ANTIOXIDATIVE PROPERTY AND REDUCING ACTIVITY OF MABOLO

(Diospyros blancoi) FRUIT EXTRACTS

VISAYAS STATE UNIVERSITY LABORATORY HIGH SCHOOL

Visca, Baybay City, Leyte

ANDRES, NATALIE B.
OGALESCO, RHEA JENNY A.
TABADA, SARAH AURORA W.
IV-
SY 2010 – 2011

Candelario L. Calibo
Adviser
 Review of Literature

Antioxidants
Antioxidants are molecules that can neutralize free radicals by accepting or

donating an electron to eliminate the unpaired condition. Typically, this means that the

antioxidant molecules becomes a free radical in the process of neutralizing a free radical

molecule to a non-free radical molecule. But the antioxidant molecule may be very large

(allowing it to “dilute” the unpaired electron), it may be readily neutralized by another

antioxidant and/or it may have another mechanism for terminating its free radical

condition. Molecules with loosely-held hydrogen atoms can use those hydrogen atoms

like electrons to neutralize free radicals. The hydrogen atoms are called reducing

equivalents, and the molecules having such hydrogen atoms are said to be in a reduced

state. Vitamin C (ascorbate, AscH¯), for example, can donate a hydrogen atom to a free

radical molecule (R) thereby neutralizing the free radical while becoming an ascorbate

radical itself (Asc¯, Asc¯, in different notation). But the Asc¯ free radical is very stable

because of its resonance structure. (http://www.benbest.com/nutrceut/AntiOxidants.html).

There are two mechanisms of antioxidant action for the inhibition of oxidative reaction:

(a) Interruption of the free-radical chain mechanism and (b) function as being

preferentially oxidized – poor protection.

 METHODOLOGY

Chemicals and Reagents

Analytical grades of the following: KCl, NaOAc, Ethanol, HCl, Trolox (6-

hydroxy-2, 5, 7, 8-tetramethyl-chroman-2-carboxylic acid) and DPPH (1, 1-diphenyl-2

picrylhydrazyl) were obtained from Merck.

Collection and Preparation of Fruits

Fresh fruits were collected at the vicinity of the Visayas State University. The

fruits were washed and cleaned thoroughly in running tap water to rinse off the dirt

particles that adhere the fruit surface.

Extraction of Fruit Extracts

Crude Mabolo Fruit Extracts

The washed Mabolo fruit will be cut into halves. Twenty grams of the fleshy

part of the fruits will be scooped using a clean spoon and will be homogenized with 40

mL solution of 1% hydrochloric acid (HCl) in ethanol until it blended with the solvent.

The macerates will be transferred to a small beaker wrap with a black carbon paper to

minimize the degradation of pigments. The beaker will be covered with parafilm and will

be stored overnight in the refregirator.

 The sample will be filtered using Whatman No. 41 filter paper and the filtrate

will be centrifuged at 2500 rpm for 20 minutes. The supernatant will be transferred to a

100mL volumetric flask and will be diluted with the extracting solvent.

Determination of Free Radical Scavenging Activity

The antioxidant activity of all extracts and fractions were determined according

to the DPPH radical scavenging assay. The stock solution of DPPH (30 mg/L) was

prepared using ethanol/water solvent and measuring the initial absorbance at its max

absorbance through UV-Vis spectrophotometer. Aliquots of the sample were diluted

(1:10) with ethanol and the assay was performed following the procedure described by

Brand-Williams, Cuvelier, and the Berset (1995), with minor modifications. The diluted

sample, 0.1 mL, was pipetted into 3.9 mL of DPPH solution to initiate the reaction; it was

then shaken and left to stand at ambient temperature in the dark for three (3) hours to give

enough time for the reaction of the cellular antioxidants with DPPH. The free radical

scavenging assay was tested by reading its absorbance at the established _ using the

UV-Vis spectrophotometer. Ethanol (95%) was used as a blank. Trolox (0, 100, 200, 300,

400 and 500 IM) was used as a standard. Analysis was done in triplicate for each sample

Trolox as standard was determined by the decrease in absorbance at a reaction time.

The quantity of the sample necessary to react with one-half of the DPPH

solution is expressed in terms of the micromole equivalents of the standard Trolox (TE)

per 100g of sample or the Trolox units per 100 gm. Analysis was done in triplicate for

each sample.
Statistical Analysis

This study used Two Factor Randomized complete block design (CRD) and was

analyzed by the analysis of variance (ANOVA) and Tukey’s Honesty Significance

Difference Test.