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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: ____2015-250903______
Application Fee Received by: _____________ (PRINT Name & Sign)
BAI Certificate No.: ____________________ (For UST-IACUC) ------------------------------------------------------------------------------------------------------------------------Instructions.
(a) Please Please complete the form by giving giving all the details asked 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 of Mussaenda ustii Alejandro (Rubiaceae) 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 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 R CN AS - UST US T I A CUC CU C A nimal nimal Care and and Use State Statem ment Pr otoco tocoll R eview Form F orm
UST: US T: S037-00-F S037-00-F O01
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UST IACUC Animal Care and Use Statement Protocol Review Form
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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-2mediated 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) = 5 male rats in 1 treatment (1 treatment) =
15 male rats ___5 male rats__ 20 male rats
R CN AS - UST I ACUC Animal Care and Use Statement Pr otocol R eview Form
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UST IACUC Animal Care and Use Statement Protocol Review Form
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5 female rats in 1 treatment (1 treatment) = 5 female rats for every 1 treatment (1 treatments) =
5 female rats __ 5 female rats__ 10 female rats
For a total of 10 treatments
E.
Total Number of Animals:
30 Sprague-Dawley rats (10 female, 20 male)
Quarantine and/or acclimation or conditioning process Indicate where: UST-RCNAS Animal Handling House Facility Seven (7) days Duration
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 Water: Distilled Water
Source: Arranque Market 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 R CN AS - UST I ACUC Animal Care and Use Statement Pr otocol R eview Form
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UST IACUC Animal Care and Use Statement Protocol Review Form
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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 OECD’s 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
R CN AS - UST I ACUC Animal Care and Use Statement Pr otocol R eview Form
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UST IACUC Animal Care and Use Statement Protocol Review Form
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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 OECD’s 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. R CN AS - UST I ACUC Animal Care and Use Statement Pr otocol R eview Form
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UST IACUC Animal Care and Use Statement Protocol Review Form
May 2010
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 antiinflammatory 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.
C. Antiinflammator
Name of Drug
No. of Trials
0.9 % Saline Solution
3
Drugs
Celecoxib
3
administered
Diclofenac
3
prior to introducton of
Ibuprofen
3
carrageenan and will be
Plant Extract
3
observed for the next 4
y Assay
Procedures
will 1
be hour
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 con firm its death.
8.
Animal Adoption / Disposal
The carcasses will be placed in a properly labeled yellow plastic bag, then disposed to designated area afterwards.
R CN AS - UST I ACUC Animal Care and Use Statement Pr otocol R eview Form
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UST IACUC Animal Care and Use Statement Protocol Review Form
a.
May 2010
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 av ailable 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
R CN AS - UST I ACUC Animal Care and Use Statement Pr otocol R eview Form
UST: S037-00-F O01
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UST IACUC Animal Care and Use Statement Protocol Review Form
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May 2010
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: ________________________ Principal Investigator
Date: ______________________
Noted by: ___________________________ Adviser
Date: ______________________
_____________________________ Dean, Faculty/College
Date: ______________________
DARIA L. MANALO, DVM
Date: _____________________
Institutional Veterinarian
R CN AS - UST I ACUC Animal Care and Use Statement Pr otocol R eview Form
UST: S037-00-F O01
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