Research Defense 2023

Bacolod City National High School Anxiolytic Activity of Essential Oils of

STE Department
Research III Select Ethnomedicinal Plants in Negros
9 Pythagoras Occidental using Different Application
RCR
Kienzyl Faith F. Jurada Methods on Mice (Mus musclus) in
Lois Abegail Murillo
Keziah Marie D. Encabo Elevated Plus Maze Test
Sanz Andrey L. Rivera
Ray Hanna Dequinto
 Introduction
| Background of the study
•In the Philippines, anxiety and depression affect six million
Filipinos, ranking the country third highest in mental disorder
rates (Maravilla and Tan 2021).

•With a rising global trend in mental disorders, patients prefer

herbal products over pharmaceutical drugs due to their safety
(Kenda et al. 2022).

•In the Philippines, traditional herbal remedies, rich in bioactive

compounds, are utilized to reduce stress. This study compares
essential oils in Negros Occidental and their impact on anxiety
and depression using lab mice.
 Significance
•This study can add to the corpus of
information about alternate natural treatments
for anxiety-related diseases. •The study's findings could ultimately help the community by
offering different ways to treat anxiety, encouraging the
sustainable use of ethnomedicinal plants, and creating new
research opportunities in the area of natural product-based
•This study could lead to the discovery of medicines.
novel plant-based remedies that could
supplement or even replace current
pharmacological treatments for anxiety.
•This study can aid the medical field in identifying
which bioactive compounds are present in the selected
•This study can increase the plants to assist with reducing anxiety and stress.

understanding of the public of the

anxiolytic properties of ethnomedicinal
plants.
Introduction
| Review of Literature
Mice/Mice strains
•Similar to humans, mice dislike pungent-smelling items. Mice
have this kind of reaction to avoid due to their potential toxicity,
as well as to avoid predators. The smell of fragrance from
essential oils inhaled by both animals and humans provides calm
and relaxation (Ueno et al. 2019).

•Rodents prefer dark and/or enclosed spaces to protect themselves

from predetors and tend to show defensive behavior if they sense
the presence of danger, this be havior reflects the stress and
anxiety they are experiencing (Lezak et al. 2017)
 Introduction
| Review of Literature
Essential oils
•A study by Lizarraga-Valderrama (2020) found that
essential oils can manage psychological disorders without
adverse effects, stimulating neural pathways effectively.

• A study shows that Malunggay, Lagundi, and Ylang-ylang

reduce anxiety level while citronella are yet to be proven
(Paguia et al. 2014), (Aswar et al. 2012), and (Zhang et al.
2018).
 Introduction

| Review of Literature
•Essential oils which are proven effective in aromatherapy
and recently been discovered to aid in neurological
disorders (Fernandes et al. 2021).

Stresses
•There are many types of stress including; Chronic stress,
Life events, Traumatic life events, Daily hassles (i.e. daily
stressors), and Acute stress (Crosswell and Lockwood
2020).
Objectives Introduction

•Identify compounds present that mainly

influence their anxiolytic activity on mice to
be a potential use for stress/anxiety reliever
•Application method (of the essential
for humans.
oils) that is most effective in testing
the anxiolytic activity of mice.
•The significant difference between
each treatment (essential oils).

•The type of essential oil that is most

effective in reducing stress/anxiety
•The common bioactive compound/s found in among the four.
selected ethnomedicinal plants that mainly
influences/impacts the anxiolytic activity of
mice.
Introduction 》 Null Hypothesis (H0)
•There is no significant difference in anxiolytic activity
between the compounds present in extracted essential oils of

Hypotheses
theses ylang-ylang, citronella, moringa, and lagundi.

•There are no significant differences in anxiolytic activity

between different application techniques and treatments of
essential oils.
》 Alternative Hypothesis (H1)
•Specific compounds present in extracted essential oils of ylang-ylang,
citronella, moringa, or lagundi exhibit significant anxiolytic activity,
indicating their potential for human stress/anxiety relief.

•There are significant differences in anxiolytic activity among different

application techniques and treatments of essential oils, suggesting variations
in their effectiveness
.
Introduction
|Variables
Independent Dependent
•Type of Essential oils •Stress level of the mice
•Stressors (forced swim, restraint, elevated •Anxiolytic activity of essential oils
platform, and temp) •Performance of the mice after exposure
•Method of applying the essential oil

Control Variables
•Amount of essential oil
•Amount of time the subjects are
exposed to stress
Introduction
|Conceptual Framework
Methodology
|Research Design
• A comparative-experimental research approach will be used to evaluate the anti-anxiety effects of
Lagundi, Citronella, Ylang-ylang, and Malunggay essential oils.

• Using a randomized block design to take into account potential confounding factors and keep study
validity.

• By raising variability between groups while decreasing variability within groups, the randomized
approach helps identify significant differences in results.

• Soxhlet extraction technique is used to get essential oils.

• Combining exact parameters with astute observations to provide reliable data collecting.
 Methodology
|Limitations
•Study focuses on house mice, limiting
generalizability to other mammalian species.

•Potential differences in physiological and behavioral

reactions across species not explored.

•Immediate effects studied, neglecting long-term

consequences like tolerance, cumulative effects, or
toxicity from prolonged exposure.
 |Settings

•The experiment will be conducted in a laboratory. All of the factors that affect the mice such as light,
temperature, and the surrounding noise will be manipulated to suit the experiment on how the
researchers see fit.

• After a week of the acclimation of mice and a day of finding and thorough selection of plants. The
extraction of essential oil will also be conducted in a separate room inside the laboratory.

•The researchers will be provided with the equipment to be used in the experiment.

•Extraction will be conducted for a week and another week for the acclimation of mice which includes
obtaining permits and finding animal facilities to legally use the vertebrate.

•As estimated by the researchers, the main experiment will be conducted for 2 weeks and the overall
methodology alone will take about 4 weeks.
 Procedures
•Selection of Local Ethnomedicinal Plants.
•Extracting the essential oils
•Acclimation and Preparation
•Running the Elevated Plus Maze Test
•Post Experiment Care
•Ylang-ylang, citronella, moringa, and lagundi are the
selected plants and the researchers will ask for the Selection of
assistance of the experts at the Bureau of Plant
Industry to ensure the safe conduct of experiments.
Characterization of Local
•The plants will be bought from a local plant nursery
Ethnomedicinal Plants.
and the researchers will consider the physical
characteristics and specific plant parts for extraction.

•The parts of the plants that will be used are washed,

dried by microwave with paper napkins to absorb
moisture, and powdered for extraction (Babu et al.
2018) (Preparation of Plant Specimens for Deposit as
Herbarium Vouchers, n.d.).
Extracting the essential oils [Soxhlet extraction].

•85 grams of dried plants and 90% ethanol solvent will be used for the Soxhlet
extraction and it will be heated at 100-140°C and will last for 6-8 hours or 16
cycles.

•The essential oil will be separated with solvent using the rotary
evaporation technique (Singh 2022).

•The essential oils will be divided into 4ml for aromatherapy, 6ml for direct
application, and 5ml reserved for compound resting for 15 grams per plant kind
and will be stored in labeled vials.
 Acclimation and
Preparation of Mice.

•The mice will be obtained from an animal facility approved by the Bureau of Animal Industry and will be transported to the chosen lab following the rules and
regulations of transporting live animals by Department of Agriculture Administrative Order no. 19

•The researchers will only obtain 20 male mice that weigh between 20-25g.

• The researchers will acquire permits for transporting the animals used if necessary/required; the researchers will ensure that the vehicle used meets the standard to
maintain the safety and condition of the mice.

•The researchers will handle the mice following the National Institutes of Health Guide for Care and Use of Laboratory Animals.

•The mice will be fed using the standard rodent pellet diet and consume liquid; the mice will be placed in a vivarium or any suitable shelter throughout the ride.

•Before experimentation, the mice will be placed in their cages with nesting(wood shavings) to keep them warm and cardboard tubes for their spatial
compartmentalization.
 Acclimation and
Preparation of Mice.
•Cage dimension: 330cm sq. enclosure space, 70cm sq. floor space, and 12 cm height following the
minimum height for cages.
•Since mice are sensitive to light, the researchers will modify the environment, having only low-
level light to avoid possible stress to mice.
•The researchers will wait for about 3 days or more before using the mice after shipping to avoid
any problems in the health of the mice.
•The mice will be placed in an environment with ambient temperature (25°-26°C) before the
experiment.
•To ensure the health and condition of the mice, the researchers will conduct observations and
evaluations of the mice.
Running the Elevated Plus Maze Test
(including exposing the mice to stressors)

•Maze dimension: center(5cm x 5cm), closed arms (15cm x 5cm x25 cm), open arms (25cm x 5cm).

•The researchers will place a camera between the arms at a 45-degree angle. 3 cameras will be used.

•The mice will be stressed out before putting them in the maze.

•20 mice will be divided into 4 groups with 4 mice each; the remaining 4 mice will be in the control group in which there will be no
treatments applied in them.

•Each group will be applied with different treatments using different application methods.

•The researchers will be experimenting with 4 sessions, exposing 4 mice to 1 stressor a day; the remaining 1 for control.

•After stressing, the mice will receive their own designated EOs

•The researchers will observe the behavior of the mice in

the EPM.
 •The researchers will observe the mice daily to ensure that there are no
health complications present.

•In any occurrence of abnormalities in mice, the researchers will

immediately call a clinical veterinarian to check up on the mice.

•After the researchers check and observe the condition of the mice post-
experiment, the researchers will adopt them as no euthanization will be
conducted.

•All data acquired from the recording will be used together with personal
observations from the researchers for data analysis.
Post Experiment Care for
•The information gathered will be evaluated and analyzed thoroughly and
accurately to provide eligible results. Mice
 Methodology
|Data Analysis
• The statistical tools one-way and two-way ANOVA will be utilized

• To the development for in-depth analysis and interpretation, the researchers will methodically
arrange the data collected for both the experimental and control groups.

• The effects of various essential oils and application methods on the anxiety-related behavior of
mice will be compared using a variety of statistical techniques, including two-way ANOVA.

• The synergistic effects of essential oil blends and application methods will be highlighted by the
two-way ANOVA, which will aid in evaluating the significant effects of these factors and their
interactions.
 Methodology
|Data Analysis
• To evaluate the influence of the stressor as an independent variable on the anxiety-related behavior of
mice, one-way ANOVA will be used.

• The statistical significance of observed variations in variables such as the length of time spent in open
arms, the length of time spent in enclosed arms, the number of entries into open arms, and the total
distance traveled will be determined using these statistical techniques.

• The detailed statistical studies will demonstrate the effectiveness of different application methods and
the potential anxiolytic activity of essential oils, especially when taking into account the presence of a
stressor.

•The ultimate objective is to assess the data analysis findings and make pertinent inferences about the
study.
Methodology
|Ethical Considerations
•Researchers' safety is ensured through guidance from professionals and veterinarians.

•Restraint methods are applied if mice do not maintain docility during the experiment.

•Researchers will be wearing disposable one-time-use Personal Protective Equipment (PPE) for safety.

•Assistance from adults/professionals for preparing protective clothing and comprehensive lab disinfection.

•Mice contained in a vivarium accessible to researchers during the experiment.

•Post-experiment, the cage, and experimental area were thoroughly disinfected for safety precautions.
References
Aswar, Manoj, Abhijeet A. Bidkar, Kajal Gujar, and Tanay G. Athawale. 2012. “Anxiolytic Like Effects of Leaves
Extract of Vitex Negundo (L.)_(Fam:-Verbaceae) in Elevated Plus Maze Test.” ResearchGate, July.
https://doi.org/10.18311/jnr/2012/268.

Babu, A. K., G. Kumaresan, V. Antony Aroul Raj, and R. Velraj. 2018. “Review of Leaf Drying: Mechanism and
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References
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References
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“Preparation of Plant Specimens for Deposit as Herbarium Vouchers.” n.d. University of Florida Herbarium
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