Antioxidant effects of Moringa oleifera seed oil against oxidative stress induced by alloxan in rats.

Background: Type I diabetes mellitus is a metabolic syndrome manifested with chronic hyperglycemia due to insufficient insulin secretion. Hyperglycemia in diabetes mellitus has been associated with the overproduction of reactive oxygen species (ROS) along with inflammatory mediators. There is no allopathic cure for T1DM; therefore, novel strategies for cost-effective plant origin treatments are needed to be formulated with fewer side effects. The research aims to analyze the potential effects of Moringa oleifera (MO) seed oil, tracking certain biochemical markers and antioxidant enzymes in Alloxan induced diabetic rats. Methodology: Eighteen male Wistar rats having weights of 180-220 g were distributed into 3 groups (n=6). Group I: served as Control group, Group II: served as Alloxan treated and Group III: served as Alloxan + MO treated. Diabetes was induced via intraperitoneally administered Alloxan dissolved in 0.9 % NaCl solution (120 mg/kg body weight). In group III, rats were also given 1.5 ml/kg body weight of MO seed oil daily from 4 th day of Alloxan administration. The rats were decapitated just after the 21st day for biochemical and antioxidant assessments. Results: Treatment with MO seed oil has shown a significant decrease (p<0.05) in plasma glucose, serum urea, blood urea nitrogen (BUN), creatinine, alkaline phosphatase (ALP) and alanine aminotransferase (ALT) levels. In contrast, aspartate aminotransferase (AST) levels were not-significant (p>0.05) as compared with untreated control groups. Insulin and total protein levels had a significant increase (p<0.05) in Alloxan+MO treated group compared to the Alloxan treated group. Also, the antioxidant enzyme activities of superoxide dismutase (SOD), glutathione reductase (GSH), and malondialdehyde (MDA) were significantly increased (p<0.05), whereas catalase (CAT) activity was not-significant (p>0.05) in Alloxan+MO treated group as compared with Alloxan treated group. Conclusion: Our study showed that MO seed oil is a potent anti-oxidative hypoglycemic agent capable of improving other clinical conditions related to either oxidative stress or diabetes mellitus, such as the hepatic, renal and pancreatic functions.


Introduction
Type I diabetes mellitus is defined by chronic hyperglycemia developed because of insulin deficiency or inadequate insulin secretion 1 . In diabetes mellitus, hyperglycemia is linked with enhanced production of ROS and inflammatory mediators, which ultimately leads to oxidative stress. One source of ROS is free endogenous radicals formed by pancreatic beta cells in response to some cytokines 2 and facilitates the decimation of cell membranes along with cellular lipids, which in turn damages many biological molecules producing inflammation, degradation of β-cells and ultimately leads to cell death via direct reaction 3 . Unfortunately, overproduction of ROS has resulted in extremely detrimental effects, as it alters the antioxidant mechanism, inducing a series of deleterious events, inactivates many antioxidant enzymes, promoting protein glycation, resulting in diabetic complications 4 such as ketoacidosis, nephropathy, retinopathy, neuropathy, arthritis, coronary artery disease, cardiac disease, cancer, DNA and protein alterations (leading to replication errors), lipid peroxidation, and early aging 5,6 .
Various hypoglycemic allopathic medications have been formulated but have been found to display some serious side-effects, prompting the demand for the emergence of indigenous and affordable herbal products for the management and treatment of diabetes mellitus. MO, a multifunctional plant, appertain to the family Moringaceae. It is acknowledged as the "Miracle-Tree" because of its having diverse applications and benefits 7 . Essentially, all parts of the MO plant have been stated to strengthen a broad range of biological functioning, including anticancer, antiinflammatory, antioxidant, anti-hyperglycemic, hepatoprotective and neuroprotective activities [8][9][10][11][12][13] . These properties are attributes of proteins, carbohydrates, tannins, glycosides, fatty-acids such as oleic acid, palmitic acid, arachidonic acid, ascorbic acid and naturally occurring antioxidants such as tocopherols, flavonoids such as quercetin, chlorogenic acid and kaempferol, phenolics and carotenoids 14 .
However, the seeds are drawn scientific attention as MO seed kernels incorporate appreciable oil content (more than 40 %) with a high-quality fattyacid composition (oleic acid greater than 70 %) and a noticeable resistivity to oxidative destruction after refining 15 . Anwar et al. reported that the oil content of Moringa produce in Pakistan's temperate region was about 38-42 % 16 . It has a pale yellow color with a pleasant nutty flavor. Its oxidative stability is better than that of other oleic acid-rich oils such as olive, high oleic sunflower, macadamia, meadowfoam, hybrid safflower, and safflower, apricot and almond oils 17 . The oil contains oleic, behenic, stearic, arachidic and palmitic acid along with tocopherols that are accountable for its antioxidant activity.
A very limited work done has been done on MO seed oil in treating DM that's why the present study focuses on the potential of MO seed oil to act as an exogenous source of antioxidants to protect against diabetes mellitus induced by Alloxan in male Wistar rats. The study further examined specific diabetic biochemical parameters in the liver, kidney and pancreas. Whereas, oxidative stress markers were estimated in the liver.

Experimental Animals
Eighteen male Wistar rats (180-220 g) were subjected to the acclimatization period of 3-4 days before the start of the experiment. The rats were grouped according to their weights (n=6) and were housed in a room with a moderate temperature of 22-24°C and a 12-hour light-dark cycle with free access to both water and food prepared as per their daily requirements. All the ethical guidelines and animal protocols used in the experiments were according to the universally recognized principles for laboratory use and vigilance in the animalbased study given by the Health Research Extension Act of 1985 and the ethical guidelines of International ERB.

Extraction of MO Seed Oil
Moringa kernels had been procured from a local market in Karachi. The Moringa seeds were dehusked from the MO kernels. Dry seeds of Moringa oleifera were crushed then passed through a mesh strainer (40-mesh sieve). The seed powder was used to extract the oil 18 . 10 g of Moringa seed powder was weighed for seed oil and laid on filter paper creased carefully. The filter paper which contained the sample was then introduced into the Soxhlet apparatus. The weights of the filter paper, along with the sample, were noted. Then 200 ml solvent (hexane) was assessed using a measuring beaker and then transferred along with the sample into a 500 ml rounded bottom container and heated at 60°C for 5 hours after which the sample (seed powder along with filter paper) was moved to the air oven for 15 minutes to remove moisture at 100°C. This sample was then weighed again, and the difference was measured as sample weight before extractionsample weight after extraction divided by the preliminary sample weight, multiplied by 100 to give the percent of oil yield. The seed oil was obtained by evaporating solvent through further heating at lower temperatures until all the solvent evaporated, leaving behind the extracted oil 18 .

Diabetes Induction
Diabetes in rats was induced by injecting Alloxan Intraperitoneal (Sigma-Aldrich Chemical co., USA) prepared freshly in 0.9 % NaCl to over-night fasted rats at a dose of 120 mg/kg of bodyweight 19 . Diabetes induction was confirmed on the fourth day by assessing blood glucose levels by tail prick method and observing hyperglycemic states such as polyuria, polyphagia, polydypsia and weight loss. Rats having >200 mg/dl blood glucose levels were considered as diabetic. MO oil administration was commenced from the fourth day, and this was observed as the first day of treatment.

Study Design
The Wistar rats were arranged into three trial groups (n=6). Group I: served as Control group, Group II: served as Alloxan Treated group (120 mg/kg of body weight of Alloxan was once injected for diabetes induction) and Group III: served as Alloxan + MO seed oil Treated group (1.5 ml/kg of body weight). Following 21 days, the rats were sacrificed. Blood was acquired through cardiac puncture and centrifuged at 2500 rev/min (rpm) for 5 min. The serum obtained was carefully stored at −80°C for biochemical analysis, whereas the liver was also isolated from each rat for antioxidant assessments.

Biochemical Analysis
Glucose by glucose oxidase (GOD)-peroxidase (POD) method 20 , Insulin by enzyme linked immunosorbent assay (ELISA) 21 , Urea and BUN by Urease-Glutamate dehydrogenase (GLDH) kinetic UV method 22 , Creatinine by Jaffe's method 23 , Total Protein by Biuret method 24 , ALT by Kinetic UV method 25 , AST 26 and ALP 27 by the colorimetric method were analyzed according to their respective kits manual.

Antioxidant Enzymes Assessments
Assessments of CAT, SOD, GSH were also carried out according to the methods of Sinha, Kono, Carlberg and Mannervik respectively [28][29][30] . MDA, a lipid peroxidation product, was also assessed by the Ohkawa et al. method 31 .

Statistical Analysis
In the current study, the results were proposed as Mean ± SEM (Standard Error of Mean). The statistical analysis was done by independent T-test using SPSS 16.0. One-way ANOVA was done to test the significance of the experimental groups. Pvalues < 0.05 were referred as statistically significant whereas, p-values > 0.05 were referred as non-significant.

Results
Alloxan treated rats have shown a rapid decline in their body weights whereas, the control and Alloxan+MO treated rats have shown a progressive increase in their body weights throughout the trial period, as shown in Table 1. Prior to the experiment, all rats had normal glucose and insulin levels, but after Alloxan administration, the insulin levels declined because of pancreatic beta-cell death that leads to higher glucose levels in Alloxan treated group. These levels were significantly (p<0.05) reversed by the MO seed oil, as shown in Table 2. Treatment with MO seed oil significantly (p<0.05) reduced the concentrations of serum urea, creatinine and BUN in the Alloxan+MO treated group, which were found to be elevated in Alloxan treated group because of the kidney damage, whereas the reduction in total protein level in Alloxan treated group was overcome by MO seed oil in Alloxan+MO treated group as shown in Table 3 evaluating the potential of MO seed oil as nephroprotective. Alloxan treated group showed high levels of ALT, AST and ALP enzymes, indicating liver damage. Treatment with MO seed oil significantly (p<0.05) reduced ALT, ALP and non-significantly (p>0.05) reduced AST levels in Alloxan+MO treated group as shown in Table 4, evaluating the potential of MO seed oil as hepatoprotective. Alloxan administration leads to oxidative stress resulting in significantly (p<0.05) high MDA and reduced SOD and GSH levels, CAT was also reduced but non-significantly (p>0.05) in Alloxan treated group, which were overcome by MO seed oil in Alloxan+MO treated group as shown in Table 5 evaluating the potential of MO seed oil as anti-oxidative.

Discussion
Diabetes is a deliberating condition that affects the overall body resulting in massive health issues. Therefore, its management is necessary to avoid its associated complications. Alternative medicines have shown limitations in their multiple side effects; thus, novel treatment strategies are required, which are more productive, have fewer side effects and are cost-effective. MO is well known because of its antioxidant, antihyperglycemic, anti-cancer and anti-inflammatory activities.
In our study, intraperitoneal administration of Alloxan established type 1 diabetes mellitus in about 3 days. As Alloxan is toxic, it initially destroys some beta cells of the pancreas that may extend to damage kidneys and liver. It also generates ROS, which further exaggerates the destruction of beta cells by which insulin secretion ceases leading to a hyperglycemic state confirmed by elevated glucose levels in the rats of groups II & III. Then MO oil was administered in group 3 using oral gavage for a period of consecutive 17 days. Rats treated with MO seed oil had significantly lower circulating levels of glucose and elevated insulin levels compared to Alloxan treated diabetic group (Table 2). MO treated diabetic rats (Group III) also showed an increase in body weight, which was decreased after diabetes induction compared with Alloxan treated group. The body weights were continued to decrease throughout the trial period (Table 1). These results were analogous with the previous studies 32 .
Serum biochemical parameters such as creatinine, urea, ALT, AST, etc., were also determined using commercial kits. Any increase in urea, creatinine and BUN levels indicates impairment of renal function. Elevation in urea and creatinine, along with elevated blood glucose levels, damages the kidney because protein excretes in urine result in lower serum levels of total protein 33 . In this study, levels of urea, BUN and creatinine were reduced significantly (p<0.05) after treating with MO seed oil in the Alloxan + MO Treated group, which were elevated after diabetes induction as compared with the Alloxan treated group (Table 3) which indicates impairment in renal function. Consequently, the kidney function gets badly affected because protein excretes in urine result in lower serum levels of total protein 33 . Also, a reduction in total protein was noted in the Alloxan treated group, which might be due to the kidney damage caused by Alloxan's reactive species. While in the Alloxan + MO Treated group, total protein was found to be increased, indicating that MO oil restores kidney function, thereby decrease protein excretion in urine ( Table 3).
The ALT, AST and ALP are liver enzymes. Elevated levels of these enzymes result in hepatic injuries 34 . Alloxan treated group showed high AST levels, ALT, and ALP enzymes that might because of their leakage from distorted hepatocytes or bile ducts compared to Alloxan +MO treated group (Table  4). These results are parallel with the previous studies 35 .
MO seed oil is also found to be efficacious against oxidative stress induced by Alloxan in rats. CAT activity was found significantly decreased (p<0.05) in Alloxan-induced diabetic rats when compared with normal controls (Table 5) whereas nonsignificantly increased (p>0.05) in Alloxan+MO treated rats when compared with Alloxan, treated diabetic group (Table 5). The levels of SOD and GSH were decreased in the Alloxan treated group compared with the control group (Table 5). In contrast, SOD and GSH concentrations were significantly increased (p<0.05) in the Alloxan+MO treated group when compared with the Alloxan treated diabetic group (Table 5). Increased levels of CAT, GSH and SOD against ROS produced by Alloxan induction is owing to the presence of large amounts of antioxidants such as tocopherols, phenolic compounds, and flavonoids, specifically quercetin, kaempferol and chlorogenic acid in MO seed oil that have scavenging potential against ROS. It was also found that Alloxan induction in group II rats markedly increased hepatic MDA levels, which is indicative of increased oxidative stress: these findings correlate with the previous studies 36 . Group treated with the Alloxan+MO seed oil showed significantly lower MDA levels than the Alloxan treated diabetic group (Table 5). In the present study, Moringa seed oil acts as a nutritional (exogenous) antioxidant source that tends to assist the antioxidant enzymes in maintaining hepatocytes' integrity. Our study confirms that MO seed oil may be of greater medical benefit if used as a dietary supplement for diabetic patients 11 .

Conclusion
This study's experimental evidence based on biochemical assessments and antioxidant levels strongly suggests that MO seed oil is a potent dietary supplement that lowers ROS and may be very much effective against elevated glucose levels. Its prolonged use might have beneficial effects on the cure and management of diabetes mellitus and its associated complications. But still, there is a need for further studies on the maximum extraction of MO oil, isolating its polyphenols, flavonoids and other compounds in the absolute form to determine the exact mechanisms and possible pathways through which moringa seed oil exerts its anti-diabetic and anti-oxidative activity on a cellular level. A clinical trial is highly recommended, considering the complexity of diabetes.

Conflicts of Interest
The authors clearly declared that there is no conflict of interest among them.