St.

Paul University Philippines

Tuguegarao City, Cagayan 3500 26

Chapter 2

METHODOLOGY

This chapter gives a brief description of materials and

methods that will be used to prepare plant sample and screen

for its fracture healing effect. It also includes a brief

description of materials and methods that will be used to

characterize active compounds. The details of the preparation

of reagents and the methods are presented below.

I. Research Design

The study is an experimental design which involves

randomized control block design consisting of one (1) control

group consisting of positive control group and one (1)

experimental group with four concentrations of Ulasimang-bato

(Peperomia pellucida) aqueous extract with concentrations of

100 mg/kg, 200 mg/kg, 400 mg/kg, and 500 mg/kg. There were

three (3) replicates per treatment. The study also utilized

Calcium + Vitamin D3 + Minerals (Caltrate Plus) as positive

control.

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II. Subjects of the Study

The subjects in this research study were 15 hamsters

which gathered from Cauayan, Isabela. These hamsters were

composed of 4 female and 11 male. The following are the weight

of Hamster:

Table 1: Weight of the Hamsters

Different Replicate Weight (kg)

Treatment
1 0.0525
1 2 0.0513
3 0.0541
1 0.0545
2 2 0.0535
3 0.0555
1 0.069
3 2 0.0557
3 0.0557
1 0.0704
4 2 0.0717
3 0.0728
1 0.120
5 2 0.117
3 0.119

III. Data Collection Procedures and Instrumentation

The following procedures for the study will be utilized

A. Plant Materials

Ulasimang-bato (Peperomia pellucida) plant was

collected in Abulug, Cagayan. The plant was selected

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because of its availability in the area aside from their

medicinal potentials. The plant was then identified by

comparing it with the herbarium specimens at the

University of Santo Tomas, España Blvd, Sampaloc, Manila,

Metro Manila. The plants was washed with water and air

dried in shade for about three weeks. The air-dried leaves

must be pounded and kept in closed plastic container at

room temperature.

B. Preparation of Plant Extracts: The plant materials was

extracted through the process of decoction.

B.1. DECOCTION

1. In this process the whole fresh plant was cleaned,

cut into pieces and dried under a shade at room

temperature.

2. The decoction was carried out by boiling 100 g of

the powder in 1.5 L of distilled water for 10 min.

3. After the decoction filter the liquid through a

cloth, squeezing very well to obtain pure liquid.

4. Decoction was prepared and kept at room temperature

for 3 days.

(Florence NT, Huguette ST, Hubert DJ, et al., 2017)

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Tuguegarao City, Cagayan 3500 29

C. Preparation of the Positive Control (Caltrate Plus)

Dissolved Caltrate plus in 35 mL of water then when

it is fully dissolved it is ready to administer and was

stored at room temperature and was used for a day.

D. Qualitative analysis of the mineral composition of

Ulasimang-bato (Peperomia pellucida)

Under standard procedures, evaluation of the

presence of some of the selected minerals namely calcium,

magnesium, potassium, sodium, and phosphorus in the

aqueous plant extract was done.

D.1. Test for Calcium ions

To 10 drops of solution, add aqueous ammonia to make

the solution basic. Then add (NH 4)2C2O4 (ammonium oxalate)

solution dropwise. A white precipitate should form. (Birk

J, 2018)

D.2. Test for Magnesium ions

To 10 drops of solution, add 2 drops of magnesium

reagent, 4-(p-nitrophenylazo) resorcinol. Then add 6 M

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NaOH dropwise. A "blue lake" -- a precipitate of Mg(OH)2

with adsorbed magnesium reagent -- forms. (Birk J, 2018)

D.3. Test for Potassium ions

Potassium ions are identified by the violet color

it produces in a flame test. (Chemistry LibreTexts: Flame

Tests, 2018)

D.4. Test for Sodium Ions

Sodium ions are identified by the intense yellow

color it produces in a flame test. (Chemistry LibreTexts:

Flame Tests, 2018)

D.5. Test for Phosphate ions

To 10 drops of solution, add a few drops of silver

nitrate solution. If the solution under study contains

phosphate ions the formation of a yellow precipitate is

observed.

E. Oblique Fracture Method

1. 15 hamsters were bought with different weight and size

at the Cauayan, Isabela and was delivered from Cauayan

to Tuguegarao. These hamsters were brought in Santiago

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City to perform the oblique fracture method in care of

Dr. Charlie Foronda, DVM at Isabela Animates Veterinary

Clinic and Supplies.

2. Before the experimental procedure the Hamster were

weighted first and was classified according to their

different weight. After weighing, the experimental

procedure started.

3. The hamsters were pre-anesthetized using an intra-

peritoneal injection of ketamine (75 mg/kg)

4. After administering ketamine, the oblique manual fracture

was done by breaking the right knee specifically in the

tibia fibula of the hamster manually by hands.

5. The hamsters were given xylazine (25 mg/kg) to sedate the

test subjects before x-ray.

6. After the x-ray, the test subjects were given splint to

minimize the exacerbation of the fractured area.

F. Bioassay

1. The day after the experimental procedure, the injured

hamster were brought back to Tuguegarao City for the

administration of different doses.

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2. The administration of the positive control and the

different doses of the plant extract through the use

of 1 mL syringe will begin the next day after the

induction of the fracture.

3. The positive control (Caltrate Plus) and the aqueous

extract of Ulasimang-bato (Peperomia pellucida) at

doses 100 mg/kg, 200 mg/kg, 400 mg/kg and 500 mg/kg

was administered orally for 14 consecutive days.

G. Evaluation

1. After 2 weeks of treatment, the hamsters will be

subjected to x-ray at Santiago City to check for the

formation of callous or to check for the healing process

of the bone.

2. The x-ray was read by Dr. Cherry Canceran, DVM at Animal

Option Veterinary Clinic, Tuguegarao City for the

assistance and verification of the x-ray examination

result.

3. After the said fracture healing process, the hamsters

were in physically good health.

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H. Data Analysis

Table 2. CALLOUS SCORING

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