In Vitro Antioxidant effect of Green Tea Polyphenol Epigallocatechin-3-Gallate (EGCG) in Protecting Cardiovascular Diseases

 

K. Pramila¹, A. Julius²

¹Research Scholar, Bharathiyar University, Coimbatore.

²Professor and Head, Department of Biochemistry, Sree Balaji Dental College and Hospital, Pallikaranai,

Bharat Institute of Higher Education and Research (BIHER)

 

ABSTRACT:

The aim of this study focused on the beneficial effect of EGCG which possess direct antioxidant potential in scavenging the free radicals. Therefore, EGCG was considered as one of the natural antioxidant in improving the quality of life by preventing cardiovascular diseases.

 

 

 

INTRODUCTION:

Free radicals play an important role in triggering cardiovascular diseases (CVD) like atherosclerosis, diabetic cardiomyopathy, heart failure etc., with large number of risk factors like hypercholesterolemia, hypertension, diabetes, stress (Bahorunet al;Ceriello A). Oxidative stress was considered as a primary or a secondary cause for diabetic cardiomyopathy (Droge W).Therefore, modulation of oxidative stress signifies an important approach for the treatment of diabetic cardiomyopathy. EGCG in green tea act as a dietary antioxidant by scavenging reactive oxygen species (ROS) and also have the ability to chelate metal ions such as iron which contribute to antioxidant activity (Rice-Evans et al; Brownet al).

 

The aim of this study was used to evaluate the 2,2 Diphenyl-1-Picrylhydrazyl (DPPH) scavenging effect and strong reducing capacity ofFerric reducing antioxidant power (FRAP) with electron donating potential of EGCG.

 

MATERIALS AND METHODS:

(-)-Epigallocatechin-3-Gallate (EGCG) was used for invitro analysis for evaluating the antioxidant property of EGCG using 2,2 Diphenyl-1-Picrylhydrazyl (DPPH) and Ferric reducing antioxidant power (FRAP) assay.

 

In vitro antioxidant assay:

(a) 2, 2 Diphenyl-1-Picrylhydrazyl (DPPH):

The free radical scavenging activity of EGCG was evaluated using 2,2 Diphenyl-1-Picryl Hydrazyl (DPPH) radical scavenging assay using the method of Blois (1958) with slight modification in the concentration of the EGCG sample (Blois MS). The DPPH radical was considered as a stable organic nitrogen radical, which possess deep purple colour. This assay measures the reducing ability of antioxidants toward DPPH. This decoloration assay was reported by Brand-Williams and co-workers (Brand-Williams,et al). This antioxidant assay was based on the measurement of the loss of DPPH colour at 515nm after reaction with test compound like EGCG (Bondet, V et al) and the reaction was monitored using spectrophotometer. Ascorbic acid was used as standard antioxidant compound. The percent inhibition ability of the DPPH was calculated using the equation as given below.

 

Radical scavenging activity (% inhibition) = ([A₀–A₁]/A₀ ×100)

 

Where

A₀ was the absorbance of the control and A₁ was the absorbance of the sample. All the samples were analysed in triplicate.

 

(b) Ferric reducing antioxidant power (FRAP):

Ferric reducing antioxidant power (FRAP) was determined by the method described by Benzie and Strain (1996) with slight modification in the concentration of the sample (20 - 100µg/ml) for the measurement of reducing power of EGCG (Benzie IF and Strain JJ). The reaction measures reduction of ferric 2,4,6 tripyridyl-s-triazine (TPTZ) to a coloured product (Benzie, I. F. F and Szeto, Y. T). Ascorbic acid was used as standard antioxidant compound (Benzie, I. F. F). From the standard curve of ascorbic acid, ascorbic acid equivalent FRAP (µM) was calculated.

 

RESULTS:

Graph-1 and 2 shows Invitro antioxidant activity of EGCG using 2,2 Diphenyl-1-Picrylhydrazyl (DPPH) and Ferric reducing antioxidant power (FRAP) assay.

 

 

Graph-1: Bar graph showing % DPPH radical scavenging activity of EGCG at 515nm

 

 

Graph-2: Ferric reducing antioxidant power (FRAP) of EGCG using standard ascorbic acid.

EGCG have strong direct antioxidant property in scavenging the free radicals. EGCG significantly scavenged the DPPH, a stable organic nitrogen radical. Graph-1 shows the increased scavenging effect of DPPH with increased concentration of EGCG as indicated. The antioxidant property was greater with increased concentration of ascorbic acid and EGCG.

 

Graph-2 shows the electron donating potential of EGCG with Ferric reducing antioxidant power (FRAP) and found that EGCG have strong reducing power capacity. The FRAP assay of dietary polyphenols epigallocatechin-3-Gallate (EGCG) showed that the absorption was slowly increased along with the standard ascorbic acid.

 

The above graph–1 and 2 shows that EGCG had a very strong potential in scavenging the free radicals by transferring hydrogen atom or by donating the electrons.

 

DISCUSSION:

In general antioxidant protect cellular damage caused by oxidative stress by both direct and indirect pathways. Direct antioxidant method involves in scavenging free radicals and reactive oxygen species (ROS) either by donating hydrogen or electrons. The antioxidant bioactivities of EGCG was considered as a main contributor for cardio protective properties

 

The usage of natural antioxidants like EGCG acquiring ample attention when compared to synthetic antioxidants which was found to be carcinogenic (Machlin, L.J. and, Bendich,). The effectiveness of antioxidants depends on the number of hydroxyl group present in their aromatic rings of natural compound (Brewer, M.S). The efficacy increases as the number of hydroxyl group increase, since EGCG possess many number of hydroxyl group in their aromatic rings, it was considered as one of the most potential natural antioxidant agents (Karori, et al). Many reports confirmed that the pathology of diabetic cardiomyopathy was linked with increased generation of ROS due to hyperglycaemia and hyperlipidaemia. At physiological condition, ROS was considered to be beneficial for cellular protection. Due to external stress factors like diabetes or obesity, the levels of ROS was elevated which leads to pathological condition (Nakamura, et al). Auto oxidation of glucose, redox balance shift, impaired antioxidant defence system and glycation of antioxidant enzymes are the sources of ROS production in diabetes mellitus (DM)condition (Chopra, et al, Sakai et al, Fakhruddin et al).

 

DPPH and FRAP assays were done for evaluating the antioxidant activities of EGCG. The DPPH assay was most popular spectrophotometric method for evaluating the antioxidant capacity of any compounds. The degree of free radical quenching in DPPH assay was dose dependent and it was increased with increase in sample concentration. This statement revealed a fact that, EGCG have the ability to scavenge free radical through hydrogen transfer reaction. (Sivakumar et al). FRAP assay was an important indicator of reducing potential of an antioxidant compound, which can break free radical chain by donating the hydrogen atom (Othman et al, Kumar et al). Antioxidant potential of EGCG was gradually increased with increasing concentration of samples (FRAP measured as ascorbic acid equivalent).

 

CONCLUSION:

This study showed the ability of EGCG as a natural antioxidant to prevent or counteract reactive oxygen species production by scavenging the free radicals and by metal chelating activity. Antioxidants in general was very useful in preventing, managing or treating many pathological conditions.

 

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Received on 24.11.2018           Modified on 04.01.2019

Accepted on 16.01.2019         © RJPT All right reserved