CHAPTER II

RESEARCH METHODOLOGY
This chapter presents the design, methods and procedures that
will be used in obtaining all the data needed in the In vivo study of the
hypolipidemic

effect

of

Patola

leaves

(Luffa

cylindrica,

Fam.

Cucurbitaceae) formulated as capsule in albino rats.

RESEARCH DESIGN
This study employs experimental design of research to determine
the

hypolipidemic

(Cucurbitaceae)

activity

leaves

of

preformulated

extract

against

Luffa

cylindrica

cholesterol-induced

hyperlipidemic albino rats. Experimental design is the process of

planning a study to meet specified objectives. Planning an experiment
properly is very important in order to ensure that the right type of data
and a sufficient sample size and power are available to answer the
research questions of interest as clearly and efficiently as possible
(https://support.sas.com/resources/papers/sixsigma1.pdf).
A clear definition of the details of the experiment makes the
desired statistical analyses possible, and almost always improves the
usefulness of the

results. The overall data collection and analysis plan considers how the
experimental factors, both controlled and uncontrolled, fit together into
a mode that will meet the specific objectives of the experiment and
satisfy

the

practical

constraints

of

time

and

money.

(https://support.sas.com/resources/papers/sixsigma1.pdf)

Duration and the locale of the study

The

phytochemical

screening

of

the

leaves

and

the

preformulation study of patola leaves extract that will be utilized

in the study will be conducted at Lorma Colleges Pharmacy Laboratory,
5th floor of the Nursing Arts Building, Carlatan, City of San Fernando, La
Union. Experimentation proper will also be conducted at the Lorma
Colleges animal house. This study will be performed during the School
Year 2016-2017 and will cover the First Semester for the preliminary
test and throughout the Second Semester for the experimentation
phase.

Test animals
Experiments will be performed on twenty-four healthy adult male
albino rats (150-200g). The test animals will be grouped into four for
the determination of cholesterol level, where six albino rats in each

group will receive the following treatment schedule. Group 1 will be

given normal diet and water. Group 2 will be given cholesterol enriched
diet and water. Meanwhile, Group 3 will be given cholesterol enriched
diet, water and standard drug (Atorvastatin 3mg/kg Bw/day) while
Group 4 will be given cholesterol enriched diet, water and extract of
Luffa cylindrica leaves.
The animals will be housed in Lorma Colleges Animal House,
Carlatan, City of San Fernando, La Union where uniform temperature
25 degrees Celsius with uniform humidity will be maintained. The test
animals will be free from disturbances and will be given unlimited
access of diet and water. The test animals will be kept and observed in
the animal house for fourteen days for the acclimatization period so
that the test animals can adjust to the new environment prior to the
experimentation phase.
Materials and procedures
A Plant Material
Luffa cylindrica leaves will be gathered from Barangay
Santiago Norte, City of San Fernando, La Union.
B Preparation of The Extract
Preparation of the extract will be done by cleaning the
leaves and decoction of Luffa cylindrica. It will be boiled in a
specified volume of water for a defined time; it is then cooled
and strained or filtered.

C Preliminaries of Phytochemical Screening

Phytochemical Screening or Preliminary Test was done
before major discoveries of molecules or drug entities were
known. It was used to provide concrete knowledge and research
to what plant active constituents have potential to benefit
mankind
(http://rxistsource.blogspot.com/2012/10/phytochemicalscreening.html).
Preliminary phytochemical screening for proteins, saponins,
flavonoids, polyphenols and phytosterols were carried out on the
100% extract based on the procedures.
D Determination of Cholesterol level
A cholesterol screening measures your level of high-density
lipoprotein

(HDL)

cholesterol,

low-density

lipoprotein

(LDL)

cholesterol and triglycerides.

The researchers will be seeking the expertise of a licensed
Medical Technician in the examination of the blood of albino rats.
The blood samples will be send to the Lorma Colleges Medical
Laboratory Department for testing. The cholesterol levels will be
taken every seven days during the acclimatization period. The
data collected will serve as the baseline readings for future
comparisons. Healthy albino rats should have the normal
cholesterol

level

of

40-130

(http://www.ratfanclub.org/values.html).
E Induction of Hyperlipidemia in albino rats

mg/dL

The researchers will increase the cholesterol level of albino

rats by feeding a high cholesterol fat diet. 1% cholesterol will be
dissolved in 10% warmed ground nut oil and this oil solution was
added slowly in to powdered mixture to obtain homogeneous soft
cake. This cholesterol rich (High Fat Diet) preparation will be
molded in the shape of pellets of about 1g each and fed for 8
weeks by 100g/day/rat for the induction of hyperlipidemia.
F Determination of Hypolipidemic Activity
Body weights of the albino rats will be measured at base
line on the 4th week and 8th week. Overnight fasting blood
samples will be collected in EDTA coated tubes (3mg/ml blood) at
base line from the tails of the albino rats also on the 4th week
and 8th week.
G Preformulation
a pH
A pH strip will be used to determine the Luffa cylindrica
leaves extract. The pH will be calibrated using a reference of
buffer (at pH 5-7), until there will be obtained constant
readings.
b Specific Gravity
Specific Gravity is the heaviness of a substance compared
to that of water and it is expressed without units. To
determine the specific gravity at 25C, the pycnometer will be
used. It will be determined by dividing the weight of the
sample (expressed in gram) by the weight of the water (in
mL).

Treatment of Data
The researchers will be using the following statistical tools to
answer the following problems presented in the study:
Problem number 1: The mean values for pH of a solution can be
determined using the following equation: pH = -log[H +] where [H+] is
the concentration of H+ ions, in mol/L. And to get the specific gravity
value the formula is Specific gravity = Weight of the Substance /
Weight of the equal amount of water.
Or para mas short?
To determine the mean values for pH and specific gravity of the
different concentrations of the extract pH and specific gravity will be
computed.
Problem number 2: To identify what concentration does the
Patola extract exhibit the highest hypolipidemic activity is through
result of the blood samples that will undergo for testing.
Problem number 3: The one-way analysis of variance (ANOVA)
and post hoc analysis were employed to determine if there are
significant differences among the treatments.