Int J Curr Pharm Res, Vol 9, Issue 5, 90-96Original Article


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

V. SUGANYA, V. ANURADHA*, M. SYED ALI, P. SANGEETHA, P. BHUVANA

Department of Biochemistry, Mohamed Sathak College of arts and Science, Sholinganallur, Chennai, Tamil Nadu, India
Email: srisugan20@gmail.com

Received: 24 May 2017, Revised and Accepted: 22 Jul 2017

ABSTRACT

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.

Keywords: Astaxanthin, Anti-diabetic, DNSA, Starch-iodine, Acarbose, α glycosidase

© 2017 The Authors. Published Published Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijcpr.2017v9i5.22147

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 anti-inflammatory [6, 7] and antidiabetic activities [8], as well as antioxidative effects [9-12]. 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.

MATERIALS AND METHODS

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:

% inhibition = [(O.D. of control–O.D. of test sample)/O.D. of control] × 100

α amylase enzyme assay (Starch-Iodine color assay method)

Screening of test samples for α-amylase inhibitors was carried out according to [25, 26] with slight modification based on the starch-iodine 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 α-amylase 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:

% inhibition = [(O.D. of control–O.D. of test sample)/O.D. of control] × 100

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:

% inhibition = [(O.D. of control–O.D. of test sample)/O.D. of control] × 100

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:

% inhibition = [(O.D. of control–O.D. of test sample)/O.D. of control] × 100

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.

RESULTS

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 α-amylase 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.

Table 1: In vitro Alpha-amylase inhibitory activity (DNSA method) of standard drug acarbose

Content Concentration (µg/ml) mean±SD percentage IC 50 values
Blank - 0.00
S1 250 32.3±0.100 690.830
S2 500 51.1±0.058
S3 750 66.2±0.053
S4 1000 82.9±0.100
S5 1250 99.2±0.058

Table 2: In vitro Alpha-amylase inhibitory activity (DNSA method) percentage for different concentration of test samples

Concentration

(µg/ml)

Non-encapsulated astaxanthin

mean±SD percentage

ME 1 mean±SD

percentage

ME 2 mean±SD

percentage

ME 3 mean±SD

percentage

ME 4 mean±SD

percentage
250 19.53±0.115 19.37±0.100 19.79±0.058 19.95±0.058 20.32±0.100
500 35.04±0.100 34.83±0.153 35.30±0.058 35.57±0.058 36.04±0.115
750 56.46±0.058 56.31±0.058 56.83±0.100 57.10±0.115 57.57±0.115
1000 73.77±0.100 73.56±0.058 74.35±0.153 74.62±0.058 75.04±0.100
1250 87.97±0.100 87.81±0.153 88.92±0.058 89.08±0.058 98.82±0.115
IC 50 Values 685.169 687.703 679.168 675.780 669.129

Among all the test samples (both encapsulated and non-encapsulated astaxanthin) the ME 4 at 1250 μg/ml concentration, had the highest amylase inhibition of 89.82% followed by ME 2 and ME 3 with inhibition of 88.92% and 89.08% respectively. ME 1 and non-encapsulated astaxanthin showed the inhibition of 87.81% and 87.97% at concentration 1250 μg/ml. The standard drug acarbose showed the percentage of inhibition 87.55% at concentration 1250 μg/ml when compared with the test samples. The graph was represented in Graph 1 and Graph 2.

The IC 50 values were also calculated from the percentage of inhibition by each samples. The IC 50 values of standard drug acarbose was 690.830 μg/ml which is compared with test samples such as non-encapsulated astaxanthin, ME 1, ME 2, ME 3 and ME 4 that possessed 685.169 μg/ml, 687.703 μg/ml, 679.168 μg/ml, 675.168 μg/ml and 669.129 μg/ml respectively (table 1 and table 2). Thus, all the test samples showed highest α-amylase inhibition at different concentration (250 μg/ml to 1250 μg/ml) than standard drug acarbose.

Graph 1: In vitro Alpha-amylase inhibitory activity (DNSA method) standard drug acarbose

Graph 2: In vitro Alpha-amylase inhibitory activity (DNSA method) for different concentration of test samples

Table 3: In vitro Alpha-amylase inhibitory activity (Starch-Iodine color assay method) of standard drug acarbose

Content Concentration (µg/ml) mean±SD percentage IC 50 values
Blank - 0.00 658.755
S1 250 19.84±0.211
S2 500 35.01±0.037
S3 750 60.07±0.091
S4 1000 75.28±0.042
S5 1250 94.34±0.103

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

Concentration

(µg/ml)

Non-encapsulated astaxanthin

mean±SD percentage

ME 1 mean±SD

percentage

ME 2 mean±SD

percentage

ME 3 mean±SD

percentage

ME 4 mean±SD

percentage
250 18.37±0.115 19.20±0.093 20.53±0.113 19.84±0.141 21.17±0.191
500 32.55±0.132 36.93±0.051 34.12±0.121 33.23±0.072 35.40±0.116
750 55.10±0.051 55.64±0.003 57.61±0.213 56.23±0.063 57.56±0.053
1000 71.54±0.033 72.82±0.001 72.43±0.115 71.54±0.041 73.36±0.051
1250 84.64±0.001 85.57±0.012

  1. ±0.043

 86.66±0.004 88.08±0.158
IC 50 Values 714.438 690.224 689.056 699.113 675.573

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.

When we compared the nonencapsulated and encapsulated astaxanthin, the maximum activity at 1250 μg/ml was exhibited by ME 4 (88.08%) followed by ME 3 (86.66%) and ME 2 (85.92%). Non-encapsulated astaxanthin and ME 1 test sample exhibit 84.64% and 85.57% which is similar to other test samples. Standard drug acarbose possessed very high inhibition of above 94.34% at concentration 1250 μg/ml which is greater than that of the test samples. At concentration 250 μg/ml the test samples such as non-encapsulated astaxanthin, ME 1, ME 2, ME 3 and ME 4 possessed 18.37%, 19.20%, 20.53%, 19.84% and 21.17% respectively. The standard exhibit maximum of 19.84% when compared with all other test samples (Graph 3 and Graph 4). The IC 50 values of standard drug, non-encapsulated astaxanthin and encapsulated astaxanthin was 658.755 μg/ml, 714.438 μg/ml, 690.224 μg/ml, 689.056 μg/ml, 699.113 μg/ml and 675.573 μg/ml respectively.

Graph 3: In vitro Alpha-amylase inhibitory activity (Starch-Iodine color assay method) of standard drug acarbose.

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

Table 5: In vitro Alpha-glucosidase inhibitory activity of standard drug ascorbose

Content Concentration (µg/ml) mean±SD percentage IC 50 values
Blank - 0.00 674.687
S1 250 16.67±0.158
S2 500 38.02±0.058
S3 750 61.98±0.208
S4 1000 71.30±0.238
S5 1250 88.76±0.118

The results of the α-glucosidase are summarized in table 5 and table 6. All the test samples showed the varying effect on α-glucosidase activity. The standard drug showed maximum inhibition with the highest value of 88.76% seen at 1250 μg/ml concentration. Among the test sample, the highest value was obtained by ME 474.30% and ME 3 70.83% at concentration 1250 μg/ml. Compared to these test samples non-encapsulated astaxanthin, ME 1 and ME 2 possessed 68.96%, 64.93% and 67.46% (Graph 5 and Graph 6) respectively.

Table 6: In vitro Alpha-glucosidase inhibitory activity percentage for different concentration of test samples

Concentration

(µg/ml)

Non-encapsulated astaxanthin

mean±SD percentage

ME 1 mean±SD

percentage

ME 2 mean±SD

percentage

ME 3 mean±SD

percentage

ME 4 mean±SD

percentage
250 14.79±0.178 13.44±0.113 14.37±0.167 14.79±0.153 15.22±0.123
500 25.61±0.143 24.58±0.134 25.28±0.153 26.45±0.124 28.89±0.146
750 46.54±0.243 43.73±0.234 45.60±0.145 49.20±0.153 50.23±0.126
1000 59.93±0.232 56.55±0.156 58.38±0.178 62.41±0.156 65.26±0.174
1250 68.96±0.134 64.93±0.145 67.46±0.103 70.83±0.183 74.30±0.126
IC 50 Values 869.760 923.286 889.683 838.895 802.093

Graph 5: In vitro Alpha-glucosidase inhibitory activity of standard drug acarbose

Graph 6: In vitro Alpha-glucosidase inhibitory activity for different concentration of test samples

The IC 50 values were also evaluated from the percentage of inhibition by each test samples. Test samples such as non-encapsulated astaxanthin, ME 1, ME 2, ME 3 and ME 4 showed IC 50 values of 869.760, 923.286, 889.683, 838.895 and 802.093 μg/ml along with standard drug acarbose that possessed 674.687 μg/ml.

In vitro Alpha-glucosidase inhibitory activity (Alternative method)

The result of In vitro Alpha-glucosidase inhibitory activity (Alternative method) was given in table 7 to table 8. All samples showed maximum inhibition at 1250 μg/ml and least inhibition at 250 μg/ml. At concentration 1250 μg/ml the test samples such as non-encapsulated astaxanthin, ME 1, ME 2, ME 3 and ME 4 produced 88.68%, 83.11%, 85.53%, 84.37% and 89.94% along with standard i.e. 90.21%. The least inhibition at 250 μg/ml of concentration were recorded by test samples i.e. 18.96%, 17.61%, 16.98%, 18.15% and 19.95%. The standard drug also exhibits similar percentage of inhibition 19.32%. The graph was plotted against the percentage of inhibition and concentration for both standard and test samples indicated in Graph 7 and Graph 8.

Table 7: In vitro Alpha-glucosidase inhibitory activity (Alternative method) of standard drug acarbose

Content Concentration (µg/ml) mean±SD percentage IC 50 values
BLANK - 0.00 656.436
S1 250 19.32±0.141
S2 500 39.71±0.017
S3 750 58.22±0.043
S4 1000 75.83±0.221
S5 1250 90.21±0.107

Table 8: In vitro Alpha-glucosidase inhibitory activity (Alternative method) percentage for different concentration of test samples

Concentration

(µg/ml)

Non-encapsulated astaxanthin

mean±SD percentage

ME 1 mean±SD

percentage

ME 2 mean±SD

percentage

ME 3 mean±SD

percentage

ME 4 mean±SD

percentage
250 18.96±0.189 17.61±0.043 16.98±0.191 18.15±0.231 19.95±0.097
500 37.35±0.119 35.22±0.057 36.12±0.117 36.66±0.145 38.27±0.063
750 55.71±0.113 53.46±0.061 53.37±0.102 54.27±0.173 56.33±0.037
1000 71.97±0.210 68.55±0.113 68.46±0.134 69.72±0.029 72.60±0.183
1250 88.68±0.173 83.11±0.182 85.53±0.165 84.37±0.075 89.94±0.197
IC 50 Values 684.879 725.811 719.134 710.211 672.294

Graph 7: In vitro Alpha-glucosidase inhibitory activity (Alternative method) of standard drug acarbose

Graph 8: In vitro Alpha-glucosidase inhibitory activity (Alternative method) for different concentration of test samples

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 non-encapsulated 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.

CONFLICT OF INTERESTS

Declare none

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