Evaluation of Polyphenols' Inhibitory Activity of α-amylase in Cinnamon

Oula Alzeina¹, Lina soubh²

¹Department of Analytical and Food Chemistry, Faculty of Pharmacy, Damascus University, Damascus, Syria.

²Department of Analytical and Food Chemistry, Faculty of Pharmacy, Damascus University, Damascus, Syria.

*Corresponding Author E-mail: loloalzein22@gmail.com

Abstract:

Purpose: Polyphenolic Compound has gained great importance in recent years due to its many benefits on human health, Hence the interest in studying the food content of these compounds. Polyphenols are significant inhibitors of the α-amylase enzyme, one of the most important targets for the development of therapeutic compounds for diabetes. The aim of this study was to: Determination of total Polyphenols by Folin-ciocalteu colorimetric assay using spectrophotometer, The research also evaluates the inhibitory activity of α-amylase for Polyphenols in cinnamon using The most common method of α-amylase activity assay Which include DNS reagent (3,5-dinitro salicylic acid). Methanol 50% was used by assistance of a moving water bath to extract plants. Results: Cinnamon surpassed as an antioxidant, its phenolic content and its inhibitory activity of the α-amylase enzyme. Conclusion: The results showed that α-amylase inhibitors can be used from natural sources such as Polyphenols found in many plants in the treatment of type 2 diabetes.

KEY WORDS: Polyphenols, Cinnamon, Inhibitory Activity Of α-Amylase, DDPH, DNS.

INTRODUCTION:

Diabetes is one of the most common diseases. According to WHO reports, the number of people with diabetes increased from 108 million in 1980 to 422 million in 2014, and statistics indicate an increase in the incidence of obesity and lifestyle changes (1). In the search for Auxiliary and alternative treatments, natural herbs, rich in phenols are the most important alternative treatments, characterized by the absence of side effects in addition to being with cheap prices and accessible to everyone. Poly phenols are organic compounds found in more than 8,000 species exist today, where they are found in many plant foods, which vary in composition and properties. There are many analytical methods for the study of phenols. However, colorimetric method is the fastest and best method to give a general idea of the food content of phenols. Polyphenols are primarily antioxidant compounds that have anti-mutagenic, anticancer, anti-inflammatory, antibacterial and antiviral activities. They are used in the treatment of many diseases such as heart disease - High blood pressure - high. In diabetes, they are significant inhibitors of the enzyme α-amylase, which is one of the most important targets for the development of antidiabetic compounds (2- 3).

The α-amylase enzyme is responsible for hydrolyzing complex sugars to glucose and other monosaccharides in small intestine. When α-amylase is inhibited, less glucose absorbs because sugars are not hydrolyzed and this would be helpfu l in treating postprandial hyperglycemia (2-3).

Because of the irritating digestive effects of α-amylase inhibitor drugs, many studies have looked for alternatives with fewer side effects. Many medicinal plants, rich in phenols are known for their sugar-lowering effectiveness. They are less expensive alternatives and show little side effects, For example: cinnamon. (2-4-5-6).

The aim of this study is to: Determination of total Polyphenols. The research also evaluates the inhibitory activity of polyphenols for α-amylase in cinnamon by using the most common method of α-amylase activity assay Which include DNS reagent (3,5-dinitro salicylic acid).

MATERIAL AND METHODS:

Chemicals and Reagents:

Folin-Ciocalteu reagent from (Fluka), methanol from (Panreac) from (Sigma- Aldrich) were purchased. Standard gallic acid, DNS regent (3, 5-dinitrosalcylic acid), maltose, porcrine pancreas α-amylase were purchased from (Titan biotech). Soluble starch, sodium potassium tartarate, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride, sodium hydroxide, sodium carbonate were purchased from (Panreac).

Plant material:

Cinnamomum zeylanicum (Lauraceae).

Preparation of plant extract:

Dry and Grounded samples (5g) were extracted with 100 ml appropriate solvent (methanol 50%) in a sealed packages and macerated for 72 hours at room temperature, then placed in a rotary water bath (at 50°C for 30 min. Each preparation was filtered through a sterilized Whatman No.1 filter paper, and the extracts were collected in Volumetric flasks and diluted to 100 ml with the appropriate solvent.

Determination of total phenolic content (TPC):

Folin-ciocalteu colorimertic method was used to determine total phenolic content (Singleton et al., 1999) (7,8) and expessed as mg of gallic acid equivalent per 100g dry matter. 100mg/100ml stock of gallic acid was prepared for the standard curve. Firstly, 1ml of extracts and 9ml distilled water were added to 1ml folin reagent, the mixture was shaken frequently for 5min then neutralized with 10ml 7% sodium carbonate (w/v), the final mixture volume was brought up to 25ml with distilled water. The absorbance was measured at 755nm wavelength using spectrophotometer after 40min. the polyphenolic content was determined as gallic acid equivalents using the following linear equation based on the calibration curve (Figure1):

Figure 1. Gallic acid standard curve.

y=0.0387x+0.0087, R2=0.9982

y is the absorbance and x is gallic acid equivalents (mg).

Determination of α-amylase activity:

Principle:

The α-amylase activity is measured, (according to Bernfeld method), using a colorimetric method with 3,5-dinitrosalicylic acid (DNS) reagent. In this method, starch is converted by α-amylase into maltose. Maltose released from starch is measured by the reduction of (DNS). Maltose reduces the pale yellow colored alkaline (DNS) to the orange-red colored one. The intensity of the color is proportional to the concentration of maltose present in the sample, which absorbance is measured using spectrophotometer at 494nm wavelength (9).

Enzyme Assay:

α-amylase activity was determined using 0.5ml of starch 1% and 0.5ml α-amylase enzyme in test tube. For blank tube, 0.5ml starch and 0.5ml buffer were mix well and incubated at 25°C for 3min. And the reaction was stopped by adding 1ml of DNS reagent, incubated in a boiling water bath for 5min and cooled to room temperature. Then add 10ml distilled water, mix well and read the absorbance at 494nm against blank. The released micromoles maltose was determined using maltose standard curve. The enzyme activity was expressed as µmole/min/ml (10).

α-amylase activity= µmole maltose formed x dilution factor / incubation time.

Determination of maltose standard curve:

The standard calibration curve (Figure 2) was prepared using serial dilutions of maltose (0.5-1-1.5-2and 2.5µM/ml). Six test tubes were taken, five of them were labeled 1 to 5 for different maltose concentration and one was labeled blank. 1ml of different concentrations of maltose was added in 5 test tubes and 1ml distilled water was taken in the blank tube. 1ml DNS reagent was added to each tube and mixed well. Then all tubes were incubated in a boiling water bath for 5 min and cool them to room temperature. 10ml distilled water were added to all test tubes and the content was mixed well. Absorbance was read at 494nm against blank and a graph was plotted for values of maltose concentration against corresponding absorbance (10).

Figure 2. Maltose standard curve

α-amylase inhibition activity assay

Procedure:

To 0.5ml of (25-50-100-200-300-400 µg/ml) plant extract was add 0.5ml enzyme, mixed well and the contents were incubated at 25°C for 4min. Next starch solution was added (0.5ml), mixed well and incubated at 25°C for 4min. After that 1ml DNS was added and incubated in boiled water bath for 5min. 10ml of distilled water were added then absorbance of the mixture was measured using spectrophotometer at 494nm and the inhibition activity% of α-amylase enzyme was calculated using the formula (11) as shown in table (1)

Inhibition Activity % = (Ac-As)/(Ac) x100

Ac: absorbance of 100% enzyme activity (only solvent with enzyme+starch).

As: absorbance of test sample (enzyme+starch+extract).

Table 1. composition of solution in α-amylase inhibition activity assay.

Control	Sample	(Blank for sample)	(Blank for control)	Solution
-	0.5	0.5	-	Sample solution
-	-	0.5	0.5	buffer
0.5	0.5	-	-	enzyme
0.5	0.5	0.5	0.5	starch
1	1	1	1	DNS
Boiling for 5 min
10	10	10	10	Distilled water

Calculation of IC50:

The concentration of the extract that inhibits 50% of the enzyme activity was determined by plotting percent inhibition (Y axis) versus log extract concentration (X axis) and calculated by logarithmic regression analysis from the mean inhibitory values (12).

RESULTS AND DISCUSSION:

Polyphenolic compounds are primarily antioxidant compounds, as well as their active importance as significant inhibitors of the enzyme α-amylase, are the most important targets for the development of therapeutic compounds for diabetes.

The amount of the total phenolics was investigated by Folin-ciocalteu method. The total phenolic content was expressed as gallic acid equivalents (mg gallic acid/100g). Total Phenol Content (TPC) of the plant extracts are shown at Table (2), showed total phenol content with average (TPC) 4871.737 mg/100g.

Table 2. Total phenolic acid (TPC) of different extract samples of cinnamon mg/100g, as gallic acid equivalent.

(TPC)( mg gallic acid/100g)	Sample	(TPC) ( mg gallic acid/100g)	Sample
4914.32	C7	4947.4	C1
4992.87	C8	4860.57	C2
4906.047	C9	4761.4	C3
4852.3	C10	4695.19	C4
4963.928	C11	4624.91	C5
4879.173	C12	4891.58	C6

The α-amylase inhibitors act as an anti-nutrient that obstructs the digestion and absorption of carbohydrates. Our finding reveals that tested plant samples, cinnamon exhibited good α-amylase in vitro inhibitory activity. The reaction mechanism involved in inhibition of α-amylase by plant polyphenols is conformational changes in structure. It has been reported that plant phenolic compound modulate the enzymatic breakdown of carbohydrate by inhibiting amylase.

The inhibitory activity of the extracts of Cinnamomum zeylanicum on α-amylase was investigated and the results are shown in table (3). In α-amylase inhibition assay, cinnamon showed (IC50= 145.88 ± 24.57mg/l).

Table 3. The percent inhibition of α-amylase by different extract of Cinnamon at varying concentrations.

IC50	Sample	IC50	Sample
143.021	C8	139.316	C1
179.887	C9	125.9	C2
180.302	C10	128.588	C3
125.314	C11	147.571	C4
128.588	C12	170.608	C5
162.56	C13	151.705	C6
187.07	C14	137.088	C7

CONCLUSION:

the results indicate that out of the three plant extracts, cinnamon exhibited good %inhibition of α-amylase activity. This Results is corresponded with the results of TPC and antioxidant activity, which the cinnamon contents surpass with its TPC and antioxidant activities. And by comparing the IC50 for the Acarbose=19.85mg/l (A. G. A. W. Alakolanga et al. 2015) used in the treatment of diabetes with the values obtained with plant extracts, we conclude the importance and feasibility of using these plant extracts (or their active compounds) as a safe alternative to pharmaceutical compounds used in the treatment of diabetes.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 23.07.2019 Modified on 07.08.2019

Research J. Science and Tech. 2019; 11(3):174-178.

DOI: 10.5958/2349-2988.2019.00026.3