Antioxidative Property and Reducing Activity of Mabolo
Transcript of Antioxidative Property and Reducing Activity of Mabolo
ANTIOXIDATIVE PROPERTY AND REDUCING ACTIVITY OF MABOLO (Diospyros blancoi) FRUIT EXTRACTS
VISAYAS STATE UNIVERSITY LABORATORY HIGH SCHOOLVisca, Baybay City, Leyte
ANDRES, NATALIE B.OGALESCO, RHEA JENNY A.TABADA, SARAH AURORA W.
IV-SY 2010 – 2011
Candelario L. CaliboAdviser
Review of Literature
AntioxidantsAntioxidants 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.