CHAPTER III

METHODOLOGY

Preparation of Materials
The materials to be used will be powderized P. pentandrum, test tubes, Wattman’s filter

paper #01, distilled water, Hydrochloric acid, solution of iodine in Potassium iodide, olive oil,

1% NH3 solution, 0.1% FeCl3, The following will also be prepared: Nutrient Agar (NA),

Erlenmeyer flasks, cotton, recycled paper, rubber band, autoclave, petri plates, micropipette,

stainless metal spreader, paper discs, Streptomycin, Vernier caliper, pipettor and brine shrimps’

eggs.

Treatments and Experimental Design

The study will be conducted to determine the phytochemical constituents that might have

potential in antibacterial property of Pittosporum pentandrum, as well as the cytotoxicity of

Pittosporum pentandrum in order to identify if it has any potential toxic agents.

Antibacterial Analysis
T1 - Aqueous solution (S1 and S2)
T2 - Ethanol solution (S1 and S2)
T3 - Streptomycin
T4 - No use of treatment
Cytotoxicity Analysis
T1 – 1 ppm of P. pentandrum (S1 and S2)
T2 – 10 ppm of P. pentandrum (S1 and S2)
T3 – 100 ppm of P. pentandrum (S1 and S2)
T4 – 1,000 ppm of P. pentandrum (S1 and S2)
T5 – 10,000 ppm of P. pentandrum (S1 and S2)
T6 – Control
T7 – Pure extracts
 All treatments will be laid out in Completely Randomized Design (CRD) under
laboratory condition.

Research Locale and Plant material

The study will be conducted at DOST – Laboratory, Department of Biological Sciences,

College of Arts and Sciences, Central Luzon State University. P. pentandrum will be obtained

from the vicinity of Nueva Vizcaya.

Research Procedures

Ethanol Extraction

Twenty grams (20g) of powderized P. pentandrum will be soaked in a 95% ethanol for

48 hours at room temperature prior filtration. Filtration will be done using Wattman’s filter paper

#01. The filtered extracts will be subjected to rotary evaporator at 200 rotation per minute (rpm)

and will be done at the Chemistry Department, CAS, CLSU.

Aqueous Extraction

Fifty grams of the powderized P. pentandrum will be soaked in sterile distilled water for

48 hours at room temperature. The extracts will be filtered using Wattman’s filter paper #01.

Filtered extracts will then be subjected to hot bath for two (2) hours at 75-80°C.
 Phytochemical Evaluation of P. pentandrum Extracts

Chemical tests for the screening and identification of bioactive chemical constituents in

the medicinal constituents under study will be carried out in aqueous extracts using the standard

procedures as described by Sofowara (1993), Trease and Evans (1989) and Harborne (1973) as

cited by Amin Mir (2013).

Alkaloids

Extracts will be dissolved individually in dilute Hydrochloric acid and will be filtered.

Filtrates will be treated with Wagner’s reagent (Solution of Iodine in Potassium Iodide).

Formation of a reddish brown colored precipitate will indicate the presence of alkaloids.

Saponins

Two (2) grams of powdered sample of each sample will be boiled together with 20 mL of

distilled water in a water bath and will be filtered. 10 mL of the filtered sample will be mixed

with 5 mL of distilled water in a test tube and will be shaken vigorously to obtain a stable

persistent froth. The frothing will be mixed with 3 drops of olive oil and for the formation of

emulsion will indicate the presence of saponins.

Flavonoids

Two to three (2-3) drops of 1% NH3 solution will be added to the aqueous extract of each

sample in a test tube. A yellow coloration will be observed if flavonoids compound is present.

Tannins
0.5g of powdered sample of each plant will be boiled in 20 mL of distilled water in a test

tube and will be filtered. 0.1% FeCl3 will be added to the filtered samples and will be observed
for brownish to green or a blue to black coloration which will indicate the presence of tannins.

Green coloration will indicate the presence of gallotannins while brown coloration will indicate

the presence of pseudotannins.

Glycosides

One (1) mL of concentrated H2SO4 will be prepared in test tube. Five (5) mL of aqueous

extract from each plant sample will be mixed with 2mL of glacial acetic acid containing 1 drop

of FeCl3. The above mixture will be carefully added to 1mL of concentrated H 2SO4 so that the

concentrated H2SO4 is underneath the mixture. If cardiac glycoside is present in the sample, a

brown ring will have appeared indicating the presence of the cardiac glycoside constituent.

Antibacterial Assay

Preparation of Culture Media and Pour Plating

Twenty-eight (28) grams of Prepared Nutrient Agar (NA) will be mixed with one (1) liter

of distilled water. It will be heated until homogenous mixture is obtained. Approximately 300

mL of the prepared medium will be dispensed onto a clean Erlenmeyer Flasks, plugged with

sterile cotton and wrapped with recycled paper and sealed with a rubber band. The medium will

be sterilized for 15 minutes at 15lbs/in2,121°C using autoclave. After sterilization, these will

allow to cool for several minutes until ready for pour plating. Approximately 15mL sterile

nutrient agar will be aseptically dispensed into sterile petri plates then allow to cool and solidify

prior to inoculation of bacterial samples.

Spread Plating

Bacterial inoculums of E. coli and S. aureus will be loaded to sterile petri plates using

spread plating method. 150 µL of each inoculum will be aseptically transferred using

micropipette, stainless metal spreader will be used to spread the inoculums onto the plates in a

circular motion.

Disc Diffusion Method

Antibacterial activities of Pittosporum pentandrum will be done following the Kirby-

Bauer Method against E. coli and S. aureus. Six (6) mm of sterile paper discs will be soaked on

the ethanol and aqueous extracts for about 30-40 minutes to allow absorption of the extracts. The

discs will be aseptically inoculated onto the plates with the test organisms with labels.

Plates will be incubated for 24-hours at room temperature. Zones of Inhibition will be

measured using a Vernier caliper after 12, 18 and 24 hours of incubation.

Brine Shrimp (Artemia salina) Lethality Assay

Source of Brine Shrimp

Brine shrimp (Artemia salina) nauplii will be obtained from Bureau of Fisheries and

Aquatic Resources (BFAR) located at San Juan, CLSU Compound, Science City of Munoz,

Nueva Ecija.

Preparation of Saline Solution

Thirty-four (34) grams of commercialized salt will be dissolved in a one liter of distilled

water for the nauplii to hatch. The solution will be supplied with aerator and sufficient lighting
and will be left for 24 hours to hatch. After 24 hours, hatched eggs will be collected and will be

transferred into a clean 40-mm petri plates with treatments.

Treatments with the hatched nauplii will be observed for 6, 12, 18, and 24 hours and

mortality results will be recorded respectively. LC 50 will be used to evaluate the lethality of the

concentrates against the nauplii.

Data Gathering

The following data will be gathered throughout the study:

1. Qualitative representation on the presence and absence of biochemical compounds using

the traditional phytochemical analysis;

2. Zones of inhibition by Pittosporum pentandrum against E. coli and S. aureus using

ethanol and aqueous extracts after 8, 16 and 24 hours of incubation, and

3. Number of alive nauplii after 6, 12, 18 and 24 hours of observation.

Statistical Analysis

All treatments will be laid out in a Completely Randomized Design (CRD) under

laboratory conditions with three (3) replication per treatment. Analysis of Variance (ANOVA)

will be used to compare means which was carried out using Duncan’s multiple range test

(DMRT). All test significance was compared at 5% level of significance.

Regression analysis will be used to determine the probit and LC50 of the solution. All

data will be subjected to a statistical tool via SPSS v.20 software.