Chapter III

Methodology
This chapter presents the methods of research and procedure used in conducting the study.
It includes the Research Design, Procedure Flowchart, Materials and Instrumentation,
Experimentation General Procedure, and the Statistical Analysis.

Research Site Location and Duration

The study will take place on January and February 2018. The Akapulko leaves will be
gathered within General Santos City. The Akapulko (Cassia lata) Leaf extract will be air dried
for 24 hours and then the sample will be soaked on ethanol for 48 hours . The sample will be
filtered for rotavap. The Akapulko (Cassia lata) extract will then be in General Santos Doctors
Medical School Inc. Laboratory for Thin Layer Chromatography. Phytochemical Analysis,
Microbial Assay, Stability Testing of the Akapulko (Cassia lata) extract will also be in the same
place. DPPH Assay and Formulation of Antibacterial Ointment of Akapulko (Cassia lata) extract
will be conducted by the researchers at the GSDMSFI laboratory.
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Research Design

Akapulko Leaf Bioassay

Extract
R1 Antibacterial

T1 R2
Zone of Inhibition
R3

R1 Phytochemical Analysis

T2 R2
Thin Layer Chromatography
R3

R1 DPPH assay
T3 R2
Ointment Formulation
R3
R1 Stability Testing
T4 R2

R3

R1
T5 R2

R3

Legend:

Treatment1- Cefoxtamine

Treatment 2-Sterilized Distilled Water

Treatment 3- 100% Akapulko Leaf Extract

Treatment 4-75% Akapulko Leaf Extract with 25%

Sterilized Distilled Water

Treatment 5- 50% Akapulko Leaf Extract Extract with

50% Sterilized Distilled Water
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Procedure Flow Chart

Gathering and Extracting the Akapulko (Cassia lata)

leaves

Antibacterial Screening

Phytochemical Analysis
of the Plant Extract

Thin Layer Chromatograph Test

of the Plant Extract

Plant EXtract

DPPH Assay of the Plant Extract

Ointment Formulation of

the Plant Extract

Stability Testing of the Plant

Extract
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Materials and Instrumentation

Table 1

List of Raw Materials and Laboratory Equipment for Pure Leaf Extract Extraction and
Bioassay.

Extraction Ointment Formulation Stability Testing

Solvent Laboratory Equipment Laboratory Equipment

Ethanol Stirring Rod Stirring Rod

Laboratory Equipment Raw Materials Beaker

Mask Petroleum Jelly Erlenmeyer Flask

Gloves Akapulko (Cassia lata) leaf pH meter

extract
Safety Goggles Raw Materials

Filter Paper Formulated Ointment

Funnel

Raw Materials

Akapulko (Cassia lata) leaf

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Experimentation General Procedure

This is to discuss about the different processes undergone by the Akapulko (Cassia lata)
leaf extract throughout the study. This talks over the general procedure of the study from the
extraction of plant material up to the formulation of ointment and the evaluation of ointment.

Laboratory Tests
This subchapter will give indication on the different tests including the procedures that will
be done in the laboratory by the Researchers.

Procedure in Gathering and Extracting the Akapulko (Cassia lata) leaf

The plant materials will be gathered within, General Santos City. After gathering
the Akapulko (Cassia lata) leaf , the samples will be taken and then cleansed with water
and will be cut into smaller pieces. About 2kg of Akapulko leaves will be gathered.
After gathering, the processes for the extraction will be done. The sample will be
air dried for 24 hours in a room temperature. After air drying, it will be soaked to 3L ethanol
for 48 hours. Using the filter paper and the funnel, after the 48 hours soaking, the sample
will be filtered and will be placed in a clean container. The filtered raw material will be at
GSDMSFI laboratory for the extraction using the rotary evaporator.
After the extraction using the rotary evaporator, Microbial Assay and
Phytochemical Screening will be done on the extract. Some of the extract will be also later
be used for the Thin Layer Chromatography Test. Those tests will be conducted by the
researchers, assisted by laboratory staffs.
Procedure for Ointment Formulation
This procedure will be done in the laboratory of General Santos Doctors Medical
School Foundation Inc. This will be done with the assistance of a laboratory staff.
In this process, the 20g petroleum jelly will be put in a beaker. Then the 5g
Akapulko (Cassia lata) leaf extract will be added. Using the stirring rod, the petroleum
jelly and the extract will be mixed thoroughly until the extract will be equally distributed
within the mixture to form a 5.34g ointment with 25% concentration of Akapulko (Cassia
lata) leaf extract.
The 5mL of the Akapulko (Cassia lata) leaf extract will be for TLC.
Procedure for Stability Testing
This is to test if an ointment can last for a long duration and is still safe after a given
time, which may be either short term or long term. The stability test that will be done on
the formulated ointment will be the short term stability test.
The short term stability testing includes the evaluation of ointment through
continuously checking its pH level, consistency and color within a month. This test will be
done by the researchers.
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Procedure for Antioxidant assay of Akapulko (Cassia lata) Leaf Extract

Dissolved ethanolic extracts in absolute ethanol and water extract in
distilled water. Diluted each sample for at least 3 concentrations (two-fold
dilutions). Prepared 6x10-5M of DPPH in absolute ethanol. Then, transferred 500
l of each sample solution into an eppendorf tube with each concentration tested in
triplicate. Transferred 500 l of DPPH solution to mix with sample solution. Then
shake and stand at the room temperature for 30 minutes. Lastly, measured the
absorbance at 520 nm, using a mixture of 500 l sample solution and 500 l
absolute ethanol as blank.

Statistical Analysis
There will be only two parameters that will be quantitatively evaluated, the Antibacterial
Activity and the Antioxidant Property. The statistical tool that will be used to evaluate the
antibacterial and antioxidant properties of the Akapulko (Cassia lata) leaf extract is the One Way
Analysis of Variance or One Way ANOVA, and Tukey test will be used.