Evaluation of Blood Glucose, serum insulin levels and some Biochemical parameters of Vigin Coconut oil in experimental Diabetic Male wistar Rats

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Evaluation of Blood Glucose, serum insulin levels and some Biochemical parameters of Vigin Coconut oil in experimental Diabetic Male wistar Rats

https://doi.org/10.21203/rs.3.rs-7905792/v1

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Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia. Virgin coconut oil (VCO), extracted from fresh coconut kernel without chemical refining or high heat has been promoted as a dietary supplement for people with various ailments including diabetes and has garnered scientific interest for its potential therapeutic benefit. This study was aimed at evaluating the antihyperglycemic effects of virgin coconut oil (VCO) and some biochemical parameters in experimental diabetic rats. A total of 25 adult Male rats were randomly assigned into 5 groups, with 5 rats each. Group 1 were the normal control and given 1ml/kg of distilled water, groups II to V were induced with diabetes using 150mg/kg Alloxan intraperitoneally, these were subsequently given 1ml/kg of distilled water, 5ml/kg, 10ml/kg and 15ml/kg of VCO orally daily for four weeks respectively. The results shows the fasting glucose in all the VCO treated groups were statistically (P ≤ 0.05) lower when compared with the diabetic control group. Also the insulin levels in these groups was statistically higher (P ≤ 0.05) when compared to the diabetic control. Furthermore, histological analysis shows more viable β cells, with increased population and more regularly shaped islets cell having a relatively organized connective tissue sheets, when compared with the normal control group. The higher cholesterol and triglycerides levels observed in the diabetic control group was significantly lower (P ≤ 0.05) in all the VCO treated group when both compared with diabetic control. The serum levels of HDL was statistically higher (P ≤ 0.05) in all the treatment groups, when compared with both normal and diabetic control groups, while no statistical difference (P \(\:\ge\:\) 0.05) was observed in the LDL levels even though a slight decrease in serum levels was observed in the 15ml/kg treated group when compared with the diabetic control. The study concludes virgin coconut oil lowered blood glucose, increases serum insulin levels and significantly restore β cell mass. Furthermore, it lowers serum total cholesterol, triglycerides and it increase serum high density lipoprotein levels in Alloxan-induced diabetic Male Wistar rats.

Physiology

virgin Coconut oil

diabetes mellitus

Wistar rats

Alloxan

insulin

lipid profile

Diabetes mellitus is a pervasive metabolic disorder characterized by chronic hyperglycemia, insulin resistance, and dysfunctional β-cell insulin secretion (Zheng et al., 2018). The term was first used in the second century to describe the condition based on the two common symptoms; excessive thirst and excessive urination. Chronic hyperglycemia is the hallmark and the final common factor of this condition resulting from several and diverse disease processes, which lead to either total or relative deficiency of insulin (Rudy and Richard, 2014). Superimposed on the disorders of carbohydrate, fat, and protein metabolism are diabetic-specific microvascular lesions in the retina, renal glomerulus, and peripheral nerve, if left untreated, it leads to renal failure, erectile dysfunction, blindness, coronary arterial disease, and increased risk of cancers (Moini, 2019). Diabetes mellitus represents one of the world’s most common diseases according to World Health Organisation (WHO) global report in 2019. The number of people living with diabetes and its prevalence are on the rise in all regions of the world. In the year 1980 for example about 108 million which represent about 4.7% of the word population, were affected. In 2000, an estimated 171 million adults were affected worldwide (Debmandal and Mandal , 2011). This figure rises steadily to affect more than 422 million adults in 2014 (Leandro et al., 2017). Recent data obtained from WHO showed that the global prevalence of diabetes in 2019 was estimated to be about 9.3% accounting for about 463 million people (Moini, 2019). According to the International Diabetes Federation (IDF), it is projected that diabetes mellitus will increase to afflict about 629 million people by 2045 (Moini, 2019).

There has been an increasingly interested in medicinal plants and herbal preparations because of their dependable pharmacological activities and accessibility to the general public, which makes them highly useful in the treatment of a wide range of illnesses (Esterhuyse et al., 2015). Despite vast advances in clinical management of diabetes, late-onset complications remain a big challenge to control particularly in our society. Oral antidiabetic medications commonly used to manage diabetes, which include metformin (Met), thiazolidinediones, alpha-glutamyl transferases, and sulphonyl urease, can cause weight gain or in some cases weight loss, hypoglycemia and coupled with other adverse gastrointestinal side effects, highlighting the need for alternative and effective therapies with fewer or minimal side effects. Furthermore, natural diet based product-derived drugs are more available, affordable and easily consumed with fewer side-effects compared to conventional therapies. Research is increasingly leaning towards the discovery of new antidiabetic drugs from natural products targeting pathways or components associated with anti diabetes potential. (Elbakry and Elremaly, 2023). Virgin coconut oil (VCO) is a natural plant source saturated oil, extracted from the fresh kernel of the coconut fruits (Cocos nucifera L.) it is processed without chemical refining, has demonstrated potential anti-diabetic properties. It is rich in medium-chain fatty acids (MCFAs), particularly lauric acid, VCO is reported to improve lipid metabolism, enhance insulin sensitivity, and exert potent anti-inflammatory and antioxidant effects (DebMandal & Mandal, 2011; Marina et al., 2009). Virgin Coconut oil has been promoted as a dietary supplement for people with diabetes mellitus. However, there was scarcity of information regarding its insulinotropic potential also there have been few formal scientific studies to validate its health benefits particularly on the β-cells, the need to investigate this effects using animal model.

DRUGS, CHEMICALS AND REAGENTS

Alloxan Monohydrate (Sigma- Aldrich) with CAS Number: 2244-11-3 , Rat insulin ELISA kits with catalog number 90010 (Crystal Chem. USA) with aprecision index < 10%, Eosin (1%) and 5% Negrosin stains were purchased from Cardinal Scientific supply from Kaduna, Nigeria. Physiological saline, distilled water, ketamine, diazepam and 10% Form-aldehyde, distilled water were used for the study.

INSTRUMENTS AND OTHER MATERIALS

Syringes and niddle, plastic cages oral cannular, masking tape, markers, disposable gloves, disposable, electronic weighing balance, HM-Lux microscope, (Germany), glass slides and cover slip, electronic blender, EDTA sample bottle, Petri- dishes, tissue forceps surgical blades, scissors, scalpel, centrifuge, refrigerator, Denley BS400 centrifuged machine (England), spectrophotometer (Spectro V-16 (MRC), blood glucometer and strips (Accu-chek Advantage II, Roche Diagnostic GmbH Gremany).

ETHICAL CONSIDERATIONS

Ethical approval was obtained from Ahmadu Bello University committee on animal use and care with this approval number obtained (ABUCAUC/2021/077).

EXPERIMENTAL ANIMALS.

Twenty-five (25) apparently healthy Male Wistar rats between the ages of 6 – 10 weeks, weighing from 90 g – 120g were used for this research. The rats were obtained from the Department of Human Physiology, Faculty of Basic Medical sciences, College of Medical Sciences, Ahmadu Bello University Zaria, Nigeria. The rats were housed in plastic cages and kept in the animal house of the Department of Human Physiology, Faculty of Basic Medical sciences, College of Medical Sciences, Ahmadu Bello University Zaria. They were fed with standardard pellet diet and water ad libitum.

SOURCE OF COCONUT FRUIT

Coconut fruits were obtained from a local market at samara Zaria, Kaduna state, Nigeria. It was taken to herbarium unit of Department of Biological science, Ahmadu Bello University Zaria for identification. Voucher number was given.

PREPARATION OF VIRGIN COCONUT OIL

Coconut oil was prepared according to the method of Hayatullina et al., (2012) with some modifications. The coconuts was broken open manually using a hammer, its meat scrapped from the shell and cut into a small piece using a sharp paring knife. Water was added to the cut pieces and grinded using an electric blending machine into viscous slurry and thereafter it was squeezed through cheese cloth to obtain coconut milk which was stored into wide bore rubber container. The container containing the squeezed coconut milk was stored in refrigerator and left for at least 5 to 6 hours to allow the coconut milk and oil to separate into a layer of curd which appears at the top of the container and water underneath. Thereafter, the hardened curd was scooped out using a serving spoon, this contains both coconut oil and coconut milk, and the underneath water was discarded. The mixture of coconut milk and oil was returned back to the refrigerator for another 2-3 hours, this separate the coconut oil from the coconut milk. Thereafter, the curd was brought out and left at room temperature for at least 20 to 30 minutes, using a spoon, the pure Coconut oil was scooped and the obtained coconut oil was decanted into a clean bottle with a plastic screw cap and stored at room temperature and used for the study.

INDUCTION OF DIABETES

Diabetes was induced intraperitoneally using Alloxan monohydrate (Sigma Co, USA) at a dose of 150 mg/kg of body weight using needle and syringe, as described by the method of Szkudelski (2001). Only animals with blood fasting glucose ≥ 200 mg/dL in two successive determinations (measured at one week interval after diabetes induction) were used. Diabetic animals that died during the post-induction period or at follow-up were replaced to avoid compromising the final number of rats in the sample group.

The animals were randomly divided into five groups with five rats per each group.

Group I (n = 5 rats ). This group served as normal control group. The rats were given 1ml per kg body weight of distilled water daily orally for a period of four (4) weeks distilled water.

Group II (n = 5 rats). This group served as diabetic control. The rats were administered 1ml per kg body weight of distilled water daily orally for a period of four (4 weeks).

Group III (n= 5 rats). These diabetic test group were administered with 5mls/kg of coconut oil daily orally for four (4) weeks. (Iranloye et al., 2013) as modified.

Group IV (n= 5 rats). This group is also a diabetic test group, the rats were administered with 10mls/kg of coconut oil daily orally for four weeks (Iranloye et al., 2013) as modified.

Group V (n= 5 rats). The group is a diabetic test group, they received 15mls/kg of coconut oil daily orally for four week (Iranloye et al , 2013) as modified.

DETERMINATION OF BLOOD GLUCOSE LEVELS

After 72 hours of Alloxan injection blood samples was obtained from the tail vein of the rats and the fasting blood glucose levels was determined as a base line. Subsequently, the blood glucose levels was determined once every week for the period of 4 weeks using standard kit Accu-CHEK Active glucometer. For every blood glucose determination, blood samples were obtained from tail vain, following overnight fasting.

SAMPLE COLLECTION

At the end of the 4 weeks of treatment, the rats were sacrifice, 24 hours after the last day of administration, after anaesthetizing the rats with ketamine and diazepam at 50mg/kg and 10mg/kg respectively. Two (2mls) of blood samples was collected from the sacrificed rats through cardiac puncture and transferred into an EDTA bottles. The abdomen was opened wide and the pancreas was harvested, weigh and fixed in 10% formaldehyde for histological examination. The blood samples in the sample bottles was centrifuge at 3000 revolutions for 10 minutes to obtain the serum. The serum was collected and transferred into a plain sample bottle using needle and syringe, which was later used for analysis of lipid profile, insulin.

DETERMINATION OF SERUM LIPID PROFILE

This was determined spectrophotometrically, using enzymatic colometric assay kits (Randox, Northern Ireland) as follows:

Determination of Serum Total Cholesterol: The serum level of total cholesterol was quantified after enzymatic hydrolysis and oxidation of the sample as described by method of Stein (1987). Briefly, 1000 µl of the reagent was added to each of the sample and standard. This was incubated for 10 minutes at 20-25 ⁰C after mixing, and the absorbance of the sample (A _sample)andstandard (A _standard)was measured against the reagent blank within 30 minutes at 546 nm. The value of total cholesterol present in the serum was expressed in mg/dl. Total Cholesterol concentration = A sample /A standard x 196.86 mg/dL.

Determination of Serum Triglyceride: The serum triglyceride level was determined after enzymatic hydrolysis of the sample with lipases as described by Tietz (1990). Briefly 1000 µl of the reagent was added to each sample and standard. This was incubated for 10 minutes at 20-25 ⁰C after mixing, and the absorbance of the sample (A _sample)andstandard ( A _standard)was measured against the reagent blank within 30 minutes at 546 nm. The value of triglyceride present in the serum was expressed in mg/dl.

Triglyceride concentration = A sample/A standard x 194.0 mg/dL.

Determination of Serum High-density Lipoprotein Cholesterol: The serum level of high-density lipoprotein Cholesterol (HDL-C) was measured using the method of Wacnic and Albers (1978). Low-density lipoproteins (LDL and VLDL) and chylomicron fractions in the sample was precipitated quantitatively by addition of phosphotungstic acid in the presence of magnesium ions. The mixture was allowed to stand for 10 minutes at 20-25 ⁰C, and centrifuged for 10 minutes at 4000 g. The supernatant represented the HDL-C fraction. The cholesterol concentration in the HDL fraction, which remained in the supernatant, was determined. The values of HDL-C wereexpressed mg/dL.

Determination of Serum Low-density Lipoprotein Cholesterol: The serum level of LDL-C was measured according to the protocol of Friedewald et al. (1972) using the equation below LDL-C = TC - (TGL/5 + HDL-C). The value was expressed in mg/dL.

DETERMINATION OF SERUM INSULIN LEVELS

The method of assay was based on the manufacturer’s manual.

The Insulin ELISA is a two-site enzyme immunoassay utilizing the direct sandwich technique with two monoclonal antibodies directed against separate antigenic determinants of the insulin molecule. 95µL diluents with 5µL of the sample specimens were pipetted into the sample well and incubated overnight at 4^oC, then followed by the addition of 100µL peroxidase-conjugated anti-insulin antibodies. The insulin present in the sample binds to the anti-insulin antibodies bound to the sample well, while the peroxidase-conjugated anti-insulin antibodies also bind to the insulin at the same time. After washing to remove unbound enzyme-labeled antibodies, 100µL TMB-labeled substrate solution was added and it binds to the conjugated antibodies. Acid was added to the sample well to stop the reaction, and the colorimetric endpoint was read on a microplate spectrophotometer set 450/630nm wavelength. This determine the serum insulin concentration.

HISTOLOGICAL ANALYSIS OF THE PANCREAS

The pancreas harvested was washed within a physiological saline solution (0.9% NaCl) for the removal of the blood, which might obstruct the process of fixation. Samples were allowed to remain in fixative (10% Neutral buffered formalin) for 24 hours (Cardiff et al., 2014).. The dehydration and clearing of the tissues were processed routinely and embedded in paraffin wax. About 5 microns thickness sections were prepared with microtome and mounted on clean glass slides and then they were deparaffinized in xylene twice for 5 minutes and then rehydrated with graded alcohol and stained with hematoxylin and eosin (H&E) dye according to the method of Fischer et al, (2008). The stained sections were examined and photographed by using microscope. Pancreas of diabetic group was compared with pancreas of the different treatment groups for histological changes.

STATISTICAL ANALYSIS

The data obtained from the study were expressed as mean + standard error of mean (S.E.M). The significance of differences among the group was assessed using one way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparism. Repeated measure ANOVA was used to compare the mean fasting glucose within the treatment groups. P values less than or equals to 0.05 was considered as statistical significance. All data were analyzed using IBM SPSS Version 20.0.

Hyperglycemia was detected one week after 150 mg/kg intraperitoneal injection of Alloxan (table 1) indicating that the induction of diabetes was successful (FBS greater then the base line values) when comparing the normal rats with diabetic control group. This support previous studies and reports by Lenzen et al,(1996), Jorns et al, (1997) and Szkudelski (2001), which showed that Alloxan induced experimental diabetes by selective β-cell death. The proposed mechanism through which it exert this effect is that; Alloxan is an unstable, cytotoxic glucose analogue, it rapidly accumulate within pancreatic β-cells through glucose transporter 2 (GLUT2), once it is taken up by the cells it is reduced to dialuric acid with intracellular and redox cycling which generate reactive oxygen species such as hydrogen peroxide, superoxide radicals and hydroxyl radicals. Alloxan also has the ability to depolarize pancreatic β-cells that further opens voltage dependent calcium channels and stimulates calcium inflow into β-cells. The increased concentration of Ca²⁺ion contributes to supraphysiological insulin secretion that along with ROS has been noted to cause damage of β-cells (Lenzen, 2008; Etuk, 2010), and the ensuing state of hyperglycemia. Furthermore, it is also shown that due to its central 5-carbonyl group that reacts with thiol groups, Alloxan inhibit pancreatic thiol enzymes such as glucokinase (Danilova et al., 2017). This selectively inhibits glucose-induced insulin secretion through the oxidation of glucokinase thus impairing glucose-sensing ability of beta cell. Couple with the fact pancreas β- cells have a low antioxidative defense capacity, thus the generated ROS, high concentration of Ca²⁺ and low energy (ATP) further generate a hostile environment leading to the death of these cells. Four weeks oral administration of coconut oil decreased the fasting blood glucose levels amongst the diabetic treatment groups when compared with diabetic control. This study support previous reports by Iranloye, et al. (2013) which showed that coconut oil has a hypoglycemic effects. On the other side, this findings contradict the work of Durasevic et al, (2019) who reported that coconut oil supplementation bring no change in weekly glycaemia in diabetic Wistar rats.

Insulin is a small protein that is synthesized by the β-cell, it lowers glucose levels partly by suppressing glucose output from the liver, both by inhibiting glycogen breakdown (glycogenolysis) and by inhibiting gluconeogenesis (Umar, 2016). The statistical lower insulin levels (P ≥ 0.05) observed in table 2 following the four weeks oral administration of virgin coconut oil could be due to the high content Medium-Chain Fatty Acids (MCFAs), primarily lauric acid (C12:0). Lauric acid, was reported to have insulinotropic properties and also partly due presence of polyphenols in the VCO which was also reported to enhances sensitivity to insulin and reduces insulin resistance. Djurasevic et al, (2018), has shown that virgin coconut oil supplement increases the fecal abundance of probiotic such as Lactobacillus, Allobaculum and Bifidobacterium species which potentially increased production of short-chain fatty acids in the colon and caecum during anaerobic fermentation of non-digestible dietary fibres, with the acetate, propionate, and butyrate representing 95% of the whole SCFAs content. These Short chain fatty acids was reported to significantly affect glucose homeostasis through improved gut barrier function through the stimulation of release of glucagon-like peptide 1 (GLP-1) and PYY, reduced inflammation, and improved insulin secretion and sensitivity (Qin et al.,2012; Zhao et al., 2018; Canfora, et al., 2019). Furthermore, Acetate and butyrate, may also play a role in maintaining β-cell function, by modulation of cytotoxic T cells action (Chambers et al., 2018). Scientific reports has shown that deficiency in SCFAs synthesis has been associated with diabetes pathophysiology (de Groot et al., 2021). It was shown that supplementation with the dietary fibres increased amount of plasma propionate, which causes a reduction in post-prandial insulin release and improved glucose homeostasis through improved pancreatic β-cell function (Durasevic et al., 2019). This study is further supported by the findings of Garfinkel et al. (1992) who worked on perfused isolated pancreatic tissue and reported that medium chain fatty acids ( such as lauric acid and capric acid) significantly increase insulin secretion. The possible mechanism for the stimulatory effect of the medium chain fatty acids on insulin secretion may be due to the polyunsaturated fatty acid-induced enhancement of insulin secretion involved in the fatty acid oxidation, since it is known that medium chain fatty acids in general are very rapidly oxidized. It seems possible that the very potent effect of capric and lauric acids on insulin secretion may be related to their rapid and complete oxidation. It can also be speculated that, it may also be related to the generation of an optimal signal transduction/energy coupling for the process of exocytosis which is a necessary step for insulin release.

Diabetes mellitus itself decreased serum HDL concentration and increased serum triglycerides and total cholesterol concentration, causing a rise in the atherogenic index as noted in the diabetic control group (table 3) in comparison with normal controls group. However, oral administration of virgin coconut oil within the four weeks treatment periods, statistically increased ( P ˂ 0.5) the serum HDL levels and decreased the total cholesterol and triglycerides levels, these in fact decrease the atherogenic index noted in the diabetic treated groups in comparison to the diabetic control group. The result of our study shows no effects on LDL levels. Paz et al. (2018), who worked on human subjects reported that administration of virgin coconut oil statistically reduced HDL levels but has no effects on serum total cholesterol, triglycerides and LDL. The statistical lower serum triacylglycerol levels (P < 0.05) observed in the treated group when compared with the diabetic control group might probable due to the presence of high content of MCFAs ( particularly lauric acid), MCFAs are absorbed directly into the portal vein and transported to the liver, where they are preferentially oxidized for energy (via beta-oxidation) rather than being stored as fat or incorporated into lipoproteins, this increased energy expenditure and fat-burning can help reduce the overall circulating levels of triglycerides as noted in our treatment group. Our findings is on the contrary with work of Durasevic et al. (2019) who reported that oral virgin coconut oil supplementation in rats increases the atherogenic index by increasing the serum triglycerides, total cholesterol, high density cholesterol, and non-high density cholesterol concentrations, leading to a drop in the TC:HDLC ratio. On the other hand, Dosumu et al. (2012) who worked on the therapeutic potential of coconut oil reported that it does not adversely alter serum lipid levels. The statistical decrease (P˂ 0.05) in serum cholesterol levels observed in this study could be due to high polyphenol content of the virgin coconut oil (Nevin and Rajamohan, 2006). The high polyphenol content is capable of maintaining the normal levels of cholesterol and other lipid parameters in tissues and serum and also increased the concentration of HDL cholesterol. The suggested mechanisms of actions used by polyphenols include trapping of reactive oxygen species in aqueous media such as serum and interstitial fluid thereby inhibiting LDL oxidation, reversal of cholesterol transport and reducing intestinal absorption of cholesterol.

Plate A shows a photomicrograph sections of the normal control rats pancreas which shows an organized pattern of the islet cells, they are arranged in trabecular and acinar pattern with abundant eosinophilic cytoplasm and centrally placed small nucleus, each cells separated by thin loose connective tissue with thin vessels. The islets covering connective tissue sheet is well distinguishable giving the islets their typical spherical shape.

Plate b present Pancreatic tissues of Alloxan induced diabetic animals. It showed massively decreased β cells mass and the islet cells are irregular in shape with degenerated entire connective tissue sheet

Plate 1 shows the pancreas treated with10ml/kg of VCO, there is a marked improvement with significant restoration of islets of Langerhans, regular and increase mass of islets cells and restored islets shape and connective tissue sheet. Significant mass of these Islet cells restored their rounded shape while few of them were still with elongated shape. The islets covering connective tissue was also relatively restored and regain its normal texture. The degeneration of the islet cells seen in the diabetic control group was due to toxic effects of Alloxan as reported above, this was evident by increased levels of blood glucose, and marked decreased in serum insulin. This agrees with the report of Szkudelski (2001), who reported that intravenous, intraperitoneal or subcutaneous injection of Alloxan induced selective necrosis of the β cells. It was reported by Migliorini et al. (2014) that the pancreatic epithelium contains multipotent progenitors which possess the inherent potential to give rise to all pancreatic cell types such as the ductal, endocrine and exocrine lineages following injury or prolonged metabolic stress. Improvement in the histological pictures observed may be possible that virgin coconut oil might have reactivated some embryonic programs (through mechanisms not covered by this study) and cause differentiation of the adult ductal epithelium, (which serve as potential facultative progenitors) into the endocrine lineage (i.e the islet cells) or it is also be likely that virgin coconut oil might have caused some neogenesis and proliferation of pre-existing β-cells in other to restore the β-cell mass.

The study concludes virgin coconut oil lowered blood glucose, increases serum insulin levels and significantly restore β cell mass. Furthermore, it lowers serum total cholesterol, triglycerides and it increase serum high density lipoprotein levels in Alloxan-induced diabetic Male Wistar rats.

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Table 1 to 3 are available in the Supplementary Files section.

Plate 1 is available in the Supplementary Files section.

The authors declare no competing interests.

Pancreashistology.jpg
Plate 1 : Effect of Virgin Coconut Oil on pancreas histology
glycemicchange.jpg
Table 1: Time- Dependent Glycaemic change in Alloxan-induced Diabetic Male Wistar rats treated with Virgin coconut oil
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Table 2: Effect of Virgin Coconut Oil Treatment on serum insulin levels
lipidprofile.jpg
Table 3 : Effect of Virgin Coconut Oil Induced change on lipid profile
Table4.1bpercentageGlycaemicChangeAmongtheTreatmentGroups12.gif
Table 4: Coconut Oil Induced change on lipid profile

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Evaluation of Blood Glucose, serum insulin levels and some Biochemical parameters of Vigin Coconut oil in experimental Diabetic Male wistar Rats

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Abstract

INTRODUCTION

EXPERIMENTAL DESIGN

RESULTS AND DISCUSSIONS

CONCLUSION

References

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Supplementary Files

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