METHODOLOGY

Location and Duration of the Study

The study was conducted from January to May 2018. Preparation of

samples and reagents needed for the analyses was done at the Chemistry
Laboratory of the Department of Chemistry, College of Arts and Sciences,
Central Mindanao University, University Town, Musuan, Bukidnon.
Spectrophotometric analyses of the total phenolic content, total antioxidant
activity and DPPH radical scavenging activity of the leaf and stem methanolic
extracts of B. mutica were performed in the NSRC and NPRDC of CMU.

Equipment, Apparatus and Chemicals

Laboratory Equipment

The equipment used in this study include osterizer, refrigerator,top

loading balance, analytical balance (Shimadzu AX200), hot plate, rotary
evaporator, vortex machine, oven, centrifuge machine, autoclave, and
microplate reader.

Apparatus and Materials

The materials and apparatus used in this study include scissors,

amber glass bottles, aluminum foil, magnetic stirrers, Whatman No. 1 filter
papers, eppendorf tubes, beakers, Erlenmeyer flasks, stirring rods,
micropipettes, detergent, parafilms, wash bottles, volumetric flasks,
micropipette tips, ziploc container bags, 96-well plates, plastic baskets, and
funnel.
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Chemicals and Reagents

The chemicals used in this study include distilled water, absolute

methanol, 0.1 mM DPPH, L-ascorbic acid, gallic acid, 0.6 M sulfuric acid, 28
mM sodium phosphate, 4 mM ammonium molybdate, 10 % Folin-Ciocalteu
reagent, and 10 % Na2CO3.

Sampling Site

The leaf and stem samples of B. mutica were obtained randomly from
Kalumihan, Kitaotao, Bukidnon with geographical coordinates of 7.6404° N,
124.9948° E as shown in Figure 8.

Sampling Site
7.6404° N, 124.9948° E

Figure 8. Aerial View of Kalumihan, Kitaotao, Bukidnon (Google Maps,

2017).
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Sample Collection

Ten kilograms of the fresh and healthy leaves of B. mutica and

another ten kilograms of its stems were collected in the morning before
sunrise. The collected samples were placed in plastic baskets and were
immediately washed with tap water. A sample of the whole plant was
submitted to the Botany Section of the University Museum of Central
Mindanao University for sample identification and authentication.

Sample Preparation

Preparation of samples was done using the method provided by

Ahmed et al. (2005) and Wijekoon et al. (2011).
The leaves of B. mutica were washed thoroughly with distilled water
in order to remove all foreign substances that accumulate at the surface,
followed by drying with paper towels. The same procedure was done for the
stems of B. mutica. The leaf and stem samples were separately cut into small
pieces of uniform size and were air dried for three weeks. The dried leaf and
dried stem samples were subjected to size reduction using an osterizer until
coarse powder samples were obtained. The pulverized leaf sample was
mixed thoroughly and was stored in ziploc container bags prior to extraction.
The same was done for the pulverized stem sample.

Preparation of Extracts

Solvent extraction was done as described by Wijekoon et al. (2011)

and Gondi et al. (2015). Five replicates were made for the pulverized leaf
and pulverized stem samples of B. mutica.
One hundred grams (100 g) of the pulverized leaf sample were mixed
with 1000 mL of absolute methanol. Similarly, one hundred grams (100 g) of
the pulverized stem sample were mixed with 1000 mL of absolute methanol.
Each mixture was extracted using a magnetic stirrer at 1150 rpm for 3 hours
 22

at room temperature. The extract obtained was then filtered using Whatman
No. 1 filter paper. The residue that remained in the filter paper was subjected
to re-extraction twice by placing the residue again to the same flask and
following the same procedure except that the amount of solvent added was
reduced to 500 mL. The filtrates collected after three consecutive extractions
were then pooled and collected in 1000-mL Erlenmeyer flasks which were
covered with aluminum foil to prevent exposure to light. The filtrates obtained
were concentrated under reduced pressure using a rotary evaporator at 40
°C. The concentrated extracts were placed in pre-weighed sterilized vials,
air-dried under the fume hood for an hour and stored in the refrigerator until
further use.
The percent extraction yield was calculated using Equation 1 below.

w1
% Extraction Yield = x 100 Equation 1
w2

where
w1 = weight of extract
w2 = weight of dried sample

Preparation of Sample Stock Solution

A 2000 mg/L sample stock solution for the leaf and stem methanolic
extracts of B. mutica was prepared by dissolving 0.2000 g of the
concentrated leaf and concentrated stem extracts in absolute methanol and
diluting each to 100 mL. The prepared solutions were then placed in 120-mL
sterilized amber bottles and were stored in the refrigerator until further use.
 23

Total Phenolic Content Determination

Preparation of the Calibration Curve

A 300 mg/L stock solution of the standard gallic acid was prepared by
dissolving 0.03 grams of the standard gallic acid in absolute methanol and
diluting it to 100 mL. Various concentrations (0, 7.5, 15, 30, 60, 75, and 90
mg/L) were prepared from the stock solution as working standards for the
calibration curve.
The calibration curve was constructed by plotting the concentration of
the working standards (gallic acid) against their corresponding absorbance
reading using the least square method. The linear regression equation of the
line derived from the calibration curve was used to determine the total
phenolic content of the leaf and stem methanolic extracts of B. mutica.

Preparation of the Test Sample Solution

A 1000 mg/L test sample solution was prepared from the stock
sample solution by adding 5 mL of the stock sample solution in 5 mL of
absolute methanol in a vial.

Assay

The total phenolic content of the leaf and stem methanolic extracts of
B. mutica was determined using the method described by Ainsworth and
Gillespie (2007).
A 200 μL test sample solution was placed in an eppendorf tube. The
sample was then added with 200 μL of 10 % Folin-Ciocalteu reagent in
absolute methanol and 800 μL of 10 % Na 2CO3. The reaction mixture was
incubated for two hours at room temperature. Thereafter, the reaction
mixture was then centrifuged for 2 minutes at 11 rpm. A 200 μL reaction
mixture was then placed in each of the three wells of a 96-well plate. The
 24

absorbance was measured at 750 nm using a microplate reader. The same

procedure was done for the working standards (gallic acid) and the blank
(absolute methanol). The total phenolic content, expressed as gallic acid
equivalent (GAE) per gram extract was calculated using Equation 2:

A
Total Phenolic Content relative to GA = Equation 2
B

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

Total Antioxidant Activity Determination

Preparation of the Calibration Curve

A 300 mg/L stock solution of the standard L-ascorbic acid was

prepared by dissolving 0.03 grams of the standard L-ascorbic acid in
absolute methanol and then diluting it to 100 mL. Various concentrations
were prepared from the stock solution as working standards for the
calibration curve which include 0, 15, 30, 60, 90, 105, 150, 210, 255, and
300 mg/L, respectively.
The calibration curve was constructed by plotting the concentration of
the working standards (L-ascorbic acid) against their corresponding
absorbance reading using the least square method. The linear regression
equation of the line derived from the calibration curve was used to determine
the total antioxidant activity of the leaf and stem methanolic extracts of B.
mutica.
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Preparation of the Test Sample Solution

A 1000 mg/L test sample solution was prepared from the stock
sample solution by adding 5 mL of the stock sample solution in 5 mL of
absolute methanol in a vial.

Assay

The total antioxidant activity of the leaf and stem methanolic extracts
of B. mutica was determined by employing the method described by Prieto
et al. (1999).
A 200 µL test sample solution was placed in an eppendorf tube
followed by the addition of 600 µL of the reagent solution (0.6 M sulfuric acid;
28 mM sodium phosphate; and, 4 mM ammonium molybdate). The reaction
mixture was then incubated at 95 °C for 90 minutes. Then, the reaction
mixture was allowed to cool at room temperature. After cooling, the reaction
mixture was centrifuged for 3 minutes at 11 rpm. A 200 µL reaction mixture
was then placed in each of the three wells of a 96-well plate. Then the
absorbance was measured at 695 nm using a microplate reader. The same
procedure was done for the working standards as well as for the blank
(absolute methanol). Total antioxidant activity, expressed as mg L-ascorbic
acid equivalent (AAE) per gram extract was calculated using Equation 3:

A
Total Antioxidant Activity relative to AA = Equation 3
B

where
A = L-ascorbic acid concentration of the test sample solution
derived from the calibration curve, mg AAE/L; and
B = concentration of the test sample solution, g extract/L
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DPPH Radical Scavenging Activity Determination

Preparation of the Standard Samples

A 100 mg/L stock solution of the standard gallic acid was prepared by
dissolving 0.01 gram of the standard gallic acid in absolute methanol and
then diluting it to 100 mL. Moreover, a 100 mg/L stock solution of the
standard L-ascorbic acid was also prepared by dissolving 0.01 gram of the
standard L-ascorbic acid in absolute methanol and then diluting it to 100 mL.
Various concentrations were prepared from each of the two stock
standard solutions. For the standard gallic acid, various concentrations of
0.20, 0.40, 0.60, 1.00, 2.50, 5.00 and 10.0 mg/L were prepared while for the
standard L-ascorbic acid, various concentrations of 1.0, 2.0, 4.0, 6.0, 8.0,
10.0 and 15.0 mg/L were prepared.

Preparation of the Test Sample Solutions

For the leaf test sample solution, various concentrations (25, 50, 100,
250, and 500 mg/L) were prepared from the 2000 mg/L leaf stock sample
solution.
For the stem test sample solution, various concentrations (100, 250,
500, 600, and 750 mg/L) were prepared from the 2000 mg/L stem stock
sample solution.

Assay

The DPPH radical scavenging activity of the leaf and stem methanolic
extracts of B. mutica was determined by employing the method described by
Miser-Salihoglu et al. (2013).
A 150 µL test sample solution were pipetted into each of the five wells
in the 96-well plate. Then, 50 µL of 0.1 mM DPPH in absolute methanol was
added into each well. The reaction mixture was then incubated for 30
 27

minutes in the dark at room temperature. The absorbance was measured at

517 nm using a microplate reader. The same procedure was applied for the
standards (L-ascorbic acid and gallic acid) as well as with the negative
control (absolute methanol).
The DPPH reduced expressed as % DPPH scavenged was
calculated using Equation 4:

Abs control – Abs sample

% DPPH Scavenged = ( ) x 100 Equation 4
Abs control

where
Abs control = Absorbance of the negative control; and
Abs sample = Absorbance of the sample

EC50 Determination

The EC50 value refers to the minimum concentration of the sample

that is able to scavenge the initial DPPH concentration by 50%. A calibration
curve was made by plotting the log values of the various concentrations of
the sample against the corresponding % DPPH scavenged. An equation of
the line of the form y = mx + b was obtained from the calibration curve where
y = % DPPH scavenged, m = slope, x = log value of concentration, and b=
intercept. The equation of the line was used to determine the EC50 value
through setting the % DPPH scavenged to 50 %. Finally, the EC50 value was
calculated by taking the antilog of the obtained log value of the test sample
concentration responsible for scavenging 50 % of the DPPH initial
concentration.
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Statistical Analysis

Analyses of the leaf and stem methanolic extracts of B. mutica were

carried out in five replicates with five trials each. For the standards used in
the determination of the total phenolic content and the total antioxidant
activity, only three replicates were made. For the standards used in the
determination of the DPPH radical scavenging activity, only four replicates
were made. The data obtained were statistically analyzed using t-Test at
0.05 level of significance. Correlation among total phenolic content, total
antioxidant activity and EC50 value for the DPPH radical scavenging activity
of the leaf and stem methanolic extracts of B. mutica was determined using
Pearson’s correlation. The software used for statistical analysis was SPSS
(Statistical Package for the Social Sciences) version 24. Grub’s test was
used to carry out the test for outliers.
Figure 9 shows the schematic workflow for the extraction and
subsequent chemical analysis of the leaves and stems of B. mutica. All
experiments were done in five replicates with five trials each.
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Sample Collection

Sample Preparation

Solvent Extraction

TPC Determination Antioxidant Activity Evaluation

TAA Assay

DPPH Assay

Figure 9. Schematic workflow for the extraction and chemical analysis of the
leaves and stems of B. mutica.