Methodology al (2009). Ten (10) grams of

Methodology

Collection
and Extraction of Plant Materials

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            Leaves of Gmelina arborea, Merremia peltata
Merr., and Ficus septica Burm F. will be collected from
Malinao, Tabaco City, Albay. A sample of collected plants will be sent to Bicol
University College of Agriculture and Forestry (BUCAF) for authentication. The
collected plants will be rinsed with distilled water to remove dirt; and will
be air-dried for forty-eight (48) hours. Then it will be cut into pieces using
scissors. After cutting, size will be reduced to powder a blender at maximum
speed for five (5) minutes. The plants will be subjected to an aqueous
extraction by decoction following the protocol of Koffi et. al (2009). Ten (10)
grams of powdered form of the plant will be mixed with 1 L of distilled water
and will be boiled for forty-five (45) minutes or until the volume was reduced
to 0.25 L. Then it will be filtered using cheesecloth. The filtrate will be
collected and the residual mac will be added with 1 L of distilled water and
will be boiled again for another forty-five (45) minutes. This process will be
repeated three more times using the residual marc. The overall collected
filtrate will be the total aqueous extract with a concentration of 10 g/L. The
extract will be kept in a refrigerator at 4°C
until use. Materials to be used in this extraction and storage will all be
sterilized to avoid contamination.

 

Experimental Animals

            Female albino mice of ICR strain
will be used for this experiment. The test animals will be purchased from Food
and Drugs Administration, Alabang, Muntinlupa City. It should be at least 6
weeks old and its weight must be within the interval of

 of the mean initial weight of the test
subjects. The temperature in the experimental animal room will be maintained at
22°C (

3°C) and with a
relative humidity of at least 50-60%. The lighting of the room will be
artificial with a 12:12 dark and light cycle. The test animals will be housed with
a maximum of 2 animals per cage. Wood shavings will be used for beddings. The
mice will be provided with unlimited access to drinking water, however, when it
comes to food, a conventional rodent laboratory diet will be used. The test
animals will be kept in their cages for 5 days prior to dosing for
acclimatization to the laboratory conditions (OECD, 2008).

 

Plant Extract Administration

            The plant extract will be
administered in a single dose by a gavage using a stomach or a suitable
intubation cannula. The gavage tube will be inserted through the mouth into the
stomach or the lower esophagus. The tube will be placed next to the mouse
adjacent to the last rib. The position of the tube adjacent to the tip of the
nose determined the distance. The feeding tube will be advanced to the oral
cavity to insure the administration of the extract into the stomach. The test
animals will undergo 3-4 hours of fasting prior to dosing and another 1-2 hours
after dosing, only food uptake will be withheld.

 

Acute Oral Toxicity Study

Acute
oral toxicity will be conducted according to the limit dose test of Up-and-Down
Procedure as described in the Organization for Economic Cooperation and
Development guidelines (OECD, 2008).
The test will be conducted to determine the median lethal dose (LD50) of the
extracts using a computer-guided statistical program (AOT425statPgm) at a limit
dose of 2000 mg/kg body weight/oral route and. Five female mice will be
selected using systematic randomized technique. Mice will be observed for 24-48
hours for any signs of toxicity, mortality, change in body weight and behavior.

 

Sub-chronic Repeated 60-day Oral Toxicity Study

            Animals will be selected randomly
and distributed into three groups having 5 animals per group for each treatment.
The aqueous extract of G. arborea, M.
peltata, and F. septica will be
labeled as Treatment 1 (T1), Treatment 2 (T2), and Treatment 3 (T3),
respectively. The control group will be divided into two test groups – the
untreated and the vehicle; which will be receiving 0.5 mL of olive oil daily. The
concentration per treatment to be used is still unknown since it is dependent
on the result of the acute toxicity test that was previously conducted. The
table below shows the different treatment groups and number of animals to be
used per test groups. The animals will be observed for changes in behavior,
lethargy,

Table 1. Different
Treatment Groups

TREATMENT

TEST GROUPS

NUMBER of ANIMALS

CONTROL

Untreated

5

Vehicle

5

TREATMENT 1

Low Dose

5

Medium Dose

5

High Dose

5

TREATMENT 2

Low Dose

5

Medium Dose

5

High Dose

5

TREATENT 3

Low Dose

5

Medium Dose

5

High Dose

5

 

Blood Collection

            A day after the last administration
of extract (61st day), experimental animals will be sacrificed
through cardiac puncture. Before the collection of blood, animals will be
deprived of food but not water overnight, to minimize the effect of recent meal
that may have on the test results (Chilukoti, 2015). The mice will be anesthetized using
Zoletil® (40-80 mg/kg) to be administered intraperitoneally. A 3 mL syringe and
22-gauge needle will be used. The mouse will be held by the scruff of skin
above shoulders and a needle will be inserted 5 mm from the center of the
thorax towards the animal’s chin, 5-10 mm deep, holding the syringe 25-30
degrees away from the chest. The mouse will be laid on their backs and push the
syringe vertically through sternum with needle perpendicular to the chest wall (Hoff, 2000). To locate the
beating heart, the thumb and forefinger will be placed near the left chest wall
to feel the heartbeat. The needle will be withdrawn without removing it from
under the skin. As the blood appears on the syringe, it should be hold steadily
as pulling back the plunger to obtain the maximum blood available. When it
stops flowing, the needle should be rotated and pull it out gently (Hoff, 2000). The collected blood
will be placed on EDTA tubes to avoid coagulation of blood (Rushdi, 2016).

 

 

Hematological and Biochemical Analysis

            The collected blood samples will be
subjected to hematological analysis using an automated hematological analyzer
to be sent to Albay Doctors Hospital (Legazpi City, Albay). Hematological
parameters to be evaluated will be the complete blood count (red blood cell,
white blood cell, and platelets), and hemoglobin concentration.

            The
biochemical analysis will determine the serum parameters such as the
concentration of total cholesterol, low density lipoproteins (LDL), high
density lipoproteins (HDL) and triglyceride level. As well as the concentration
of urea, creatinine, and glucose content in blood.

 

Statistical Analysis

            The data that will be obtained from
this experiment will be subjected to an Analysis of Variance (ANOVA) to
determine if there are significant differences between treatments and control.
The test will be followed by Tukey HSD and Post Hoc Test using statistical
software (IBM SPSS Statistics v.25).