Therapeutic Applications of Coconut Water Mitigates Diabetes Mellitus in Oryctolagus cuniculus (Rabbit)

Diabetes mellitus (DM) is a pandemic disease and causes significant contribution to human mortality across the globe. Since, natural products have been proved to be safe and effective remedies in human healthcare practice; therefore, the present study has investigated the anti-diabetic potential of coconut water against the diabetic rabbits. Rabbits were intoxicated with streptozotocin (80 mg/kg body weight) and diabetes mellitus was induced. To counteract initial hypoglycemia, 10% of glucose was administrated in drinking water for 24 hours. One group was kept as negative control, another was diabetic and un-medicated control, two groups were fed with coconut water dose 2.2g/kg and 4.1g/kg, and last group was medicated with Metformin. Several analyses such as body weight, physical activities, urination, and different biochemical properties and characterization of blood samples were performed. The intoxicated rabbits were characterized by weight loss, frequent urination, sluggish physical activities, low level of high-density lipoprotein, and elevated levels of blood glucose, cholesterol, and triglyceride. Upon administration of coconut water at a dose rate of 2.2 g/kg and 4.1 g/kg for two weeks, all the parameters became normal and/or near to normal when compared to positive control. Therefore, we recommend that coconut water can be developed as a nutraceutical or functional food for the management of diabetes and its associated complications.


Introduction
Diabetes mellitus (DM) is an epidemic disease and causes severe problem to human health [1]. By definition, diabetes mellitus is a group of metabolic imbalances, results in hyperglycemia, which is either caused by abnormal production of insulin or its harmful physiology [2]. Diabetic patients are at more risk to cardiac, peripheral arterial and cerebrovascular diseases [2,3]. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs including eyes, kidneys, nerves, heart, and blood vessels [4]. DM also disturbs function of liver, therefore, in blood serum the level of glutamic-oxaloacetic transaminase (SGOT) and Serum Glutamic Pyruvic Transaminase (SGPT) increases [5]. There is usually persistent thirst, polyuria, blurred vision, and weight loss followed by ketoacidosis or nonketotic hyperosmolar condition that leads to stupor, coma and, if untreated causes death [6]. In 1993, the World Health Organization (WHO) Diabetes reporting group published global estimates for the prevalence of diabetes from 75 communities in 32 countries [7].
Worldwide, the prevalence rate of obesity, diabetes, and other metabolic syndromes is high [8,9]. There have been several previous estimates of the number of persons with diabetes. Prevalence of diabetes among adults (age 20-79 years) will increase to 7.7% alarmingly high for developing countries where diabetes patients will increase to 69% by 2030 [10]. According to World Health Organization (WHO), diabetes will be the 7th leading cause of death in 2030 [11]. Currently, Pakistan occupies 7th position of high-risk diabetes-suffered countries [12]. The current prevalence of type 2 diabetes (T2D) in Pakistan is 11.77% [13]. Furthermore, according to National Survey (2016-17), 26% of Pakistan population is suffering from diabetes. Therefore, prevention of diabetes mellitus needs serious attention in health policy. By far the most popular approach to treat T2D is glycemic control in an attempt to reduce complications and death. In the past, several drugs for T2D including oral anti-diabetic agents (OAAs), insulin, and incretin-based drugs have been developed.
For instance, sulfonylurea-type drugs, insulin sensitizers such as TZDs (e.g., rosiglitazone and pioglitazone) and a biguanide, metformin, can directly lower insulin resistance and, subsequently, blood glucose. Glucose (re)absorption is viewed as an alternative way to lower blood glucose level. Inhibitors of Sglt 2 and glucosidase such asdapagliflozin, empagliflozin, and acarbose, inhibit the activity of sodium-glucose cotransporter-2 and glucosidases, respectively.
Such inhibition leads to decrease glucose (re)absorption via the renal tubules and the intestine, respectively [14,15]. The available anti-diabetic drugs have limited rolesand cause serious sideeffects. For instance, metformin [16], pioglitazone [17], and SGLT-2 inhibitors [18] cause lactic acidosis, edema and heart failure, and dehydration, urinary and genital infections,respectively. Lowering blood glucose and lipid profiles by using natural products of plant origin as a possible therapeutic measure has become a promising scientific investigation. It has been reported that essential oils may be effective against type 2 diabetes mellitus. The daily consumption of olive oil had positive effect on fasting blood glucose and lipid profiles of healthy controls [19].

Experimental studies have shown that Hibiscus rosasinensis
Linn. ethanol flower extracthas positive effect on blood glucose and lipid profile in streptozotocin induced diabetes in rats [20]. Aloe vera gel extract has hypoglycemic, hypotriglyceridemic properties in STZ-diabetic rats [21]. The natural Egyptian Morus alba root bark extracthas protective effects in diabetes by decreasing glucose level, moderate lipid peroxidation, and preservation of pancreatic β cell integrity [22]. CW is a rich source of essential metabolites including sugars, sugar alcohols, vitamin C,folic acid,free amino acids, phytohormones (auxin, cytokinin), enzymes (acid phosphatase, catalase, dehydrogenase, diastase, peroxidase, RNA -polymerases) and growth promoting factors [23]. Quantitative difference in the composition of the active components available in coconut water are 95.3 % water, 0.005% nitrogen, 0.56% Phosphoric acid, 0.25% Potassium, 0.69% Calcium oxide, 0.59% Magnesium oxide, 0.5% Iron, 0.8% reducing sugar and a total sugar of 2.08% [24]. Since, the coconut water has several medicinal properties such as hypocholesterolemic, antihypertensive, cardio protective, hepatoprotective, and hypolipidemic properties [25,26]. Therefore, the current study has evaluated the therapeutic potential of CW against the diabetic rabbits.

Selection of Animals
Oryctolagus cuniculus (Rabbit) was selected as experimental animals. Rabbits were purchased from local markets and divided into several groups on the basis of their body weights.

Induction of Diabetes Mellitus (DM)
Streptozotocin was used for the induction of diabetes mellitus in rabbits. The rabbits were made diabetic by injecting streptozotocin via peritoneum route [27]. Streptozotocin dose was administrated body weight dependent. To counteract initial hypoglycemia, glucose was given orally, also 10% glucose was provided in drinking water for next 24 hours [28].

a.
Group A was medicated with coconut water at the dose rate of 2.2g/kg body weight.
b. Group B was medicated with coconut water at the dose rate of 4.1g/kg body weight.
c. Group C was kept as null control without any treatment.

Collection of Blood Samples
Blood samples were collected from all the groups with the following schedule; Day zero, day 03, day 08, day 13, and day 18.
Blood samples were collected, stored in slant position and serum was oozed out upon clotting. Serum was collected in falcon tubes and was analyzed for glucose, triglyceride, cholesterol, HDL and LDL levels.
Analysis of biochemical parameters. Blood samples were analyzed for different parameters using their respective standard available kits (Semi Auto Chemistry Analyzer).

Statistical Analysis
The results obtained were analyzed statistically. Mean and standard deviation were determined. All the values of different groups were compared at different days using ANOVA. Online software, Prism, and Demo v05 were used for statistical analysis (www.graphpad.com).

Induction of Diabetes Mellitus
All the rabbits of group A, B, D and E were intoxicated with streptozotocin (80mg/kg). Diabetes mellitus was confirmed on day 3rd of the experiment. Our results suggested that diabetic-induced rabbits lost their body weight (Table 1). Two groups (A and B) were treated with coconut water at dose rate of 2.2g/kg and 4.1g/ kg, while, one group (E) was medicated with 14mg/kg metformin HCl. Our results observed significant increase (P<0.0001) in the body weight of diabetic treated groups (Table 1). Upon treatment of diabetic rabbits with CW, it was observed that the treated rabbits were gaining body weight (Table 1). Conversely, significant decrease (P<0.0001) was observed in the body weight of positive control (Table 1). Our results observed that that coconut water and metformin increase the body weight in diabetic groups (Table 1).

Evaluation of Low-Density Lipoprotein (LDL) in Blood
The healthy samples showed normal concentration of low-

Discussion
Diabetes mellitus (DM) can mainly be attributed to the sedentary life style and calorie-rich diet. DM is linked with abnormal lipid metabolism and is considered as a major factor for the development of atherosclerosis and cardiovascular complication [29]. STZ causes hyperlipidemia along with hyperglycemic condition [21,30,31].
Previously, it was investigated that STZ causes diabetes by rapid depletion of beta-cells, which leads to reduction of insulin secretion [32]. In rats, intravenous injection of STZ (70 mg/kg of body weight) induces diabetes mellitus [33]. Moreover, Mozaffari et al., induced DM in rats by injecting STZ at dose rate of 90 mg/kg of the body weight [31]. In present study, STZ was used as DM inducer at the dose rate of 80 mg/kg body weight. Our results confirmed that STZadministrated rabbits lost their body weights, which is the ultimate symptom of diabetic rabbits [34][35][36] and virgin coconut oil have significant hypoglycemic potential [37].
Additionally, a couple of studies have reported that mature coconut water and coconut kernel protein reduced the blood glucose level of diabetic rats [38,43]. Therefore, our study recommends that coconut water decreasedthe blood glucose level of diabetic rabbits.
Our results observed elevated level of cholosterol in STZ-induced diabetic rabbits. Our results are in strong agreement that STZ accumulates high concentration of cholesterol diabetic rats [41,44].
We observed that CW reduced the blood cholesterol level of diabetic rabbits. Previously, palm kernel oil and coconut oil were used to reduce the total cholesterol levels of diabetic rats [45,46]. Our study also observed that diabetic rabbits were symptomized by elevated levels of triglycerides. Increase in triglyceride concentration is an essential indication of diabetic subjects [41,44,47]. Our study confirmed that CW caused a signifcant reduction in the concentration of triglyceride level of diabetic rabbits.Other studies have also reported that coconut oil and coconut water reduces triglyceride level and have anti-diabetic activities [45,46,48].
STZ administration decreases HDL levels in animals [30,44]. Our study observed that CWincreased HDL level in CW-treated rabbits.
Same results werealsodocumented by several other reports [48][49][50]. Finally, the present study investigated that STZ increased the LDL level STZ-intoxicated rabbits. STZ has been reported to cause a significant increase in LDL concentration which leads to DM [41,44,51]. Our study investigated that CW reduces LDL level in diabetic rabbits. other studies have also observed that coconut water was given to diabetic rats and same reduction in LDL level were found [48,52]. In another study, the synergistic effects of coconut oil and olive oil has demonstrated to reduce the LDL level [53]. Overall, our study demonstrated that CW has anti-diabetic potential and could be used as a nutraceutical or functional food for the management of diabetes and its associated complications.