Thesis

COMPARATIVE STUDY OF THE EFFICACY OF OILS
COMPARATIVE STUDY OF THE EFFICACY OF OILS OF

Azadirachta indica and Zingiber officinale IN FORMULATION
WITH COMMERCIAL MOSQUITO REPELLENT
AGAINST Aedes aegypti (DIPTERA: CULICIDAE)
Dayrit, Kenneth G.1,2,3,4

De Padua, Alexandra Nicole, N.1,2,3,4
Gomez, Nio V.1,2,3,4
Gutierrez, Paula Giselle P.1,2,3,4
Hulleza, Nathalie C.1,2,3,4
Martinez, Maria Lorenz M.1,2,3,4
Christina G. Sabroso, RPh, MSPharm2,3,4,5
Bachelor of Science in Pharmacy
College of Pharmacy
Research Development and Innovation Center
Our Lady of Fatima University
Research Adviser
March 2015
OUR LADY OF FATIMA UNIVERSITY
COLLEGE OF PHARMACY
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Endorsement Page
This thesis entitled "Comparative Study of the Efficacy of Oils of Azadirachta indica and
Zingiber officinale in Formulation with Commercial Mosquito Repellent against Aedes aegypti (Diptera:
Culicidae)" prepared and submitted by Kenneth G. Dayrit, et al, in partial fulfilment of the requirements
for the degree of Bachelor of Science in Pharmacy has been examined and now recommended for oral
examination.
This is to certify that Kenneth G. Dayrit et al, is ready for the oral examination.
__________________________________
Christina G. Sabroso, RPh, MSPharm
Name of Faculty Adviser
Panel of Examiners
Approved by the committee of Oral Examination with the grade of ________.
Dean Olive M. de Vera, RPh, MSPharm

Chairperson
Angelita A. Rodriguez, RPh, MSPharm, PhD
Jobelle S. Abrio, RPh
Member
Member
Examined and approved in the partial fulfillment of the requirements for the Degree of Bachelor of
Science in Pharmacy
Michael Joseph S. Dio, RN, MAN, PhD

Director
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Certificate of Originality
We, hereby, declare that this thesis entitled, "Comparative Study of the Efficacy of Oils of
Azadirachta indica and Zingiber officinale in Formulation with Commercial Mosquito Repellent against
Aedes aegypti (Diptera: Culicidae)" is our own work. The researchers made the thesis in their own
knowledge and there is no previous published works or study about the said topic. If there will be ideas
coming from others' work, we will acknowledge them in our study. We also declare that the content of
the thesis is our own work, however, we may ask some help and assistance when it comes from
presentation, instrumentation, and documentation.
Kenneth G. Dayrit
Principal Investigator
Alexandra Nicole N. De Padua

Member
Nio V. Gomez
Member
Paula Giselle P. Gutierrez

Member
Nathalie C. Hulleza
Member
Maria Lorenz M. Martinez

Member
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Acknowledgement
The researchers would like to express their deepest appreciation to those who have extended
their generous support, time, assistance, concern, and encouragement through the entire period of the
research work until the time of its completion.
To Dean Olive M. de Vera for encouraging the researchers to uplift their knowledge and interest
towards a better research foundation.
To Ms. Cristina G. Sabroso, Research Adviser, for all the advice, encouragement, endless
motivation, suggestion, knowledge, and expertise imparted to the researchers.
To Danilo N. Tandang, Senior Researcher of National Museum, for the authentication of the
botanical specimens.
To Dr. Angelita A. Rodriguez and Ms. Jobel S. Abrio, Panelists, for evaluating the study and
for giving valuable insights and recommendations to the researchers.
To Ms. Alicia G. Garbo, Principal Investigator at the Insectary of STD-ITDI DOST, for assisting
the researchers in conducting laboratory evaluation.
To Ms. Jewel M. Refran, RDIC Statistician, for her professional interpretation of the data to
come up with a significant statistical result.
To the parents of the researchers, for their never-ending love and support.
Most of all to God Almighty, for giving the researchers the never-ending patience, the courage,
the wisdom to conduct the study and the divine inspiration to continue despite of all the shortcomings.
K.G.D.
A.N.N.D.P.
N.V.G.
P.G.P.G
N.C.H.
M.L.M.M.
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Table of Contents
First Title Page.... ...
Endorsement Page..
Certificate of Originality
Acknowledgement.
Table of Contents...
List of Appendices..
List of Figures.
List of Plates.......
List of Tables......
List of Selected Journals.
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Second Title Page...

Abstract..
1.0 Introduction
1.1 Background of the Study.
1.2 Statement of the Problem.
1.3 Research Hypotheses...
1.4 Significance of the Study.
1.5 Scope and Delimitation
2.0 Review of Related Literature and Study
2.1 Local Literature
2.1.1 Ginger..
2.1.2 Neem...
2.1.3 Aedes aegypti..
2.2 Foreign Literature
2.2.1 Ginger..
2.2.2 Neem...
2.2.3 Essential oilGinger oil.
2.2.4 Fixed oilNeem oil
2.2.5 Extraction of essential oil.
2.3 Local Study
2.3.1 Ginger..
2.3.2 Neem...
2.4 Foreign Study
2.4.1 Ginger..
2.4.2 Neem...
3.0 Research Method
3.1 Research Design..
3.2 Research Locale...
3.3 Population and Sampling.
3.3.1 Ginger oil and Neem oil...
3.3.3 Volunteers...
3.4 Research Ethics
3.4.1 Volunteers on laboratory evaluations..
3.4.2 Risk minimization...
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3.4.3 Risk
3.4.3.1 Potential risk from exposure to treatments...
3.4.3.2 Potential risk of exposure to mosquito bites.
3.4.3.3 Potential risk of exposure to mosquito-borne disease..
3.4.4 Nature and magnitude of all expected benefits.
3.4.5 Right to refuse or withdraw..
3.4.6 Right to privacy.......
3.5 Research instruments
3.5.1 Fourier Transform Infrared Spectrometer (FTIR)
3.5.2 Hydrosteam Distillation set-up
3.6 Data collection
3.6.1 Preparation of Ginger oil.
3.6.2 Preparation of Neem oil...
3.6.3 Product Formulation
3.6.4 Laboratory Evaluation (Arm-in-cage set-up)...
3.6.4.1 Test considerations..
3.6.4.2 Test proper...
3.7 Data analysis
4.0 Results
4.1 Organoleptic testing.
4.2 Fourier Transform Infrared Spectrometer (FTIR) Analysis Result
4.2.1 Neem oil..
4.2.2 Ginger oil.
4.3 Physicochemical characterization...
4.4 Repellency...
5.0 Discussion....
6.0 Conclusion
7.0 Recommendation..
8.0 References
9.0 Glossary of Terms and Abbreviations ..
10.0 Appendices
Appendix
A Certifications
A.1 Permission and Counseling..
A.2 Authentication of Ginger..
A.3 Certification of Ginger Distillation...
A.4 Certification of FTIR
A.5 Certification of Ethical Review
A.6 Certification of Statistical Analysis..
A.7 Certification of Proofreading
B Research Plates...
C Ethics
C.1 Informed Consent (Engl.).
C.2 Informed Consent (Fil.)
D Research Budget.
E FTIR Results
E.1 FTIR of Neem Oil ...
E.2 FTIR of Ginger Oil...
F Computations and Figures..
G Timeline.
H Authors...
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List of Figures
Figure
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Paradigm of the Study........

Simulacrum of the Study
Major components of Ginger oil ...
Azadirachtin's chemical structure..
Salannin's chemical structure
Geraniol's chemical structure....
Citral's chemical structure.
The repellency of essential oils (100% concentration) to Aedes Mosquitoes...
Azadirachta indica.
Ginger (Zingiber officinale)..
Flow Chart of the Study.
Preparation for Aedes aegypti
Flow Chart of the Laboratory Evaluation..
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List of Plates
Plate
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Hydrosteam Distillation.
TENSOR27Spectrometer of Bruke Optics...
Physicochemical characterization..
Substances used in the Laboratory Evaluation...
Arm-in-cage set-up.
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List of Tables
Table
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Treatments to be used on laboratory evaluation (arm-in-cage set-up)...

Results of the organoleptic testing Ginger oil and Neem oil.
The IR Absorption values of Neem oil..
The IR Absorption values of Ginger oil.
Results of physicochemical characterization.
Results of repellency..
Overall comparison of the results of the repellency using Friedman's Two-way ANOVA
Comparison of the mean CPT to mean %P within treatments using Pairwise comparison
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List of Selected Journals

1 Ansari, M.A., P. Vasudevan, M. Tandon, & R.K. Razdan (2000). Larvicidal and mosquito
repellent action of peppermint (Mentha piperata) oil. Bioresource Technology, 71,
267
2 Boonyuan, W., Grieco, J. P., Bangs, M. J., Prabaripai, A., Tantakom, S., &
Chareonviriyaphap, T. (2014). Excito-repellency of essential oils against an Aedes
aegypti (L.) field population in Thailand. Journal of Vector Ecology, 39(1), 112-122
3 Maia, M. F. and Moore, S. J. (2011). Plant-based insect repellents: A review of their
efficacy, development and testing. Malaria Journal, 10(1). Retrieved September 5,
2014, from http://www.malariajournal.com/content/10/S1/S11
4 Mishra, A. K., Singh, N. and Sharma, V. P. (1995). Use of neem oil as a mosquito repellent
in tribal villages of Mandla District, Madhya Pradesh, India. Indian Journal of
Malariology, 32, 99103
5 Moore, S. J., Lenglet, A., & Hill, N. (2002). Field evaluation of three plant based insect
repellents against malaria vectors in Vaca Diez Province, the Bolivian Amazon.
Journal of the American Mosquito Control Association, 18, 107110.
6 Pandian R. S., Dwarakanath S. K., & Martin P. (1989). Repellent activity of herbal smoke
on the biting activity of mosquitoes. Journal of Ecobiology, 1(2); 87-89.
7 Pandian, R. S., Manoharan, A. C. & Pandian, R. S. (1995). Herbal smoke a potential
repellent and adulticide for mosquitoes. Insect Environment, 1: 1415
8 Pitasawat, B., Choochote, W., Tuetun, B., Tippawangkosol, P., Kanjanapothi, D., Jitpakdi,
A., & Riyong, D. (2003). Repellency of aromatic turmeric curcuma aromatic under
laboratory and field conditions. Journal of Vector Ecology, 28, 234-240.
9 Sharma, V. P., Ansari, M. A., & Razdan, R. K.. (1993). Mosquito repellent action of neem
(Azadirachta indica) oil. Journal of the American Mosquito Control Association, 9,
359.
10 Tawatsin, A., Asavadachanukorn, P, Thavara, U., Wongsinkongman, P., Bansidhi, J.,
Boonruad, T., ...Mulla, M. S. (2006). Repellency of essential oils extracted from
plants in Thailand against four mosquito vectors (Diptera: Culicidae) and
oviposition deterrent effects against Aedes aegypti (Diptera: Culicidae). Southeast
Asian Journal Tropical Medicine and Public Health, 37(5). Retrieved September 5,
2014, from http://webdb.dmsc.moph.go.th/ifc_nih/applications/files/repellency.pdf
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COMPARATIVE STUDY OF THE EFFICACY OF OILS OF

Azadirachta indica) and Zingiber officinale IN FORMULATION
WITH COMMERCIAL MOSQUITO REPELLENT
AGAINST Aedes aegypti (DIPTERA: CULICIDAE)
Gomez, Nio V.1,2,3,4
Christina G. Sabroso, RPh, MSPharm2,3,4,5
College of Pharmacy
Research Adviser
COLLEGE OF PHARMACY
COMPARATIVE STUDY OF THE EFFICACY OF OILS OF Azadirachta

indica and Zingiber officinale IN FORMULATION WITH COMMERCIAL
MOSQUITO REPELLENT AGAINST Aedes aegypti (DIPTERA:
CULICIDAE)
Gomez, Nio V. 1,2,3,4
Cristina G. Sabroso, RPh, MSPharm2,3,4,5
1
2
College of Pharmacy
3
4
5
Research Adviser
ABSTRACT
One of the prevalent concerns in the tropical and subtropical areas is dengue transmission. The
most vital precautionary measure has been focused on personal protection and control intervention. The
use of repellent seems to be the fundamental method of personal protection against annoyance and
infection. The study sought to formulate a herbal pump spray repellent that contains Ginger oil and Neem
oil. The formulated herbal was evaluated by laboratory evaluation (arm-in-cage set-up) through which
complete protection time (CPT) and percent protection (%P) were determined. The results showed that
formulated herbal (2% Ginger oil:5% Neem oil) is an effective repellent and is more effective than
formulated herbal (2% Neem oil:5% Ginger oil) in %P. The formulated herbal (2G:5N) provided mean
%P of 80.98 and a complete protection time (CPT) of 30 to 60 minutes. Analysis of variation exhibited
among experimental, positive, and negative group was analyzed by conducting Friedman's Two-way
ANOVA of the mean CPT and %P. Further data were analyzed by Pairwise comparison to compare the
positive and negative control with the experimental group. In all mean CPT, there is a significant
difference between the negative control and the experimental group. While in mean %P, the negative
control is significantly different to formulated herbal (2G:5N), commercial herbal (citronella), and
commercial synthetic (7.5% DEET). Also, %P of 2G:5N is not significantly different to commercial
synthetic and herbal. It suggests that 2G:5N is comparable to commercial synthetic and herbal. The study
is commendable in providing evidence for the potential of oils contained in the formulated herbal in
developing novel herbal repellents against mosquitoes.
Key words Aedes aegypti, Formulation, Ginger, Mosquito, Neem, Repellent
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1.0
Introduction
1.1
Background of the Study
In the Philippines mosquito-borne infection, specifically dengue, has been a prevalent cause of
morbidity and mortality that causes widespread concern in our times. Dengue has been called the most
dreaded mosquito-borne viral disease in man. The World Health Organization defined dengue as "the
most rapidly spreading mosquito-borne viral disease in the world. It is a febrile illness that affects infants,
young children, and adults with symptoms appearing 3-14 days after the infective bite." Among the
clinical cases in the Philippines, dengue is reported as a leading cause of childhood hospitalizations
(Oishi et al., 2006). Dengue transmission is due to the common vector female Aedes aegypti. Dengue
infection rates are higher in outdoors and during daytime. Dengue outbreaks have also been attributed
to Aedes albopictus, Aedes polynesiensis, and several species of the Aedes scutellaris complex. Each of
these species has a particular ecology, behavior, and geographical distribution (WHO, 2014).
Dengue transmission heighten health risk to over billions of people primarily in tropical and
sub-tropical areas. According to the World Health Organization, approximately fifty million dengue
infections are reported worldwide every year and 2.5 billion people live in dengue endemic countries.
Since the 1940s, the risk of contracting dengue infection has increased dramatically. This is due to the
huge number of global travel, population growth and urbanization, poor sanitation and hygiene,
ineffective mosquito control, and a growing range of both virus and vector. There is also an increase in
surveillance and official reports of dengue cases (Dengue transmission, 2014; Seng et al., 2009).
There is no available vaccine for preventing this infection. Personal protection and control
intervention against mosquito bites are currently the most vital precautionary measures in reducing
transmission of dengue virus and improving quality of environment and public health. This measure may
limit the disease-related morbidity and mortality. The study sought to formulate alternative approaches
yielding mosquito control effectively. These have resulted in an urge to look into local plants as potential
nontoxic and economical-friendly pump spray repellent.
Repellents are practical and economical means of preventing transmission of these infections to
humans. Plant-based repellents are extensively used because this is they may offer accessible and
affordable protection with reduced toxicity from mosquito bites among poorer communities. These were
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also preferred because plants are perceived as safe and trustworthy agent in mosquito bite prevention
(Maia & Moore, 2011).
The study have made considerable efforts to promote the use of herbal repellents. The study
have conducted laboratory evaluation on the repellent activity of a formulated herbal. The dose of
formulated herbal was assessed at predetermined concentrations2% Ginger oil:5% Neem oil and 5%
Ginger oil:2% Neem oil. Repellency testing was conducted wherein five test treatmentsformulated
herbal (2G:5N), formulated herbal (2N:5G), commercial herbal (HomeLife Citronella Twist Spray
Lavander), commercial synthetic (repellent lotion with 7.5% DEET), and negative control (formulated
herbal without the oils)were used against Ae. aegypti.
1.2
Statement of the Problem
The study aims to compare the oils of Neem (Azadirachta indica) and Ginger (Zingiber
officinale) in formulation with commercial mosquito repellent against Aedes aegypti (Diptera:
Culicidae).
Specifically, the study sought to establish an answer to the following questions:
1. What are the different functional groups present on the
a. Essential oil of Z. officinale as determined by the FTIR
b. Fixed oil of A. indica as determined by the FTIR
2. Are there significant differences among formulated herbal (2G:5N), formulated herbal
(2N:5G), commercial synthetic (7.5% DEET), and negative control in their
a. Complete Protection Time (CPT)
b. Percent Protection (%P)
1.3
Research Hypotheses
The following hypotheses were formulated for testing in the research:

1. There are no significant differences among formulated herbal (2G:5N), formulated
herbal (2N:5G), commercial synthetic (7.5% DEET), and negative control in their CPT.
2. There are no significant differences among formulated herbal (2G:5N), formulated
herbal (2N:5G), commercial synthetic (7.5% DEET), and negative control in their %P.
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1.4
Significance of the Study
In a tropical country like the Philippines, Filipinos consider mosquitoes as nuisance not only
because of the uncomfortable feeling they left on the skin after biting but also to the fatal effects it may
carrymild-to-severe allergic reactions and disease transmission. The study have focused on the
development of a product that is made from a combination of fixed oil and essential oil from two different
plants (A. indica and Z. officinale) respectively. The study also allows the people who live in places that
have abundant supply of the said plants to utilize them and make herbal mosquito repellents.
To the Community or Consumers. The study aims to create awareness on advantage of using
an herbal mosquito repellent over synthetic products and specifically to provide an effective repellent
comparable to the commercially available mosquito repellent.
To the Patient. The study will help the patient to acquire information on utilization of an
alternative herbal mosquito repellent that could be a precautionary measure in preventing outbreak or
lowering the casualties of mosquito-borne pathogen transmission.
To the Manufacturers. The study will provide an idea to the manufacturing firms for further
evaluation and validation of the formulated herbal as a potential herbal mosquito repellent.
To the Government Officials. The study may be able to induce government officials to utilize
and improve the formulated product for distribution and/or production for further economic income of
the country, thereby saving resources spent for importing such products.
To the Pharmacy Students. The study will contribute on the innovation of ideas for the students
with regards to the repellency property of the oils present in the plant samples.
To the Other Researchers. The study will serve as a reference and a basis for future objectives
to support, improve, and furnish additional data that would be beneficial to fulfill the study and advances
it to the next level prior to the increasing demands of the society.
1.5
Scope and Delimitation
Scope. The study covered comparative study of the efficacy of oils of Meem
(Azadirachta indica) and Ginger (Zingiber officinale) in formulation with commercial mosquito
repellent against Aedes aegypti (Diptera: Culicidae). Ginger was collected from La Trinidad,
Benguet while Neem oil was obtained from Swanson Health Products. An equivalent of 3000
grams of fresh rhizome of the Z. officinale was weighed. Upon semi-drying of rhizome, 2200
grams was weighed. The fresh semi-dried rhizome had undergone hydrosteam distillation. The
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fixed oil and essential oil were subjected to organoleptic test, FTIR analysis, and
physicochemical characterization (specific gravity, refractive index, and saponification value).
After collecting the oils, formulation of herbal was conducted. Subsequently, the
researchers have conducted laboratory evaluation on the repellent activity of the formulated
herbal. The dose of formulated herbal was assessed at predetermined concentrations2%
Ginger oil:5% Neem oil and 5% Ginger oil:2% Neem oil. Repellency testing was conducted
wherein five treatmentsformulated herbal (2G:5N), formulated herbal (2N:5G), commercial
herbal (HomeLife Citronella Twist Spray Lavander), commercial synthetic (repellent lotion
with 7.5% DEET), and negative control (formulated herbal without the oils)were used against
Ae. aegypti through arm-in-cage set-up. Laboratory evaluation (arm-in-cage set-up) was mainly
guided by modified EPA guidelines (OPPTS 810.3700). Adjuncts and modifications to the
guidline were extracted from WHO guidelines (WHO/HTM/NTD/WHOPES/2009.4), Tawatsin
et al. (2006), and Patent WO 2014137811 A1. The laboratory evaluation was conducted at the
insectary of Standards and Testing Division (STD), Industrial Technology Development
Institute (ITDI), DOST Compound, Gen. Santos Ave., Bicutan, Taguig City. Mosquitoes were
reared and maintained at the insectary of the STD-ITDI. Biotic factors considered: A) Mosquito:
5-7 days old, nulliparous female Aedes aegypti B) Volunteers 18-55 years old (2 males, 2
females), body temperature 97.5 (36.4) to 98.8 F (37.1C). Abiotic factors considered:
Evaluation area A) 27 2 degree Celsius air temperature, B) 70-80% relative humidity, C) Time
of evaluation 9:00AM-4:00PM, D) Arm-in-cage (303030 cm) cage with same mosquito
density (80). Comparative parameters were of percent protection (%P) and complete protection
time (CPT). The data was collected and treated with Friedman's Two-way Analysis of Variance
(ANOVA) with subsequent Pairwise comparison to obtain significant results.
Delimitation. The study does not cover parameters like heterogeneity of the volunteers
(e.g., gender, age, geographic differentiation, blood typing), and of the environment (different
sizes of cage, different insect species, mosquito densities, field evaluation) in evaluating the
efficacy of the herbal pump spray. Acute toxicity of the formulated herbal on the mosquito and
other animals was not covered; only empirical evaluation of toxicity (observed irritation upon
application) on volunteers has been performed. Stability of the product was not also covered.
The study was started on July 2014 and has ended on March 2015.
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Figure 1. Paradigm of the Study. It shows the entire framework of procedures and methods within
which the study takes place.
Figure 2. Simulacrum of the Study. It shows the independent variables (left) and
dependent variable (right) involved in the research.
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2.0
Review of Related Literature and Study
2.1
Local literature
2.1.1
Ginger
Ginger (Zingiber officinale) is described as an erect, smooth plant rising from

thickened, very aromatic rootstocks. The leafy stems are 0.4 to 1 meter high. The leaves
are distichous, lanceolate to linear-lanceolate, 15 to 25 centimeters long, and 2
centimeters wide or less. The scape rising from the rootstocks is erect, 15 to 25
centimeters high, and covered with distant, imbricate bracts. The spike is ovoid to
ellipsoid, and about 5 centimeters long. The bracts are ovate, cuspidate, about 2.5
centimeters long and pale green. The calyx is 1 centimeter long or somewhat less. The
corolla is greenish-yellow, and its tube is less than 2 centimeters long, while the lip is
oblong-obovate and slightly purplish (Bureau of Plant Industry, 2011)
Ginger is one of the earliest important species grown in the Western hemisphere
reported to be a native of Southeast Asia. Ginger (Zingiber officinale Rosc.) which is
popularly known as luya, luy-a, and kabasi in the Philippines is grown as an important
spice crop. It is used as a raw material in the production beverages, perfumes and
medicines. Due to its penetrating flavor, it is largely used for cooking and the
preparation of preserves, candy, and pickles (Department of Agriculture Regional Field
Office X, 2014).
Ginger's primary scientific is Zingiber officinale Rosc. Some of its alternative

are Amomum zingiber Linn. and Zingiber blancoi Hassk. Its local names in the
Philippines are the following, agat (Pang.); basing (Ilk.); gengibre (Sp.); laial (Sbl.);
laiya (If.); laya (Ilk., Bon., Ibn., It.); luya (Tag.). The rhizomes of Ginger are used as a
condiment, being one of the most popular flavoring agents known. Ginger ale and
Ginger beer, also made from the rhizomes, are refreshing drinks, Tahu, or salabat, a
native popular beverage, is also prepared from the rhizomes. The pungency is due to the
pungent principle, mainly zingerone and shogaol it contains, while the aroma is given
by the volatile oil. They enter into confectionery, Ginger beers, Ginger champagnes,
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and other beverages. In the East and Malaya fresh Ginger plays an important part in
curcy (Bureau of Plant Industry, 2011).
The Ginger family is noted for its volatile oils, which are concentrated mainly
in their rhizomes or underground roots. Besides the familiar luya or Ginger, other plants
from this family used medicinally include dilaw (Cucurma domestica Valet, Cucurma
longa L.), luya-luyahan (Cucurma zedoaria (Berg.) Rosc.), gisol (Kaempferia galanga
L.), kamia (Hedychium coronarium Koenig), and langkawas (Alpinia galanga L.). The
rhizomes of luya contain 1-3% volatile oil, mainly gingerone, phellanderene, camphene,
cineol, borneol, and citral. Also present is gingerol, a non-volatile oil responsible for
Ginger's distinctive odor. Gingerol is found in the resin (Tan, 1980).
Dry Ginger contains 1 to 3 percent volatile oil and 50 percent starch; its other
constituents are fiber, protein, resin, fixed oils, etc. Two well-known by-products are
Ginger essence and oil. The characteristic aroma of Ginger is due to the volatile oil
content of about 3 percent. Its probable chief components are the sesquiterpene
zingiberene, the terpenes of D-camphene and phellandrene, and the alcoholic zingiberol,
although several other components have been reported present in small amounts. The
pungency of Ginger is due to an ether soluble non-volatile substance known as gingerol,
a mixture of phenolic compounds containing the ketone zingerone. The Ginger is found
low in amino acid but rich in potassium. It is widely used as essential flavoring in the
preparation of European and Japanese dishes (Department of Agriculture Regional Field
Office X, 2014).
2.1.2
Neem
The Neem or "margosa" (Azadirachta indica meaning "Free Tree of India") is

described as an evergreen tree native to South Asia and Southeast Asia belonging to the
Meliaceae Family. It is known in India as "Nature's Drug Store" and "Village Pharmacy"
for its many health benefits. The leaves, bark and seeds of the Neem have been sources
of remedies for thousands of years in the Ayurvedic traditional medicine of India.
Laboratory studies have found more than 100 bioactive compounds in the Neem that
have beneficial applications in human health and agriculture. Scientific research has
confirmed its traditional use for medical use. There are more than 500 reports on the
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10
Neem as "one of the most widely used medicinal herbs in the world." Drinking the
decoction of Neem leaves can relieve arthritis and rheumatism because of its antiinflammatory effect. It also stimulates the immune system, improves liver function, and
cleanses the blood. The 10% water extract of the young leaves was found by researchers
to have anti-viral properties believed to be attributed to the bioactive compounds nimbin
and nimbinine. The same decoction is used externally as head wash for hair loss, lice
infestation and dandruff. Eating 8-10 fresh young leaves of the Neem every morning
with an empty stomach for 24 days is recommended for people (except pregnant women
and children) with hyperacidity, constipation, hypertension and diabetes. Although no
negative side effects are reported for the consumption of fresh and dried Neem leaves,
they should be taken in moderation and with the doctor's advice. Neem leaf juice is used
for the treatment of biliousness (bad digestion) and snake bites. By boiling 40-50 Neem
leaves for 20 minutes in 0.25 liter of water, an astringent and antiseptic can be prepared
for mouth and body wash. The bitter-tasting bark of Neem with bioactive compounds
has anti-inflammatory, anthelminthic, anti-emetic, antacid, antipyretic and analgesic
properties. The oil from Neem seeds (Figure 9) has nimbidin with anti-bacterial action.
Neem oil also has sodium nimbinate which is a spermicidal agent used extensively in
India for family planning. For piles or hemorrhoids, four powdered Neem seeds mixed
in warm water and drank with an empty stomach every morning for a week is said to
stop the bleeding. In agriculture, the leaf extract of Neem with azadirachtin, an
insecticide, is used for controlling biting and chewing insect pests. Dried Neem leaves
are also utilized for the protection of stored food grains against insect infestation.
Soaking fresh fruits and vegetables for a few minutes in a water solution of Neem leaf
extract extends their shelf life. The application of crushed Neem seed and oil in the
breeding areas of mosquitoes inhibits their egg-laying for a week. The Neem is
commonly propagated by means of its seeds which can be directly planted in the ground
or initially grown in containers as seedlings for transplanting. It can grow in clayey,
sandy and rocky soils with full sunlight and good drainage. With its deep tap roots, the
tree can extract calcium from the ground and help neutralize acidic soils through the leaf
litter. The Neem tree grows fast and can attain a height of 15-20 meters and lifespan of
150- 200 years. It starts bearing fruits after 3-5 years from planting and becomes fullbearing at 10 years of age with 30-50 kilos of fruits per year (Guerrero, 2012).
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11
In the Philippines, Azadirachta indica can be accessed in the UPLB Herbal

Garden, Institute of Biological Sciences, University of the Philippines, Los Banos
(Department of Agriculture of the Philippines, 1995). It is known for its insecticidal
properties (pang-lamok) than for its medicinal applications. In India, it is considered the
most useful traditional medicinal plant, and commercially beneficial as each part of the
tree has some medicinal property. The seed yields a bitter fixed oil known as "Oil of
Margosa" or neem oil. Seeds yield a fixed oil of glycerides and bitter compounds
including nimbin, nimbidin and nimbidol. Bark and leaves contain tannin and oil. Seed,
leaves, bark and root yield varying amounts of alkaloid (L>B>S>R), flavonoid (LBSR),
saponin (LSBR), phenols (BRSL), Mg (SBLR), phytate (SLBR) and tannin (LBSR).
Various extracts of seeds yielded alkaloid, amino acid, carbohydrate, glycoside, inulin,
mucilage, tannin, steroid, triterpenoid, flavonoid. In the rural areas, burning of leaves
and seeds used as mosquito repellent. Fresh seed oil has a strong garlic odor and used
as ingredient for insect sprays (StuartXchange, 2014).
2.1.3
Aedes aegypti
Aedes aegypti is the main vector of dengue or dengue fever in the Philippines.
It is characterized by being a small black mosquito with white stripes on its back and
legs. When an Aedes mosquito bites and feeds on the blood of a person with dengue, it
acquires the dengue virus. The virus proliferates within the mosquito and after eight to
eleven days, the mosquito becomes infective to humans and remains so for the rest of
its life, which can be anywhere from 15-65 days. When an infective mosquito bites a
human, it inadvertently injects the dengue virus into the person. Incidentally, only the
female Aedes mosquito bites and it does so because animal blood is needed for proper
development of its eggs. Also, the female dengue mosquito loves to bite during the day
and has a flight range of up to 300 meters (Gonzales, 2013).
Dengue (pronounced as DENGgae) is a term derived from the phrase "ki denga
pepo", meaning "cramp-like seizure caused by evil spirit". The term was an attempt to
describe victims of the then still unknown disease during outbreaks in Swahili, East
Africa, and in the Caribbean in 1880s. Dengue fever or its potentially fatal form known
as dengue hemorrhagic fever (DHF), is a febrile viral disease that affects countries in
tropical and sub-tropical regions where warm temperature and high relative humidity
COLLEGE OF PHARMACY
12
favor the breeding and proliferation of Aedes aegyti mosquitoes, also known as tiger
mosquitoes. Dengue fever is caused by four serologically related virus types i.e., dengue
1, dengue 2, dengue 3, and dengue 4 under the Family Flaviviridae. The disease is
transmitted by day-biting female Aedes aegypti mosquitoes that have previously bitten
persons and have the virus in their blood stream. These persons may or may not show
any sign of illness but are unknowingly ready sources of infection. Outbreaks
resembling the signs and symptoms of dengue disease have been reported throughout
medical history. Benjamin Rush coined the term breakbone fever during its first
reported case in 1789 due to the victims manifested physical symptoms such as myalgia
(muscle pains due to overstretching) and arthralgia (joint pains). The spread of dengue
is attributed to the expanding geographic distribution of the four flaviviruses by Aedes
aegypti and Aedes albopictus mosquitoes, the WHO explained. In urban areas like in
Metro Manila and some districts of Quezon City, Aedes aegypti is the most predominant
species of mosquito vector. Filipino scientist Nelia P. Salazar, currently a consultant of
DOH's Research Institute for Tropical Medicine said that Aedes aegypti breeds in
different ubiquitous water-holding containers such as unused or junk tires, drums, jars,
bottles, tree holes, roof gutter, and flower vases among others. She added that unclean
urban areas are generally the favorite habitat of these virus carriers although these can
also be found in better residential districts, schools, and other public places. More so,
crowding contributes to increased man-vector contact since the mosquitoes prefer to
stay in domestic and peridomestic habitats. Humans are the main amplifying hosts of
the viruses. Once the virus enters the victim's body, the virus settles and replicates in
various target organs like lymph nodes (responsible for cleansing human body tissues
and associated with the reproduction of white blood cells that fight foreign bodies like
bacteria and viruses) or liver (an important organ of digestive system). Upon release
from these organs, the virus spreads through the blood infecting the white blood cells
and causing the release of substances that trigger a chain of physiological reactions
affecting the capillaries (the smallest blood vessels). The capillary walls become prone
to bleeding or hemorrhage in various tissues and organs. Blood platelets and coagulation
factors are mobilized by the body to contain the bleeding, hence, the depletion of
platelets as one of DHF's clinical manifestations. Dengue victim then suffers fever
reaching 106 degrees Fahrenheit or 41.11 degrees Celsius with severe headache, joint
and muscular pains, and rashes (red spots) lasting a few days. WHO statistics show that
during dengue epidemics, attack rates among those at risk (mostly children 10 years
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13
below are observed to be the most susceptible age bracket) are often 4050% but may
reach 8090%. An estimated 2.5% of the cases will be fatal. Without proper and
immediate medical supportive therapy, the rate could reach as high as 20%. The
repression and mitigation of this viral infection is yet to be accomplished. It will
continue to be a threat to human lives (Department of Science and Technology, 2008).
According to the article written by Eduardo Gonzales (30 July 2013), dengue
can be prevented by controlling its mosquito vector or protecting people from mosquito
bites. Measures to control the mosquito are most effective if they are done on a
community basis. Screen your house. Alternately you can use mosquito nets, mosquito
repellents, mosquito coils ("katol") and mats, and mosquito patches that one sticks on
outer clothing. Isolate persons with dengue fever in a screened room for at least five
days from the onset of symptoms. Eliminate all possible breeding places of mosquitoes
in your neighborhood. Fill potholes; cover water containers and septic tanks; do not
allow empty cans, soft drink bottles, spare tires, etc. to accumulate water; ensure that
drains and gutters are not clogged and that water flows freely in sewage lines; cut tall
grass, etc. Dispose garbage properly and regularly.
2.2
Foreign literature
2.2.1
Ginger
Zingiber officinale Roscoe (Zingiberaceae) (Figure 10). Common name: Luya (Tag.),
Ginger (Engl.). Ginger is one of the most important and most widely used spices worldwide.
Due to its universal appeal, Ginger has spread to most tropical and subtropical countries from
the ChinaIndia region, where Ginger cultivation was prevalent probably from the days of
unrecorded history (Ravindran & Nirmal Babu, 2005). By hydrodistillation of the fresh rhizomes
of Ginger from the Philippines, Anzaldo et al. (1986) obtained 0.2 to 1.0 percent oil yield. By
using TLC, GC, and IR spectroscopic data, 10 components were identified, with citral being the
major component. Geraniol and linalool were also present. Physicochemical constants of the oil
were also reported (Ravindran & Nirmal Babu, 2005).
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14
Ginger has 1-3 percent volatile oilscomplex (hydrocarbons, sesquiterpenes, and

numerous monoterpene hydrocarbons, alcohols, and aldehydes, e.g., phellandrene, camphene,
geraniol, neral, linalool, D-nerol) (Barnes, 2007).
From Maria Lis-Balchin (2006), the major components of ginger oil was tabulated
(Figure 3). Minor components were also mentioned like sesquiterpenes including zingiberol,
zingiberenol, ar-cucurmene, -sesquiphellandrol (cis and trans). It was stated, in-text cited by
Lawrence (1976-2001), that there is wide variation in the composition of ginger oils from
different origins.
%
-Pinene
Camphene
8.3
-Phellandrene
9.6
Linalool
0.8
Borneol
0.8
Neral
1.4
Geranyl acetate
0.9
-Zingiberene
29
-Bisabolene + -farnesene
14
-Sesquiphellandrene
9.9
Figure 3. Major components of ginger oil
Toxicity of ginger oilA) Clinical data (none documented; it lacks clinical safety and
toxicity data; stated to be non-irritating and non-sensitizing (none at 4%-human) although
dermatitis may be precipitated in hypersensitive individuals; phototoxicity is not that
significant), B) Preclinical data (it is stated that it is of low toxicity with acute LD50 values (rat,
by mouth; rabbit, dermal) reported to exceed 5 g/kg (Barnes, 2007; Lis-Balchin, 2006).
Purity of Zingiber officinaleIt contains not less than 42 percent of starch, 8 percent of
crude fiber, not more than 1 percent of lime (CaO), not less than 12 percent of cold water extract,
nor more than 7 percent of total ash, not more than 2 percent of ash insoluble in hydrochloric
acid, nor less than 2 percent of ash soluble in cold water (Kraemer, 1920).
COLLEGE OF PHARMACY

2.2.2
15
Neem
The Neem (Azadirachta indica) is described as a common tree in towns and villages in
India. Its distinctive leaves and sprays of small, white, sweet smelling flowers are a familiar
sight in avenues and gardens. It is sometimes called 'Nature's Pharmacy', because of its many
uses as a mild antibiotic, pesticide, and insect repellent. At least 35 active chemical principles
have been found in its leaves, bark and seeds. The use of Neem as a pesticide and the practice
of cleaning one's teeth with Neem twigs have already been mentioned in Footsteps, and there
are many other uses for this tree. For example, fresh green leaves mixed with grain in closed
containers will keep the grain free from pests for two to three months. Farmers in Pakistan know
this, and regularly plaster the inner surfaces of large storage bins for wheat with a mixture of
mud and Neem leaves. Neem leaves dried in books and kept at the bottom of drawers and in
woollen clothing, keep away silverfish and moths (Reuben, 2013).
In India, Neem is known for its use and is more utilized in rice cultivation. Neem is also
called 'arista' in Sanskrit- a word that means perfect, complete and imperishable'. The Sanskrit
name 'nimba' comes from the term 'nimbatisyasthyamdadati' which means 'to give good health'.
The seeds, bark and leaves contain compounds with proven antiseptic, antiviral, antipyretic, antiinflammatory, anti-ulcer, and antifungal uses. Neem is recognized today as a natural product
which has much to offer in solving global agricultural, environmental, and public health
problems. Researchers worldwide are now focusing on the importance of Neem in the
agricultural industry (Lokanadhan et al., 2012).
The Neem (A. indica) was described by Iwu (2014) as a shady tree with an evergreen
crown; it grows up to 25 m high in some places but occurs in West Africa mostly as a mediumsize tree. It has rough, dark brown bark with wide, shallow longitudinal fissures separated by
flat ridges. The bole is short and stout. It is easily confused with Melia azedarach, an Asian tree,
which has also been introduced to other tropical parts of the world; references to A. indica in
very old literature should be viewed with caution. The leaves are compound, imparipinnate, each
comprising 5-15 leaflets; they are very diagnostic and measure about 6 m long and 2 cm broad.
The tree bears many flowered panicles, mostly in the leaf axils; sepals are ovae-, sub-, or
bicullar, about 1 cm long, with sweet-scented white oblanceolate petals. It produces yellow
drupes, which are ellipsoid, glabrous, and 12-20 cm long. Neem-based consumer products used
COLLEGE OF PHARMACY
16
in health care and for cosmetic purposes appear to be well tolerated. Histopathological exams
showed that toxicity was observed only in very high doses and after chronic use.
Neem is a tree indigenous to the East Indies and rather widely distributed in the tropical
countries of Asia and to some extent cultivated (Kraemer, 1920). The Latin name is derived
from the Persian "azad darkht i hindi," which means "free tree of India" (National Research
Council, 1992; Willcox et al, 2004). It is used in India and the eastern colonies of Great Britain,
as a simple bitter, replacing gentian and quassia (Kraemer, 1920).
The Meliaceae family is characterized by the presence of limonoid triterpenes, many of

which are biologically active against insects. From the Asian species Azadirachta indica and
Melia azedarach, two limonoids have been commercialized: azadirachtin in the US and
toosendanin in China. They were outstandingly effective against insects (Hammad, 2008; Isman
et al., 1997 and references cited therein, inter alia; Mata et al., 2001). Neem oil is extracted from
seed kernels, leaves, bark, flowers, and wood. Neem oil is broad-spectrum insect poison,
repellent, and feeding deterrent (Bradley et al., 2009). Neem has been used since antiquity as an
insect repellent for both people and food crops. Neem oil vs DEET. Neem oil is an excellent
skin moisturizer while DEET is not recommended for repeated application to the skin, around
the face or on the hands of small children. Neem oil is a natural vegetable oil while DEET is not
recommended to be sprayed on furniture, plastics, watch crystals, leather and painted surfaces
including automobiles. DEET may actually dissolve all synthetic fabrics but nylon. Neem oil
has been used safely for centuries while DEET is a synthetic chemical that has only been used
for a short time and may pose future unknown health risks. Many researchers believe DEET to
be partly responsible for the devastating effects of Gulf War Syndrome. Neem is a healing herb
that is famous for its wound healing properties. Cuts, scrapes and poison oak and ivy can be
salved with Neem oil lotions. DEET products contain warnings against getting them in open
sores or on damaged skin (Conrick, 2009). As cited by Cook (2013), a study by the US National
Research Council neem oil is more effective than DEET. The results were confirmed by
scientists at the Malaria Institute in India and in research cited in the Journal of the American
Mosquito Control Association. Neem is a plant that grows in India.
Azadirachtin (Figure 4) is a tetranortriterpenoid that is utilized as a highly active feeding

deterrent and growth regulator; used experimentally as insect control agent (Kar, 2007). Neem
is effective against the mosquito in two ways, as a larvicide, and as a repellent. Neem extract is
COLLEGE OF PHARMACY
17
an important ingredient of some herbal shampoo, and Neem oil is used in hair oils, body lotions,
creams and mosquito repellent preparations (Puri, 1999). Neem is also utilized in pet care
formulas as an external herbal flea and insect repellent (D'Arcy, 2004).
Neem protects itself from the multitude of pests with a multitude of compounds. These
compounds belong to a general class of natural products called "triterpenes"; more specifically,
"limonoids." New limonoids are still being discovered in neem, but azadirachtin, salannin,
meliantriol, and nimbin are the best known and, for now at least, seem to be the most significant.
Salannin (Figure 5) found in Neem leaves, seeds and seed oil is a safer and more effective insect
repellent than the widely used chemical ingredient called DEET (N,N-diethyl-m-toluamide)
currently in most commercial repellents (National Research Council, 1992; Prakash & Rao,
1997).
Figure 4. Azadirachtin's chemical structure

2.2.3
Figure 5. Salannin's chemical structure
Essential oilGinger oil
Since the olden times, essential oils are known to mankind for their medicinal value.
This purports them as an innovative and dominant natural plant products. Essential oils have
long been the popular source of perfume and fragrance essences. They have been used
commercially as flavors in foods and beverages. They continue to be of great use and interest to
man until the present day. Significantly, essential oils were used in folk medicine as healer of
both body and mind since time immemorial (Djilani & Dicko, 2012).
Essential oils, also called volatile or ethereal oils, refer to a large class of natural
aromatic substances found in various flowers, leaves, seeds, roots, bark, wood, resin, and the
rinds of some fruits. These substances resemble oils in appearance but they are generally light,
non-greasy, and highly volatilemeaning they evaporate readily. Essential oils, therefore, are
chemically distinct from, and should not be confused with, fatty oils. Essential oils are usually
clear (rarely colored) liquid characterized by a strong odor. They are highly concentrated
COLLEGE OF PHARMACY
18
substances isolated from aromatic plants by several extraction methods; the most commonly
utilized are steam distillation and hydro-distillation. The levels of essential oils found in plants
range from 0.01 to 15 wt % of the total (Cavalcanti et al, 2013; Glaser, 1995). The total essential
oil content of plants is generally very low and rarely exceeds 1%, but in some cases, for example
clove (Syzygium aromaticum) and nutmeg (Myristica fragrans), it reaches more than 10%. Many
oils contain over 50 individual compoundsthese can generally be identified using gas
chromatography and mass spectrometry (GC/MS) (Djilani & Dicko, 2012; Pengelly, 2004).
Essential oils are typically named after the plants from which they are derived-for
example, peppermint oil and orange oil-and are called "essential" because they tend to represent
the natural "essence" of the plant based on various characteristics such as odor and taste.
Essential oils and their derivatives are widely used as flavors and fragrances, and some are used
for their chemical or biological activity (Glaser, 1995).
Essential oils are secondary metabolites that act as protection of plants that have
properties of antibacterial, antiviral, antifungal, and insecticide properties. Due to these
properties, essential oils have been largely employed in pharmaceutical and cosmetic industries.
In recent years, importance of essential oils as biocides and insect repellents has also increased
(Cavalcanti et al, 2013).
Essential oils are known major oil constituents imparting characteristic odors of plants
thus often acts as olfactory attractants or repellents to herbivorous or pollinating insects; they
are responsible for the repellency property in a commercial insect repellent products (Hattendorf,
2007). Geraniol (Figure 6) and citral (Figure 7), which are both present in the rhizomes of
Ginger, were the two essential oils that showed 2-3 hours of repellency against Aedes mosquitoes
(Moore, 2006; see Figure 8).
Geraniol (or Lemonol; IUPAC: 3,7-Dimethyl-2,6-octadien-1-ol) is an olephenic terpene

alcohol constituting the major portion of oil of rose and oil of palmarosea. It is also found in
many volatile oils, for instance: citronella, lemon grass etc. Citral is one of the two important
members of the alipathic terpene aldehydes. Citral's IUPAC name is 3,7-Dimetyl-2,6-octadienal;
(C10H16O). Citral from natural sources is a mixture of two geometric isomers Geranial and Neral
(Kar, 2007).
COLLEGE OF PHARMACY
Figure 6. Geraniol's chemical structure

Compound
Duration of Protection (h)
Terpenene
Citronellal
<1
Limonene
-Pinene
Citronellol
12
Eugenol
12
Linalool
12
-Terpeneol
12
Geraniol
23
Citral
23
19
Figure 7. Citral's chemical structure
Source: From USDA, Agricultural Research Service United States Department of Agriculture Handbook,
Washington, DC, 1967
Figure 8. The Repellency of Essential Oils (100% Concentration) to Aedes Mosquitoes
2.2.4
Fixed oilNeem oil
Various oils present in natural extracts have been classified as fixed oils or high boiling
oils, and essential or volatile oils. Very popular fixed oils are Neem oils (nonedible), coconut,
gound nut, soya, sunflower, mustard, etc. oils (edible) (Mukhopadhyay, 2005).
Neem is traditionally used as an insect repellent. In India, a field test with Anopheles
culicifacies using topical neem oil at 2% strength in a base of coconut oil provides 100%
protection against biting for 12-hour period. Also, in India, it is believed that the fumes of dried
Neem leaves help to prevent malarial fevers; termed as fumigants, mechanism of repellency is
that when the fresh leaves are burned, oils volatilized (Moore et al, 2006; Willcox et al, 2004).
COLLEGE OF PHARMACY

2.2.5
20
Extraction of essential oil
The extraction of essential oils from plant material can be achieved by various methods,
of which hydrodistillation, steam and steam/water distillation are the most common method of
extraction. Other methods include solvent extraction, aqueous infusion, cold or hot pressing,
effleurage, supercritical fluid extraction and phytonic process. This later process has been newly
developed; it uses refrigerant hydrofluorocarbons solvents at low temperatures (below room
temperature), resulting in good quality of the extracted oils (Bowles, 2003; Da Porto et al., 2009;
Djilani & Dicko, 2012; Hunter, 2009; Lahlou, 2004; Margaris et al., 1982; Martnez, 2008;
Pourmortazavi & Hajimirsadeghi, 2007; Surburg & Panten, 2006).
Volatile oils are usually obtained by distillation of the plant parts containing the oil, the
method depending on the condition of the plant material. Three types of distillation are used by
industrial firms: water or hydrodistillation (e.g., Clevenger hydrodistillation method), water and
steam distillation, and direct steam distillation. Hydrodistillation is applied to plant material that
is dried and not subjected to injury by boiling such as turpentine oil. Water and steam distillation
is employed for substances either dried (e.g. cinnamon and clove) or fresh (peppermint and
spearmint) that may be injured by boiling. Steam distillation is conducted for materials with
certain components of a volatile oil tend to hydrolyze where as other constituents are
decomposed by the high temperatures (Joubert, 2004; Hung, 2008; Varro et al., 1970).
Steam distillation employs either water, wet steam, or dry steam. The oil and water
vapour are passed into a condenser. The oil is separated automatically from the water phase.
Water distillation is a mild and slow process but it yields a superior product. In what is called
wet stem distillation, the plant is placed on a grid in the still. There may be water below the grid
or it may accumulate during the process. Steam is introduced from an outside source into the
still. Initially sufficient water condenses in the cool charge to wet it slightly. External heat may
be applied to limit the build-up of water and wetting of the charge. Steam distillation is a
reasonably rapid method and can be used for most oils with the exception of those containing
high concentrations of low volatility components. In dry steam distillation, the plant is placed
on a grid in the still. Direct steam is applied and outside heat supplied by a steam jacket is
maintained at a temperature sufficient to prevent any water condensation. Care must be
exercised to prevent charring (creation of hot spot in the jacket) and channeling (result of hole
in the charge which prevent contact of stem with the entire charge) (Khomasurya, 1999).
COLLEGE OF PHARMACY
21
Hydrosteam distillation was utilized on the study. Other types of distillation were
disregarded because of time constraint and solvent-consuming methods that yields poor
penetration of the tissues by the solvent and also possible destruction of thermolabile
compounds. Advantages of conventional extraction methods result from basic, inexpensive and
simple equipment to operate (de Castro & da Silva, 1997; de Castro & Garcia-Ayuso, 1998;
Szewczyk & Bogucka-Kocka, 2012).
Chemicals can protect humans from mosquito feeding in three different ways, namely
irritation, repelling, or killing (Grieco et al. 2007). These are often carried out to determine the
toxicity of a plant part. Usually animal models such as mice, guinea pigs or rabbits are often
employed (Doughari, 2012).
2.2.6
Aedes aegypti
The transmission of the dengue virus between humans is primarily the work of the
"yellow fever mosquito," Aedes aegypti. A small, dark mosquito with white markings, Aedes
aegypti thrives in all but the coldest climates worldwide, including much of the southern United
States. Typically, 8-12 days after the female mosquito feeds on an infected human, it can pass
the virus to another human. Aedes aegypti, which commonly bites during daylight, is uniquely
adapted to living in and around human habitations, where it lays eggs in artificial containers,
like pet water bowls, vases, and discarded plastic trash. Although commonly thought of as a
disease of the tropics, dengue is no stranger to the United States. The first detailed description
of the disease was written by Dr. Benjamin Rush, signer of the Declaration of Independence,
who studied cases during a 1780 epidemic in Philadelphia. Dengue disappeared from the
continental U.S. by the early 20th century and it remained relatively rare everywhere until after
World War II. Since 1952, however, when the first cases of dengue hemorrhagic fever were
described in the Philippines, dengue has grown to pandemic proportions and again constitutes a
threat to the U.S. There is not yet a dengue vaccine and controlling the mosquito "vector" is the
only method for preventing transmission (Centers for Disease Control and Prevention, 2010).
COLLEGE OF PHARMACY

2.3
22
Local study
2.3.1
Ginger
In the stability studies of essential oils from some Philippine plants Zingiber officinale
was spearheaded by Brandares et al. (1987) Extraction of ginger oil by hydrosteam distillation
gave the highest yield (0.5-1.0%). Water distillation method was discontinued because of heavy
frothing and stream distillation, too, because of the low yield and for economic reasons since it
necessitates use of a more complicated set-up.
Ginger oil is a yellow to orange liquid possessing the characteristic aromatic odor, but
not the pungent flavor or "bite" of the spice. The piquant nature is due to the oleoresin which
has been removed by distillation. The oil is lighter than water with a specific gravity of 0.8803
and a refractive index of 1.4772 at 25 degrees Celsius. It gives average ester and acid numbers
of 14.96 and 1.57, respectively. The oil is soluble in 70-95% ethyl alcohol, the degree of
solubility increasing with the concentration of the solvent (Brandares et al., 1987).
Anzaldo et al. (1986) identified the chemical components of local ginger oil. The results
confirmed the presence of the following in the ginger oil sample: limonene, -pinene, -pinene,
phellandrene, cineole, linalool, geraniol, terpineol, caryophyllene, and citral.
2.3.2
Neem
In the Philippines, a study aimed to produce mosquito spray using Neem leaves extract.
It aimed to lessen the lives of growing mosquitoes in our environment and to utilize Neem into
useful products. The middle parts of the leaves or the greenness leaves were used. This will be
soaked in 90% ethanol with 2:4 ratios, leaves: ethanol. The mixture will stand overnight. And
then after getting the Neem extract, this will be combined to different volume (mL) of water.
The researchers recorded the results and got the ratio of compound which is most effective. And
the researchers concluded that there are no side effects on the skin of the pigs. The result of the
treatment showed that there is no significant difference among treatments meaning that all the
concentrations have the same effect on the number of mosquito that bite the piglets. The least
number of mosquitoes that bite the piglets was observed during the first 1.5 hours and the most
COLLEGE OF PHARMACY
23
was observed at 3 hours after application of the treatments. This implies that the effectivity of
the extract is reduced as time passes (Galang & Gervacio, n.d.).
A study was also conducted by Parugrug and Roxas (2009) regarding evaluation of the
insecticidal action of five locally available plants namely: Azadirachta indica (Neem),
Cymbopogon citratus (Lemon Grass), Lantana camara (Lantana), Ocimum basilicum (Basil)
and Tagetes erecta (African marigold) against maize weevil, Sitophilus zeamais Motsch
following the repellency, adult mortality and antioviposition and growth inhibition tests. Results
revealed that all test materials exhibited repellency action against maize weevil. Within 96 hours
of exposure, powdered leaves of Neem and lantana were noted to be highly repellant while
powdered leaves of lemon grass, basil, and African marigold were observed to be moderately
repellant against maize weevil.
There was also an invention of an insect repellent made from the neem tree by a Filipina.
She is Ms. Ma. Carlita Rex-Doran, a prolific scientist who was entitled The Herbal Doctor in
the book of Malang, V. L., Inventions and Innovations A Glimpse of Filipino Legacy (1998).
She first popularized the gugo (an indigenous vine) shampoo, tawas (alum) roll-on deodorant,
sarsaparilla anti-aging cream, cucumber facial toners, and neem insect repellent. She is also a
consistent DOST (Department of Science and Technology) awardee and in DOST roster of
Filipino Inventors. She have won a silver medal for product named Bioneem during the 25th
Salon International Exhibition of Inventions in Geneva, Switzerland in 1997 ("Bioneem", 2008).
2.4
Foreign study
2.4.1
Ginger
An investigation of the behavioral responses of Aedes aegypti(= Stegomyia aegypti) to

various concentrations of essential oils (2.5, 5, and 10%) extracted from hairy basil (Ocimum
americanum Linn), Ginger (Zingiber officinale Roscoe), lemongrass (Cymbopogon citratus
Stapf), citronella grass (Cymbopogon nardus Rendle), and plai (Zingiber cassumunar Roxb)
were conducted using an excitorepellency test chamber. The study concluded that the essential
oils from native plants tested, and likely many other extracts found in plants, have inherent
repellent and irritant qualities that should to be screened and optimized for their behaviormodifying properties against Aedes aegypti and other biting arthropods of public health and pest
COLLEGE OF PHARMACY
24
importance (Boonyuan et al., 2014). There was also a study that aims to investigate plants used
traditionally against repelling hematophagous insects in Laos. Lemongrass (Cymbopogon
citratus) and Ginger (Zingiber officinale) were used in the research to pilot a topical repellent
that was tested in vivo on Aedes aegypti under controlled conditions. The formulations elicited
about 60 minutes of full protection but when combined, a potential additive effect was noted,
prolonging the efficacy by nearly 50% (Schubert, 2014).
From Campbell (2004), it was cited that many species within the Zingiberaceae family
are repellent to mosquitoes and other insects. Washings of macerated Zingiber officinale deter
the Asian armyworm Spodoptera litura from feeding on shoots of groundnut, Arachis hypogea
(Sahayaraj, 1998). The aromatic turmeric Curcuma aromatica repels Ae. togoi up to 3 h
(Pitasawat et al., 2003), and both Curcuma longa and Zingiber officinale repel Aedes aegypti for
up to 1 h (Tawatsin et al., 2001; Trongtokit et al., 2005).
2.4.2
Neem
Neem is known for its insecticidal properties and low toxicity to mammals. These have
particularly attracted scientists in chemistry, pharmacology and agriculture, and many bioactive
compounds have been identified in the plant such as nimbin (anti-inflammatory), nimbidin
(antibacterial, anti-ulcer), nimbidol (anti-tubercular, anti-protozoan), gedunin (anti-malaria,
anti-fungal), sodium nimbinate (diuretic, anti-arthritic) and salannin (repellent) (Brahmachari,
2004; Xuan et al., 2004; Subapriya & Nagini, 2005; Bhattahcharyya et el., 2007; Gahukar, 2012;
Kato-Noguchi, 2014).
Neem is traditionally used as an insect repellent. Extracts of several plantsNeem

(Azadirachta indica), basil (Ocimum basilicum), (Mentha piperata), and lemon eucalyptus
(Corymbia citriodora)have been studied as potential mosquito repellents and have
demonstrated good efficacy against some mosquito species (Sharma et al., 1993; Ansari et al.,
2000; Trigg and Hill, 1996). In Sri Lanka, 69 percent of households burn Neem seeds and leaves
in clay pots to repel mosquitoes in the evening (Konradsen et al., 1997; Willcox & Chamberlain,
2004). Neem has been shown that when applied topically at 2 percent strength in a base of
coconut oil, Neem oil provides 100 percent protection against biting by all Anopheles species
during a 12-hour period (Sharma et al., 1993). In India, it is believed that the fumes of dried
Neem leaves help to prevent malarial fevers (Nargas and Trivedi, 1999); this may be through
COLLEGE OF PHARMACY
25
their mosquito repellent action. In Kenya, Snow et al. (1992) found that 3 percent of households
burned Neem leaves to repel mosquitoes. In other studies as well Neem (Azadirachta indica)
and turmeric was effective (Ansari & Razdan, 1995). In India, for repelling insects or for socalled purification of air, premises are often fumigated with a mixture of Neem leaves, oleogum resin, particularly that of Commiphora wighti, and sulfur. Pandian et al. (1989) and Pandian
& Manoharan (1995) observed that with smoke from dry leaves of Neem, the landing and biting
rates of mosquitoes were reduced considerably. In a field trial in a village in west India, a mixture
of 0.5, 1 or 2 percent Neem oil in coconut gave 79.65, 96.07, and 98.03 percent protection
respectively against Anopheles culicifaciesin an all-night biting test. Two percent Neem oil
provided 75 percent protection against other types of mosquito (Kant and Bhatt, 1994). Similar
results were obtained by Mishra et al. (1995) with 14 percent Neem oil in coconut oil, when
applied to human volunteers in a tribal village. Dua et al. (1995) applied a Neem cream to see if
it can provide protection against mosquitoes. One application of the cream was effective in 68
percent of the population for four hours. Prakash et al. (2000) recorded 66.7 percent protection
after 9 hours using 2 percent Neem oil diluted in mustard oil. Again, numbers of mosquitoes
were low, and mosquitoes were caught using an unprotected collector and a bait wearing
repellent. In a test in the Bolivian Amazon with high densities of An. darlingi (mean = 71
mosquitoes/man-hour), 2 percent Neem oil in ethanol provided only 56.7 percent protection 3
and 4 hours after application (Moore et al., 2002).
COLLEGE OF PHARMACY
26
Figure 9. Azadirachta indica

Left: A. indica Leaves and Fruits from http://www.hibiscuscoastseconds.co.za/wpcontent/uploads/2013/11/Azadirachta_indica_leaves__fruits.jpg
Right: A. indica Seeds from
http://explorepharma.files.wordpress.com/2010/09/2607242300_974df4397c.jpg
COLLEGE OF PHARMACY
27
Figure 10. Ginger (Zingiber officinale)

Left: from http://allison-beriyani.deviantart.com/art/Ginger-Zingiber-Officinale-406475029
Right: http://www.nurseriesonline.us/articles/Growing-Ginger.html#.VBM8cvmSySo
COLLEGE OF PHARMACY

3.0
Research method
3.1
Research design
28
The research made use of the quantitative approach and experimental research design to evaluate
the efficacy of a herbal pump spray repellent which was determined using laboratory evaluation (armin-cage set-up) mainly guided by modified EPA guidelines (OPPTS 810.3700). Adjuncts and
modifications
to
the
guideline
were
extracted
from
WHO
guidelines
(WHO/HTM/NTD/WHOPES/2009.4), Tawatsin et al. (2006), and Patent WO 2014137811 A1. The

study comparatively evaluated the effectiveness of five test treatmentsformulated herbal, commercial
herbal (HomeLife Citronella Twist Spray Lavander), commercial synthetic (lotion with 7.5% DEET),
and negative control (formulated herbal without the oils)against Ae. aegypti.
According to Khalid et al. (2012), quantitative approach of research, through the use of complex
statistical model, uses different techniques of quantitative analysis from giving simple description of the
concerned variables to creating statistical relationships among variables. The design permitted the
researcher to identify cause and effect relationships between variables and to distinguish placebo effects
from treatment effects (Anastas, 1999). Also, an experimental research design supported the ability to
limit alternative explanations and to infer direct causal relationships in a research (Trochim, 2008). It
was mentioned by Khalid et al. (2012) that accepting and rejecting hypotheses will be based on the
experiments. Since the study uses experimentations to know if there are significant differences among
the treatments, this kind of research design is applicable.
Figure 11. Flow Chart of the Study
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3.2
29
Research locale
Prior to distillation, the plant material to be used on herbal formulation was authenticated. A
specimen of the Ginger was validated at the National Museum of the Philippines. The extraction of
Ginger was conducted in Chemicals and Energy Division (CED), Industrial Technology Development
Industry (ITDI), DOST Compound, Gen. Santos Ave., Bicutan, Taguig City. The Neem oil and Ginger
oil were stored at 15C (refrigerator temperature) in light-resistant tight amber containers to avoid
excessive loss or gain of moisture and to avoid degradation from light exposure. The Neem oil and
Ginger oil underwent the processes Fourier Transform Infrared Spectroscopy (FTIR) analysis and
physicochemical characterization simultaneously. The FTIR analyis was conducted at De La Salle
UniversityManila while physicochemical characterization was conducted at the laboratories of the
Graduate School Building of the Our Lady of Fatima University (OLFU)Valenzuela Campus. After
which, the study formulated a herbal pump spray repellents by combining the Neem oil and Ginger oil.
There were two formulated herbals in two predetermined concentrations.
The laboratory evaluation (arm-in-cage set-up) was conducted at the insectary of Standards and
Testing Division (STD), Industrial Technology Development Industry (ITDI), DOST Compound, Gen.
Santos Ave., Bicutan, Taguig City. Collection of related literature and studies were conducted at the
OLFU Library, National Library, and ITDI-DOST Library.
3.3
Population and sampling
3.3.1
Ginger oil and Neem oil
The plant sample used in the research was the rhizomes of Zingiber offinale (Ginger)
that were collected in La Trinidad, Benguet. Three (3) kilograms of rhizome Ginger was
collected for the study.
The Neem oil from seed was obtained from Swanson Health Products.
3.3.2
Aedes aegypti
For the laboratory evaluation (see Figure 12), adult (5-7d old) nulliparous, bred, female
Aedes aegypti used in all tests were obtained from a laboratory colony maintained at the
COLLEGE OF PHARMACY
30
insectary of the STD-ITDI. The mosquito larvae were reared at 27 2 degree Celsius, 70-80%
relative humidity, and photoperiod 14:10 hours (light:dark). Adult mosquitoes were maintained
in standard cages, provided 10% sucrose, and bloodfed periodically on mice. Before performing
the test, the mosquitoes were not blood fed and were starved for 12 hours immediately. Test
mosquitoes were established to be free of disease as they were reared from egg to adult at the
STD-ITDI insectary.
Figure 12. Preparation for Aedes aegypti
3.3.3
Volunteers
2 males and 2 females (nonpregnant, nonlactating)

Age: 18-55 years old
Body temperature: 97.5F (36.4C) to 98.8F (37.1C).
Volunteers are healthy (physically and psychologically fit) and literate

individuals. The volunteers do not have dependent relationship on the principal
investigator. Subsequently, they were validated by the principal investigator of the
institution where the laboratory evaluation was conducted to ensure eligibility (medical
history check-up, body temperature, pregnancy testing, skin irritation on treatments, and
abstinence of tobacco smoking 12 hours prior to trials). A treatment will be applied to
the test area which is your elbow to the wrist. The test area was washed with unscented
soap, sprayed with 70% ethanol until thoroughly damp and then dried with a clean paper
towel. This is done to remove interference on resulting data.
COLLEGE OF PHARMACY
31
One arm will be treated with a treatment and the other remained untreated,
serving as the control. Control arm was cleansed using the same method. During testing,
white latex gloves were worn to protect the hands from mosquito bites. Exposures will
end at the treatment's confirmed efficacy failure.
Volunteers were recruited by a principal investigator of the laboratory

evaluation. Subsequently, they were validated by the principal investigator to ensure
eligibility (medical history check-up, body temperature, pregnancy testing, skin
irritation on treatments, and abstinence of tobacco smoking 12 hours prior to trials).
Confidentiality of the results on the medical validation has been maintained. Prior to
trials, the volunteers have given the informed consent to the principal investigator.
Volunteers have not been coerced into giving consent, and the consent has been given
freely and voluntarily. Personal information gathered on a volunteer will be kept
confidential.
3.4
Research ethics
The study ensured that all issues concerning ethicality were addressed. The volunteers
for the laboratory evaluation were recruited by the institution where the laboratory evaluation
was conducted. Written consents were obtained from each test volunteer before trial. Ethical
clearance was approved by the OLFUIERC (Institutional Ethics Review Center).
3.4.1
Volunteers on laboratory evaluations
Each volunteer has been exposed to mosquito bites between 9:00AM and
4:00PM for every replicate of treatment. This includes preliminary checking of
aggressiveness (landing of 10 mosquitoes in 60s) of mosquitoes to biting that will
land on their exposed arms. The laboratory evaluation is done in 8 replicates on four
volunteers. Thus, each treatment was tested twice by each volunteer in different fresh
female mosquito batches. Only one replicate per treatment together with the control per
volunteer per day was done. In other words, the volunteer will be exposed once with the
first treatment. Same number of exposure goes with the other four (4) treatments. The
volunteer will receive five (5) different kinds of treatment with a total number of five
COLLEGE OF PHARMACY
32
(5) exposures in a day. The volunteer will repeat this process in another day, thus, the
volunteer will experience ten (10) exposures to conclude him/her from the laboratory
evaluation. All volunteers will undergo the same process within two (2) days.
3.4.2
Risk minimization
Test mosquitoes were established to be free of disease as they were reared from egg to
adult at the STD-ITDI insectary. The volunteer will be provided with vitamin C (500mg) as it
can aid in improving immunity and preventing dengue hemorrhagic fever. The volunteer will
have to take the medicine twice a day within the week prior to the test and the week after and
sign a form to confirm that he/she have taken the medicine; the medicine will be paid by the
researchers. If the volunteer were to become sick at the course of the laboratory evaluation, the
he/she will be given a medicine and immediately he/she will no longer be part of the laboratory
evaluation.
The volunteer can leave the laboratory evaluation at any time without explanation. It is
the volunteer's choice to take part. The volunteer's participation in this laboratory evaluation is
entirely voluntary. It is the volunteer's choice whether to participate or not. The volunteer may
change his/her mind later and stop participating even if he/she agreed earlier.
3.4.3
Potential risk
3.4.3.1 Potential risk from exposure to treatments
The potential risk of the laboratory evaluation in exposure to treatments

is that the volunteer may experience skin irritation. This is most unlikely to
happen because pure oil were incorporated not its active constituents, thus, it is
less concentrated compared with that of repellents commercially available.
Study shows that at 4% of ginger oil is stated to be nonirritating and
nonsensitizing while the toxicity of Neem oil was observed only in very high
doses; 30% of neem oil causes subdermal toxicity in albino rats (Qadri, 1984;
Barnes, 2007; Lis-Balchin, 2006).
COLLEGE OF PHARMACY
33
3.4.3.2 Potential risk of exposure to mosquito bites
The potential risk of the laboratory evaluation in exposure to mosquito

bites is that the volunteer may be made uncomfortable by mosquito bites and
may produce flat tiny red spots almost like a dot from a red felt-tip pen but soon
it will disappear without incident.
3.4.3.3 Potential risk of exposure to mosquito-borne disease
The potential risk of the laboratory evaluation in contracting mosquitoborne disease is that the volunteer may get a risk of getting dengue fever in a
very low probability as the test mosquitoes were reared. The volunteer will be
given protective clothing to make sure mosquitoes can bite only on the his/her
arms, where he/she can catch them before they have time to bite. If the volunteer
do become ill at any time during the laboratory evaluation or after a week, the
he/she will receive treatment from Fatima University Medical Center (FUMC)
inclusive all medications and hospitalization bills related to mosquito-borne
disease findings. Upon the guaranteed hospitalization, the researchers are not
required to compensate the volunteer's accrued unpaid sick days from
employment.
3.4.4
Nature and magnitude of all expected benefits
The laboratory evaluation is to serve the researchers which means that there will
be no direct benefit on volunteers. However, the volunteers do not have financial
obligations on all testing and intervention instead the volunteer will receive just
compensation (transportation and meals worth 500 pesos) after the laboratory
evaluation.
3.4.5
Right to refuse or withdraw
The volunteer do not have to take part in this laboratory evaluation if the
volunteer do not wish to do so. The volunteer may also stop participating in the
COLLEGE OF PHARMACY
34
laboratory evaluation at any time he/she choose. It is the volunteer's choice and all of
his/her rights will still be respected.
3.4.6
Right to privacy
Confidentiality of the results on the validation will be maintained. Prior to

testing, the volunteer have given the informed consent to the principal investigator. The
volunteer have not been coerced into giving consent, and the consent has been given
freely and voluntarily. Personal information gathered on the volunteer will be kept
confidential.
3.5
Research instruments
3.5.1
Fourier Transform Infrared Spectrometer (FTIR)

The TENSOR27Spectrometer of Bruke Optics was used in the research
for the chemical makeup of the fixed oil and essential oil. An infrared spectrum
represents a fingerprint of a sample with absorption peaks which correspond to the
frequencies of vibrations between the bonds of the atoms making up the material. The
model of the instrument used in the research was located in the Chemistry Laboratory
of De La Salle UniversityManila.
3.5.2
Hydrosteam distillation set-up
The hydrosteam distillation set-up was used in the research. It was located in
the Pharmaceutical Laboratory Chemical and Energy Division of DOST.
3.6
Data collection
3.6.1
Preparation of Ginger oil
The fresh rhizomes of the Zingiber officinale (Ginger) were properly washed
with water. An equivalent of 3 kilograms of the Ginger was weighed. The fresh
rhizomes were semi-dried to lose moisture content present of the sample. After semi-
COLLEGE OF PHARMACY
35
drying, the sample's weight is 2.2 kilograms. The rhizome were cut into small pieces,
packed into a muslin cloth and directly subjected to the hydrosteam distillation at a
temperature lower than 100C for 12 hours.
3.6.2
Preparation of Neem oil
The Neem oil was obtained from Swanson Health Products due to timeconstraint and lack of resources.
3.6.3
Product formulation
Formulated herbal
Ingredients A
50 mL (3 SFs)
Neem oil
SG=0.9213
2.71 mL (5%)
1.09 mL (2%)
Ginger oil
SG=0.8811
1.13 mL (2%)
2.84 mL (5%)
Sodium lauryl sulfate
0.4%
0.200 g
Potassium sorbate
0.1%
0.0500 g
Ingredients B
Glycerin
0.0946% SG=1.26
0.0375 mL
Xanthan gum
0.13%
0.0650 g
Citric acid, anhydrous 0.125%
0.0625 g
Vanillin
1.00%
0.500 g
Distilled water, q.s.ad
to 100%
qs 50 mL
Procedure:
1. Ingredients A and Ingredients B were mixed separately.
2. Ingredients B phase was mixing of the glycerin, citric acid, drops of water, and
vanillin and then xanthan gum was slowly added with an electric beater until
homogenous. The ingredients A were subsequently homogenized, oils are being
added last, to be mixed only at least 1 min (to prevent yielding heat that may corrode
the oils).
COLLEGE OF PHARMACY
36
3. The homogenous solution was put into a pump spray.

Neem oil, Ginger oilactive ingredients
Xanthan gumshear stabilizer
SLSanionic surfactant
Citric acidstabilizer, pH modifier
Potassium sorbatepreservative
Vanillinfixative
Glycerinemulsifier
Deionized watervehicle
List of ingredients other than the Active Ingredients (Neem oil and Ginger oil)1
1
http://www.repel.com/~/media/Repel/Files/Labels/Natural/011423941146.ashx
3.6.4
Laboratory evaluation (arm-in-cage set-up)
Laboratory evaluation (arm-in-cage set-up) was mainly guided by modified

EPA guidelines (OPPTS 810.3700). Adjuncts and modifications to the guidline were
extracted from WHO guidelines (WHO/HTM/NTD/WHOPES/2009.4), Tawatsin et al.
(2006), and Patent WO 2014137811 A1.
3.6.4.1 Test considerations
The room condition is maintained at 272C temperature and 70-80%

relative humidity throughout the test. Investigators and volunteers avoided
exhaling in to the test cage; introduction of CO2 could bias mosquitoes towards
biting.
3.6.4.2 Test proper (see Figure 13)
For each test, there will be five test treatments (see Table 1) to be tested
on four volunteers (2 males and 2 females). All volunteers were provided
written informed consent before beginning the laboratory evaluation.
Volunteers between the age of 18 to 55 years participated and had no known
allergy to insect bites, herbal-, and DEET-containing products. The timing of
the laboratory evaluation was during the daytime from 9:00AM to 4:00PM.
Tests for each treatment were conducted once a day only. The test is done in 8
replicates on groups of four volunteers. Thus, each treatment was tested twice
by each volunteer in different mosquito batches. Only one replicate per
treatment together with control per volunteer per day was done.
COLLEGE OF PHARMACY
37
For each test, eighty (80) nulliparous, host-seeking adult female

mosquitoes aged 5-7d were placed in a square 303030 cm cage with a sleeved
opening at the front for insertion of the volunteer's forearm. On each testing
day, each subject used a separate test cage containing new batch of mosquitoes;
test mosquitoes were used only once and destroyed immediately after each
testing day.
Table 1. Treatments to be used on laboratory evaluation (arm-in-cage set-up)

Treatments
Experimental
Formulated herbal (2G:5N)

Formulated herbal (2N:5G)
Positive
Commercial herbal (HomeLife Citronella Twist Spray Lavander)

Commercial synthetic (Lotion with 7.5% DEET)
Negative
Formulated herbal without the oils
Each test is done in two mosquito cages. The test cage is made up of
metal frame of about 30 cm per side with a solid bottom. The left and right side
is made of glass for viewing during testing, and a stainless screen mesh on all
other sides with a fabric sleeve for access on front side. One cage was used for
testing the treatment and the other cage was used for control.
At the beginning of each test, the readiness of mosquitoes to bite was

confirmed by exposing untreated forearm of the volunteer into the cage. If at
least ten mosquitoes landed in 30 seconds or less, then subsequent expositions
were continued. A landing was defined as a mosquito resting on the surface of
the volunteer's arm for >2s. If at any time fewer than 10 mosquitoes land on the
untreated control forearm within one minute, all mosquitoes are to be removed
from all cages and fresh mosquitoes are to be added to each cage.
One arm was treated and the other remained untreated, serving as the
control. About 1 mL of the treatment in a pipette was evenly applied to the
volunteer's arm from wrist to elbow: area covered = 600 cm2. During testing,
white latex gloves were worn to protect the hands from mosquito bites.
Volunteers avoided rubbing their arms when inserting them into or removing
COLLEGE OF PHARMACY
38
them from the cage and between exposure periods. After 30 minutes of the
allowed time to dry of the treatment, the treated arm was inserted for a 3-minute
exposure period and repeated at 30-minute interval. The occurrence of one
landing followed by another in a 3-minute exposure at a given time interval
concluded the test for the treatment. Then, CPT and %P can be determined.
Figure 13. Flow Chart of the Laboratory Evaluation
COLLEGE OF PHARMACY

3.7
39
Data analysis
Data were encoded using IBM Statistical Package for the Social Sciences (SPSS)
version 21 software statistical support. It used one-way ANOVA to analyze the data. The mean
values were calculated for each parameter and Tukey test was used to compare and to determine
the significant differences of the groups.
4.0
Results
4.1
Organoleptic testing
Table 2 shows the result of the organoleptic testing of the essential oil Z. officinale (Ginger oil
and fixed oil of the A. indica (Neem).
Table 2. Results of the organoleptic testing of the Ginger oil and Neem oil
Properties
Ginger oil (Z. officinale)
Neem oil (A. indica)
Odor
Aromatic, characteristic odor
Nutty garlic-like
Color
Light-yellow (clear)
Dark greenish brown
Consistency
Oily
Syrupy
4.2
Fourier Transform Infrared Spectrophotometer (FTIR) Analysis Result
4.2.1 Neem oil
The FTIR Absorption values of Neem oil were shown in Table 3. The wavenumbers
recorded corresponds to a specific atomic bonding that denotes to the functional groups present.
The absorption values recorded correspond to the following functional groups: Alcohol,
Aromatic ring, Alkyl group, Ketone/ Ester/ Carboxylic acid and Amide group.
COLLEGE OF PHARMACY
40
Table 3. The IR Absorption values of Neem oil1

% Transmittance
Wavenumbers (cm-1)
Functional group present
89%
3471.85
Alcohol
39%
3005.86
Aromatic ring
0%
2924.99
Alkyl group
1%
2854.22
Alkyl group
0.5%
1745.43
Ketone/ Ester/ Carboxylic Acid

Carbonyl stretch
88%
1656.36
Amide
Interpreted using the table of IR Absorption by UCLA.
4.2.2 Ginger oil
The FTIR Absorption values of Ginger oil were shown in Table 4. The wavenumbers
recorded corresponds to a specific atomic bonding that denotes to the functional groups present.
The absorption values recorded correspond to the following functional groups: Amine, Aromatic
ring, Alkyl group, Carboxylic acid, Ketone/ Ester, Amide group, and Alkanes (methyl and
methylene)
Table 4. The IR Absorption values of Ginger oil1
% Transmittance
Wavenumbers (cm-1)
Functional group present
96%
3469.41
Amine
46%
3008.70
Aromatic ring
4%
2925.98
Alkyl
11%
2854.90
Alkyl
93%
2729.44
Carboxylic acid
95%
2677.09
Carboxylic acid
7%
1745.47
Ketone/ Ester/ Carboxylic Acid
87%
1651.60
Amide
39%
1460.57
Alkanes (methyl and methylene)
Interpreted using the table of IR Absorption by UCLA.
COLLEGE OF PHARMACY
4.3
41
Physicochemical characterization
Table 5 shows the result of the physicochemical characterization of the essential oil Z.
officinale (Ginger) and fixed oil of the A. indica (Neem).
Table 5. Results of physicochemical chacterization
Properties
4.4
Ginger oil (Z. officinale)
Neem oil (A. indica)
Refractive index
1.344
1.349
Specific gravity
0.8811
0.9213
Saponification value
150.33
174.62
Repellency
Table 6 shows the result of the repellency of the treatments by conducting laboratory
evaluation (arm-in-cage set-up). The mean CPT ( after 8 replicates were 30, 30, 60, 30, and 0
minutes for formulated herbal (2N:5G), formulated herbal (2G:5N), commercial synthetic (7.5%
DEET), commercial herbal (citronella), and negative control respectively. The mean %P after 8
replicates were 70.19%, 80.98%, 85.55%, 75.55%, 8.94% for formulated herbal (2N:5G),
formulated herbal (2G:5N), commercial synthetic (7.5% DEET), commercial herbal (citronella),
and negative control respectively.
Table 6. Results of repellency
Treatment
Formulated
herbal
(2N:5G)
Formulated
herbal
(2G:5N)
Replicate (R)
R1
R2
R3
R4
R5
R6
R7
R8
Mean
SD
30
70.19
2.51
30
80.98
3.64
CPT (in min)

L0
L30
L60
%P
30
12
4
5
66.67
30
11
3
6
72.73
30
10
3
7
70.00
30
12
4
6
66.67
30
10
3
5
70.00
30
10
3
6
70.00
30
11
3
8
72.73
30
11
3
6
72.73
CPT (in min)

L0
L30
L60
%P
30
10
2
5
80.00
30
12
2
3
83.33
30
12
2
5
83.33
30
11
2
4
81.82
30
10
2
3
80.00
30
11
3
4
72.73
30
12
2
3
83.33
30
12
2
3
83.33
COLLEGE OF PHARMACY

Commercial
synthetic
(7.5% DEET)
42
CPT (in min)

L0
L30
L60
L90
L120
%P
30
12
2
2
83.33
30
13
1
1
92.31
60
11
0
3
3
72.73
90
10
0
0
1
1
90.00
60
12
0
2
2
83.33
60
10
0
1
2
90.00
90
11
0
0
2
2
81.82
60
11
0
1
1
90.91
CPT (in min)

L0
L30
L60
%P
30
11
3
5
72.73
30
10
3
4
70.00
30
12
2
5
83.33
30
12
4
6
66.67
30
10
3
5
70.00
30
12
2
5
83.33
30
12
2
5
83.33
30
12
3
4
75.00
CPT (in min)

0
0
0
0
0
0
0
L0
10
12
11
10
11
10
12
L30
9
10
10
11
10
10
10
L60
%P
10.00 16.67
9.09
0.00
9.09
0.00
16.70
L0 = untreated arm; L30 = treated arm at 30 minutes; L60 = treated arm at 60 minutes
%P = 100 [(first landing after untreated1 untreated) 100]
1
First landing values were underlined.
0
10
9
10.00
Commercial
herbal
(citronella)
Negative
control
60
22.68
85.55
6.57
30
75.55
6.87
8.94
6.35
The overall comparison using Friedman's Two-way Analysis of Variance (ANOVA) was shown
in Table 7. The computed p-value were 6.1410-6 for mean CPT and 3.4310-5 for mean %P.
Table 7. Overall comparison of the results of the repellency using Friedman's Two-way ANOVA
COLLEGE OF PHARMACY
43
Table 8 shows the comparison of the mean CPT and mean %P within treatments for their relative
significance with respect to each other using Pairwise comparison.
Table 8. Comparison of the mean CPT to mean %P within treatments using Pairwise comparison
5.0
Discussion
The research was undertaken to evaluate the efficacy of a formulated herbal with Ginger oil and
Neem oil as a potential mosquito repellent. Identification tests were performed including
physicochemical characterization and instrumental assay. It was also supported by the Fourier Transform
Infrared Spectrometer result which shows the functional groups present in the Ginger oil and Neem oil.
COLLEGE OF PHARMACY
44
The Neem oil and Ginger oil contains ketone/ ester/ carboxylic acid carbonyl stretch and aromatic ring
which is their characteristic functional group of triterpenoids (Neem) and sesquiterpenes (Ginger). The
repellency testing conducted by the researchers was laboratory evaluation (arm-in-cage set-up) wherein
five treatmentsformulated herbal (2G:5N), formulated herbal (2N:5G), commercial herbal (HomeLife
Citronella Twist Spray Lavander), commercial synthetic (repellent lotion with 7.5% DEET), and
negative control (formulated herbal without the oils)were used against Aedes aegypti through arm-incage set-up. The results showed that formulated herbal (2% Ginger oil:5% Neem oil) is an effective
repellent and is more effective than formulated herbal (2% Neem oil:5% Ginger oil) in %P. The
formulated herbal (2G:5N) provided mean %P of 80.98 and a complete protection time (CPT) of 30 to
60 minutes. In all mean CPT, there is a significant difference between the negative control and the
experimental group. While in mean %P, the negative control is significantly different to formulated
herbal (2G:5N), commercial herbal (citronella), and commercial synthetic (7.5% DEET). Also, 2G:5N
is not significantly different to commercial synthetic and herbal. It suggests that %P of 2G:5N is
comparable to commercial synthetic and herbal. In this test, the researchers came up with the result that
the oils can provide mosquito repellency comparable to commercial synthetic and herbal mosquito
repellent.
6.0
Conclusion
The researchers will now be able to conclude the following:
1.
There is a significant difference between the negative control and the experimental group
formulated herbal (2G:5N), formulated herbal (2N:5G), commercial synthetic (7.5% DEET)
in their CPT.
2.
There are no significant differences among formulated herbal (2G:5N), commercial

synthetic (7.5% DEET), and commercial herbal (citronella).
3.
In general, the results identified in the study are helpful for the community who live in such
places that are highly reported of dengue cases. They can use the formulated herbal as a
precautionary measures in preventing outbreak or lowering the casualties of mosquito-borne
pathogen transmission. Evidence of repellency of the incorporated oils of Neem
(Azadirachta indica) and Ginger (Zingiber officinale) was able to present by the research
which makes it a source for developing novel herbal repellents against mosquitoes.
COLLEGE OF PHARMACY

7.0
45
Recommendation
The researchers were able to meet the main purpose of the study, which is to evaluate the
formulated herbal as a potential mosquito repellent by looking for the significant differences among
treatments. But there are still some areas that could be further improved on. The research design can be
improved by making the laboratory evaluation in randomized, double-blinded, and crossover method.
Future researchers are recommended to study on the minimum effective dose of the said oils. They can
conduct other design for the evaluation such as field evaluation. It is also recommended to know if there
is synergism of repellency of the two plant variables. The future researchers may also want to consider
product development and reformulation specially in the aspects of stability-compatibility.
8.0
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http://www.nurseriesonline.us/articles/Growing-Ginger.html#.VBM8cvmSySo
9.0
Glossary of Terms and Abbreviations

Abiotic factors Pertaining to repellents: non-biological variables that may influence
repellency, e.g., air quality, humidity, light, temperature, wind (Moore, 2006, p. 32)
Aerosol Extremely fine spray droplets suspended in air. The WHO classifies spray droplets as
fine aerosols < 25mm, coarse aerosols 2550 m, mists 50100 m, fine sprays 100200 m, medium
sprays 200300 m and coarse sprays >300 m. (Moore, 2006, p. 32)
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Arthropods Invertebrate Phylum Arthropoda. Creatures with exoskeleton (consisting of

chitin) and jointed legs. The blood-feeding arthropods are either insects (Class Insecta) or mites/ticks
(Class Arachnida, Order Acari). Numerous other groups of animals affect humans directly through bites
or envenomation (e.g., snakes, scorpions, spiders, and wasps) (Moore, 2006, p. 32)
Bioassays Standard methods and procedures for replicated comparative testing of effects on
biological materials (Moore, 2006, p. 33)
Biotic factors Pertaining to repellents. Biological variables that may influence repellency,
such as physiological condition of the insect (e.g., level of hunger, activity cycle) or the host (e.g., rates
of exhalation and sweating) (Moore, 2006, p. 33)
Bite The act of penetrating human skin by the mouthparts of an insect or other arthropod with
ingestion of blood, typically associated with abdominal swelling and color change (Moore, 2006, p. 33)
Biting rate The number of bites/person/time period (e.g., 12 bites/hour), as a measure of
population density in relation to humans, for any given species of biting arthropods, or group of species
at a particular place and time. For ethical reasons, especially where vector-borne disease risks must be
considered, it is customary to intercept the attacking insects before they actually bite (possibly increasing
catch efficiency); the results are therefore reported in terms of the landing rate rather than the biting
rate (Moore, 2006, p. 33)
CDC Centers for Disease Control and Prevention (Moore, 2006, p. 34)
Compatible Ingredients that retain their individual properties when mixed together
(Moore, 2006, p. 34)
Complete protection time (CPT) The time from application of a repellent until efficacy
failurefor example, the time from application until the first efficacy failure event confirmed within 30
minutes by a second similar event (EPA, 2010, p. 6)
Concentration Proportion of a given ingredient in a formulation (Moore, 2006, p. 33)
Confirmed event One landing, probe, or bite followed by another similar event within 30
minutes. The first event is confirmed by the second; the second event is the confirming event
(EPA, 2010, p. 6)
DEET N,N-diethyl-3-methylbenzamide (originally known as N,N-diethyl-meta-toluamide)
(Moore, 2006, p. 35)
Dose determination A testing procedure used to estimate a typical consumer dose of a
repellent (EPA, 2010, p. 6)
Empirical evidence Based on practical experience rather than scientific proof (Collins
English Dictionary, 2014)
EPA U.S. Environmental Protection Agency (Moore, 2006, p. 36)
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Essential oils Terpenes and other volatiles obtained from plants by steam distillation or
pressing, they are hydrophobic and mostly aromatic (Moore, 2006, p. 36)
Formulation Defined chemical product mixture, usually meaning the commercialized version
of a special formula, sometimes requiring dilution before use (Moore, 2006, p. 37)
Human subject A living individual about whom an investigator conducting research obtains
either data through intervention or interaction with the individual or identifiable private information. By
this definition, human subjects were referred to as volunteers in the study (EPA, 2010, p. 6)
Insectary
place
where
living
insects
are
kept,
bred,
and
studied
(Collins English Dictionary, 2014)

Insect Any member of the arthropod Class Insecta. The named derived from the Latin
insectum for having been cut, referring to the articulated body; adults typically with three pairs of legs
(hexapod) (Moore, 2006, p. 36)
Insect repellent Usually first line of defense because they require no large equipment, no
organized effort of community vector control, and they distribute the responsibility for protection to the
individual (Moore, 2006, p. 36)
Landing The act of a flying or jumping insect or other arthropod alighting on human skin
without probing or biting (EPA, 2010, p. 5)
Nulliparous Being a female that has not borne offspring (Merriam-Webster, 2015)
Probe The act of penetrating human skin by the mouthparts of an insect or other arthropod
without ingestion of blood (EPA, 2010, p. 6)
Questing The behavior of ticks or chiggers actively seeking a host (EPA, 2010, p. 6)
Repellent, repellant For insects, something that causes insects to make oriented movements
away from its source; a product intended to disrupt the host-seeking behavior of insects or other
arthropods, driving or keeping them away from treated human skin (Moore, 2006, p. 40)
Replicate repeated experimental observation of the same test across different groups (Chegg, 2015).
Risk assessment In context of human health, estimating the probability of adverse effects
resulting from defined exposure to known chemical hazard (Moore, 2006, p. 40)
Specifications Standard descriptions of products for quality control purposes. For repellents
and other pesticides, international specifications are prepared by the FAO and/or WHO, then adopted by
the FAO/WHO Joint Meeting on Pesticide Specifications (Moore, 2006, p. 41)
WHOPES World Health Organization Pesticides Evaluation Scheme, responsible for
assessments, specifications and recommendations for pesticides (including repellents) used for public
health pest and vector control on behalf of Member States of the United Nations (U.N.)
(Moore, 2006, p. 43)
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Appendices
Appendix A
Certifications
Appendix A.1
Permission and Counseling
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Appendix A.2
Authentication of Ginger
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Appendix A.3
Certification of Distillation of Ginger
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Appendix A.4
Certification of FTIR
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Appendix A.5
Certification of Ethical Review
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Appendix A.6
Certification of Statistical Analysis
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Appendix A.7
Certification of Proofreading
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Appendix B
Research Plates
Hydrosteam Distillation
TENSOR27Spectrometer of Bruke Optics
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Physicochemical characterization
(L-R) Refractometer, Analytical Balance (Volumetric Flask), Titration Set-up for Saponification
Substances used in the Laboratory Evaluation
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Arm-in-cage setup
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Appendix C
Ethics
Appendix C.1
Informed Consent (Engl.)
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Appendix C.2
Informed Consent (Fil.)
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Appendix D
Research Budget
EXPENSES
COST
TRANSPORTATION
4 000.00
PLANT MATERIAL
600.00
PLANT AUTHENTICATION
240.00
FTIR
1 200.00
HYDRODISTILLATION
2 000.00
ETHANOL EXTRACTION
2 100.00
PHYTOCHEMICAL SCREENING
1 200.00
LABORATORY EVALUATION
15 000.00
INGREDIENTS (INERT AND ACTIVE)
4 500.00
DOCUMENTATION
2 500.00
PROPOSAL DEFENSE
1 500.00
FINAL OD
2 600.00
TOTAL
37 440.00
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Appendix E
Results of FTIR
Appendix E.1
FTIR of Neem Oil
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Appendix E.2
FTIR of Ginger Oil
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Appendix F
Computations and Figures
Percentage Yield of the Essential Oil of Zingiber officinale (Ginger) rhizomes

Data:
Weight of plant sample
Weight of dried plant sample
Weight of the oil
% Yield of the Essential Oil
=
=
=
=
3000 grams
2200 grams
1.30 grams
0.06 %
Calculation:
% Yield = [(Weight of the oil) / (Weight of the dried sample)] 100
% Yield = [(1.30 g/ 2200 g)] 100
% Yield = 0.06
Sample Computations for Percent Percentage of Two Replicates of Different Treatment

Replicate 4 of Formulated Herbal (2G:5N)
L0 (untreated arm)
=
11 landings
First landing after untreated
=
2 landings
%P = 100 [(first landing after untreated untreated arm) 100]
= 100 [(2 11) 100] = 100 18.18
= 81.82%
Replicate 4 of Negative Control
L0 (untreated arm)
First landing after untreated
=
=
10 landings
11 landings
%P = 100 [(first landing after untreated untreated arm) 100]

= 100 [(11 10) 100] = 100 110
= -10% 0%
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Appendix G
Timeline
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Appendix H
Authors
Kenneth G. Dayrit, Ken for short is 20 years old, born on the

15th of September 1994. He lives at Porac, Pampanga. He is a
computer enthusiast. He finished his secondary education at
Holy Family Academy High School (2007-2011) and currently
studying at Our Lady of Fatima University-Valenzuela taking
up Bachelor of Science in Pharmacy.
Alexandra Nicole N. De Padua, Alex for short is 19 years old,

born on the 7th of June 1995. She lives in Tiaong, Guiguinto,
Bulacan. She likes reading books. She finished her secondary
education at Balagtas National Agricultural High School (20072011) and currently studying at Our Lady of Fatima
University-Valenzuela taking up Bachelor of Science in
Pharmacy.
Nio V. Gomez is 19 years old, born on the 19th of March 1995.

He lives in Balintawak, Quezon City. He likes playing
badminton. He finished his secondary education at Balingasa
High School (2007-2011) and currently studying at Our Lady of
Fatima University-Valenzuela taking up Bachelor of Science in
Pharmacy.
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Paula Giselle P. Gutierrez, Pau for short is 20 years old, born on

the 9th of March 1994. She lives in Brentwood Village, Mabalacat,
Pampanga. She likes singing. She finished her secondary
education at Holy Family Academy (2007-2011) and currently
studying at Our Lady of Fatima University-Valenzuela taking up
Bachelor of Science in Pharmacy.
Nathalie C. Hulleza, Nat for short is 20 years old, born on the

12th of September 1994. She lives in Bagong Bario, Caloocan
City. She likes dancing. She finished her secondary education
at Janiuay National Comprehensive High School (2007-2011)
and currently studying at Our Lady of Fatima UniversityValenzuela taking up Bachelor of Science in Pharmacy.
Maria Lorenz M. Martinez, Lorenz for short is 19 years old,

born on the 18th of April 1995. She lives in Novaliches, Quezon
City. She likes reading books and traveling. She finished her
secondary education at San Jose High School (2008-2011) and
currently studying at Our Lady of Fatima UniversityValenzuela taking up Bachelor of Science in Pharmacy.
COLLEGE OF PHARMACY

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COMPARATIVE STUDY OF THE EFFICACY OF OILS

COMPARATIVE STUDY OF THE EFFICACY OF OILS OF

Dayrit, Kenneth G.1,2,3,4

Bachelor of Science in Pharmacy

Research Development and Innovation Center

Our Lady of Fatima University

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

Dean Olive M. de Vera, RPh, MSPharm

Angelita A. Rodriguez, RPh, MSPharm, PhD

Jobelle S. Abrio, RPh

Michael Joseph S. Dio, RN, MAN, PhD

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

Alexandra Nicole N. De Padua

Paula Giselle P. Gutierrez

Maria Lorenz M. Martinez

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

Second Title Page...

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

Paradigm of the Study........

Treatments to be used on laboratory evaluation (arm-in-cage set-up)...

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

List of Selected Journals

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

COMPARATIVE STUDY OF THE EFFICACY OF OILS OF

Bachelor of Science in Pharmacy

Research Development and Innovation Center

Our Lady of Fatima University

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

COMPARATIVE STUDY OF THE EFFICACY OF OILS OF Azadirachta

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

Background of the Study

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

Statement of the Problem

The following hypotheses were formulated for testing in the research:

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

Significance of the Study

Scope and Delimitation

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

Review of Related Literature and Study

Ginger (Zingiber officinale) is described as an erect, smooth plant rising from

Ginger's primary scientific is Zingiber officinale Rosc. Some of its alternative

OUR LADY OF FATIMA UNIVERSITY

COMPARATIVE STUDY OF THE EFFICACY OF OILS

The Neem or "margosa" (Azadirachta indica meaning "Free Tree of India") is

OUR LADY OF FATIMA UNIVERSITY