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. IVSY 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 (6hydroxy-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.