Chapter 3

METHODOLOGY

Sample Collection and Identification

The samples will be randomly collected in the vicinity of Sitio Handuman, Mt. Nebo,
Valencia City, Bukidnon 35km and 700m away from Central Mindanao University. The collected
sample (leaves) will be brought to the Chemistry Laboratory and the leaf and stems will be
submitted to the Botany Section of the University Museum for sample identification and
authentication. The preparation of concentrated extract of the leaves of D. meyeniana will be
done in Chemistry Laboratory of the Department of Chemistry for the spectrophotometric
analyses of the DPPH radical scavenging activity and total phenolic content (TPC) determination
on the leaf ethanolic extract of D. meyeniana are to be performed in NSRC and NPRDC of
Central Mindanao University (CMU), University Town, Musuan, Maramag, Bukidnon.

Figure2. Aerial view of the sampling site (Google Map, 2019)

Sample Preparation

The collected samples were gently cleaned with a soft brush to remove accumulated dirt
in the upper and baseline of the leaf (Casio and Johnson, 2017). Further preparation of the leaf
samples was adapted from the method describe by Ahmed et al. (2005) and Wijekoon et al.
(2011). The leaves of D. meyeniana will be washed thoroughly with distilled water to eliminate
the foreign substances that accumulates at the surface and baseline of the sample, followed by
drying to a paper towel. Samples will undergo sizing and air drying in preparation to pulverizing.
The pulverize sample will be stored in ziploc container bag in preparation for extraction. With
the test solution of 1000mg/L test sample prepared from the stock sulotion.
The percent extraction yield was calculated using the equation 1 below.

w1
% Extraction Yeild = w2 x 100 Equation 1

where,
w1 = weight of concentrated extract
w2 = weight of the sample

Research Locale
The preparation of other reagents, extract, and filtration in DPPH radical scavenging
activity and total phenolic content (TPC) and the preparation of the stock solution on its leaf
ethanolic extraction are to be done in the Chemistry Laboratory of the Department of Chemistry.

Standard Sample and Test Sample Solution

Total Phenolic Content

The researchers will provide various concentrations in dual treatment for the filling of
two 300m/L stock solutions in preparation for the gallic acid standards and for L-ascorbic acid of
leaf. The researchers will also provide few concentrations for the test sample solutions as leaf
stock solutions.

DPPH Radical Scavenging Activity

The researchers will provide various concentrations in dual treatment for the filling of
two 100 m/L stock solutions in preparation for the gallic acid standards and for L-ascorbic acid
of leaf. The researchers will also provide few concentrations for the test sample solutions as leaf
stock solutions.

Experimental Procedure

The whole procedures are to be done in the study is depicted in Figure 3 which shows the
schematic workflow for the extraction and subsequent chemical analysis of the leaves of D.
meyeniana

Sample collection

Sample Preparation

Sample Extraction

TPC Determination Antioxidant Activity

Evaluation

DPPH Assay

Figure 3. Schematic workflow for the extraction and chemical

analysis of the leaves D. meyeniana

Total phenolic Content Determination (TPC)

Assay Preparations

The total phenolic content of the leaf ethanolic extract of D. meyeniana was determined
using the method previously describe by Ghorbani et. al (2014), Sowndhararajan and Kang
(2003), and Islam et. al(2014).
 The total phenolic content, expressed as gallic acid equivalent (GAE) per gram extract
and to be calculated using Equation 2.

A
Total Phenolic Content (TPC) relative to GAE= B x 100 Equation 2

where,
A = gallic acid concentration to the test solution solution derived from the given
calibration curve, mg/L; and
B = concentration of the test sample solution, g/L

Antioxidant Activity

The total antioxidant activity (TAA), expressed as L-Ascorbic acid equivalent (AAE) per
gram extract and to be calculated using Equation 3.

A
Total Phenolic Content (TPC) relative to GAE= B x 100 Equation 2

where,
A = L-Ascorbic acid concentration to the test solution derived from the given calibration
curve, mg/L; and
B = concentration of the test sample solution, g/L

DPPH radical scavenging activity determination

The DPPH radical scavenging activity of the leaf ethanolic extract of D. meyeniana will
be determined upon employing the method describe by Ghorbani et al (2014) and
Sowndhararajan and Kang (2003).

The DPPH radical scavenging activity reduced express as % DPPH scavenged will be
calculated using the Equation 4.
DPPH Scavenged = [(Abs control – Abs S)/ Abs control] x 100 Equation 7

where,
Abs S = Absorbance of the sample, and
Abs control = Absorbance of the negative control

EC50 Determination

The EC50 value will refer to the Equivalent Concentration of the sample necessary to
reduce the initial DPPH concentration by 50% by taking its antilog.

Statistical Analysis
The data that will be obtained will be statistically analyzed using the t-Test at 0.05 level
of significance. Correlation among the Total Phenolic Content (TPC), DPPH radical scavenging
activity and of the EC50 and Total Antioxidant Activity (TAA) will be determined by using the
Pearson’s correlation.