An In vitro Evaluation of Antidiabetic Activity of Alpinia galangal and Alpinia calcarata

Background: The herbs, genus Alpinia calcarata and Alpinia galanga that underneath the family Zingiberaceae are rhizomatous and extremely aromatic. The study is to investigate the anti-diabetic activity of Alpinia galanga and Alpinia calcarata in-vitro. Material and Methods: The inhibitory effect of Alpinia galanga and Alpinia calcarata on α-amylase and α-glucosidase activities were evaluated. Results: The results revealed that both Alpinia galanga and Alpinia calcarata inhibited α-amylase and α-glucosidase activities in a dose-dependent manner (200–1000 μg/mL). However, Alpinia calcarata possess better antidiabetic activity than Alpinia galangal. Conclusion: The presence of phenolic and other phytochemical content in the herbs might be the reason for their ability to inhibit α-amylase and α-glucosidase activities. Thus, the drug formulating from the herbs, Alpinia galanga and Alpinia calcarata could be part of the potential alternative for synthetic anti-diabetic drug.


INTRODUCTION
According to World Health Organization, the term Diabetes Mellitus (DM) is outlined as a metabolic disorder of multiple etiology characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in hypoglycemic agent (insulin) secretion hypoglycemic agent action, or both [1]. DM is one of the most prevalent disease that affects many folks round the world [2]. As expressed by World Health Organization, the population of diabetic patient can increase over three hundred million folks, most likely in the year 2025 [3]. DM is known as hypo insulinemia, hyperglycemia, hyperlipidemia and hyper aminoacidemia [4]. It is classified as type-1, type-2 and Gestational Diabetes Mellitus (GDM). Type-1 DM is additionally named as autoimmune diabetes, that due to extermination of β-cell islets in exocrine gland via response -mediate, resulting in deficiency of hypoglycemic agent (insulin). Type-2 DM is conjointly named as ketoacidosis-resistant diabetes, that due to resistance of hypoglycemic agent or uncommon secretion of hypoglycemic agent [5]. 90% of polygenic disorder cases add up to Type-2 DM and remainder of the ten percent cases add up to Type-1 DM and GDM [6]. Currently, insulin and some synthetic oral hypoglycemic agents such as sulfonylurea, meglitinides, incretin mimetics, thiazolidinediones and biguanides are used for treating hyperglycemia [7]. Although the synthetic drug act as the first line of therapy, they will turn out with some adverse impact and drug interaction [8] e.g., sulfonamides hinders excretion or metabolism of antidiabetic drug sulfonylurea thus causing hypoglycemia. Rifampicin speed up the rate of metabolism so as to decrease symptom impact [9]. On account of the high price and adverse impact of artificial medicine, medicine from natural plants is designed to treat the un wellness [2,10] and it is appropriate for any age and sexes [11]. Medicinal plants have bioactive compound like polyphenols, phenol, flavanoid, terpenoid. These are helpful to prevent the damage by oxidation due to formation of reactive oxygen species (ROS) [12]. These phytochemicals within the medicinal plants have medical care worth like anti-diabetic, anti-inflammatory, antihyperglycemic etc [13]. Therefore the usage of the inhibitor is helpful for the treatment of diabetes, since it's a reasonably oxidative stress associated diseases [14]. So treating these diseases with the drug from medicinal herbs has been approved by World Health Organization [6,15]. Several Indian plants are studied to treat different forms of diabetes and been reported in numerous scientific journals. The present study was done with plants extract of Alpinia galanga and Alpinia calcarata to prove their anti-diabetic properties.

Alpinia galanga and Alpinia calcarata
The herbs, genus Alpinia calcarata and Alpinia galanga that underneath the family Zingiberaceae, are rhizomatous and extremely aromatic [16]. In Indonesian and Thai cuisines, these herbs are used for preparation and that they posses healthful worth like antimicrobial, antiulcer, anti-spasmodic, anti-inflammatory and anti-diabetic [17]. Alpinia galanga, conjointly referred to as greater galanga, is a source of ascorbic acid, iron, fat-soluble vitamin and sodium. These herbs have bioactive compounds like galangin, saponins, terpenoids, phenolics, carbohydrates, quercetin, alkaloids, glycosides, emodin, phytosterols, galango, beta-Sitosterol, flavonoids. These bioactive compounds have some therapeutic worth like antineoplastic, hypoglycemic, gastro protective, hypo lipidemic, antifungal and anti-inflammatory activities. Studies have shown that ethanolic extract of Alpinia galanga possess sturdy inhibition action against α -amylase and αglucosidase [18] whereas the methanolic extract of Alpinia galanga shows gentle inhibition action against αamylase and α-glucosidase and scavenging activity against DPPH (2,2,1-diphenyl-1picrylhydrazyl) radical [19]. The genus Alpinia calcarata rhizome have several medicinal value such as antibacterial, antifungal, anti-diabetic, antioxidant , aphrodisiac, antifungal, gastro protective, anthelmintic and anticancer activity [20]. Hence the decoction of this rhizome can be used to treat respiratory ailments, stomach ache, asthma, cough, rheumatism, bronchitis, diabetes [21]. Analysis have shown that the ethanolic and hot water extract of Alpinia calcarata has considerable repressing action against αamylase and α-glucosidase [21,22] and strong free radical scavenging activity against DPPH [23]. Data are available that Alpinia calcarata and Alpinia galanga have ellagic acid and gallic acid. Alpinia calcarata has ellagic acid which is double the amount of ellagic acid present in Alpinia galanga whereas the Alpinia galanga has four times higher amount of gallic acid than Alpinia calcarata [24,16].

α-amylase and α-glucosidase Inhibitor
In the blood, the predominant supply for glucose is starch [25] .The dietary starch is hydrolyzed by two major enzymes. They are α-amylase and αglucosidase. Alpha-amylase breakdown the starch into maltose. Then it is further breakdown to glucose [26]. The quicker digestion of starch to glucose can ends up with the elevation of glucose level in the blood which is termed as Post Prandial Hyperglycemia [27]. By inhibiting the hydrolyzing enzyme concerned with the digestion of carbohydrates like α-amylase and αglucosidase, the digestion of sugar is delayed, this can eventually decrease Post Prandial Hyperglycemia [7,28]. This is one of the effective ways to control type-2 diabetes mellitus [29]. The bioactive component present in the medicinal plants and herbs can act as inhibitors of alphaamylase and alpha-glucosidase with minimal side effects [5,30]. Because of the above reason and economical consideration, inhibitors from natural plants have earned popularity [31].

Aim of the Study
The aim of the study was to investigate the antidiabetic effect of Alpinia galanga and Alpinia calcarata by in-vitro studies by inhibiting the alpha amylase and alpha glucosidase in order to minimize the toxicity and side effects of the inhibitors currently used to control hyperglycaemia.

Objectives
To determine the anti-diabetic activity of Alpinia galanga and Alpinia calcarata by In-vitro studies.

MATERIALS AND METHODS
This study was conducted in the research laboratory at department of Biochemistry, Chettinad Academy of Research and Education (CARE), Kelambakkam.

Preparation of Plant Extract
Aqueous alpinia galanga and alpinia calcarata extract was prepared from locally available alpinia roots. The alpinia roots were peeled on crushed ice and 50 g of alpinia galanga and alpinia calcarata rhizome were cut into small pieces and homogenized in 75 ml cold, sterile 0.9% Sodium chloride in the presence of some crushed ice. The homogenization was carried out in a blender at high speed using 2 min bursts for a total of 12 minutes. The homogenized mixture was filtered three times by cheese cloth (very little material was retained on the cheese cloth). The filtrate was centrifuged at 2000 relative centrifugal force for 10 min and the clear supernatant fraction was made up to 100ml with normal saline. The concentration of this extract preparation was considered to be 500mg/ml. The aqueous extract was stored in small samples at 0°c for further use [32].

Porcine Pancreatic Alpha Amylase
Inhibition Assay by Using Starch

PPA -Porcine pancreatic amylase solution
The IC 50 values were determined from plots of percentage inhibition versus log inhibitor concentration and were calculated by non-linear regression analysis from the mean inhibitory values. Acarbose was used as the reference alpha amylase inhibitor. The entire test was performed in triplicate.

Alpha -Glucosidase Inhibition Assay
The IC 50 values were determined from plots of percentage inhibition versus log inhibitor concentration and were calculated by non-linear regression analysis from the mean inhibitory values. Acarbose was used as the reference alpha glucosidase inhibitor. All the test were performed in triplicate.

Statistical Analysis
All of the statistical analyses were performed using Graph Pad Prism statistical. The data were expressed as mean ± SEM for here experiments in each group. The IC50 values were estimated by nonlinear curve-fitting and presented as their respective 95% confidence limits using graph pad prism software. Iodine reagent 600 μl 600 μl 600 μl Read absorbance of test and control against the reagent blank at 620nm

RESULTS
The ability of Alpinia galanga and Apinia calcarata to inhibit α-amylase and α-glucosidase activity in vitro was investigated and the result is presented in

DISCUSSION
Management of the blood glucose level is an essential approach in the control of diabetes complications. Inhibitors of carbohydrates hydrolysing enzymes have been helpful as oral hypoglycemic medicines for the control of hyperglycemia exclusively in patients with type-2 diabetes mellitus. Inhibition of these enzymes holds of carbohydrate digestion and extends the total carbohydrate digestion time, leading to a decrease in the postprandial plasma glucose rise [34].  An effective strategy for type 2 diabetes management is the strong inhibition of αglucosidase and mild inhibition of pancreatic αamylase, which was achieved by plant extracts [35].

CONCLUSION
The result of this study shows that the both Alpinia galanga and Alpinia calcarata exhibits appreciable inhibition againsts α-amylase and αglucosidase but it indicates that the Alpinia calcarata possess better anti-diabetic activity than Alpinia galanga. Alpinia calcarata and Alpinia galanga are important medicinal plants of great deal and are useful for its various properties by a number of pharmaceutical companies and general public. Still a lot of scope is there for research on these plants to explore it further for the well-being of humans. Thus, it can be concluded from the above study that its antidiabetic properties might be due to the presence of high content of phenolic compounds and other phytochemicals present in this plant. So the preparation of drug from these herbs might be a potential alternative for synthetic anti-diabetic drug.

CONSENT
It is not applicable.