Chapter III

Methodology
This chapter presents the methodology employed in the study. The chapter consist of
the nature of the study, research design, experimental animals, research instruments,
experimental procedures, and data quantification & analysis.

Research Design
This study will utilize a true experimental pretest-post-test design to determine the
antihyperlipidemic effect of Ashitaba (Angelika keiskei) ethanol extract to diet-induced
hyperlipidemic rats. The design enables the researchers to have complete control on the
extraneous variables and confidently predict that the observed effect by the manipulation of the
independent variables. The researchers will randomly assign the subjects to either the
experimental or the control group. The effect of the dependent variable on both the groups will
be seen before the treatment. The treatment will be carried out on the experimental group only,
and after-treatment observation of dependent variable on the dependent variable. This design
involves observation of 3 groups: negative control group and positive control, and an
experimental group.
27 rats within fixed weight range will be selected, weighed then marked for individual
identification. The rats were divided into 3 groups each contains 3 in each namely A, B & C.
Groups A, B & C fed equally with atherogenic diet to induce hyperlipidemia for 15 days. Group
A serves as negative control group receives atherogenic diet and vehicle, Group B served as
positive control group receives atherogenic diet and administered with standard drug
simvastatin 10mg/kg body weight and Group C served as test group receives atherogenic diet
and administered with test extract at a dose of 400mg/kg body weight, once daily for 6 days
orally through gastric intubation. During these days, all the groups received atherogenic diet
equally. The control group animals received hyperlipidemic diet and vehicle. Finally after 6
days blood samples are collected from all the groups within veterinary assistance and the blood
samples is subjected to lipid profile screening.
Results for the lipid profile screening will be validated by a statistician.

Experimental Animals/Population

The characteristics of the rats that were included in the study were rats that already have
hyperlipidemia as baseline, rats with 1-3 months of age, weighing 300g-1kg, a variety of
Common Rabbit, rats having a health certificate and rats of either sex were used for the study.
The participants that were not Common Rats or the rats that have different colors were
excluded. New born rats were also excluded in the study.
 Bioethical Consideration
All animals used in the study were handled properly in accordance with the Guide for
the Care and Use of Laboratory Animals Eight Edition (2011).

Sampling Procedure
The sampling method that was used in the study is the Simple Random Sampling which
is the simplest type of random sample. The population in this study are the Common Rats and
they are being divided into 3 groups, the treatment group, the controlled positive group and the
controlled negative group.

Research Instruments
I. Research Equipment
1.1 Test tubes/Vials containers for the blood samples for the laboratory analysis.
1.2 Syringe for extraction of blood samples from test animals
1.3 Dropper tool for administration of Ashitaba leaves extract to test animal
1.4 Volumetric Flask used in the maceration process of the Ashitaba leaves to extract the
active component.
1.5 Sealed Glass Container storage container for Ashitaba leaves extract
1.6 Rotary Evaporator used for concentrating the extract

Data Gathering Procedure

Stage 1 Permission
1.1 Secure a certificate of permission from the Deans Office/laboratory in-charge
stating their approval for the utilization of the PHARMACY laboratory where
experimental procedures is conducted.
1.2 Secure a medical certificate from the Veterinarian stating that the population (rats)
are of good health condition to undergo experiments.
Stage 2 Preparation for the experiment
2.1 Rats housed in wooden cages for 15 days
2.1.1 The cages are equipped with pellet trays and water trays
2.1.2 Cages are kept more or less 25C room temperature
2.1.3 Hyperlipidemia was induced in the rats by feeding them with
 Cholesterol mixed with pellet diet (Cholesterol (4%), Crude
Protein (11%), Calcium (1%), Phosphorus (0.4%), Moisture (12%)).
2.2 Allowing the rats to adjust to the new environment
2.3 Preparation of Ashitaba leaves extract
2.3.1 Collection of Ashitaba leaves
2.3.2 Authentication by a taxonomist
2.3.3 Leaves are washed, the leaves of the Leaves were hand leavesed, air
dried under shade and powdered to coarse consistency in a
blender. The powder is passed through a 40# mesh particle size
and stored in an air tight container at noon temperature.

2.4 Preparation of Dose for Ashitaba extract

2.4.1 The powdered material of Ashitaba (Angelika Keiskei)
is taken and mixed and is extracted with mixture of alcohol
and water (1:1) by adopting simple maceration procedure at
room temperature for seven days in a conical flask with
occasional shaking and stirring.
2.4.2 The extract will be filtered and concentrated to dryness by means
of evaporation to avoid the decomposition of the natural
metabolites.
2.4.3 The extract will be preserved in a refrigerator till further use.
2.4.4 Hydro-alcoholic extract was formulated as suspension is
distilled water. The strength of the suspension was
according to the dose administered and was expected as
weight of the dried extract.
Stage 3 Data Gathering
1. Induction of Hyperlipidemia diet to rats
3.1.1 (9) White Rats will be fed with high cholesterol diet for 15 days.
3.1.2 After 15 days the lipid profile will be screened in the laboratory
by a medical technologist.
3.1.3 The rats will be divided into 3 groups the negative control,
 experimental control, and the positive control group.
2. Administration of Simvastatin
3.2.1 Preparation of Simvastatin made to have 100mg/kg dose
3.2.2 Pulverizations of simvastatin
3.2.3 Dilution of Simvastatin in 1.5 mL distilled water
3.2.4 Inducing Simvastatin orally by use of syringe
3.2.5 Lipid profile is screened after 6 days

3. Administered of apple extract

3.3.1 Preparation and grounding of Ashitaba (Angelika keiskei) The leaves were
extracted and were macerated.
3.3.2 Extract were mixed in water by 1:1 ration forming
a hydro alcoholic mixture.
3.3.3 Dosage frequency of 400 mg/kg of Ashitaba (Angelika keiskei)
hydro alcoholic extract were induced to rats to
determine its antihyperlipidemic effect given once daily for 6
days orally.
3.3.4 Lipid profile was screened after 6 days.
4. Collection of Blood
3.4.1 Blood collection with veterinary assistance 0.5 1 mL whole
blood was extracted per rabbit.
3.4.2 Collection samples centrifuged at 2000rpm for the various
lipid manners.
5. Biochemical Analysis
3.5.1 Serum samples are to be submitted to the laboratory for cholesterol
assays.