Assay of total phenolics

Assay of total phenolics

Introduction

The antioxidant effect is mainly due to the phenolic components, such as flavonoids(pieta, 1998), phenolic acids, and phenolic diterpenes (shahidi, janitha & wanasundara, 1992). The antioxidant activity of phenolic compound s is mainly due to their redox properties, which can play an important role in absorbing and neutralizing free radical, quenching singlet and triple oxygen, or decomposing peroxides (Osawa, 1094). Many photochemical posses significant antioxidant capacities that may be associated with lower incident and lower mortality rates of cancer in several human population (Velioglu et al, 1998).

Principle

Total phenol content in the extract was determined with Folin-Ciocalteu reagent. Extract (200mg/ml) was mixed with 400 ml of the Folin-Ciocalteu reagent and 1.5 ml of 20% sodium carbonate. The mixture was shaken thoroughly and made up to 10 ml using distilled water. The mixture was allowed to stand for 2 h. Then the absorbance at 765 nm was determined. These data were used to estimate the phenolic contents using a standard curve obtained from various concentration of gallic acid (Yu et al., 2002). Total phenol content was expressed as mg of gallic acid equivalent.

Materials and method:

Total phenolic content of heliotropium indicum extractives was measured employing the methods as described by Skerget et al. (2005) involving Folin Ciocalteu reagent as oxidizing agent and gallic  acid as standared ( Majhenic et al, 2007).

Materials

1.    Folin-Ciocalteu reagent (10 fold diluted)

2.    Na2Co3 solution (7.5%)

3.    Tert-butyl-1 hydroxytoluene(BHT)

4.    Ascorbic acid

5.    Methanol

6.    Chloroform

7.    Carbon tetra chloride

8.    n-hexene

9.    UV –spectrometer

10. Vial

11. Beaker (100 & 200 ml)

12. Test tube

13. Pipette (1ml)

14. Pipette (5ml)

15. micro pipette (50-200µl)

16. Distilled water

Table: Composition of Folin-Ciocalteu reagent

SL. NO.	Component	Percent
1.	Water	57.5
2.	Lithium sulfate	15.0
3.	Sodium Tungstate Dihydrate	10.0
4.	Hydrochloric Acid>=25%	10.0
5.	Phosphoric Acid 85% solution in water	5.0
6.	Molybdic Acid Sodium Dihydrate	2.5

Standard curve preparation

Gallic acid was used here as standard. Different gallic acid solution were prepared having a concentration ranging from 100 µg / ml to 0µg / ml. 2.5 ml of Folin-Ciocalteu reagent (diluted 10 times with water) and 2.0 ml of Na2Co3 (7.5 % w/v) solution was added to 0.5 ml of gallic acid solution. The mixture was incubated for 20 minutes at room temperature. After plotting the absorbence in ordinate against the concentrstion in abscissa a linear relationship was obtained which was used a standard curve for the determination of the total phenolic content of the test samples.

Sample preparation

2 mg of the extractives was taken and dissolved in the distilled water to get a sample concentration of 2 mg / ml in every case. The samples along with their concentration for the total phenolic content measurement are given.

Table: Test samples for total phenolic content determination

SL. NO.	Plant  part	extractives	Conc .      (mg / ml)
1.	Leaves, flower	Methanolic extract	2.0

Total phenolic compound analysis

To 0.5 ml of extract solution 9conc. 2 mg/ml), 2.5 ml of Folin-Ciocalteu reagent (diluted 10 times with water) and 2.0 ml of Na2Co3 (7.5 % w/v) solution was added. . The mixture was incubated for 20 minutes at room temperature. After 20 minutes the absorbance was measured at 765 nm by UV-spectrophotometer and using the standard curve prepared from gallic acid solution with different concentration, the phenols content of the sample were expressed as mg og GAE ( gallic acid equivalent) / gm of the extract. 

If you have any  opinion Please, comments ...

Data accumulated & compiled by:

Dewan Pavel (M. PHARM)

       Mundipharma (Bangladesh) Pvt. Ltd.

       Officer, Quality Assura