Protocol form for research proposals to be submitted to the institutional animal ethics committee for new experiments.

1. IAEC Form B (updated Feb 2023)
Form B (per rule 8(a))
APPLICATION FOR PERMISSION FOR EXPERIMENTS INVOLVING ANIMALS
PART B
Protocol form for research proposals to be submitted to the Institutional Animal Ethics
Committee for new experiments or extension of ongoing experiments using animals other
than non-human primates
1. Title of project / dissertation / thesis: Protective action of ethanolic extract
of Rosmarinus officinalis L. in gastric ulcer prevention induced by ethanol in rats.
2. Principal Investigator:
a. Name : Mrs. Komal Chandra
b. Qualification : M. Pharm
c. Designation : Assistant Professor
d. Dept / Div/ Lab : Department of Pharmaceutical Sciences, Bhimtal
e. Telephone No. : 9042612345
f. E-mail: : Komalchandra@gmail.com
g. Experience in the field (years) : 5 years
3. List of Co-investigators (provide all details for all such individuals)
a. Name : Nikita Bankoti
b. Qualification : B. Pharm
c. Designation : Student
d. Dept / Div/ Lab : Department of Pharmaceutical Sciences, Bhimtal
e. Telephone No. : 9087645367
f. E-mail: : nikita.bankoti007@gmail.com
g. Experience in the field (years) : NA
4. Source of funding (please enclose proof – IRB approval letter (mandatory) and
sanction letter of funding agency (if applicable)- NA
5. Duration of the project
a. Number of months : 3 Months
b. Date of initiation (proposed) : 1 May 2024
c. Date of completion (proposed) : 1 August 2024

2. IAEC Form B (updated Feb 2023)
6. Plan of study including brief introduction and background; methodologyand
expected outcomes (include all relevant details; word limit: 1000 words)
Note: Please ensure that the rationale of the study and methodology to be used
are clearly presented without using technical terms and scientific jargon as far
as possible. Avoid using abbreviations without defining them at first mention.
Objective: Protective action of ethanolic extract of Rosmarinus officinalis L. in
gastric ulcer prevention induced by ethanol in rat.
 Chemical, apparatus and general procedure.
 Animals selection
 Obtaining of ethanolic extract of R.officinalis.L (eeRo)
 Quantification the ethanolic extract constituents of R.Officinalis.L by HPLC-
DAD
 The Experimental protocol and ethanol-induced gastric lesions method.
 The gastric lesion index.
 Histopathology
 Biochemical analysis
 Statistical analysis
 Result, Discussion and conclusion.
Anti-ulcer activity
Model for evaluation: Ethanol-induced gastric lesions method
Sr.No. Groups Treatment Animals
per group
1. Control group
This group received only a daily dose of vehicle A
i.e. Distilled water(98%)+ Tween80(2%) – 15ml/kg
along the treatment time.
06
2. eeRo control
group
It received only eeRo at 1000 mg/kg (15 mL/kg),
only one dose every 24 h, during every experimental
period
06
3. Omeprazole
group
(omep+etOH)
The animals of this group got only one omeprazole
dose (30 mg/kg, 2 mL/kg, p.o.), 1 h after ethanol
administration (70%, 2 mL/kg, p.o.) given to animals
only once.
06
4. 500+etOH
The animals of this group were submitted to eeRo
treatment at 500 mg/kg, every 24 h, they received
only one ethanol dose (70%, 2 mL/kg, p.o.), 1 h after
eeRo administration
06
5. 1000+etOH
The animals of this group were submitted to eeRo
treatment at 1000 mg/kg, respectively, every 24 h,
they received only one ethanol dose (70%, 2 mL/kg,
p.o.), 1 h after eeRo administration
06
6. Ethanol group
(etOH)
It received vehicle A, every 12 h, along the
experimental period. Furthermore, this group got
only one ethanol administration (70%, 2 mL/kg,
p.o.), 1 h after last vehicle dose.
06

3. IAEC Form B (updated Feb 2023)
7. Animals required
a. Species and strain : Wistar Rats
b. Age and weight : Young and Adult/ 270-320gm
c. Gender : Male
d. Number to be used (Provide full details of sample size calculation) Please
provide a table with year-wise break-up and number of animals per
experimental group: 36
Group: No. of Animals:
1. Control group Six animals
2. eeRo control group Six animals
3. Omeprazole group (omep + etOH) Six animals
4. 500 + etOH Six animals
5. 1000 + etOH Six animals
6. Ethanol group (etOH) Six animals
e. Number of days each animal will be housed: 3 Months
f. Proposed source of animals: Animal House of Department of Pharmaceutial
Sciences, Kumaun University, Bhimtal.
8. Rationale for animal usage (provide details as appropriate under the following
headings).
a. Why is use of animals necessary for this study?
On the basis of the literature survey, for the evaluation of the anti-ulcer activity
of the Rosmarinic Acid, the models required the use of animals, so it become
necessary.
b. Whether similar study has been conducted on in vitro models? If yes, describe
the leading points to justify the requirement of animal experiment.
Wistar rats show some physiological similarities to humans, thus making them a
reliable model that closely mimics human physiology and more accurately
replicates human diseases. The choice of Wistar rats for this procedure is based
on their demonstrated favourable response to peptic ulcer.
c. Why is the estimated number of animals essential (justification of sample size
calculated)?
The selection of the no. of animals for this activity is on the basis of the literature
survey. And, for the statistical justification, we have selected a min no. of animals
per group (n=6) total no. of animals used are 36.
d. Have similar experiments been conducted in the past in your establishment?
If yes, justify why new experiment is required.
Similar experiments were conducted in past using several plant extracts, poly
herbal preparation, nutraceutical etc. and minimum of 6 animals in each group
were taken in their experiments. To explore the anti-ulcer potential of the
substance, its necessary to conduct the experiments.

4. IAEC Form B (updated Feb 2023)
e. Have similar experiments been conducted by any other organization in same
or other in vivo models? If yes, enclose the reference and justify why new
experiment is required.
No, because the selected drug is approved for different therapeutic use and we are
first to conduct and evaluate in-vivo anti-ulcer activity for the selective drug.
9. Description of the procedures to be used.
Evaluation Parameters
a. List and describe allinvasive and potentially stressful non-invasive
procedures that animals will be subjected to in this study. NA
b. Furnish details of injections / substances: Omeprazole, Ethanolic extract of
Rosmarinic acid (eeRo), Distilled water (98%)+ Tween80 (2%), Ethanol (etOH).
 Doses: 500 OR 1000mg/kg of (eeRo)
 Sites: Oral administration
 Volume: As per body weight
c. Blood withdrawal details: Yes
 Volumes: 1-3ml
 Sites: Retro-orbital plexus
 Frequency: once a week
d. Radiation (dosage and schedules): NA
10.Please provide brief description of similar studies from in vivo/In vitro(from other
animal model) on same/similar test component or line of research. If, enough
information in available, justify the propose reasons. NA
11. Does the protocol prohibit the use of anesthetics or analgesics for the conduct of
painful procedures (procedures which cause more pain than that associated with
routine injection or blood withdrawal)? If yes, please provide an explanation
and justification for this. NA
12. Describe post-experimentation procedures
a. Euthanasia (specific method): Decapitation.
b. Method of carcass disposal after euthanasia: NA
c. Rehabilitation (name and address of facility where the animals are proposed
to be rehabilitated): NA
13. Animal transportation methods
It will be decided by Department of Pharmaceutical Sciences, Bhimtal, Kuman University,
Nainital.

5. IAEC Form B (updated Feb 2023)
14. Use of hazardous agents (use of recombinant DNA-based agents or potential
human pathogens requires documented approval of the Institutional Biosafety
Committee (IBSC). For each category, the agents and the biosafety level
required, appropriate therapeutic measures and the mode of disposal of
contaminated food, animal wastes and carcasses must be identified).
a. Radionucleotides (AERB): NA
b. Microorganisms / Biological infectious Agents (IBSC): NA
c. Recombinant DNA (RCGM): NA
d. Any other Hazardous Chemical / Drugs: NA
e. Any other give name: NA
If your project involves use of any of the above hazardous agents, attach a copy
of the minutes of IBC granting approval.

6. IAEC Form B (updated Feb 2023)
Protective action of ethanolic extract of Rosmarinus officinalis L. in gastric ulcer
prevention induced by ethanol in rats
Investigators’ declaration
1. I certify that I have determined that the research proposal described here is not
unnecessarily duplicative of previously reported research.
2. I certify that I am qualified and have adequate experience in the experimentation on
animals.
3. For procedures listed under item 10, I certify that I have reviewed the
pertinentscientific literature and have found no valid alternative to any procedure
described herein which may cause less pain or distress.
4. I will obtain approval from the IAEC/ CPCSEA before initiating any changes in this
study.
5. I certify that performance of experiment will be initiated only upon review and
approval of scientific intent by appropriate expert body (Institutional Review Board /
funding agency / other body).
6. I certify that I will submit appropriate certification of review and concurrence for
studies mentioned in point 15
7. I shall maintain all the records as per format (Form D) and submit to Institutional
Animal Ethics Committee (IAEC).
8. I certify that, I will not initiate the study before approval from IAEC/ CPCSEA
received in writing. Further, I certify that I will follow the recommendations of IAEC/
CPCSEA.
9. I certify that I will ensure the rehabilitation policies are adopted (wherever required).
Signature of Principal Investigator: Signature of Co-investigators:
Name: Mrs. Komal Chandra Name: Nikita Bankoti
Assistant Professor Date: 26 April 2024
Note: The Principal Investigator must sign at the bottom of all the pages of this proposal.

7. IAEC Form B (updated Feb 2023)