CHAPTER III

METHODOLOGY

This chapter contains the methodology of the anti-rheumatic effect of panyawan

(Tinospora rumphii) pure leaf extract as an herbal liniment. This also includes the

research-design, preparation of materials, general procedure, data gathering techniques,

and statistical tools which are necessary information in conducting this study.

Research Design

The study observed the experimental type of research. Figure 1 illustrates the

process in the conduct of the study. It shows the variables of anti-rheumatic effect of

panyawan (Tinospora rumphii) that were subjected to the test.

The conduct of the experiment engaged the two treatments done. Each treatment

has one replicate. The following treatments are: T 1 uses commercial liniment as the

positive control and T2 uses the pure leaf extract form panyawan (Tinospora rumphii)

leaves as the experimental treatment. There are two rats in each treatment. The

dependent variable
 Pure leaf extract of
Bioactivity
panyawan(Tinospora
rumphii) leaves

Anti-rheumatism
T1 R1
Healing the part
where rheuma is
present

T2 R1

Anti-inflammtory

Healing the part

where rheuma is
present

Legend:

T1- Commercial liniment

T2- Pure leaf extract of panyawan(Tinospora rumphii) leaves

Rn- Replicates

Figure1. Research Design

Preparation of Materials

In making the liniment, the first step the researcher will do is to prepare the

necessary materials (flask, mortar and pestle). The most necessary materials needed are

the panyawan leaves, which should be in a fresh when gathered, as goes for the

wintergreen as well. The researchers will use female induced lab rats. The eucalyptus

powder should have a fine texture when bought in the market.

The rest of materials to be used in making the liniment are scissors, foil, beaker, pan,

strainer, tong, winter green, eucalyptus powder and mineral oil. All of these shall be

provided by the researchers, solely. It is necessary to prepare all these materials before

proceeding to asking for permission and conducting the experiment, for these materials

are basic needs for this study to be successful.

General procedure

a. Asking Permission

The researchers are willing to ask permission to the principal, subject

teacher, and to the assigned facilitators of the area in which the study shall be

conducted (research laboratory) by means to pursue the trial on the researchers’

study beforehand. Letters will be distributed to the respective persons. Once the

researchers gained all the approval needed, thus the experiment be conducted

under supervision.

b. Collection of Specimen

First, the panyawan leaves, which will be collected from the local markets

of General Santos City. The eucalyptus powder will also be collected from
 local supermarkets within the city. On the other hand, the lab rats will be

bought from the local pet stores in the city. The researchers will assure that

the experiment will cause the rats no more than momentary pain or distress to

the lab rats. The rest of the materials wile solely be provided by the

researchers themselves.

c. Preparation of Treatment

The researchers will prepare 250 mg eucalyptus powder, 250mg of mineral

oil, 15g of wintergreen and leaves of the panyawan to be mixed to prepare the

said specimen. It should all be cleaned with water (mineral water).

d. Preparation of Set-up Experiment

The researchers will follow these steps to prepare the set-up experiments

Pour the mixture of solvent and desired compound in a round bottom flask.

Best results are achieved when the flask is filled less than half full of the

solution. Then, fill the rotovap cold traps with dry ice. Next, attach a glass or

"bump trap" which prevents any solution from entering the main part of the

rotovap, then secure with a Keck clip. With a Keck clip, attach the flask and

bump trap to the adapter portion of the roto-evaporator. Finally, lower the flask

into the water bath, because this helps to prevent the flask from disconnection.

The researchers will use the formaldehyde. It is an anti-inflammatory assay

method that uses induced mice paw edema. The injection of formaldehyde

under the plantar Apo neurosis of the hind paw of the mice or the rat causes

an inflammatory reaction which can be reduced by anti-inflammatory

 substances. It will determine how effective the treatment could be and if it can

fully heal rheumatism.

e. Rotary Evaporization

The researchers will use these few steps to attain the pure leaf extract of the

panyawan leaves.

First, soak the panyawan leaves in a 100% isopropyl alcohol. Then, start

the rotation of the machine. Different speeds are preferable for different volumes.

Slowly start increasing the vacuum. The vacuum is at the proper strength when:

1) condensation of the solvent can be seen on the cold finger or in the receiving

flask, or 2) the solvent begins to bubble. Then, turn on the heat for the water bath.

Recall from general chemistry that vacuum reduces the boiling point of the

solvent, so significantly lower temperature is needed to evaporate the solvent

using a rota-vap than at STP. Soon, adjust the vacuum setting as needed. When

all solvent has been removed turn off the vacuum and return the flask to

atmospheric pressure. Then, stop the rotation. Raise the flask from the bath and

remove the flask from the adapter. If there is more solvent to remove it can be

added it to the same flask and the procedure is repeated. Remember to empty the

receiving flask when the evaporation is complete.

Data gathering techniques

The researchers aim to create a liniment able to treat rheumatism. In this

experiment, an effective liniment that, when simply tested on the lab rats, can give
 proficient results as to treating rheumatism. A liniment that is also secure and efficient.

The researchers will analyze the rat after taking the treatment and find out if it heals the

rheumatism.

Statistical tool

The statistical tool to be used in the conducting of this research is chi-square

because it is used with two or less variables.

Liniment Anti-rheumatic effect

Commercial liniment

Panyawan liniment