Mandal et al. World Journal of Pharmaceutical Research
IN VITRO ALPHA AMYLASE INHIBITORY ACTIVITY OF
ETHANOL AND HOT WATER EXTRACT OF POLYHERBAL
FORMULATION
*
Ajay Mandal and P. Jagan Mohan Reddy
Department of Biotechnology Engineering, Acharya Institute of Technology, Bangalore-560107, India.
ABSTRACT
In vitro α-Amylase inhibitory activity of polyherbal preparation, a combination of six selected medicinal plants. The antidiabetic potential of the aqueous extract and ethanolic extract of Ocimum sanctum, Withania somnifera, Gymnema sylvestre, Tinospora cordifolia,
Phyllanthus emblica and Coleus forskohlii. Diabetes has become a common global health problem that affects >170million people worldwide. It is one of the leading causes of death and disability. It is estimated that by 2030, the number will rise to 366 million (www.who.int). The majority of diabetes (~90%) is type 2 diabetes (T2D) caused by a combination of impaired insulin secretion from pancreatic beta cells and insulin resistance of the peripheral target tissues, especially muscle and liver. Traditional Medicines obtained from medicinal plants are used by about 40-60% of the world’s population. Though there are many approaches to control diabetes and its secondary complications, herbal formulations are preferred due to lesser side effects and low cost. In the present study focused on screening of ethanolic and hot water extract of polyherbal formulation for antidiabetic activity with respect to enzymatic assay as such and α- amylase inhibition assay. Polyherbal formulation (PHF) extracts of two different organic solvents exhibited significant reduction in alpha-amylase activity. Acarbose standard antidiabetic drug which was used as positive control at concentrations 10-100µg/ml showed PPA inhibitory activity from 18.75% to 58.60% with an IC50 value of 83.19µg/ml [Table-17 and Graph-15].PHF of hot water extract at concentration10-100µg/ml showed maximum inhibitory effects on Alpha-Amylase activity from 23.44% to 88.22% with an IC50 value of 41.84µg/ml respectively [Table-18 and
Volume 5, Issue 1, 968-978. Research Article ISSN 2277– 7105
Article Received on 01 Nov 2015,
Revised on 22 Nov 2015, Accepted on 12 Dec 2015
*Correspondence for
Author
Ajay Mandal
Department of
Biotechnology
Engineering, Acharya
Institute of Technology,
16]. PHF of Ethanolic extracts at concentrations 10-100µg/ml showed moderate inhibitory effects on Alpha-Amylase activity from 17.05% to 70.05% respectively with an IC50 value of 66.62µg/ml. [Table-19 and Graph-17]. Out of two solvent selected the hot water extract solvent showed maximal inhibition indicates its maximal therapeutic efficacy.
KEYWORDS: Polyherbal formulation (PHF), Phytochemicals, Alpha-Amylase inhibition and Acarbose.
INTRODUCTION
In humans, the digestion of starch involves several stages. Initially, partial digestion by the salivary amylase results in the degradation of polymeric substrate into shorter oligomers. Later on in the gut these are further hydrolyzed by pancreatic α-amylase into maltose, maltotriose and small malto-oligosaccharides.[1] The digestive enzyme α-amylase) is responsible for hydrolyzing dietary starch to maltose, which breaks down to glucose, prior to absorption. Inhibition of the α-amylase should reduce the unfavorable high postprandial blood glucose peak in diabetes. α-amylase inhibition is also a useful target in obesity[9,10] Pancreatic α-amylase hydrolyses the 2-chloro-4- nitrophenol α-D - maltotrioside (CNP-G3) to release 2-chloro-4-nitrophenol and form 2-chloro-4- nitrophenol α-D - maltoside (CNPG2), maltotriose and glucose. The rate of formation of the 2-chloro-4-nitrophenol can be measured at 405nm.[2]
MATERIAL AND METHODS Collection of plant material
The present study is aim to screen the synergetic Antidiabetic potential of six selected antidiabetic plants of western ghat. The plant materials were collected from western ghat and Bangalore rular zone. In the polyherbal preparation, leaves of Tinnospora cordifolia, ocimum sanctum, Gymnema sylvestre were selected, where as in the plants like Withania somnifera
and Coleus forskohlii, the root part of the plant were selected, where as in the plant
Phyllanthus emblica, the fruit portion of the plant was selected to prepare the polyherbal formulation to screen the antidiabetic and antioxidant therapeutic value through in vitro and in vivo studies.
PREPARATION OF PLANT’S EXTRACTS
petroleum ether and n-hexane. Cold water extracts was obtained by adding distilled water to the crushed material in ratio 1:4 and kept in rotary shaker for 24h at 30 degree Celsius and at 130 rpm respectively. After 24 h it was filtered and the resulting extract was stored in refrigerator. Hot water extracts was obtained by soxhlet extraction were 400ml of distilled water to 100 grams of plant powders. Similarly medicinal powders were extracted using organic solvent in ratio 1:3 by soxhlet extraction.[3,4]
METHODS
Test of extracted material from different part of plant (phytochemical analysis)
Test for the presence of different Bio-components such as alkaloids, flavonoids, glycosides, saponins, phenols, steroids and terpenoids were performed using standard procedures.
Table: 1 Phytochemical Test
Sl.no Test performed Observation Inference
1.
Test for carbohydrates:
Molisch test: To 1mlof extract add 2 drops of Molisch reagent mix well. Then add 3ml of concentrated sulphuric acid along the sides of the test tubes keeping the tube in an
inclined position.
Formation of purple to violet ring appears at the junction of two liquids.
Carbohydrates present.
2.
Test for glycosides:
Keller killani test: To 1ml of extract add 0.4ml of glacial acetic acid cool then add 2 drops of ferric chloride solution and 2ml of concentrated sulphuric acid by sides of the tube
Formation of reddish brown colour at junction of two liquid layer
Indicates presence of cardiac
glycosides.
3.
Test for alkaloids:
Mayer’s test: Extract +4-5 ml of dilute HCL (0.1N) shake well and add Mayer’s reagent.
White or yellow cream precipitate
Presence of alkaloids.
4.
Steroids and triterpenoid test:
Salkowski test: To 2ml of extract add 2ml of chloroform and few drops of concentrated sulphuric acid by sides of test tube
Red colour at lower layer.
Yellow colour at lower layer. Steroids present. Triterpenoid present. 5. Flavonoid test
Shinoda test: Extract with fragments of magnesium ribbon and concentrated Hydrochloric acid
Shows pink scarlet red or green to blue color after few minutes
Flavonoid present.
6.
Phenol test:
Ellagic acid test: Extract plus 3drops of 5% Sodium nitrate solution
Muddy or niger brown precipitate
Phenol present
7.
Tannins test:
Ferric chloride test: Extract plus 1% ferric chloride solution. Blue color Green color Hydrolysable tannins Condensed tannins 8.
Test for saponin:
Froth test: To 2ml of extract add 1ml of distilled water shake well
Alpha-Amylase inhibition assay
In humans, the digestion of starch involves several stages. Initially, partial digestion by the salivary amylase results in the degradation of polymeric substrates into shorter oligomers. Later on in the gut these are further hydrolyzed by pancreatic α-amylase into maltose, maltriose and malto-oligosaccharides. The digestive enzyme (α-amylase) is responsible for the hydrolyzing dietry starch (maltose), which breaks down into glucose prior to absorption. Inhibition of α-amylase can lead to reduction in post prandial hyperglycemia in diabetic contion.[5,6]
Principle
Alpha-amylase activity can be measured in-vitro by hydrolysis of starch in the presence of α-amylase enzyme. This process was quantified by using iodine, which gives blue color with starch. The reduced intensity of blue color indicates the enzyme induced hydrolysis of starch into monosaccharides. If the extract possesses α-amylase inhibitory activity, the intensity of blue color will be more. In other words, the intensity of blue color in test sample is directly proportional to α-amylase inhibitory activity.[7]
MATERIAL AND METHODS
The hot water and ethanolic extract of polyherbal formulation were serially diluted to get required concentration to perform the alpha amylase inhibition assays.
Materials required
DNS (3, 5 Dinitrosalicylic acid):
Enzyme: -amylase (EC 3.2.1.1) (Type VI-B: From porcine pancreas, 500,000 units [19.6 units/mg solid and store at 2- .
Positive control: Acarbose and store at room temperature.
Strach solution 1%.
Phosphate buffer: 0.1M.
UV-Spectrometer.
Preparation of working solutions Phosp ate u er M C
39.0 ml of 0.2 M monobasic sodium phosphate and 61.0 ml of 0.2 M dibasic sodium phosphate and diluting to a total volume of 200 ml, to make a 0.1 M phosphate buffer of the required pH 7.0 at room temperature.
Enzyme (0.48412units/ml)
2.65mg of -amylase is made up to 100 ml with 0.1 M phosphate buffer pH 6.9. (Enzyme units are lot specific).
Starch 1%
Dissolving 1.0 gm soluble starch, in 100 ml 0.02 M sodium phosphate buffer, pH 6.9 with 0.006 M sodium chloride and gentle boil to dissolve. Cool and bring volume to 100 ml, with water, if necessary. Incubate at 25°C for 4-5 minutes prior to assay.
DNS (3, 5 Dinitrosalicylic acid)
Dissolving 1.0 gm of 3,5-dinitrosalicylic acid in 50 ml of reagent grade water. Add slowly 30.0 gms sodium potassium tartrate tetrahydrate. Add 20 ml of 2 N NaOH. Dilute to a final volume of 100 ml with reagent grade water. Precaution was followed to protect from carbon dioxide and store no longer than 2 weeks.
(a)Positive control
Stock 1: (1mg/ml): 50mg of Acarbose dissolved in 50ml of 0.1M Phosphate buffer, pH 6.9.
Stock 2: Diluted to a concentration of 2.5µg/ml with 0.1M Phosphate buffer, pH 6.9.
Working stock: Diluted to a concentration of 0.25µg/ml with 0.1M Phosphate buffer, pH 6.9.
(b) Sample preparation
A sample stock of 310µg/ml was prepared for the sample by dissolving 3.1mg of the sample in 10ml with 0.1M Phosphate buffer, pH 6.9. Further dilutions were made as required with 0.1M Phosphate buffer, pH 6. 9.
Positive control
Stock- 50 mg of Acarbose in 50 ml was prepared to set 0.1M phosphate buffer.
Procedure
assay condition. The enzyme inhibitory activity was expressed as decrease in units of glucose liberated.[8] Plant extract concentrations ranging from 10-100µg was incubated with 1ml of 1unit PPA (Porcine Pancreatic Alpha-Amylase) enzyme for 30minnutes at 37°C. After incubation 1ml of 1% buffered starch was added and the mixture was further incubated for 10minutes at room temperature. The reaction was stopped by adding 1ml DNS reagent and the contents were heated in boiling water bath for 5minutes. Blank was prepared without plant extract and enzyme which was replaced with equal quantity of 0.1M phosphate buffer. Control representing 100% enzyme activity without plant extract was also included. The absorbance was read at 540nm using UV Spectrophotometer. Standard antidiabetic drug Acarbose was used as positive control. The antidiabetic property was determined through inhibition of alpha amylase which was expressed as percentage of inhibition and calculated by following equation.
% Inhibition = Absorbance of control – Absorbance of test Absorbance of control ×100
IC50 values of Acarbose and Polyherbal extracts of hot water and ethanolic solvents were determined from plots of percentage inhibition versus concentration (µg/ml). IC50 value is defined as the concentration of extract required to inhibit 50% of PPA (Porcine Pancreatic Alpha-Amylase) activity.
RESULTS
Phytochemical Screening of the Polyherbal Formulation
As a first part of study from the phytochemical analysis, it is evident that both hot water and ethanolic solvent showed presence of carbohydrates, glycoside, alkaloids, sterioids, triterpenoids, flavonoids, phenol, saponins and tannins. Out of the phytochemical, tannins showed maximum, which is further screened to know the type of tannin which indicated the presence of hydrolysable tannins.
Invitro Porcine Pancreatic Alpha-Amylase inhibition assay
from 23.44% to 88.22% with an IC50 value of 41.84µg/ml respectively [Table-2 and Graph-2]. PHF of Ethanolic extracts at concentrations 10-100µg/ml showed moderate inhibitory effects on Alpha-Amylase activity from 17.05% to 70.05% respectively with an IC50 value of 66.62µg/ml. [Table-3 and Graph-3]. Out of two solvent selected the hot water extract solvent showed maximal inhibition indicates its maximal therapeutic efficacy.
Table-1: Percentage inhibition and IC50 Values of Acarbose on Alpha-Amylase.
Graph-1: Acarbose standard drug IC50 Value graph.
Table-2: PHF showing maximum inhibitory effects on alpha-amylase activity.
Standard drug (Acarbose)
Concentration(µg/ml) % Inhibition IC50 Value
10 18.75
83.19µg/ml
20 22.41 40 29.73 60 38.74 80 48.27 100 58.60
Polyherbal formulation
Extract Concentration(µg/ml) % Inhibition IC50 Value
Hot water
10 23.44
41.84µg/ml 20 41.23
Graph-2: PHF Hot water extract IC50 value graph.
Table-3: PHF showing moderate inhibitory effects on alpha-amylase activity.
Polyherbal formulation
Extract Concentration(µg/ml) % Inhibition IC50 Value
Ethanol
10 17.05
66.62µg/ml 20 26.01
40 32.33 60 41.10 80 61.11 100 70.05
Graph-3: PHF Ethanol extract IC50 Value graph.
DISCUSSION
there is a growing interest in herbal remedies due to the fewer side effects associated with these therapeutic agents.[9] This has led to an increase in the demand for natural products with antihyperglycemic activity having few side effects. The polyherbal formulation was formulated using the petroleum ether, ethanol, methanol, hot water, cold water and hexane extracts of the leaves of Tinospora cordifolia, leaves of Ocimum sanctum, roots of Withania somnifera, roots of Coleus forskohlii, leaves of Gymnema sylvestre and fruits of Phyllanthus emblica which are mixed properly in equal quantity.
The treatment goal of diabetic patients is to maintain near normal levels of glycemic control, in both fasting and post-prandial conditions. Many natural sources have been investigated with respect to suppression of glucose production from the carbohydrates in the gut or glucose absorption from the intestine.[10] Alpha-amylase catalyses the hydrolysis of alpha-1,4-glycosidic linkages of starch, glycogen and various oligosaccharides. Alpha-glucosidase further breaks down the disaccharides to simple sugars, readily available for intestinal absorption. The inhibition of their activity in the digestive tract of humans is considered to be effective tool to control diabetes. Polyherbal formulation (PHF) extracts of two different organic solvents exhibited significant reduction in alpha-amylase activity. Acarbose standard antidiabetic drug which was used as positive control at concentrations 10-100µg/ml showed PPA inhibitory activity from 18.75% to 58.60% with an IC50 value of 83.19µg/ml [Table-1, graph-1]. PHF hot water extract at concentration10-100µg/ml showed maximum inhibitory effects on Alpha-Amylase activity from 23.44% to 88.22% with an IC50 value of 41.84µg/ml respectively [Table-2, graph-2]. PHF Ethanolic at concentrations 10-100µg/ml showed moderate inhibitory effects on Alpha-Amylase activity from 17.05% to 70.05%, respectively with an IC50 value of 66.62µg/ml [Table-3, graph-3].
The literature reports reveal that alkaloids, carbohydrates, polysaccharides and saponin glycosides present in the plant extract known to possess Flavonoids possess antioxidant and antidiabetic activity.[11,12,13,14] The present investigation also observed antidiabetic potential of test extract may be due to the presence of similar Phytoconstituents which was evident by preliminary phytochemical screening.
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