in triplicate.

Hydroxyl radical-scavenging activity

The hydroxyl radical scavenging activity was determined according to the colorimetric deoxyribose oxidation by the Fenton reaction leading to malondialdehyde (Chung et al., 1997). The hydroxyl radicals was generated from the Fe3+/ascorbate/EDTA/H2O2 system in the non site-specific assay or Fe3+/ascorbate/H2O2 in the site-specific assay. The reacting mixture for the deoxyribose assay contained in a final volume of 1ml the following reagents: 200 µl KH2PO4-KOH (100 mM), 200 µl deoxyribose (15 mM), 200 µl FeCl3 (500 µM), 100 µl EDTA (1 mM), 100 µl ascorbic acid (1 mM), 100 µl H2O2 (10mM) and 100 µl sample. Reaction mixtures were incubated at 370C for 1 h. At the end of the incubation period, 1 ml of 1% (w/v) TBA was added to each mixture followed by the addition of 1ml of 2.8% (w/v) TCA. Solutions were heated on a water bath at 80°C for 20 min to develop the pink coloured malondialdehyde-thiobarbituric acid: MDA-TBA2 adduct, and the absorbance of the resulting solution (total volume = 3.0 ml) was measured at 532 nm. Mannitol, a classical hydroxyl radical scavenger was used as positive control. The inhibition ratio of the extract (%) was calculated using the following formula:

Inhibition ratio (%) = (A control 532 nm - A sample 532 nm / A control 532 nm) x 100

In vivo antioxidant properties

Animals

Adult male albino Wistar rats weighing 180 to 200g were obtained from the Central Pharmacy of Tunisia (SIPHAT, Tunisia). The animals were quarantined and allowed to acclimatize for a week prior to experimentation. The animals were handled under standard laboratory conditions of a 12-h light/dark cycle in a temperature- and humidity-controlled room. Food and water were available ad libitum. Our Institutional Animal Care and Use Committee approved the protocols for the animal study, and the animals were cared for in accordance with the institutional ethical guidelines.

Treatment

After acclimatization, the rats were divided into two batches: 16 control rats (C) drinking tap water and 16 treated-rats drinking O. vulgaris fruit extract (OE) for six weeks. Then, each group was divided into two subgroups and one of them was intraperitoneally injected (IP) daily, for four weeks, with methanol (2.37 g/kg b.wt.) according to Parthasarathy et al. (2006). After treatment, 8 rats of each group were sacrificed under anaesthesia by i.p injection of chloral hydrate.

Serum parameters

Serum samples were obtained by the centrifugation of blood at 4000 rpm for 15 min at 4°C, and were then divided into eppendorf tubes. Isolated sera were stored at -30°C until they were used for the analyses. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphate (ALP), lactate dehydrogenase (LDH), bilirubin, urea and creatinine were measured using commercial kits according to the manufacturer's directions.

Oxidative stress analysis

Thiobarbituric acid reactive substances (TBARS) measurements

Lipid peroxidation in the tissue homogenate was estimated by measuring thiobarbituric acid reactive substances (TBARS) and was expressed in terms of malondialdehyde (MDA) content which is the end product of lipid peroxidation, according to Buege and Aust (1972). In brief, 125 μl of supernatants were homogenized by sonication with 50 μl of TBS, 125 μl of TCA-BHT in order to precipitate proteins and centrifuged (1000 g, 10 min, 4 â-¦C). 200 μl of obtained supernatant were mixed with 40 μl of HCl (0.6 M) and 160 μl of TBA dissolved in Tris and the mixture was heated at 80 °C for 10 min. The absorbance of the resultant supernatant was read at 530 nm. The amount of TBARS was calculated by using an extinction coefficient of 156 x 10 5 mM-1 cm-1.

Anitoxidant enzymes studies

In liver and kidney tissues, SOD activity was determined according to the colorimetric method of Beyer and Fridovich (1987) using the oxidizing reaction of nitroblue tetrazolium (NBT); CAT activity was measured by the UV colorimetric method of Aebi (1974) using H2O2 as substrate; glutathione peroxidase (GSH-Px) activity was measured by a modification of the colorimetric method of Flohe and Günzler (1984) using H2O2 as substrate and the reduced GSH.

Histopathological studies

Pieces of liver and renal tissues were excised, washed with normal saline and processed separately for histopathological observation. The liver and kidney tissues were fixed in bouin solution, dehydrated in graded (50-100%) alcohol and embedded in paraffin. Thin sections (4 - 5 µm) were cut and stained with routine hematoxylin-eosin (H&E). The sections were examined microscopically for histopathology changes, including cell necrosis, fatty change, and ballooning degeneration (Gabe, 1968).

Statistical analysis

All values are expressed as mean ± S.E.M. The results were analyzed by one-way analysis of variance (ANOVA) followed by Tukey test for multiple comparisons using SPSS for Windows (version 11). Differences were considered significant at p < 0.05.

Results

In vitro antioxidant properties

Total phenolics (TPC) and flavonoids

The OE extracts were found to contain 107 ± 1.30 mg GAE/g of dry plant extract a total phenolic content (TPC). The flavonoid content in the studied extract was 18.70 ± 0.5 mg/g expressed as equivalent quercetin per g of dry plant extract (Quercetin, mg/g of QE) (Table 1).

Antioxidant capacities of O. vulgaris extract

The effect of O. vulgaris extracts on DPPH radical scavenging showed a dose-dependent activity that can be evaluated by the determination of the IC50 values corresponding to the amount of the fraction required to scavenge 50 % of DPPH radicals present in the reaction mixture. As shown in Table 1 the radical scavenger extract showed an IC50 = 990 ± 5.60 µg/ml, followed by water fraction. Therefore, we can conclude that this extract was able to reduce the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) to the yellow-colored diphenylpicrylhydrazine. In the β-carotene bleaching method, the degree of linoleic acid oxidation is determined by measuring oxidation products (lipid hydroperoxides, conjugated dienes and volatile by-products) of linoleic acid which simultaneously attack β-carotene, resulting in bleaching of its characteristic yellow color in aqueous solution. The antioxidant activity of O. vulgaris was evaluated using different concentrations of extracts and was compared with BHT used as reference (Table 2). The addition of the (OE) fraction and the BHT at a concentration of 800 and 1000 µg/ml prevented the bleaching of β-carotene with different degrees. Hydroxyl radical (.OH) can easily cross cell membranes, and can readily react with most biomolecules including carbohydrates, proteins, lipids, and DNA in cells, and cause tissue damage or cell death. Thus, removing .OH is important for the protection of living systems. As shown in Figure 2, the OE fraction exhibited high hydroxyl radical scavenging activity with an IC50 of 990 ± 5.60 µg/ml.

In vivo antioxidant properties

Serum biochemical parameters

The activities of various biochemical markers in normal, methanol control and treated groups were represented in Table 3. The activities of ALT, AST, ALP, LDH and bilirubin were significantly increased in methanol control group compared to normal control group. The levels of the above enzymes were significantly reversed on treatment with O. vulgaris extract. The present study showed that the administration of O. vulgaris fruit extract alone did not cause any significant alteration on the biochemical enzymes activities. Creatinine and urea were measured as an index of renal failure. Significantly higher serum creatinine and urea were observed in methanol treated group as compared to normal control. By contrast, administration with O. vulgaris fruit extract significantly decreased the levels as compared to the methanol-treated group. Whereas, no significant change was observed in OE-treated rats, as well as in OE+M rats, showing the protective effects of O. vulgaris fruit extract against changes induced by methanol treatment.

Hepatic and renal antioxidant enzyme activities

SOD, CAT and GSH-Px were measured as an index of antioxidant status of tissues (Figure 3). Significantly lower liver and renal SOD, CAT, and GPx activity were observed in rat from the methanol treated group as compared to the normal control group. Treatment with OE restored the levels of methanol to near normal. After treatment of rats with methanol plus O. vulgaris fruit extract, SOD, CAT and GPx activities were normalized to their control values. The present study shows that administration of O. vulgaris fruit extract alone does not cause any significant change on the hepatic and renal antioxidant enzyme activities. No significant change was observed in OE+M rats, showing the protective effects of O. vulgaris fruit extract against changes induced by methanol treatment.

Hepatic and renal lipid peroxidation

To explore the oxidative consequences of methanol treatment in hepatic and renal tissues of control and treated rats and to determine the possible protective effect of OE, we analyzed the lipid peroxidation as an indicator of oxidative damage. Exposure to methanol increased significantly the LPO levels in hepatic and renal tissues samples as compared to control group (Figure 4). OE treatment prevented the LPO production induced by methanol. OE alone did not change the degree of LPO formation as compared to controls.

Histopathological findings

The histological results revealed that the liver of the control (C) group showed normal hepatocytes and central vein. The liver sections of rats treated with the extract (OE) showed normal hepatocytes, nuclei and central vein. Rats treated with methanol (M) showed dilated portal tract with massive aggregation of mononuclear inflammatory cells which scattered in different spaces, hepatocytes focal necrosis and vacuolisation. Rats treated with the extract of O. vulgaris extract plus methanol (OE+M) showed prominent improvement in hepatocytes with vesiculated nuclei (Figure;;;).

The histological examination of the kidney tissues of the control animals showed normal structure of glomerulus's and renal tubules. Rats treated with the extract alone showed normal structure of glomeruli and tubules. Rats treated with methanol alone showed reduction of glomerular space, vacuolar and cloudy in epithelial cells lining, interstitial inflammatory cells, haemorrhage, cellular debris and glomerulus's hyper cellularity. Rats treated with methanol plus the extract showing improvement in tubular structure and glomerulus's (Figure;;).

Discussion

In the current study, the role of O. vulgaris fruit extract on the oxidative stress in methanol toxicity was investigated in male rats. The selective dose of methanol was literature based (Parthasarathy et al., 2006). Whereas, the selective dose of O. vulgaris extract was based on our previous work (Saoudi et al., 2011). The results indicated that phenolic estimation reveals that aqueous extract of O. vulgaris fruit contain considerable amount of polyphenolic and flavonoid coumpounds. Our results are in accordance with findings of Kuti (2003) which reported that extracts from four cactus (Opuntia species) fruit varieties marked a good source of natural antioxidants such as flavonoids and polyphenols, and consumption of Opuntia fruit or its products may contribute substantial amounts of antioxidants to the diet. The stable DPPH radical is widely used to evaluate the free radical scavenging activity in many plant extracts (Brand-Williams et al., 1995). The assessment of antioxidant activity showed that O. vulgaris fruit was able to scavenge this radical (IC50 = 990 ± 5.60 µg/ml). This result revealed that aqueous extract of O. vulgaris fruit was free radical scavengers, acting possibly as primary antioxidants. The inhibition of lipid peroxidation by addition of O. vulgaris extract can be used to improve the quality and stability of food products.

The Opuntia vulgaris fruit extract was found to have strong superoxide radical scavenging activity, which may be due to the presence of polyphenolic compounds. Also, higher peroxidation inhibiting activity of OE suggests a possible biological functionality in preventing the oxidative damage. It is well known that radicals such as nitric oxide (NO) are associated with various carcinomas and inflammatory conditions, multiple sclerosis, arthritis and ulcerative colitis (Taylor et al., 1997). The toxicity of NO increased when it reacts with superoxide radical, forming the highly reactive peroxynitrite anion (ONOO-) (Huie and Padmaja, 1993). The present study showed that the extract O. vulgaris studied has more potent scavenging activity. Methanol is toxic to the cells by the formation of free radical and increase lipid peroxidation (Kadiiska and Mason, 2000). Numerous evidences clearly demonstrated the importance of medicinal plants in the treatment of oxidative stress induced cell death (Jung et al., 2006).

The present study evaluated the effects of aqueous extract of O. vulgaris fruit on methanol-induced biochemical perturbation and oxidative stress. Also, damage to the liver after methanol exposure is a well-known phenomenon, and the obvious sign of hepatic injury is the leakage of hepatic enzymes into plasma. The increased levels of serum enzymes such as ALT, AST, ALP, LDH and bilirubin have been observed in methanol-treated rats, which indicate the increased permeability, damage or necrosis of hepatocytes. The OE administration gave a high hepatoprotective effect by reversing these changes produced by methanol. The observed decrease in the activities of these enzymes showed that OE, to some extent, preserved the structural integrity of the liver from the toxic effect. In addition, treatment with methanol induced increase in serum urea and creatinine which indicated kidney dysfunction. The results also showed that OE significantly decreased serum levels urea and creatinine. These results clearly showed that methanol has a harmful and stressful influence on the hepatic and renal tissue consistent with those reported in the literature (Parthasarathy et al., 2006). Methanol is well known to produce oxidative stress in liver and kidney by disturbing the prooxidant/antioxidant status as revealed from the altered levels of different intracellular antioxidant markers (Datta and Namasivayam, 2003; Sandhir and Kaur, 2006).

Antioxidant enzymes are considered to be the first line of cellular defense against oxidative damage. Among them SOD and CAT mutually function in the elimination of radical oxygen species. In the current study, treatment with methanol resulted in a significant decrease of antioxidant enzymes such as SOD, CAT and GSH-Px activities. Reduction in SOD activity in methanol-exposed animals may be due to the enhanced production of super oxide radical anions (Shanmugam et al., 2010). Catalase is to scavenge H2O2 that has been generated by free radical or by SOD in removal of superoxide anions. The administration of O. vulgaris fruit extract restored the activities of enzymatic antioxidant in liver and kidney. Polyphenolic compounds are present in O. vulgaris, which are powerful antioxidant properties, i.e free radical scavenging activity (Pal and Mitra, 2010). Lipid peroxidation is one of the characteristic features of increased oxidative stress associated with methanol toxicity (Sandhir and Kaur, 2006). Increased TBARS level is an index of enhanced lipid peroxidation (Devipriya et al., 2007). The oral administration of O. vulgaris extracts prior to methanol intoxication significantly lower this enhanced TBARS level in hepatic and renal tissues. Extract may suppress lipid peroxidation through different chemical mechanisms, including free radical quenching, electron transfer, radical addition, or radical recombination (Pal and Mitra, 2010). This observation directly demonstrates the anti-peroxidative and antioxidant effects of OE.

Histopathological examination of the liver and kidney revealed that methanol treatment caused abnormal ultrastructural changes in the tissues. However, pretreatment with OE could prevent the changes and could also maintain the ultra structure almost similar to that of normal control. Similar observations were reported by Xu et al. (2009). The current results clearly indicated that treatment with O. vulgaris extract did not induce any harmful effects on the animals. The changes in the liver of methanol treated rats predominant in the centrilobular region. Administration of OE to methanol treated rats reduced the liver cell damage and improved the histomorphology of the liver near to normal. In kidney show that dilated tubules with cloudy swelling in methanol treated rats. Rats administered with OE along with methanol showed near normal appearance with mildly dilated tubules with regenerating epithelium of kidney, which indicates the significant protection offered by OE against the toxic effect of alcohol. The results of functional tests together with histological observations suggest that methanol leads to serious changes in histology of liver and kidney. The O. vulgaris fruit is rich in compounds known to be strong antioxidants and it ameliorated liver and kidney damages induced by methanol. Our data indicate that O. vulgaris fruit extract has a protective action against methanol-induced toxicity as evidenced by the lowered tissue lipid peroxidation and elevated levels of biochemical enzymes and a decrease in enzymatic antioxidants in liver and kidney. Hence, our study suggests that O. vulgaris fruit extract play a beneficial role in the treatment of methanol induced tissue damage, which could be one of its therapeutic values.

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