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UST IACUC
Animal Care and Use Statement
Protocol Review Form

May 2010

University of Santo Tomas

Institutional Animal Care and Use Committee
ANIMAL CARE AND USE STATEMENT
(Protocol Review Form)
UST-IACUC Code: ___2014-1160805_______Application Fee Received by: _______________
(PRINT Name & Sign)
BAI Certificate No.: ____________________
(For UST-IACUC)
--------------------------------------------------------------------------------------------------------------------Instructions.

(a) Please complete the form by giving all the details asked for.
(b) Have the Protocol Review Form SIGNED and DATED by your respective
ADVISER/S & DEAN.
(c) Submit the accomplished Protocol Review Form to the IACUC Office at the Research
Center for the Natural Sciences (Rm. 202 TARC) for review and approval prior the
conduct of the scientific work involving animals.

RESPONSIBLE PERSON OR PRINCIPAL INVESTIGATOR [PRINT Complete

Name, Student Number & Contact Details (cell phone number and email address)]

Name

Student
Number

Email Address

Cell
No.

Concepcion, Ma. Patricia

2011-027557

pcon.07712@live.com

+639437002111

Lee, Ira

2011-027626

iherlee@yahoo.com.ph

+639151080279

Mercado, Lovelyn

2011-028061

lovelynmercado@gmail.com

+639275206861

Ordiz, Critstine

2011-027677

cristine_ordiz@ymail.com

+639177162598

Oropeza, Nicole Marie

2011-027678

oropeza.nm@gmail.com

+639173071127

Palomar, Eric

2011-036235

ericpalomar_95@yahoo.com

+639264652322

Tan, Phoebe I.E

2011-027743

phoebeieptan@gmail.com

+639266778894

NAME OF ADVISER/S &/OR CO-ADVISER/S [PRINT Complete Name, College of

Affiliation& Contact Details (cell phone number and email address)]
Asst. Prof. Mary Jho-Anne T. Corpuz
Faculty of Pharmacy
09178543896
maryjhoannecorpuz@gmail.com

Phone

PROCEDURE(S) OR TITLE OF RESEARCH/STUDY:

Formulation of antimicrobial ointment from Bidens pilosa leaf extract.

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Protocol Review Form

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Animal Care and Use Statement
Protocol Review Form

May 2010

PURPOSE/OBJECTIVES:
The researchers aim to evaluate the antibacterial property of the ethanolic and methanolic
leaf extracts of B. pilosa. Then, the extract with the more antibacterial property will be
used as an active ingredient to formulate the antibacterial ointment.
Specifically, this study seeks to answer the following questions:
1. Which among the two extracts exhibit the most antibacterial property by determining
its Minimum Inhibitory Concentration (MIC)?
a) methanol
b) ethanol
2. What is the stability of B. pilosa against light, heat, and relative humidity (RH)?
3. What is the effective concentration of the B. pilosa extract?
4. What is the most effective excipient base to be used in the formulation of the
antimicrobial B. pilosa ointment?

DURATION OR TIME FRAME:

6 months

BACKGROUND AND SIGNIFICANCE OF THE PROCEDURE OR RESEARCH:

Background of the Study
Bidens pilosa, commonly found in tropical countries, is known to exhibit antibacterial,
antidysenteric, antimicrobial, anti-inflammatory, and hypotensive activities. Malaria,
glandular sclerosis, wounds, flu, and urinary tract infections are a few of the diseases
which make use of B. pilosa as a mode of treatment (Dagawal & Ghorpade, 2011).
Results have shown that the presence of flavonoids and polyacetylenes is responsible for
the general pharmacological activities found in the said plant (Wang et al., 2010).
Another study shows that polyacetylenes also inhibit the growth of various pathogenic
organisms in the study carried out by Sundararajan et al. in 2006 (as cited in
Ezeonwumelu, J. et al. 2011). Meanwhile, Geissberger and Sequin (1991) claimed that
the constituents that impart the antimicrobial activity in B. pilosa were found to be
phenylheptatriyne, linoleic acid, and linolic acid.
In this study, B. pilosa will be formulated into an ointment. Although ointments are
bulkier and less stable than solid dosage forms, it is more convenient for the patient to
apply or use. With ointment as a formulated dosage form, it is easier to
facilitate the application of the drug on the affected part of the body. Not only is it known
to be more chemically stable than liquid dosage forms, it is also easier to handle than
bulky liquid dosage forms (Mehta, 2003).
With B. pilosa as the active ingredient, it can be deemed to be less costly. Aside from its
antibacterial property, it can also be used as an anti-inflammatory agent in cases where
both bacterial infections and inflammation occur. The constituent responsible for the antiinflammatory effect is the presence of flavonoids with its two bioactive types: centaurein
and centaureidin (Chiang et al., 2004).
Significance of the Study
This study can greatly help medical students, practitioners, pharmaceutical industries, and
organizations in prescribing, manufacturing, and promoting a new antibacterial drug.
Pharmaceutical manufacturers and innovators would benefit with this study since Bidens
pilosa is a very common plant with a wide range of medical uses. Once this antibacterial
ointment is proven to be effective, it would be their opportunity to produce this innovator
drug.
This research is also helpful to pharmacists and aspiring pharmacy students as it
encourages them find other alternatives, to discover more therapeutic substances
especially those that are abundant in our country, to exercise their profession in
formulating dosage forms, and to enhance scientific discoveries.

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Animal Care and Use Statement
Protocol Review Form

May 2010

Most importantly, this paper benefits the environment and agricultural industry by
suppressing the germination and growth of seedlings in a number of crops. If used, not
only would B. pilosa decrease in number, but it also the problems brought about by the
suppression of the germination and growth of seedlings.

DESCRIPTION OF METHODOLOGIES/EXPERIMENTAL DESIGN:

A. Type of animal used:
Albino rabbits weighing 1.5 to 2 kilograms will be used.
B. Source of the animals:
Plegaria Pet Shop
C. Reason/Basis for selecting the animal species:
The albino rabbits will be used due to its appropriate size and suitability for
assessment of hypersensitivity to the ointment.
D. Sex and number of animals:
Male; 3
E.

Quarantine and/or acclimation or conditioning process

The animals are randomly selected and marked to permit individual identification,
and will be housed in groups for five (5) days prior to the dosing to allow for
acclimatization to the laboratory conditions. As with other sequential test
designs, care must be taken to ensure that the animals are available in the
appropriate size and age range for the entire study

A. Animal care procedures

1. Cage Type & Size:
The cage is 0.90x0.60x0.45 meters made of plastic with a netted lid. The
water bottle is placed at the side of the plastic cage and the porcelain bowl is
placed on top of the bedding. The bedding is made of sterilized wooden chips
that will be placed directly into the cage to allow the absorption of the urine
and the animal to burrow.
1. Number of animals per cage:
Three (3)
\

2. Cage cleaning method:

The cages will be cleaned at least twice a week with a solution of water and
liquid detergent, then disinfected with bleach. The beddings will be changed
with every cleaning to prevent the buildup of urea.
1. Room temperature, humidity, ventilation and lighting
The temperature in the experimental animal room should be 25C ( 3C).
Although the relative humidity should be at least 30% and preferably not
exceed 70% other than during room cleaning, the aim should be 50-60%.
Lighting should be artificial, the sequence 12 hours light and 12 hours darkness
1. Animal diet and feeding
Feed: Rabbit pellets
Water: distilled water

Source: Pet shop

Source: Wilkins

A. Experimental or animal manipulation methods

1.

General description of animal manipulation methods

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Animal Care and Use Statement
Protocol Review Form

May 2010

They should be held by grasping a large fold of loose skin over the shoulders
with one hand and either supporting or grasping the rear feet with the other hand.
2.

Dosing method
Uniformly apply 5mg of the ointment onto the shaved portion of the rabbits
skin.

3.

Specimen or biological agent

The researches will use Escherichia coli, Staphylococcus aureus, and Candida
albicans as biological agents to test for the antimicrobial property of the crude
methanolic and ethanolic extract of Bidens pilosa. First, these microbial agents
would be cultured in agar plates. Then, the crude extract would be applied to the
agar plates through disk diffusion method. The researchers would use the
alcoholic extract with more antimicrobial activity in the formulation of the
ointment.

4.

Animal examination procedures and frequency of examinations

The researchers would not introduce any microbial agent to the rabbits. They
would only use the rabbits for a skin irritation test regarding the ointment. Test
animals will be shaved at the back using an animal razor 24 hours before
application of the sample. The shaved area of skin of each rabbit would be the
same area (25x25 mm). The ointment (500 mg) will be mixed and immediately
applied to two rabbits with an approximate amount of 80 mg per cm2. One rabbit
will be applied with the ointment containing the active ingrdient and the second,
with the ointment base (without the active ingredient). The third rabbit will
serve as a negative control. Both sites of application would be covered by gauze
and the back of the rabbit was wrapped with a non-occlusive bandage. The
animals would then be returned to to their cages. After 24 hours, the bandage
and the test materials would be removed and after one hour, the sites would be
examined for skin irritation. Observation of the sites would be done 24 hours
after application, and repeated at 48 and 72 hours thereafter. The reactions,
defined as erythema and edema, would be evaluated according to the scoring
system for skin reactions (see table below).
Table: Classification system for skin reactions
Reaction
Erythema
No erythema
Very slight erythema (barely perceptible)
Well-defined erythema
Moderate to severe erythema
Severe erythema (beet redness) to eschar formation
Edema
No edema
Very slight edema (barely perceptible)
Well-defined edema (edges of the area well defined by definite raising)
Moderate edema (raising approximately 1 mm)
Severe edema (raised more than 1 mm and extending beyond the area of exposure)
Total possible score for primary irritation

Score
0
1
2
3
4
0
1
2
3
4
8

5.

Use of anesthetics
Not applicable

6.

Surgical procedures
Not applicable

7.

Euthanasia
Not applicable

8.

Animal Adoption / Disposal

Once approved, the students will fill up a form for the adoption of test animals.

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Animal Care and Use Statement
Protocol Review Form

May 2010

B. Is there a non-animal model applicable for the procedure/study? If so, please

provide the reasons for not using it.
No because the hypersensitivity test is only applicable to living animals.
C. Indicate the names and qualifications of all personnel who will be responsible for
conducting the procedures.
Concepcion, Ma. Patricia
Lee, Ira
Mercado, Lovelyn
Ordiz, Cristine
Oropeza, Nicole Marie
Palomar, Eric
Tan, Phoebe I.E.
Student researchers who have passed the Animal Handling Test and have been
immunized with Tetanus toxoid.
*Attached herewith is the photocopy of the certificate of the Tetanus toxoid vaccination
and the screenshot of the animal care handling exam score.

DECLARATION BY THE RESPONSIBLE PERSON:

I ACCEPT RESPONSIBILITY FOR ASSURING THAT THE PROCEDURES/ STUDY
WILL BE CONDUCTED IN ACCORDANCE WITH THE APPROVED PROTOCOL.
I ASSURE THAT ALL PERSONNEL WHO USE THIS PROTOCOL AND WORK
WITH ANIMALS, HAVE RECEIVED APPROPRIATE TRAINING/ INSTRUCTIONS
IN PROCEDURAL AND HANDLING TECHNIQUES, AND ON ANIMAL WELFARE
CONSIDERATIONS.
I AGREE TO OBTAIN WRITTEN APPROVAL FROM THE INSTITUTIONAL
ANIMAL CARE AND USE COMMITTEE PRIOR TO MAKING ANY CHANGES
AFFECTING MY PROTOCOL. I ALSO AGREE TO PROMPTLY NOTIFY THE
IACUC IN WRITING OF ANY EMERGENT PROBLEMS THAT MAY ARISE IN THE
COURSE OF THIS STUDY, INCLUDING THE OCCURRENCE OF ADVERSE SIDE
EFFECT.
Signature over Printed Name of the Responsible Person:

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UST IACUC
Animal Care and Use Statement
Protocol Review Form

May 2010

Principal Investigators:
Concepcion, Ma. Patricia D.

Oropeza, Nicole Marie T.

Lee, Ira H.

Palomar, Eric H.

Mercado, Lovelyn G.

Tan, Phoebe I.E P.

Ordiz, Cristine A.

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Animal Care and Use Statement
Protocol Review Form

May 2010

Date: ______________________
Noted by:
_____________________________________
Adviser

Date: ______________________

MA. ELENA J. MANANSALA, RPh. MSc.

Dean, Faculty/College

Date: ______________________

DR. DARIA MANALO, DVM

Date: _____________________

Institutional Veterinarian

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