Tender Coconut Water Inhibits the Process of Lipid Peroxidation, Reduce Glucose Levels, and Increase Plasma Insulin in Pregnant Diabetic Rats

Gestational Diabetes Mellitus (GDM) is described as glucose intolerance which diagnosed during pregnancy, this happens due to the increase of oxidative stress. Globally, one in 10 pregnancies can be related to diabetes, 90% among them are gestational diabetes (GDM).1 In 2015, almost 21 million births (16,2%) were influenced by hyperglycemia during pregnancy. Among 85-90% births of women with GDM, the 10%-15% rest of these pregnancies involved mothers with type 1 or type 2 diabetes or newly detected. in the United States prevalence of GDM increased by 56%.2 After the baby is born, usually GDM disappear. The Oral Glucose Tolerance Test (OGTT) were conducted for six weeks after delivery to make sure that the blood glucose is back to normal. However, the women with GDM have potential risk to get type 2 diabetes in the future include their offsprings and must be tested for diabetes at least once in every 2-3 years.1 Babies from GDM mothers have twice higher risk of injuries during delivery, three times possibility of caesarian delivery, and four times incidence to be admitted into Neonatal Intensive Care Unit (NICU).3


INTRODUCTION
Gestational Diabetes Mellitus (GDM) is described as glucose intolerance which diagnosed during pregnancy, this happens due to the increase of oxidative stress. Globally, one in 10 pregnancies can be related to diabetes, 90% among them are gestational diabetes (GDM). 1 In 2015, almost 21 million births (16,2%) were influenced by hyperglycemia during pregnancy. Among 85-90% births of women with GDM, the 10%-15% rest of these pregnancies involved mothers with type 1 or type 2 diabetes or newly detected. in the United States prevalence of GDM increased by 56%. 2 After the baby is born, usually GDM disappear. The Oral Glucose Tolerance Test (OGTT) were conducted for six weeks after delivery to make sure that the blood glucose is back to normal. However, the women with GDM have potential risk to get type 2 diabetes in the future include their offsprings and must be tested for diabetes at least once in every 2-3 years. 1 Babies from GDM mothers have twice higher risk of injuries during delivery, three times possibility of caesarian delivery, and four times incidence to be admitted into Neonatal Intensive Care Unit (NICU). 3 GDM happen if the body of a woman unable to produce sufficient insulin during her pregnancy.
Insulin is produced by the pancreas and help body cells to use glucose from blood as energy. Insulin deficiency can increase blood glucose. High level of blood glucose can cause problem to women and the babies if it is not maintained properly, have bad effect for mothers and babies, can risk to abortion, stillbirths, premature births, and pre eclampsia and also cause oxidative stress. 4 Some micronutrients intake includes vitamin E, selenium and zinc are significantly low on women with GDM compared to heatlhy pregnant women, the selenium levels of GDM women also decreased, 5 lack of selenium can decrease the activity of glutation peroxidase (GPx) to 90% so it cause lipid peroxidation. 6 MDA levels as lipid peroxidation and oxidative stress biomarkers on women with GDM are higher compared to healthy pregnant women. 5 The increased oxidative stress on GDM can cause the increase of free radicals and decrease of antioxidant protection 7 Lipid peroxidation is significantly lowering the antioxidant capacity during the development of pregnancy. 8 The prevention and management of GDM can be done by increasing the food consumption rich in antioxidant, fruit and vegetables and also other antioxidant rich sources. 7 Tender coconut water is a natural, healthy, nutritious, provided by nature, easily obtained in tropical countries. Tender coconut water is rich in antioxidants, sources of free amino acid such as L-arginine, vitamin C, selenium and minerals which can prevent lipid peroxidation. 6 Several studies have proven that L-arginine contained in tender coconut water is significantly can lower free radicals in the body. 9 The antioxidant effect in coconut water can recover the sensitivity of insulin and has antihypertensive effect. 10 Arginine also has the effect to regenerate pancreatic β cells which cause activity from enzyme which regulates the carbohydrate metabolism and pancreatic damage back to normal. 11 However, study on tender coconut water on oxidative stress on women with GDM is still limited. This research aims to prove that tender coconut water can prevent lipid peroxidation, lower glucose level and increase plasma insulin levels in pregnant diabetic rats.

Tender coconut water
TCW used was coconut varieties viridis (green coconut) aged 5-7 months and obtained from research surroundings. During the age of 5-7 months, coconut has tender, thin, jelly like endosperm and it is edible using spoon. The dosage administered is 8ml/200grBW/day for 2 weeks.

Streptozotocin (STZ) dan Nicotinamide (NA)Induction
Rats were adapted for 7 days with the surroundings, intraperitoneally induced by STZ dosage 65 mg/kgBW and nicotinamide 230 mg/kgBW, 12 wait for 3 days then assessed for glucose level using glucometer, if the glucose level >200mg/dL rats can be used as research material. Dosage of the drug glibenclamide used 0,23mg/kgBW. 13

Administration procedures
Rats used are pregnant wistar strain rats according to the criteria which are aged 2 months old, weighed approximately 180-220g, healthy appearance, active movement, normally fed, no wound and no defects. Of 24 rats were randomly divided into 4 groups and each group consists of 6 rats. After 2 weeks blood were drawn to measure glucose, plasma insulin and MDA level (Chart 1).

Blood drawing procedure
The equipment used is sterile microhematrocrit tubes, blood vials, and sterile cottons. Blood is taken by inserting a microhematocrit tube in the ophthalmic vein in the corner of the rats' eyes' periorbita then slowly rotated until the blood comes out. Blood coming out is contained in ependrof as much as 2cc. Plug out the micro hematocrit tube if the required blood is sufficient, clean the remaining blood in the corner of the rat's eye using sterile cotton 14 . The examination of glucose levels, insulin plasma and MDA levels using ELISA method

Research location
The treatment of experimental animal and examination of MDA, glucose and insulin plasma levels was carried out in PAU Gadjah Mada University Yogyakarta.

Statistical analysis
The data from results of glucose levels, plasma insulin and MDA were tested for normality using Shapiro-Wilk and tested for homogeneity using Levene's Test. Data were normally distributed and homogenous therefore One way Anova continued with Post Hoc LSD test to determine the difference amongst groups. The determination of analysis is according to alpha 5%. 15

DISCUSSION
The result of study indicated that administration of tender coconut water dose 8mL/200grBW for 2 weeks is proven to lower the glucose levels, increasing plasma insulin level and preventing lipid peroxidation marked by the decreased of MDA levels in pregnant diabetic rats. This study was using pregnant rats induced by STZ and Na so rats become diabetic. The pathogenesis mechanism of DM is due to insulin resistance and β cell dysfunction, to get diabetic condition in wistar strain rats, it is by injecting STZ and Na combinations, this was conducted for hyperglycemic and insulin resistance to occur as well as pancreatic damage. Glucose levels before and after STZ+Na were measured, increase of blood glucose after STZ+Na become indication of DM if the blood glucose is > 200 mg/dL. 16 STZ induction resulted in toxic effect of pancreatic cell β selectively and cause diabetic condition, have interconnections with glucose parts in its chemical structrure which allows STZ to enter pancreatic cell β through Glucose transporter type 2 (GLUT-2) in plasmic membrane, pancreatic cell β induced by STZ caused the cell β apoptosis. There are three main pathways to cell apoptosis which are: (1). DNA methylation through ion carbonium formation (CH 3+ ) can cause activation from poly ADP-ribosesynthase enzyme as part of the cellular repairing mechanism and as result, there is decrease of Nicotinamide Adenid Dinukeotida(NAD + ); (2). NO Production; (3). ROS Production in free radicals. 17 The increase of oxidative stress causes diabetic problems. 1 Oxidative stress is due to the imbalances between antioxidant defense system with free radicals due to the ROS increase and caused hyperglycemia. 18 Oxidative stress has been recently recognized as a key mechanism in insulin resistance 17. Some of the mechanisms of reaction which are considered to be involved in oxidative stress Genesis are auto-oxidation glucose, protein glycation, formation of advanced glycation products and polyol pathway. The involvement of ROS during STZ metabolism is the production of uric acid as a final product of ATP degradation by xanthine oxidase of hypoxanthine. This reaction will result in radical superoxidants and hydroxyl derived from dismutase H 2 O 2 during hypoxanthine metabolism, which will accelerate the process of destruction of pancreatic cells β. Hydrogen peroxide (H 2 O 2 ) then produces free radicals such as superoxide anion (O 2 -) and radical Hydroxil (OH -). This reactive compound can lead to lipid peroxidation, resulting in the formation of hydroperotic and endotoxcide fatty acids. 17 Research in this area has revealed that there is a strong correlation between the state of oxidative stress in the body and the incidence of insulin resistance and even late stage diabetes cases. 19 Hyperglycemia can also induce pancreatic β-cell dysfunction, reduced β-cell mass, and insulin deficiency. 16 Oxidative stress can be triggered due to elevated levels of glucose and increased free fatty acids (FFA) which causes increased ROS in the mitochondria, which eventually cause insulin resistance and β cell dysfunction. 18 The occurrence of insulin resistance and β cell dysfunction can be seen in Figure 1.
The use of L-arginine is increased in pregnant woman, this is due to an increased formation of nitric oxide, supporting adaptive vasodilation of pregnancy, and the use of L-arginine by the fetus. L-arginine supplementation in foods can provide substrate sources for nitric oxide synthesis during pregnancy. 20 GDM is associated with serious complications for both the mother and child, adverse consequences on the foetus and the mother increase linearly with increasing maternal blood glucose. 1 GDM represents a defect in pancreatic β-cell function both during and after pregnancy and thus identifies a state of chronic β cell dysfunction, rather than the mere development of relative insulin deficiency in the face of rising insulin resistance during pregnancy. 1 Increased FFA levels have a positive correlation with insulin resistance and insulin resistance causing impaired glucose tolerance. 18   clinical trials, suggesting that treatment with vitamin E, vitamin C, or glutathione (GSH) may increase insulin sensitivity in individuals who are resistant to insulin and or type-2 diabetes mellitus patients.. 7 In normal pregnancy and GDM, lipid transfer from mother to fetal blood passes the placenta through the process, which is regulated by insulin level. 7 There is associations of environmental toxic/nutrient elements' exposure with GDM prevalence. 21 For example, the exposure to arsenic and cadmium during pregnancy were significantly associated with GDM. 22 This research proved that young coconut water can prevent lipid peroxidation which is characterized by decreased MDA levels, this result in accordance with the research of Zulaikhah et al, stating that young coconut water is able to lower MDA levels of gold miners exposed to mercury. 6 Human body put in place several strategies to counteract the effects of free radicals and oxidative stress, based on enzymatic (e.g., SOD, CAT, and GPx) and nonenzymatic (e.g., lipoic acid, glutathione, ʟ-arginine, and coenzyme Q10) antioxidant molecules, all of them being endogenous antioxidants. Beside these, there are several exogenous antioxidant molecules of animal or vegetal origin, mainly introduced by diet or by nutritional supplementation. 23 The lipid peroxidation process can be inhibited by compounds contained in young coconut water. L-arginine content contained in coconut water can be used as source no and no proved to be able to lower Fe and Cu, so as to inhibit Fenton reaction and inhibit the formation of hydroxyl radicals (OH -) which will eventually inhibits lipid peroxidation. 24 The role of vitamin C works synergized with vitamin E in inhibiting lipid peroxidation process. Vitamin E which is oxidized by free radicals can react with vitamin C, after getting a hydrogen ion from vitamin C will turn into vitamin E. Vitamin E in the membrane reacts with radical lipids (LOO • ) forms a radical vitamin E (Vit. E • ). The radical vitamin E reacts with vitamin C to form free radicals vitamin C (Vit. C • ). Radical vitamin C (Vit. C • ) will experience regeneration into vitamin C by involving glutathione (GSH). GSH will be oxidized to oxidized glutathione (GSSG) by the enzyme glutathione peroxidase (GPx), GSSG will be rereduced to form GSH by the glutathione reduktese enzyme (Grade) by involving NADPH as an electron donor. 25 Vitamin E (α-tocopherol) is a lipid-soluble vitamin which acts as a lipid peroxyl radical scavenger, preventing lipid peroxidation chain reactions in the cell membranes. 26 Arginine supplementation has been shown to be a promising method of improving cardiovascular function type II diabetes by increasing endothelial NO synthesis. 11 Tender coconut water contains L-arginine. 27 L-arginine novel nutritional strategy, 11 is non essential amino acid, in nitrid oxid synthetase (no synthase). This compound is kind substrates producing citrulline and NO. NO can inhibit xantin oxidase (XO), increase the levels of SOD, total levels of group (T-SH), vitamin C, total antioxidants (TAC) and inhibit free radical chain reactions through lipid peroxidation. 6 One of the processes of superoxide formation in the body is through the xanthine or hypoxantin oxidation catalyzed by the xanthine oxidase (XO) producing uric acid and superoxide, so that when XO is inhibited by L-Arginine's work then the production of superoxide Decreased due to the need of SOD to elaborate superoxide (O 2 •-) to H 2 O 2 also decreased and eventually hindered lipid peroxidation. 6 L-arginine as a precursor to nitric oxide (NO), may increase insulin secretion and reduce hyperglycemia, and this beneficial action is associated with increased formation of NO in diabetic patients. 11 Glucose levels in this study also decreased after given young coconut water in diabetic pregnant rats. Young coconut water can be used as a source of mineral such as Cu, Zn, Mn, Mg that is beneficial to the body. 27 Epidemiological studies show that most diabetics have low Mg levels. Insulin and glucose are important compounds in the metabolism of Mg. Low Intracellular Mg levels will lead to damaged kinase tyrosine activity, post-receptorial decline in insulin work, and worsening of insulin resistance in diabetic patients. Low Mg intake is associated with the development of type 2 DM. The deficiency of Mg can trigger the development of proinflammatory which causes production and excessive release of interleukin. Mg supplementation is a public health strategy to reduce the risk of diabetes. A prospective study of 85. 000 women who are followed for 18 months are representing that women who consume the highest mg have a protective factor against type 2 DM when compared to women who consume the lowest Mg (RR = 0.68). 28 Results showed that mean levels of Mg serum with DM sufferers lower if compared with non DM (patients with DM = 1.88 ± 0.28 mg/dL and non DM = 2.10 ± 0.29 mg/dL). There are differences in the significance of the mean Mg serum between patients with DM and non DM (P < 0.003). 29 The effectiveness of the antioxidant enzyme SOD can be increased by the presence of minerals such as copper (CU) and zinc (Zn) and manganese (Mn). Lacks of minerals such as Cu, Zn and Mn can decrease the activity of Cu-Zn SOD and MN-SOD. SOD so that it can cause lipid peroxidation. 6

CONCLUSION
Tender coconut water is able to inhibit the proces of lipid peroxidation, reduse glucose levels and increasing plasma insulin levels in pregnant diabetic wistar strain rats.