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IN VITRO ANTI-DIABETIC ACTIVITY OF MICROENCAPSULATED AND NON-ENCAPSULATED ASTAXANTHIN

Objective: Diabetes is a long term condition which indicates the high blood pressure. The symptoms indicates, polyuria (frequent urination), they will become increasingly thirsty (polydipsia) and hungry (polyphagia). Many drugs has been discovered for curing diabetes. Recent studies reported that the administration of astaxanthin reduces the blood pressure in the diabetic patient. Astaxanthin is a powerful antioxidant found in wide variety of aquatic living organism which has wide applications in pharmacological studies. Methods: In vitro antidiabetic study of both encapsulated and non-encapsulated astaxanthin such as DNSA method, starch-iodine color assay method and α glycosidase enzymes assay was carried out. Results: The results of the present study indicated that both encapsulated and non-encapsulated astaxanthin shows higher antidiabetic activity in all the method. Each test samples possess the best activity when compared to standard drug acarbose. Conclusion: The present study, it is concluded that both non-encapsulated and encapsulated astaxanthin exhibit good antidiabetic activity.


INTRODUCTION
Diabetes is one of the major causes of premature death worldwide. Every ten second a person dies from diabetes related causes mainly from cardiovascular complications. Metabolic disease, including dyslipidemia and diabetes, constitutes a major emerging health crisis in the world. The WHO, in the 2009 report, states that high blood plasma ranked first in the list of leading global risks for mortality and accounted for 7.5 million deaths in the world in 2004 [1]. According to reports, 415 million people worldwide were diabetic in 2015, most of them suffering from Type II diabetes [2].
Diabetes, which is diagnosed based on blood plasma hyperglycemia, has been linked to lipid overload and abdominal obesity and may synergize with these conditions to promote negative clinical outcomes [3,4]. Although the symptoms and clinical pathology and physiology of these conditions are well understood, the question of pharmacologic treatment of dyslipidemia and diabetes remains unresolved well. The marine world, due to its phenomenal biodiversity, is a rich natural resource of many biologically active compounds such as polyunsaturated fatty acids (PUFAs), sterols, proteins, polysaccharides, antioxidants and pigments. People worldwide know that marine foods participate in human health promotion. A diet rich in marine products is considered to result in a lower incidence of diabetes, cancer and obesity. To date, many of reports have also showed that bioactive compounds from marine organisms, including Fucoxanthin, Astaxanthin, Marine Collagen Peptides, Dieckol and Krill Oil, exert a positive influence on metabolic dysfunction (diabetes and obesity) [1].
Astaxanthin, a red-orange carotenoid pigment, is a biological antioxidant that naturally found in a wide variety of aquatic living organisms, such as shrimp, crab, and salmon [5]. The green micro algae Haematococcus pluvialis and the red yeast Phaffi a rhodozyma are common sources of natural astaxanthin [5]. Astaxanthin has shown various pharmacological activities, including antiinflammatory [6,7] and antidiabetic activities [8], as well as antioxidative effects [9][10][11][12]. Diabetes mellitus is strongly associated with oxidative stress, which can be a consequence of increased free radical production, reduced antioxidant defences or both [13].
Oxidative stress induced by hyperglycemia possibly causes the dysfunction of pancreatic b-cells and various forms of tissue damage in patients with diabetes mellitus. It was found that astaxanthin could diminish the oxidative stress caused by hyperglycemia in the pancreatic β cells, significantly improve glucose tolerance, increase serum insulin levels, and decrease blood glucose levels, indicating that astaxanthin might exert beneficial effects on pancreatic b-cell function and could protect pancreatic b-cells against glucose toxicity by preventing the progressive destruction of these cells [8]. The main objective of the present study is to investigate the antidiabetic activity for both encapsulated and non-encapsulated astaxanthin.

Microencapsulation of astaxanthin using different agents
Astaxanthin purchased from Rudra Bio ventures Pvt Ltd, Bangalore was encapsulated using four different agents by ionotropic gelation method. In the first method, microencapsulated astaxanthin was prepared by using sodium alginate and calcium chloride [14,15,40]. In the second method, microencapsulated astaxanthin was prepared using sodium alginate and chitosan [16]. In the third method, chitosan-Tripolyphosphate was used to produce microencapsulated astaxanthin [17,18]. In the fourth method, liposome encapsulated astaxanthin was carried out by the method [19]. These test samples (Both encapsulated and non-encapsulated astaxanthin) were used to study the antidiabetic activity using four different methods.

In vitro anti-diabetic activity α amylase enzyme assay (DNSA method)
Starch solution (0.1% w/v) was prepared by stirring 0.1g of starch in 100 ml of 16 mmol of sodium acetate buffer. The enzyme solution was prepared by mixing 27.5 mg of alpha-amylase in 100 ml of distilled water. The colorimetric reagent was prepared by mixing the sodium potassium tartrate solution and 3, 5 Di-nitro salicylic acid solution at 96 mmol concentration [20,21]. The control tube contains an only reagent and the test sample in the range of 100-500 µg/ml was prepared. From this, 500 µl of sample was mixed with 500 µl of starch solution and 500 µl of alpha-amylase solution which is incubated at 37 ° C for 10 min. The reaction was stopped by the addition of 1 ml of 3, 5 Di-nitro salicylic acids and incubated in boiling water bath for 5 min, cooled at room temperature. The reaction mixture was then diluted by adding 10 ml of distilled water. The absorbance was measured at 540 nm [22,23,24]. Control was tested by replacing test sample with DMSO. The similar procedure were also followed for the standard drug Acarbose. The percentage of inhibition was calculated using the formula: Screening of test samples for α-amylase inhibitors was carried out according to [25,26] with slight modification based on the starchiodine test. Test samples of varied concentrations in 500 μL were added to 500 μL of 0.02 M sodium phosphate buffer (pH6.9 containing 6 mmol sodium chloride) containing 0.04 units of the αamylase solution and were incubated at 37 °C for 10 min.
Then 500 μL soluble starch (1%, w/v) was added to each reaction well and incubated at 37 °C for 15 min. 1 M HCl (20 μL) was added to stop the enzymatic reaction, followed by the addition of 100 μL of iodine reagent (5 mmol I2 and 5 mmol KI). The color change was noted and the absorbance was read at 620 nm on a microplate reader. The control reaction representing 100% enzyme activity were taken. Inhibition of enzyme activity was calculated as: α glucosidase enzyme assay The inhibitory activity of α-glucosidase enzyme was determined by 1 ml solution of starch substrate (2 % w/v maltose or sucrose) with 0.2 M Tris buffer pH 8.0 and 1 ml of test samples in the range of 100-500 µg/ml were incubated separately for 5 min at 37 °C. The reaction was initiated by adding 1 ml of alpha-glucosidase enzyme (1U/ml) to it followed by incubation for 40 min at 35 °C. Then the reaction was terminated by the addition of 2 ml of 6N HCl. Then the intensity of the color was measured at 540 nm [27,20,24]. A control experiment was done by replacing the test sample with DMSO and also for a standard drug Acarbose [28,29,30]. Percentage of inhibition was calculated by the formula: α glucosidase enzyme assay (alternate method) The α-glucosidase inhibitory activity was determined according to a modified procedure [31,32]. Briefly, 50μl of 0.1M potassium phosphate buffer (pH6.9) was pre-incubated with50μl of reduce dglutathione (1 mgml -1), 20μl a-glucosidase (1Uml−1 in0.1M phosphate buffer, pH 6.9 and 20μl of test samples at37 °C for 10 min. After the incubation, 20μl pNPG was added and the mixture was further incubated at 37 °C for 30 min. The reaction was terminated by adding1 ml of 0.1Msodiumcarbonate. The absorbance of the samples and control were taken at 405 nm against a blank devoid of pNPG and sample. The control reaction (with 100% enzyme activity) contained buffer or DMSO instead of the irrespective samples while acarbose was used as a positive control. The inhibitory activity was calculated by using the following Equation:

Statistical analysis
The statistical analyses for all the experiments were done using Excel 2013 through the statistical formula. Experimental data were expressed as mean±SD and IC 50 values were calculated. The experiment was performed in triplicates for all the test samples.

In vitro anti-diabetic activity
In vitro Alpha-amylase inhibitory activity (DNSA method).
In the present study, astaxanthin was encapsulated using four different methods were investigated for their potential to inhibit αamylase activity and α-glucosidase activity. Five different concentrations viz., 250, 500, 750, 1000 and 1250 μg/ml of test samples were separately tested for the inhibition of α-amylase activity and α-glucosidase activity along with standard acarbose.

In vitro Alpha-amylase inhibitory activity (Starch-Iodine color assay method)
We investigated the encapsulated and non-encapsulated astaxanthin as well as standard drug acarbose for their α-amylase inhibitory activities using starch iodine color assay. The OD values were noted in table 3 and table 4.

Graph 4: In vitro Alpha-amylase inhibitory activity (Starch-Iodine color assay method) for different concentration of test samples
In vitro Alpha-glucosidase inhibitory activity

In vitro Alpha-glucosidase inhibitory activity (Alternative method)
The result of In vitro Alpha-glucosidase inhibitory activity (Alternative method) was given in

DISCUSSION
Lack of insulin affects the metabolism of carbohydrates, proteins, fat and causes significance disturbance of water and electrolyte homeostasis [33]. Recent advances in understanding the activity of intestinal enzymes (α-amylase and α-glucosidase both are important in carbohydrate digestion and glucose absorption) have leads to the development of newer pharmacological agents. A high postprandial blood glucose response is associated with micro-and macro-vascular complications in diabetes and is more strongly associated with the risk for cardiovascular diseases than are fasting blood glucose. α-Glucosidase enzymes in the intestinal lumen and in the brush border membrane play main roles in carbohydrate digestion to degrade starch and oligosaccharides to monosaccharides before they can be absorbed. It was proposed that suppression of the activity of such digestive enzymes would delay the degradation of starch and oligosaccharides, which would, in turn, cause a decrease in the absorption of glucose and consequently the reduction of postprandial blood glucose level elevation [34].
Alpha-glucosidase inhibitor retards the digestion of carbohydrates and slows down the absorption. Acarbose and miglitol are a competitive inhibitor of α-glucosidases and reduces absorption of starch and disaccharides [35]. Hence one of the therapeutic approaches for reducing postprandial (PP) blood glucose levels in a patient with diabetes mellitus is to prevent absorption of carbohydrate after food intake. Inhibition of these enzymes (α-amylase and α-glucosidases) reduced the high postprandial (PP) blood glucose peaks in diabetes [36]. Acarbose and Miglitol are a competitive inhibitor of α glucosidases and reduces absorption of starch and disaccharides [35]. The α-amylase inhibitors act as an anti-nutrient that obstructs the digestion and absorption of carbohydrates. Acarbose is complex oligosaccharides that delay the digestion of carbohydrates. It inhibits the action of pancreatic amylase in the breakdown of starch. Synthetic inhibitor causes side effect such as abdominal pain, diarrhoea and soft faeces in the colon.
The present study reveals that both encapsulated and nonencapsulated astaxanthin effectively inhibit both α-amylase and α-Glucosidase enzymes. Our findings were compared with other research articles. The mechanisms were investigated underlying the insulin sensitivity effects of ASX in a non-genetic insulin resistant animal model. The results showed that ASX improved insulin sensitivity by activating the post-receptor insulin signalling, i.e. enhancing the auto phosphorylation of insulin receptor-b (IR-b), IRS-1 associated PI3-kinase step, phospho-Akt/Akt ratio and GLUT-4 translocation in skeletal muscle [37].
Oxidative stress induced by hyperglycemia possibly causes the dysfunction of pancreatic β-cells and various forms of tissue damage in patients with diabetes mellitus. It was found that astaxanthin could diminish the oxidative stress caused by hyperglycemia in the pancreatic β-cells, significantly improve glucose tolerance, increase serum insulin levels, and decrease blood glucose levels [8]. Recently, [38] demonstrated that astaxanthin could substantially improve insulin sensitivity through abolishing significant elevation in both glucose and insulin levels induced by a high fat plus high fructose diet in mice.
But beyond that, the effects of astaxanthin in a metabolic syndrome animal model of spontaneously hypertensive corpulent rat,the results showed that astaxanthin markedly decreased the levels of blood glucose, triglycerides and non-esterified fatty acids, and significantly increased the levels of high-density lipoprotein cholesterol and adiponectin. It is suggested that astaxanthin ameliorates insulin resistance and improve insulin sensitivity by increasing glucose uptake, and by modulating the levels of circulating adiponectin and blood lipids [39].

CONCLUSION
To date, more and more metabolic diseases have influenced in human's health and quality of life. In the last few years, there has been a growing interest in the herbal medicine in care and management of diabetes both in developing and developed countries, due to their natural origin and less side effects.
The adverse effects of current drug treatment are not always satisfactory in maintaining normal levels of blood Glucose. Hence there is continuous thirst towards discovering or identification of bioactive compounds derived from plants and marine sources with potent antidiabetic activity. In the present study, the astaxanthin in free form and encapsulated form has been found to exhibit better antidiabetic potential by inhibition of Amylase and Glucosidase. Our other studies also proved their antioxidant, radical scavenging and anti-inflammatory potential. However, further research should go in this direction in order to show new preventive and potential therapeutic strategies against diabetes and associated disorders.