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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: ____2015-250903______ Application Fee Received by: _____________

(PRINT Name & Sign)
BAI Certificate No.: ____________________

(For UST-IACUC)
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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)]

Arvin John S. Mendoza

2012037941
09164391671
[email protected]

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

Affiliation & Contact Details (cell phone number and email address)]

JOVENCIO G. APOSTOL, MS Pharm, PhD Pharm, RPh, CPS

FACULTY OF PHARMACY
09165501925

GRECEBIO JONATHAN ALEJANDRO, PhD

DIRECTOR OF GRADUATE SCHOOL
09178654373

PROCEDURE(S) OR TITLE OF RESEARCH/STUDY:

Phytochemical Screening and Evaluation of Anti-Inflammatory Property of Mussaenda

ustii Alejandro (Rubiaceae) in Carrageenan-induced Paw Edema Sprague-Dawley Rats

PURPOSE/OBJECTIVES:

To be able to assess the concentrated ethanolic extract of the leaves of Mussaenda ustii
Alejandro if the plant possesses an anti-inflammatory property when introduced orally to the
carrageenan-induced paw edema Sprague-Dawley rats.

To identify minimum effective concentration by using OECD guidelines as reference.

DURATION OR TIME FRAME:

8-10 weeks
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BACKGROUND AND SIGNIFICANCE OF THE PROCEDURE OR RESEARCH:

Mussaenda ustii Alejandro belongs to the family of Rubiaceae. This plant is endemic to the
Philippines and has only been recently discovered in the province of Antique. The Rubiaceace family
possess different pharmacological properties and the Asian species are used in traditional Chinese
medicine for the treatment of breast cancer, fever, rheumatism, and malaria (Alejandro, 2015) According
to Ahmar et al. (2010) the ethanolic extract of this family has anti-inflammatory and hypoglycemic
properties.

In a wide variety of health conditions, Inflammation is the main cause and symptom of diseases.
Herbs and plants are anti-inflammatory to some degree, some with a more pronounced action than others
where the effect is secondary yet still an important part of the overall synergistic effect of the plant.
(Retrieved from anniesremedy.com, 2005). Plants are assumed to have bioactive compounds that may
have health beneficial effects and reduce the risk of chronic inflammatory diseases. (Strzelecka, 2005).

Cyclooxygenase-2-mediated prostaglandin is involved in inflammation and an increased

synthesis in the central nervous system is associated to allodynia and hyperalgesia. Cyclooxygenase-2-
mediated prostaglandin produces Prostaglandin which has a key role in homeostatic functions.
Furthermore, it can mediate the production of pathogenic mechanisms which include the inflammatory
response. (Guay, J., et. al., 2005) The biosynthesis of Prostaglandins is significantly elevated in inflamed
tissues, thus developing the cardinal signs of acute inflammation. (Ricciotii, E., et. al., 2012)

The study aims to investigate the secondary metabolites present in the plant extract. Also, to find
out what particular phytochemical is responsible for the anti-inflammatory activity of Mussaenda ustii
Alejandro. Futhermore, the study wants to validate and substantiate the anti-inflammatory activity of the
plant extract using an animal model. Lastly, to encourage future researchers who are interested in
developing this research or to simply serve as basis for related studies.

DESCRIPTION OF METHODOLOGIES/EXPERIMENTAL DESIGN:

A. Type of animal used: Sprague-Dawley rats

B. Source of the animals: Food and Drug Administration (FDA) Address: Civic Drive,
Filinvest Corporate City, Alabang, Muntinlupa City

C. Reason/Basis for selecting the animal species: Sprague-Dawley rats are calm and
therefore easier to handle. Another reason is that this breed of rat is used extensively in
medical research.

D. Sex and number of animals: 17 = no. of male rats, 23 = no. of female rats
Number of Animals per Treatment (Maximum of 6)

3 male rats for every 1 treatment (5 treatments) = 15 male rats

5 male rats in 1 treatment (1 treatment) = ___5 male rats__

20 male rats

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5 female rats in 1 treatment (1 treatment) = 5 female rats
5 female rats for every 1 treatment (1 treatments) = __ 5 female rats__

10 female rats

For a total of 10 treatments

Total Number of Animals: 30 Sprague-Dawley rats (10 female, 20 male)

E. Quarantine and/or acclimation or conditioning process

Indicate where: UST-RCNAS Animal Handling House Facility
Duration Seven (7) days

Conditions:
F. Animal care procedures
5. Cage Type & Size:

Metal cages with impervious flooring and minimal angles with a measure of 15 in. x
9 in. x 7 in. will be used.

Type of Bedding: Kusot or sterilized wood shavings

5. Number of animals per cage:

3 male rats for every 1 cage = 5 cages

5 male rats in 1 cage
5 female rats in 1 cage
5 female rats for 1 cage

For a total of 8 cages

5. Cage cleaning method: Beddings will be changed twice a week. Cages will be
flushed with water with the use of detergents and disinfectants also twice a week.
The water bottles and feeders will be cleaned and refilled every day.

5. Room temperature, humidity, ventilation and lighting:

*Between 20-25 degrees Celsius
*Humidity should at least be 30% and should not exceed 70%
*Artificial light; 12 hours light then 12 hours dark

5. Animal diet and feeding

Feed: Protein feeds Source: Arranque Market
Water: Distilled Water Source: Aqua Soft

A. Experimental or animal manipulation methods

5. General description of animal manipulation methods

Removal of rat from cage: hand over the back of the rat and thumb underneath the
animal and between its front legs
Oral administration: loose skin is gripped directly behind the ears then oral gavage is
inserted

5. Dosing method

Route: Oral BASED ON OECD GUIDELINES Using oral gavage needle

M. ustii extract The rats will be given 2000mg/kg BW of the plant extract. If the
rat dies, the dose will be lowered to a certain amount (e.g. 1000 mg/kg BW). If the
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new rat survives the new dose, the same amount will be administered in 4 rats. If the
casualty is 2 then the dose must be lowered again. Until such time that the dose
would cause no casualty at all.

Route: Subplantar (injection at paws) Celecoxib, Ibuprofen, and Diclofenac A

certain dosing computation used for rats will be utilized. Treatment Group: 3 male
rats will be administered with Ibuprofen (50mg/kg), Celecoxib (10mg/kg) and
Diclofenac (20mg/kg) with a total of 9 rats all of which came from the group that has
been induced with inflammation.

The concentration to be administered to the Sprague Dawley rats is quantified

using the OECDs guideline in identifying the LD50. Healthy, young, female rats
are preferred because they are generally more sensitive in LD50 tests The female
rat should be between 8 to12 weeks old and must be nulliparous and non-
pregnant. A test dose of 2000mg/kg, or exceptionally 5000mg/kg may be used
and a maximum of 5 rats is used. The first rat is given the test dose and will be
observed. If it survives, four more rats will be given the same dose and will also
be observed. If the latter rat dies and if three of the four rats die, the rats undergo a
single ordered dose progression, one at a time, at a minimum of 48-hours
intervals. Consequently, each rat is observed carefully before making a decision
on whether and how much dose will be given to the next rat. If the rat survives,
the previous dose will be increased by 3.2 times and will be administered to the
next rat. If the rat dies, the previous dose will be decreased by a similar dose
progression. The weight should fall in an interval within 20% of the mean initial
weight of any previously dosed rats.

Approximate Lethal Dose will be quantified by conducting a limit test and a main
test:

i. Limit Test:
Initially, 1 Sprague Dawley rat will be given a test dose of 2000mg/kg and the
effects to that rat will be observed. If the rat dies, proceed immediately with the
main test to determine the LD50. If the rat survives, four more rats will be given
the same dose and will be observed. If three of the rats die, the limit test will be
terminated and the main test will be performed.

*If three or more rats die, then the LD50 is lower than the test dose
*If three or more rats survive, then the LD50 is higher than the test dose

ii. Main Test:

Initially, the first rat will be dosed below the best preliminary estimate of the LD50.
If the rat survives, the second rat will receive a higher dose. However, if the first rat
dies then the second rat will receive a lower dose. All rats will individually be
observed at a 48 hour interval. The main test will be finished when 3 consecutive
rats survive at the upper bound.

5. Specimen or biological agent

Mussaenda ustii plant extract

Carrageenan suspension
Celecoxib
Ibuprofen
Diclofenac

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5. Animal examination procedures and frequency of examinations
A. Toxicology / LD50

The purpose of this toxicity testing is to identify the appropriate dosage of the
plant extract that will render a cure for the inflammation. Also, making sure that
the animal model will show little or no side effects at all. The concentration that
will be administered to the Sprague-Dawley rats is quantified using the OECDs
guidelines in identifying the LD50. Healthy, young, female rats are preferred
because they are generally more sensitive in LD50 testing. The female rat should
be between 8 to12 weeks old and must be nulliparous and non-pregnant. A test
dose of 2000mg/kg BW will be used in 5 female Sprague-Dawley rats. The first
rat is given the test dose and will be observed. If it survives, four more rats will be
given the same dose and will also be observed. If the latter rat dies and if three of
the four rats die, the rats will undergo a single ordered dose progression, one at a
time, at a minimum of 48-hour interval. Consequently, each rat is observed
carefully before making a decision on whether and how much dose will be given
to the next rat. If the rat survives, the previous dose will be increased by 3.2 times
and will be administered to the next rat. If the rat dies, the previous dose will be
decreased by a similar dose progression. The weight should fall in an interval
within 20% of the mean initial weight of any previously dosed rats.
Approximate Lethal Dose will be quantified by conducting a limit test and a main
test:

i. Limit Test:
Initially, 1 Sprague Dawley rat will be given a test dose of 2000mg/kg and
the effects to that rat will be observed. If the rat dies, proceed immediately with
the main test to determine the LD50. If the rat survives, four more rats will be
given the same dose and will be observed. If three of the rats die, the limit test
will be terminated and the main test will be performed. If three or more rats die,
then the LD50 is lower than the test dose. If three or more rats survive, then the
LD50 is higher than the test dose

ii. Main test:

Initially, the first rat will be dosed below the best preliminary estimate of
the LD50. If the rat survives, the second rat will receive a higher dose. However,
if the first rat dies then the second rat will receive a lower dose. All rats will
individually be observed at a 48 hour interval. The main test will be finished
when 3 consecutive rats survive at the upper bound.

B. Inducing of inflammation by introduction of Carrageenan

100mg of carrageenan sodium salt (BDH) will be used for the preparation of
1% carrageenan suspension. This will be sprinkled on a 10mL sodium chloride,
NaCl (0.9% w/v). The salt will be allowed to settle for 1 hour and will be stirred
after to suspend the mixture.

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After finding out the appropriate dosage for the rats and making the
carrageenan suspension, the plant extract is now ready to be tested for its anti-
inflammatory effect. The carrageenan will be induced at the right hind paw of the
Sprague-Dawley rats. The left hind paw will be injected with 0.9% w/v Saline
Solution and this will serve as control. This animal model will comprise of
different groups which are: the control which will only be tested with 0.9% w/v
Saline Solution, three reference drugs namely: Ibuprofen, Celecoxib, and
Diclofenac, and three different doses of the plant extract at 100mg/kg, 200mg/kg,
and 400mg/kg. The control group of rats will be injected with 0.9% w/v Saline
Solution at a concentration of 10mL/kg body weight. The 2nd, 3rd, and 4th group
will be tested with: Ibuprofen, Celecoxib, and Diclofenac with a 50mg/kg,
10mg/kg, 20mg/kg dosage respectively. The last 3 groups will be tested with
different dosages of the plant extract at 100mg/kg, 200mg/kg, 400mg/kg
respectively.

Name of Drug No. of Trials Procedures

0.9 % Saline Solution 3 Drugs will be
Celecoxib 3 administered 1 hour
C. prior to introducton of
Diclofenac 3
Anti- carrageenan and will be
Ibuprofen 3
inflammator observed for the next 4
Plant Extract 3
y Assay hours.

5. Use of anesthetics: Anesthetics will not be used for the entire testing.

6. Surgical procedures

No surgical procedures will be done.

7. Euthanasia

The method of euthanasia to be used is cervical dislocation so that

minimal pain or distress will be felt. This will be done in a kind manner. A
knowledgeable personnel will assess the animals and confirm its death.

8. Animal Adoption / Disposal

The carcasses will be placed in a properly labeled yellow plastic bag, then
disposed to designated area afterwards.

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a. Is there a non-animal model applicable for the procedure/study? If so, please
provide the reasons for not using it.

Unfortunately, there are no non-animal models available for this procedure.

b. Indicate the names and qualifications of all personnel who will be responsible for
conducting the procedures.

Diane Joy Diaz

-Attended the animal handling seminar and passed the exam
Contact number: 09176476274

Nigel Dolina
-Attended the animal handling seminar and passed the exam
Contact number: 09177046896

Abram Jeremy Millan

-Attended the animal handling seminar and passed the exam
Contact number: 09271530612

Francesca Isabel Montenegro

-Attended the animal handling seminar and passed the exam
Contact number: 09176550210

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7. 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:

________________________ Date: ______________________

Principal Investigator

Noted by:

___________________________ Date: ______________________

Adviser

_____________________________ Date: ______________________

Dean, Faculty/College

DARIA L. MANALO, DVM Date: _____________________

Institutional Veterinarian

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