Thesis

COMPARATIVE STUDY OF THE EFFICACY OF OILS… i

OUR LADY OF FATIMA UNIVERSITY 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)

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

De Padua, Alexandra Nicole, N.1,2,3,4

Gomez, Niño 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

1Bachelor of Science in Pharmacy

2College of Pharmacy

3Research Development and Innovation Center

4Our Lady of Fatima University

5Research Adviser

March 2015

COMPARATIVE STUDY OF THE EFFICACY OF OILS… ii


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. Diño, RN, MAN, PhD

Director

Research Development and Innovation Center

COMPARATIVE STUDY OF THE EFFICACY OF OILS… iii


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

Niño V. Gomez

Member

Paula Giselle P. Gutierrez

Member

Nathalie C. Hulleza

Member

Maria Lorenz M. Martinez

Member

COMPARATIVE STUDY OF THE EFFICACY OF OILS… iv


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.

COMPARATIVE STUDY OF THE EFFICACY OF OILS… v


Table of Contents

First Title Page...……………………………………………………………………………. …... i

Endorsement Page……………………………………………………………………………….. ii

Certificate of Originality………………………………………………………………………… iii

Acknowledgement………………………………………………………………………………. iv

Table of Contents………………………………………………………………………………... v

List of Appendices……………………………………………………………………………….. vi

List of Figures……………………………………………………………………………………. vii

List of Plates…………………………………………………………………………………....... vii

List of Tables…………………………………………………………………………………...... vii

List of Selected Journals…………………………………………………………………………. viii

Second Title Page………………………………………………………………………………... 1

Abstract………………………………………………………………………………………….. 2

1.0 Introduction

1.1 Background of the Study……………………………………………………………. 3

1.2 Statement of the Problem……………………………………………………………. 4

1.3 Research Hypotheses………………………………………………………………... 4

1.4 Significance of the Study……………………………………………………………. 5

1.5 Scope and Delimitation……………………………………………………………… 5

2.0 Review of Related Literature and Study

2.1 Local Literature

2.1.1 Ginger…………………………………………………………………….. 8

2.1.2 Neem……………………………………………………………………... 9

2.1.3 Aedes aegypti…………………………………………………………….. 11

2.2 Foreign Literature

2.2.1 Ginger…………………………………………………………………….. 13

2.2.2 Neem……………………………………………………………………... 15

2.2.3 Essential oil—Ginger oil…………………………………………………. 17

2.2.4 Fixed oil—Neem oil……………………………………………………… 19

2.2.5 Extraction of essential oil…………………………………………………. 20

2.2.6 Aedes aegypti…………………………………………………………….. 21

2.3 Local Study

2.3.1 Ginger…………………………………………………………………….. 22

2.3.2 Neem……………………………………………………………………... 22

2.4 Foreign Study

2.4.1 Ginger…………………………………………………………………….. 23

2.4.2 Neem……………………………………………………………………... 24

3.0 Research Method

3.1 Research Design…………………………………………………………………….. 28

3.2 Research Locale……………………………………………………………………... 29

3.3 Population and Sampling……………………………………………………………. 29

3.3.1 Ginger oil and Neem oil…………………………………………………... 29

3.3.2 Aedes aegypti…………………………………………………………………….. 29

3.3.3 Volunteers………………………………………………………………... 30

3.4 Research Ethics

3.4.1 Volunteers on laboratory evaluations…………………………………….. 31

3.4.2 Risk minimization………………………………………………………... 32

COMPARATIVE STUDY OF THE EFFICACY OF OILS… vi


3.4.3 Risk

3.4.3.1 Potential risk from exposure to treatments……………………... 32

3.4.3.2 Potential risk of exposure to mosquito bites……………………. 33

3.4.3.3 Potential risk of exposure to mosquito-borne disease………….. 33

3.4.4 Nature and magnitude of all expected benefits……………………………. 33

3.4.5 Right to refuse or withdraw……………………………………………….. 33

3.4.6 Right to privacy………………………………………………………....... 34

3.5 Research instruments

3.5.1 Fourier Transform Infrared Spectrometer (FTIR)………………………… 34

3.5.2 Hydrosteam Distillation set-up…………………………………………… 34

3.6 Data collection

3.6.1 Preparation of Ginger oil…………………………………………………. 34

3.6.2 Preparation of Neem oil…………………………………………………... 35

3.6.3 Product Formulation……………………………………………………… 35

3.6.4 Laboratory Evaluation (Arm-in-cage set-up)……………………………...

3.6.4.1 Test considerations…………………………………………….. 36

3.6.4.2 Test proper……………………………………………………... 36

3.7 Data analysis………………………………………………………………………… 39

4.0 Results

4.1 Organoleptic testing…………………………………………………………………. 39

4.2 Fourier Transform Infrared Spectrometer (FTIR) Analysis Result

4.2.1 Neem oil………………………………………………………………….. 39

4.2.2 Ginger oil…………………………………………………………………. 40

4.3 Physicochemical characterization…………………………………………………... 41

4.4 Repellency…………………………………………………………………………... 41

5.0 Discussion………………………………………………………………………………….... 43

6.0 Conclusion…………………………………………………………………………………… 44

7.0 Recommendation…………………………………………………………………………….. 45

8.0 References…………………………………………………………………………………… 45

9.0 Glossary of Terms and Abbreviations ……………………………………………………….. 50

10.0 Appendices

Appendix

A Certifications

A.1 Permission and Counseling……………………………………………….. 54

A.2 Authentication of Ginger………………………………………………….. 55

A.3 Certification of Ginger Distillation………………………………………... 56

A.4 Certification of FTIR……………………………………………………… 57

A.5 Certification of Ethical Review…………………………………………… 58

A.6 Certification of Statistical Analysis……………………………………….. 59

A.7 Certification of Proofreading……………………………………………… 60

B Research Plates…………………………………………………………………... 61

C Ethics

C.1 Informed Consent (Engl.)…………………………………………………. 64

C.2 Informed Consent (Fil.)…………………………………………………… 71

D Research Budget…………………………………………………………………. 78

E FTIR Results

E.1 FTIR of Neem Oil ………………………………………………………... 79

E.2 FTIR of Ginger Oil………………………………………………………... 80

F Computations and Figures……………………………………………………….. 81

G Timeline…………………………………………………………………………. 82

H Authors…………………………………………………………………………... 83

COMPARATIVE STUDY OF THE EFFICACY OF OILS… vii


List of Figures

Figure

1 Paradigm of the Study………………………………………………………………........ 7

2 Simulacrum of the Study………………………………………………………………… 7

3 Major components of Ginger oil ………………………………………………………... 15

4 Azadirachtin's chemical structure……………………………………………………….. 18

5 Salannin's chemical structure…………………………………………………………… 18

6 Geraniol's chemical structure………………………………………………………….... 20

7 Citral's chemical structure………………………………………………………………. 20

8 The repellency of essential oils (100% concentration) to Aedes Mosquitoes…………... 20

9 Azadirachta indica………………………………………………………………………. 26

10 Ginger (Zingiber officinale)…………………………………………………………….. 27

11 Flow Chart of the Study…………………………………………………………………. 28

12 Preparation for Aedes aegypti…………………………………………………………………… 30

13 Flow Chart of the Laboratory Evaluation……………………………………………….. 38

List of Plates

Plate

1 Hydrosteam Distillation…………………………………………………………………. 64

2 TENSOR®—27—Spectrometer of Bruke Optics………………………………………... 64

3 Physicochemical characterization……………………………………………………….. 65

4 Substances used in the Laboratory Evaluation…………………………………………... 65

5 Arm-in-cage set-up………………………………………………………………………. 66

List of Tables

Table

1 Treatments to be used on laboratory evaluation (arm-in-cage set-up)…………………... 37

2 Results of the organoleptic testing Ginger oil and Neem oil……………………………. 39

3 The IR Absorption values of Neem oil………………………………………………….. 39

4 The IR Absorption values of Ginger oil…………………………………………………. 39

5 Results of physicochemical characterization……………………………………………. 41

6 Results of repellency…………………………………………………………………….. 41

7 Overall comparison of the results of the repellency using Friedman's Two-way ANOVA 42

8 Comparison of the mean CPT to mean %P within treatments using Pairwise comparison 43

COMPARATIVE STUDY OF THE EFFICACY OF OILS… viii


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, 99–103

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, 107–110.

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: 14–15

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

COMPARATIVE STUDY OF THE EFFICACY OF OILS… 1


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, Niño V.1,2,3,4




Christina G. Sabroso, RPh, MSPharm2,3,4,5

1Bachelor of Science in Pharmacy

2College of Pharmacy

3Research Development and Innovation Center

4Our Lady of Fatima University

5Research Adviser



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, Niño V. 1,2,3,4




Cristina G. Sabroso, RPh, MSPharm2,3,4,5 1Bachelor of Science in Pharmacy

2College of Pharmacy 3Research Development and Innovation Center

4Our Lady of Fatima University 5Research 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



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



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 concentrations—2% Ginger oil:5% Neem oil and 5%

Ginger oil:2% Neem oil. Repellency testing was conducted wherein five test treatments—formulated

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.



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

carry—mild-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



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 concentrations—2%

Ginger oil:5% Neem oil and 5% Ginger oil:2% Neem oil. Repellency testing was conducted

wherein five treatments—formulated 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 (37.1°C). 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 (30×30×30 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.



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.



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,



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



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 anti-

inflammatory 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, antiemetic, 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 full-

bearing at 10 years of age with 30-50 kilos of fruits per year (Guerrero, 2012).



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 DENG–gae) 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



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



below are observed to be the most susceptible age bracket) are often 40–50% but may

reach 80–90%. 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 China–India 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).



Ginger has 1-3 percent volatile oils—complex (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, β-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.

%

α-Pinene 3

Camphene 8.3

β-Phellandrene 9.6

Linalool 0.8

Borneol 0.8

Neral 1.4

Geranyl acetate 0.9

α-Zingiberene 29

β-Bisabolene + α-farnesene 14

β-Sesquiphellandrene 9.9

Figure 3. Major components of ginger oil

Toxicity of ginger oil—A) 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 officinale—It 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).



2.2.2 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, anti-

inflammatory, 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 medium-

size 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



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



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

Figure 5. Salannin's chemical structure

2.2.3 Essential oil—Ginger 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 volatile—meaning 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



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 compounds—these 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).



Figure 6. Geraniol's chemical structure Figure 7. Citral's chemical structure

Compound Duration of Protection (h)

Terpenene 0

Citronellal < 1

Limonene ≤ 1

α-Pinene ≤ 1

Citronellol 1–2

Eugenol 1–2

Linalool 1–2

β-Terpeneol 1–2

Geraniol 2–3

Citral 2–3

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 oil—Neem 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).



2.2.5 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; Martínez, 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).



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).



2.3 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, α-pinene, β-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



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 behavior-

modifying properties against Aedes aegypti and other biting arthropods of public health and pest



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 plants—Neem

(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



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 so-

called purification of air, premises are often fumigated with a mixture of Neem leaves, oleo-

gum 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 1–4 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).



Figure 9. Azadirachta indica

Left: A. indica Leaves and Fruits from http://www.hibiscuscoastseconds.co.za/wp-

content/uploads/2013/11/Azadirachta_indica_leaves__fruits.jpg

Right: A. indica Seeds from

http://explorepharma.files.wordpress.com/2010/09/2607242300_974df4397c.jpg



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



3.0 Research method

3.1 Research design

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 (arm-

in-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 treatments—formulated 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



3.2 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 15°C (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

University–Manila 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



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: 97.5°F (36.4°C) to 98.8°F (37.1°C).

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.



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 OLFU—IERC (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



(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).



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 mosquito-

borne 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



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 TENSOR®—27—Spectrometer 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 University–Manila.

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-



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 100°C for 12 hours.

3.6.2 Preparation of Neem oil

The Neem oil was obtained from Swanson Health Products due to time-

constraint 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).



3. The homogenous solution was put into a pump spray.

Neem oil, Ginger oil—active ingredients Xanthan gum—shear stabilizer

SLS—anionic surfactant Citric acid—stabilizer, pH modifier

Potassium sorbate—preservative Vanillin—fixative

Glycerin—emulsifier Deionized water—vehicle

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 27±2°C 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.



For each test, eighty (80) nulliparous, host-seeking adult female

mosquitoes aged 5-7d were placed in a square 30×30×30 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



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



3.7 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.



Table 3. The IR Absorption values of Neem oil1

% Transmittance Wavenumbers (cm-1) Functional group present

1 89% 3471.85 Alcohol

2 39% 3005.86 Aromatic ring

3 0% 2924.99 Alkyl group

4 1% 2854.22 Alkyl group

5 0.5% 1745.43 Ketone/ Ester/ Carboxylic Acid

Carbonyl stretch

6 88% 1656.36 Amide

1Interpreted 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

1 96% 3469.41 Amine

2 46% 3008.70 Aromatic ring

3 4% 2925.98 Alkyl

4 11% 2854.90 Alkyl

5 93% 2729.44 Carboxylic acid

6 95% 2677.09 Carboxylic acid

7 7% 1745.47 Ketone/ Ester/ Carboxylic Acid

8 87% 1651.60 Amide

9 39% 1460.57 Alkanes (methyl and methylene)

1Interpreted using the table of IR Absorption by UCLA.



4.3 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 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

4.4 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 Replicate (R)

R1 R2 R3 R4 R5 R6 R7 R8 Mean SD

Formulated

herbal

(2N:5G)

CPT (in min) 30 30 30 30 30 30 30 30 30 0

L0 12 11 10 12 10 10 11 11

L30 4 3 3 4 3 3 3 3

L60 5 6 7 6 5 6 8 6

%P 66.67 72.73 70.00 66.67 70.00 70.00 72.73 72.73 70.19 2.51

Formulated

herbal

(2G:5N)

CPT (in min) 30 30 30 30 30 30 30 30 30 0

L0 10 12 12 11 10 11 12 12

L30 2 2 2 2 2 3 2 2

L60 5 3 5 4 3 4 3 3

%P 80.00 83.33 83.33 81.82 80.00 72.73 83.33 83.33 80.98 3.64



Commercial

synthetic

(7.5% DEET)

CPT (in min) 30 30 60 90 60 60 90 60 60 22.68

L0 12 13 11 10 12 10 11 11

L30 2 1 0 0 0 0 0 0

L60 2 1 3 0 2 1 0 1

L90 - - 3 1 2 2 2 1

L120 - - - 1 - - 2 -

%P 83.33 92.31 72.73 90.00 83.33 90.00 81.82 90.91 85.55 6.57

Commercial

herbal

(citronella)

CPT (in min) 30 30 30 30 30 30 30 30 30 0

L0 11 10 12 12 10 12 12 12

L30 3 3 2 4 3 2 2 3

L60 5 4 5 6 5 5 5 4

%P 72.73 70.00 83.33 66.67 70.00 83.33 83.33 75.00 75.55 6.87

Negative

control

CPT (in min) 0 0 0 0 0 0 0 0 0 0

L0 10 12 11 10 11 10 12 10

L30 9 10 10 11 10 10 10 9

L60 - - - - - - - -

%P 10.00 16.67 9.09 0.00 9.09 0.00 16.70 10.00 8.94 6.35

L0 = untreated arm; L30 = treated arm at 30 minutes; L60 = treated arm at 60 minutes

%P = 100 – [(first landing after untreated1 ÷ untreated) × 100] 1First landing values were underlined.

The overall comparison using Friedman's Two-way Analysis of Variance (ANOVA) was shown

in Table 7. The computed p-value were 6.14×10-6 for mean CPT and 3.43×10-5 for mean %P.

Table 7. Overall comparison of the results of the repellency using Friedman's Two-way ANOVA



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.



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 treatments—formulated 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-in-

cage 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.



7.0 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.

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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 25–50 μm, mists 50–100 μm, fine sprays 100–200 μm, medium

sprays 200–300 μm and coarse sprays >300 μm. (Moore, 2006, p. 32)



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

failure—for 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)



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 – A 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)



10.0 Appendices

Appendix A

Certifications

Appendix A.1

Permission and Counseling



Appendix A.2

Authentication of Ginger



Appendix A.3

Certification of Distillation of Ginger



Appendix A.4

Certification of FTIR



Appendix A.5

Certification of Ethical Review



Appendix A.6

Certification of Statistical Analysis



Appendix A.7

Certification of Proofreading



Appendix B

Research Plates

1 Hydrosteam Distillation

2 TENSOR®—27—Spectrometer of Bruke Optics



3 Physicochemical characterization

(L-R) Refractometer, Analytical Balance (Volumetric Flask), Titration Set-up for Saponification

4 Substances used in the Laboratory Evaluation



5 Arm-in-cage setup



Appendix C

Ethics

Appendix C.1

Informed Consent (Engl.)



Appendix C.2

Informed Consent (Fil.)



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



Appendix E

Results of FTIR

Appendix E.1

FTIR of Neem Oil



Appendix E.2

FTIR of Ginger Oil



Appendix F

Computations and Figures

Percentage Yield of the Essential Oil of Zingiber officinale (Ginger) rhizomes

Data:

Weight of plant sample = 3000 grams

Weight of dried plant sample = 2200 grams

Weight of the oil = 1.30 grams

% Yield of the Essential Oil = 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) = 10 landings

First landing after untreated = 11 landings

%P = 100 – [(first landing after untreated ÷ untreated arm) × 100]

= 100 – [(11 ÷ 10) × 100] = 100 – 110

= -10% ≅ 0%



Appendix G

Timeline



Appendix H

Authors

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 (2007-

2011) and currently studying at Our Lady of Fatima

University-Valenzuela taking up Bachelor of Science in

Pharmacy.

Niño 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.

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.



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 University-

Valenzuela 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 University-

Valenzuela taking up Bachelor of Science in Pharmacy.

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