Phytochemical Screening of Beehive Ginger (Zingiber spectabile) Leaves Extract and

its Pesticidal Property Through Brine Shrimp Lethality Assay

Science Investigatory Project

Science, Technology and Engineering Department (STE)
Jacinto P. Elpa National High School

3rd Place Best in Poster

Researchers
Hanna Louise U. Acosta
Althea Jasmine T. Arreza
Aezra V. Moral

Research Consultant

Venchie C. Badong

Research Adviser
Ms. Mary Cris G. Abao
Mrs. Ana Geran V. Millan
Mrs. Jovey Mae H. Plaza
Mr. Marvin Y. Sumastre

S.Y 2022-2023
 TABLE OF CONTENTS

Chapter I: INTRODUCTION
Definition of Key Terms 3
Abstract 5
Background of the study 6
Statement of the Problem 9
Research Objectives 9
Statement of Null Hypotheses 10
Significance of the Study 11
Scope and Limitations 12
Conceptual Framework 13

Chapter II: REVIEW OF RELATED LITERATURE 14

Chapter III: METHODOLOGY

Research Design 20
Research Procedures 20
Preparation of Plant 20
Identification of Plant 21
Statistical Data 22
Schematic Diagram of Research Procedures 23

Chapter IV: PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

Analysis of Statistical Data 23

Phytochemical components of the plant extract 24
Pesticidal Property of the plant extract 27
Mortality Rate on brine shrimp 28

Chapter V: SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Findings 31
Conclusions 32
Recommendations 32

2
 ACKNOWLEDGEMENT

We would like to extend our sincere appreciation to our almighty father for leading us to

the completion of this research proposal and to everyone that has provided support and made

significant contributions to the development of this research proposal. To our friends and

classmates that helped us throughout the time, the invaluable assistance and guidance have been

crucial at every step of the process.

First and foremost, we would like to extend our heartfelt gratitude to Mrs. Ana Geran V.

Millian for her valuable insight into this study, and her continuous support and encouragement.

Her valuable comments and insights shaped this study to its fullest and we are sincerely thankful

for her commitment to our academic growth.

To Sir Venchie Badong who assisted us with the conduction of our experiment, his

valuable response to our queries, and the patience and support he has given in shaping this study.

We are especially thankful for the knowledge he shared with us.

We would like to acknowledge the support and resources provided by our school

principal Mrs. Evelyn Bandoy and to the school library for giving us access to the actual

resources, research databases, and facilities that have been instrumental in conducting an in-

depth literature review and gathering relevant information for this proposal.

We would also like to thank ourselves for our hard work and all the sleepless nights, and

effort placed in building this research proposal.

To our parents, we are eternally grateful for their unwavering support and constant

motivation they have provided us throughout this journey.

The completion of this study would not have been visible without these individuals, we

are foremost thankful for everything they have contributed.

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 DEFINITION OF TERMS

For clarity and understanding of the study, the researcher provided operational definition

of the following terms:

Alkaloids. A bioactive component used in the fight against pest insects containing at least one

nitrogen atom.

Beehive Ginger (Zingiber Spectabile). A plant material that was used in this study to determine

its phytochemical and pesticidal properties against brine shrimp.

Brine Shrimp Lethality Assay. The inexpensive bioassay used in this study for the testing of

the efficacy of phytochemicals present in Beehive Ginger.

Gingerols. The phenol phytochemical compound found in fresh ginger that activates spice

receptors on the tongue which we will be testing for its pesticidal capability.

Median Lethal Concentration. The dose that is used in this study to test the substance that is

lethal for 50% of the animals in a dose group.

Negative control. The group in this study that does not receive any type of treatment and,

therefore, should not show any change during the experiment and will be used for comparison.

Phytochemical Screening. The method of extraction, screening and identification of the

medicinally active substances found in plants that this study will be using.

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Pesticides. A substance used for destroying insects or other organisms harmful to cultivated

plants or to animals that this study will attempt to create its organic alternative.

Pests. A destructive insect or other animal that attacks crops, food, livestock, etc. that this study

will attempt to explore on how it can be eradicated.

Paradol. One of the phenolic compounds of ginger which is an active flavor constituent of the

seeds of Guinea pepper which has the same role as gingerol in this study.

Saponnins. A substance that is found in beehive ginger that has potent insecticidal effect on both

moth and aphid.

Shogaols. One of the pungent constituents of ginger similar in chemical structure to gingerol

and its purpose in this study.

Steroids. Regulate many aspects of growth and development, and mutants deficient in

brassinosteroids are often extremely stunted and infertile.

Tannins. A substance that is found in beehive ginger that are endogenous inhibitors of the

growth of pest species and acts as an antibiotic that prevents insects from obtaining nourishment.

5
 ABSTRACT

The conflict between pests and farming productivity have been rampant ever since the
beginning of agriculture and with the impact of climate change the problem has been continuously
growing. Up to 40 percent of global crops have been destroyed and costing $220 billion in losses
(Sarkozi, 2021). Subsequently, the demand for efficient pesticides has been on the rise and with the
industrialization of the agricultural sector followed the increased chemical burden on natural ecosystems.
However, due to their known ability to cause a large number of negative health and environmental
effects, their side effects can be an important environmental health risk factor. (Hens et al., 2016) Which
is why farmers are becoming more and more aware of the potential dangers these developed chemical
pesticides can bring. Hence, in this study Beehive Ginger (Zingiber spectabile) leaves extract will be
examined for its pesticidal property which can be used as a natural counterpart to today’s synthetic
chemical pesticides and determine which phytochemicals are present that provides its pesticidal property.
In this study, phytochemical screening of the Beehive gingers leaves extract was initiated to identify the
chemical constituents of the plant. Following a cytotoxicity test using Brine shrimp lethality assay to
determine its efficacy as a pesticide in terms of its ability to cause death in the brine shrimp larvae.
Following a phytochemical examination of the plant extract, the presence of secondary metabolites such
as alkaloids, saponins, tannins, and steroids was discovered. This is evidence that the plant has the
potential to serve as an excellent source of medication. In cytotoxicity testing utilizing shrimp brine
assay, the results suggest that the plant materials explored in this study are poisonous at 500ppm,
indicating an 80 percent fatality rate. On the basis of these findings, it is possible to draw the conclusion
that the plant extract may have possessed potential pesticidal qualities.

Keywords: Phytochemical, Brine Shrimp lethality, Beehive Ginger, pesticide

6
 CHAPTER 1

INTRODUCTION

A. Background of the Study

Pests have been tormenting crops ever since the beginning of agriculture. However, due to the

continuous impact of climate change, plant pests that ravage economically important crops are becoming

more destructive and posing an increasing threat to food security, environment, destroying up to 40

percent of global crops and costing $220 billion in losses (Sarkozi, 2021). Subsequently, the demand for

efficient pesticides has been on the rise to aid this problem. With the industrialization of the agricultural

sector followed the increased chemical burden on natural ecosystems. However, due to their known

ability to cause a large number of negative health and environmental effects, their side effects can be an

important environmental health risk factor. (Hens et al., 2016) In addition, some of these pesticides

bring endocrine-disrupting effects and reproductive toxicity which is harmful to the human body

(Nicolopoulou-Stamati et al., 2016). Which is why farmers are becoming more and more aware of the

potential dangers these developed chemical pesticides can bring. Hence, in this study Beehive Ginger

(Zingiber spectabile) leaves extract will be examined for its pesticidal property which can be used as a

natural counterpart to today’s synthetic chemical pesticides.

Beehive ginger according to primary sources is a species of true ginger, native to Maritime

Southeast Asia primarily grown in the West as an ornamental plant, and has been used in South-East Asia

as a medicinal herb and has gingerol-related components. According to a study about beehive ginger's

characteristics, in the area of agricultural pest control in Sri Lanka, the powder of beehive ginger

(Zingiber spectabile) can be an effective replacement for chemical insecticides to protect stored seeds. lt

serves as a natural insect repellent and has been proven to be environmentally friendly (Chee et al.,

2014) .The phytochemical content of Beehive ginger (Zingiber spectabile) similar to (Zingiber officinale)

7
contains phytochemicals like shogaols, paradols and gingerols. Gingerol being the phytochemical

substance that causes irritation which theoretically can be used as a pesticidal agent (Mao et al., 2019). In

addition to that, volatile oils, phenols, alkaloids, and high oleoresin content are also present in the plant

which farmers in developing countries use as biopesticides for the management of plant diseases which

will be easy for them to adopt using these extracts (Kankam et al., 2020).

Plants from the genus Etlingera and Zingiber are widely used in many parts of the world as food

and medicinal sources. These genus are highly treasured for their herbal remedies in various traditional

healing systems where the rhizomes of Zingiber spp. is regarded to possess’ array of antimicrobial,

anticancer and antiinflammatory properties. (Anuar et al., 2018). Various communities around the world

have used the plant or part of the plant in their daily lives apart from its medicinal and decorative

purposes. In the area of agricultural pest control in Sri Lanka, the powder of Z. spectabile can be an

effective replacement for chemical insecticides to protect stored seeds. lt serves as a natural insect

repellent and has been proven to be environmentally friendly. There are also many reported uses of Z.

spectabile as traditional medicine and as food. The young and tender shoots are always consumed as

"ulam" or eaten fresh as salad. lt is sometimes used as extra flavoring in cooking. In traditional Malay

medicine, the leaves of Z. spectabile are pounded into a thick paste and applied to injured parts of the

body; it is used to reduce inflammation and to treat backaches and headaches. (Hoo et al., 2014).

The qualitative phytochemical screening of the extracts is performed to identify the main groups of

chemical constituents (glycosides, alkaloids, tannins, saponins, terpenoids, carbohydrates, cardiac

glycosides, anthraquinones glycosides, flavonoids, and phenols) present in the extracts using the color

reactions. Through brine shrimp lethality assay (BSLA) a simple and inexpensive bioassay used for

testing the efficacy of phytochemicals present in the plant extracts. The extracts, fractions and pure

isolated compounds are routinely evaluated in a test for lethality to brine shrimp larvae. It is based on the

killing ability of test compounds on a simple zoological organism-brine shrimp. (Wu, 2014) The nauplii

(brine shrimp) were exposed to different concentrations of plant extract for 24 hours. The number of

8
motile nauplii is calculated for the effectiveness of the extract. It is simple, cost effective and requires a

small amount of test material. The lethality of the test sample in a simple zoological organism like the

brine shrimp (Artemia salina) has been utilized by many researchers and has proven to be a useful tool in

screening various chemical compounds found in various bioactivities. (Kale et al., 2019).

B. Statement of the Problem

This study aims to examine the vulnerability of pests to the pesticidal properties that are

found in Beehive Ginger (Zingiber Spectabile).

This study will attempt to answer the following questions:

1. What are the phytochemicals present in Beehive Ginger (Zingiber Spectabile) that

provide pesticidal property?

2. What is the percentage mortality of beehive ginger (Zingiber Spectabile) plant extract

against brine shrimp?

3. What is the median lethality concentration (LC50) of beehive ginger (Zingiber

Spectabile) plant extract against brine shrimp?

4. Is there a significant difference on the median lethality concentration of beehive ginger

(Zingiber Spectabile) plant extract and negative control?

C. Research Objectives

This study aims to help farmers and gardeners protect their crops and plants against pests

using the pesticidal properties of Beehive Ginger (Zingiber Spectabile) leaves extract.

1. To determine the phytochemicals present in Beehive Ginger (Zingiber Spectabile) that provide

pesticidal property.

9
2. To measure the percentage mortality of beehive ginger (Zingiber Spectabile) plant extract

against brine shrimp.

3. To determine median lethality concentration (LC50) of beehive ginger (Zingiber Spectabile)

plant extract against brine shrimp.

4. To compute the significant difference on the median lethality concentration of beehive ginger

(Zingiber Spectabile) plant extract and negative control.

D. Statement of Null Hypothesis

1. There is no significant difference on the median lethality concentration of beehive ginger

(Zingiber Spectabile) plant extract and negative control.

E. Significance of the Study

This study aims to provide benefits for the following sectors:

Bureau of Agricultural Research. This study's findings will contribute additional information

and emphasis towards the further development of pesticides.

Department of Agriculture (DOA). This study's findings will captivate them with the potential

of Beehive Ginger’s pesticidal property and further explore its agricultural benefits for the

welfare of their sector.

10
Fertilizer and Pesticide Authority (FPA). This study's outcomes will benefit their agency with

information of Beehive Ginger’s (Zingiber Spectabile) potential as a pesticide and use it as a

basis for the future development of organic pesticides.

Students and Researchers. This study can be used as a basis for future related research

involving phytochemical screening of a plant and its characteristics.

F. Scope and Limitations

This study focuses on the analysis and the capability of Beehive Ginger’s (Zingiber

Spectabile) pesticidal property . The test to be conducted in our study is the brine shrimp

lethality assay. It is an inexpensive bioassay that tests the efficacy of the plant extract. From this,

we can determine if the plant extract of beehive ginger is applicable for pesticide use. The

limitations of our study are the limited test to be conducted to assess the efficacy of the plant

extract. A limited test is to be conducted in our study because through brine shrimp lethality

assay we can already determine the effectiveness of the phytochemicals present in beehive

ginger leaves extract and if it is really applicable for farmers to use as a pesticide.

G. Theoretical Framework

The framework of this study is anchored to the theory of Beehive ginger (Zingiber

Spectabile) as a pesticide and can be used as an alternative for today's harmful synthetic

pesticides. Phytochemicals that are commonly found in botanic pesticides include bioactive

compounds such as steroids, alkaloids, tannins, terpenes, phenols, flavonoids and resins that

possess antifungal, antibacterial, antioxidant or insecticidal properties. (Lengai et al., 2020)

11
 The phytochemical content of Beehive ginger (Zingiber Spectabile) similar to (Zingiber

officinale) contains phytochemicals like shogaols, paradols and gingerols. Gingerol being one of

the phytochemical substances that causes irritation which theoretically can be used as a pesticidal

agent (Mao et al., 2019). In addition to that these phytochemicals also have phenolic properties

that act as protective agents, inhibitors, natural animal toxicants and pesticides against invading

herbivores, nematodes, phytophagous insects, and fungal and bacterial pathogens. (Dar et al.,

2017)

H. Conceptual Framework

Independent variable Moderating variable Dependent variable

A. Behive Ginger Median Lethal

Contact Time
Plant Extract Concentration
B. Negative Control (mins) (ug/mL)

Figure 1 . The Schematic Diagram of the Study

12
 CHAPTER 2

REVIEW OF RELATED LITERATURE

Zingiberaceae family are widely used throughout the world as food and medicinal plants.

Beehive ginger, or Zingiber spectabile, is a variety of ginger that is native to Southeast Asia. It

belongs to the Zingiberaceae family, which also encompasses a number of other ginger types

such as torch ginger, Thai ginger, and cardamom. Beehive ginger plants (Zingiber spectabile) are

named for their distinct floral form that resembles a small beehive (Grant, 2020). Beehive ginger

thrives in warm, frost-free climates that are similar to its native habitat of India and China. Like

other plants in the Zingiber genus, the cone-like bracts, which are not anatomically flowers, are

much showier than the actual flowers, which protrude from the bracts. (Mcintosh, 2021).

In the tropical jungle of Davao, Mindanao, Philippines, close view of Zingiber spectabile

blossoms (Beehive ginger). Though beehive ginger can be used in cooking, it is commonly

planted for its aesthetic value. It can be dried or cut fresh and used as a dramatic addition in

flower arrangements. The leaves and rhizomes of beehive ginger have a flavor comparable to

that of common ginger.

Zingiber Spectabile is one of the many genera that occur within the family Zingiberaceae

(Chee,et.al,2010). Beehive Ginger rhizome (Zingiber Spectabile) is one of the plant sources of

pesticides (vegetable pesticides) that can be used for pest control because it contains zingeron

ketone compounds that have a spicy taste, causing the insect body to feel hot, fever and die.

13
 Varieties of ginger have gingerols, shogaols, and paradols mainly as phenolic compounds

(Siddiqui et al., 2018). Gingerols is the source of hotness of the ginger; a type of chemical

irritant, a substance that causes irritation. Shogaols are pungent constituents of ginger similar in

chemical structure to gingerol. Shogaols are important biomarkers used for the quality control of

many ginger-containing products, due to their diverse biological activities. Paradol is also found

in ginger, it is the active flavor constituent of the seeds of guinea pepper.

The brine shrimp lethality assay (BSLA) is a simple and inexpensive bioassay used for

testing the efficacy of phytochemicals present in the plant extracts. The present study determined

that the extent of lethality was directly proportional to the concentration of the extract (Kale

et.,al,2019). Brine shrimp lethality assay is an important tool for the preliminary cytotoxicity

assay of plant extract and others based on the ability to kill a laboratory cultured larvae (nauplii).

The nauplii were exposed to different concentrations of plant extract for 24 hours (Sarah et.al,

2017).

Lethal Concentration or LC values usually refer to the concentration of a chemical in air

but in environmental studies it can also mean the concentration of a chemical in water.

According to the (Organization for Economic Cooperation and Development) (OECD)

Guidelines for the Testing of Chemicals, a traditional experiment involves groups of animals In

the fight against arthropod crop pests using plant secondary metabolites, most research has

focused on the identification of bioactive molecules. Several hundred candidate plant species and

compounds are now known to have pesticidal properties against a range of arthropod pest

14
species. (Tembo et.al,2018)exposed to a concentration (or series of concentrations) for a set

period of time (usually 4 hours). The animals are clinically observed for up to 14 days. The

concentration of the chemical in air that kills 50% of the test animals during the observation

period is the LC50 value. Other durations of exposure (versus the traditional 4 hours) may apply

depending on specific laws.

Pesticides are substances that are meant to control pests. Pesticides are chemical

compounds that are used to kill pests, including insects, rodents, fungi and unwanted plants

(weeds). According to the World Health Organization (WHO), over 1000 different pesticides are

used around the world. Pesticides are used in agriculture to kill pests that damage crops.

Pesticide is any substance or mixture of substances intended for preventing, destroying,

repelling, or mitigating any pest. There are chemical pesticides available in the market, however

the possibilities are present that these pesticides can ruin the leaves of the plant and it can also

harm farmers if it is near or accidentally applied in the skin. Pesticide exposure causes immediate

irritation of the nose, throat, and skin, resulting in burning, stinging, and itching, as well as

rashes and blisters. Pesticides can induce both acute and chronic harmful health consequences,

which can occur months or years after exposure.

Pesticides are agrochemicals that are used to protect plants and humans from numerous

diseases in agricultural fields, public health programs, and urban green spaces. Their side effects,

on the other hand, can be a significant environmental health risk factor due to their proven

15
capacity to create a wide range of detrimental health and environmental repercussions

(Maipas,et.al,2016).

There are few types of pesticides in which some bring the most harm and some do not.

Synthetic pesticides being one of them which is a substance that is formulated or manufactured

by a chemical process or by a process that chemically changes a substance extracted from

naturally occurring plant, animal, or mineral sources (McCoy et. al., 2020). According to this

analysis, exposure to synthetic pesticides is associated with adverse health outcomes in

agricultural workers. The authors describe these outcomes highly related to different types of

cancers, genomic damage, oxidative stress, neurological disorders and respiratory, metabolic and

thyroid effects (Hazlegreaves, 2020). Many organic pesticides are less toxic than their synthetic

counterparts, but that doesn't mean they are safe or won't cause environmental harm. Just like

synthetic pesticides, organic pesticides are formulated to kill.

Plant pests and pathogens interfere with the growth and cause damage to cultivated and

naturally growing plants. The interference and damage result in the failure of plants to reach their

genetic potential. Plant pest and disease crop losses are a global issue (Parray, et.al, 2020). Pests

reduce plant density, cause plant stunted growth and their death, cause lower production

capacity, damage berries, and in many other ways reduce the yield or quality of agricultural

products. Agricultural pests are plants, animals, and fungi that reduce crop yields

In the fight against arthropod crop pests using plant secondary metabolites, most research

has focused on the identification of bioactive molecules. Several hundred candidate plant species

16
and compounds are now known to have pesticidal properties against a range of arthropod pest

species. (Tembo et.al,2018). Pests harm the majority of crops in our farmlands. These pests are

feasting on our crops, causing our agricultural output to collapse. To protect these crops from

pests, we developed insecticides, which proved to be quite effective in eliminating the bugs.

Mkindi, Angela et.,al (2015) Pesticidal plants are scientifically proven for their

effectiveness in controlling insect pests. Their activity is enhanced by active compounds

contained, which are known for their repellant and antifeedant potentials to the insects. Use of

pesticidal plants by local small scale farmers has been a point of concern following information

that the majority of farmers do not widely use pesticidal plants despite an indigenous knowledge

that they possess. Improvement of the technologies used by local farmers in previous times, that

are easy and effective, need to help farmers abstain from the use of synthetic pesticides that are

detrimental to the environment and to their own health. This paper reviews the potentiality of

pesticidal plants to small holder farmers. It also gives the status of pesticidal plants use, their

possible effectiveness against insect pests, persistence as well as the knowledge that indigenous

people possess in their use.

Mwine, Julius et.,al (2010) Fresh extracts from three locally accessible pesticidal plants

were tested in the field against two key cabbage insect pests on the banks of Lake Victoria in

Central Uganda, in order to establish and assess their effectiveness for future pesticidal

application. This was in response to the farmers' belief that the plants may have pesticidal

properties or have been observed to protect leafy crops from pests. Following a hint from local

farmers that the plants would be useful for pest control, locally available plants were utilized.

17
 Tlannka, Nicudemus et.,al (2020) Bambara groundnuts (Vigna subterranea (L.) Verdc.)

are an important African leguminous crop used primarily for their very nutritious grains. Many

insect pests, such as aphids, leafhoppers, foliage beetles, pod sucking bugs, red spider mites,

groundnut jassids in the field, and bruchids. Synthetic insecticides are commonly used by

smallholder farmers to manage insect pests. Synthetic pesticides, on the other hand, have the

potential to affect the environment, non-targeted animals, and human health.

18
 CHAPTER 3

METHODOLOGY

Research Design

This study employed the descriptive-quantitative research design in assessing the safety

and efficacy of beehive ginger (Zingiber spectabile) leaves crude plant extract as a natural

pesticidal agent using brine shrimp lethality assay . Quantitative determination of crude plant

extract activity is measured following standard procedures.

Sampling Design

The researchers used the non probability purposive sampling design in the collection of

beehive ginger (Zingiber spectabile) leaves plant material. The researchers harvested the beehive

ginger (Zingiber spectabile) leaves in the Tandag City Surigao Del Sur. The laboratory analysis

was carried out at WVN Research and Laboratory in Davao City.

Data Gathering Procedures

This section shows in a step-by-step manner the course of experimentation. It also

provides narrative descriptions of various aspects of the study from the collection of materials to

analysis of data.

A. Collection and Extraction of beehive ginger (Zingiber spectabile) leaves

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 The beehive ginger (Zingiber spectabile) leaves plant material was collected in Tandag

City Surigao Del Sur. About one hundred grams (100 g) of freshly washed beehive ginger

(Zingiber spectabile) leaves plant material was completely macerated in 200 mL of 95% ethyl

alcohol for 24 hours and were filtered. The crude ethanolic extract was then concentrated by

using a rotary evaporator at temperature of 600C, resulting to a thick, viscuous, and syrupy

extract.

B. Phytochemical Screening (Guevera et al., 2004)

Several tests were conducted to determine the bioactive compounds present in beehive

ginger (Zingiber spectabile) leaves extracts. To determine the presence of alkaloids,

Dragendorff’s test was conducted. Froth test for Saponins; Liebermann-Burchard for Steroid/

triterpenoid and ferric chloride test was used to determine the presence of tannins (A Guidebook

to Plant Screening: Phytochemical and Biological).

C. Cytotoxicity Brine Shrimp Lethality Assay of Beehive Ginger Extract

C.1. Preparation of Artificial Seawater

Some 3.8 grams of rock salt was weighed and mixed in 100 ml of distilled water using a

stirring rod until the salt had completely dissolved.

C.2. Hatching Brine Shrimp Eggs

A petri dish was filled with artificial seawater and sprinkled with a pinch of brine shrimp

eggs. It was placed in the lighted area of the laboratory to allow the shrimp eggs to hatch. After

48 hours, the eggs hatched into nauplii (larvae).

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C.3. Concentration of Plant Extract

Twenty five of the extract was dissolved in 5 mL methanol and labeled solution A, 0.50

ml of solution A was diluted to 10 mL methanol and labeled solution B. Volumes of 100 µL

(microliter) of solution B, 50 µL of solution A and 500 µL of solution A were transferred

accurately into separate test tubes and were labeled as: test tubes 1, 2 and 3. The test tubes were

placed inside the incubator for 500C for 24 hours to allow the methanol to evaporate. This was

done in three replications.

C.4. Bioassay

The artificial seawater was added to each test tube to bring the volume to 5 mL. Ten (10)

nauplii were transferred to each test tube using a pipettor. A drop of baker yeast solution was

introduced to each test tube as food for the nauplii. After 24 hours, the number of dead brine

shrimps were counted and recorded.

Biostatistics Used in the Study

In this study the researchers employed single factors Analysis of Variance (ANOVA) and

Least Significant Difference (LSD) tests to determine the significant difference on the pesticidal

property of beehive ginger extract in the brine lethality shrimp assay.

21
Schematic Diagram of Research Procedures

22
 CHAPTER 4

PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

This chapter presented in detail the overall data obtained from the experimentation phase

of the study. It aims to determine the pesticidal property of beehive ginger extract based on the

brine shrimp lethality assay. Inferential statistics using One-Way Analysis of Variance was

utilized to assess which of the three treatments revealed the highest mortality rate against the test

organism.

Phytochemical Screening of Plant Material

Presented in Table 1 are the results of phytochemical screening of some secondary plant

metabolites of ethanolic Beehive Ginger plant extract. The tests followed standard procedures

from University of Santo Thomas by Guevara, B. (2005).

Table 1

Phytochemical Components of Beehive Ginger Crude Plant Extract

Trial Number
Components
1 2 3
Alkaloids + + +

Saponins + + +

Steroids + + +

Tannins ++ ++ ++

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 The phytochemical investigation of the extract of the beehive ginger plant found the

presence of the secondary metabolites alkaloids, saponins, tannins, and steroids. The fact that

this type of plant contains alkaloids, saponins, and steroids demonstrates that it has the potential

to serve as an excellent source of medication. According to Gibson (2002), secondary

metabolites are molecules that are not required for the growth and reproduction of a plant.

However, secondary metabolites have the potential to operate as phytoalexins, which kill

microorganisms that the plant identifies as a threat. Tannins, particularly ellagitannins, are

endogenous inhibitors of the growth of several pest species. They serve as a chemical that acts as

an antibiotic or as a deterrent that prevents insects and aphids from obtaining nourishment

(Torawane and Mokat, 2020). Previous research has demonstrated that saponins have a potent

insecticidal effect on both the diamondback moth (Plutella xylostella) and the aphid (Aphis

craccivora) (Cui et al. 2019). On the other hand, alkaloids are one of the most diverse and

prominent families of natural products that are both important from a pharmacological and

toxicological standpoint. There have been over 10,000 reported instances of alkaloids. Since the

beginning of agriculture and the public health movement, plant extracts containing insecticidal

alkaloids as bioactive ingredients have been an essential component in the fight against pest

insects. This fight has been ongoing for millennia. While the direct application of these

chemicals has recently decreased, they continue to be used as leads for the development of

synthetic analogs and are also essential biochemical tools for the investigation of modes of

action. In recent years, the function of these alkaloids in the host plant has begun to unfold. It is

now generally believed that the ecological role of these compounds, often acting in concert with

other nonalkaloidal substances, is to provide a chemical defense against predators and pathogens

in a sustained manner through multiple biological mechanisms. This notion is supported by the

24
fact that it is now common knowledge that the role of these compounds in the ecosystem is to

provide a chemical defense against predators and pathogens (Ujvary, 199).

Screening for Level of Toxicity of Plant Extract

The percentage mortality rate of brine shrimp larvae in different concentrations of crude

Beehive Ginger were computed using the equation below and overall results are shown in table

2.

Death in test vial – death in control vial

% death = death in control vial x 100%

Table 2

Pesticidal Property of Beehive Ginger Extract using

the Brine Shrimp Lethality Assay

Percentage Mortality Mean SD

Concentration, ug/ml
T1 T2 T3

5.0 ppm 30 30 30 30.0 0.000

50 ppm 50 50 50 50.0 0.000

500 ppm 80 70 80 77.0 5.787

Negative control 0 10 0 3.3 5.774

25
 Results of the pesticidal test using the brine shrimp lethality assay have indicated the

beehive ginger possessed pesticidal property against the test organism in concentration

dependent manner. This means that the plant extract induced mortality to the shrimp larvae

depending on the concentrations of the plant extract utilized. It was clear that the higher the

concentration of the plant extract utilized the higher the mortality rate.

To determine at which concentration the mortality rate was found to be highly significant,

One-Way Analysis of Variance (ANOVA) was utilized and results are shown in table 3

Table 3

One-Way Analysis of Variance in the Mortality Rate of Brine Shrimp

Source of Sum of Mean p

Variation Squares df Square F value Remarks*

Between 8666.667 3 2888.889 173.3 .000 Significant /

Groups Reject H0

Within 133.333 8 16.667

Groups

Total 8800.000 1

*Calculation was performed at 0.05 level of significance

26
 Table 4

Post Hoc Multiple Mean Comparison Test in the Mortality Rate of Brine Shrimp

(I) Types of (J) Types of Mean Difference (I- P

Treatment Treatment J) value Remarks*

5.0 ppm 50.0 ppm -20.00000* .000 Significant

500 ppm -46.66667* .000 Significant

Negative control 26.66667* .000 Significant

50.0 ppm 5.0 ppm 20.00000* .000 Significant

500 ppm -26.66667* .000 Significant

Negative control 46.66667* .000 Significant

500 ppm 5.0 ppm 46.66667* .000 Significant

50.0 ppm 26.66667* .000 Significant

Negative control 73.33333* .000 Significant

Negative control 5.0 ppm -26.66667* .000 Significant

50.0 ppm -46.66667* .000 Significant

500 ppm -73.33333* .000 Significant

*Calculation was performed at 0.05 level of significance

Results of the statistical analysis revealed that there is an existing significant difference

(p<0.05) in the different types of concentrations of beehive ginger extract in inducing mortality

against the brine shrimp larvae. This indicates that the null hypothesis indicating that there is no

27
significant difference in the pesticidal property of the difference concentrations of beehive ginger

extract is rejected.

On this note, based on the post hoc multiple mean comparison test, the most potent

concentrations of beehive ginger extract that induces high mortality on test organisms was

500ppm. This means that the plant extract possessed potential pesticidal properties.

CHAPTER 5

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary

28
 In this study, the alternative medicinal plant was evaluated for its possible use as a

pesticide in terms of its ability to cause death in brine shrimp larvae. Concerns about the plant's

safety and effectiveness were investigated. The phytochemical screening and pesticidal analysis

using the brine shrimp lethality assay are two of the procedures that are carried out during the

testing that is performed. Following a phytochemical examination of the plant extract, the

presence of secondary metabolites such as alkaloids, saponins, tannins, and steroids was

discovered. This is evidence that the plant has the potential to serve as an excellent source of

medication. In cytotoxicity testing utilizing shrimp brine assay, the results suggest that the plant

materials explored in this study are poisonous at 500ppm, indicating an 80 percent fatality rate.

This information was obtained from the plant materials. The findings of the statistical study

showed that there is a discernible distinction (p 0.05) between the effectiveness of various

concentrations of beehive ginger extract in bringing about the brine shrimp larvae's demise at the

hands of the various types of beehive ginger extract. This suggests that the null hypothesis

stating that there is no significant difference in the pesticidal property of the different

concentrations of beehive ginger extract is not valid and should be rejected as a result of this

finding. According to the results of the post hoc multiple mean comparison test, the

concentration of beehive ginger extract that caused the highest rate of mortality in the test

organisms was 500 ppm. This was the most effective level. On the basis of these findings, it is

possible to draw the conclusion that the plant extract may have possessed potential pesticidal

qualities.

Conclusions

After thorough investigations of the study, the following conclusion has been deduced:

29
1. Beehive Ginger plant extract contains secondary metabolites: alkaloids, saponins, tannins, and

steroids. These results illustrated the fact that the plant species can be a very good source of

medicine.

2. The screening on pesticidal property using the brine shrimp lethality assay showed that

500µg/ml of crude extract is an active cytotoxic agent.

3. There is an existing significant difference (p<0.05) in the different types of concentrations of

beehive ginger extract in inducing mortality against the brine shrimp larvae.

Recommendations

1. Specific alkaloids and other secondary metabolites should be isolated for medicinal uses.

2. Investigate other therapeutic activities of the plant extract like: antipyretic, diuretic and other

medicinal activities.

3. Investigate the activity of the plant extract as an antifungal agent.

REFERENCE CITED

30
Mao, Qian-Qian, Xiao-Yu Xu, Shi-Yu Cao, Ren-You Gan, Harold Corke, Trust Beta, and Hua-
Bin Li. 2019. “Bioactive Compounds and Bioactivities of Ginger (Zingiber Officinale
Roscoe).” Foods 8 (6): 185. https://doi.org/10.3390/foods8060185.

Dar, Showket, Bashir Rather, Abdul Wani, and Mushtaq Ganie. 2017. “Chemical Science
Review and Letters Resistance against Insect Pests by Plant Phenolics and Their
Derivative Compounds.” Chem Sci Rev Lett 6 (23).
https://chesci.com/wp-content/uploads/2017/11/V6i23_91_CS292048077_Dar_1941-
1949.pdf.
‌
Lengai, Geraldin M. W., James W. Muthomi, and Ernest R. Mbega. 2020. “Phytochemical
Activity and Role of Botanical Pesticides in Pest Management for Sustainable
Agricultural Crop Production.” Scientific African 7 (March): e00239.
https://doi.org/10.1016/j.sciaf.2019.e00239.

‌McCoy, Tim. 2020. “Organic vs. Conventional (Synthetic) Pesticides: Advantages and
Disadvantages.” https://www.pubs.ext.vt.edu/content/dam/pubs_ext_vt_edu/ENTO/ento-
384/ENTO-384.pdf.

Hazlegreaves, Steph. 2020. “Influence of Synthetic Pesticides on Human Health and the Global
Ecosystem.” Open Access Government. November 17, 2020.
https://www.openaccessgovernment.org/influence-of-synthetic-pesticides-on-human-
health-and-the-global-ecosystem/97802/#:~:text=According%20to%20this%20analysis
%2C%20exposure.

“Are Organic Pesticides Safer for My Garden?” 2019. Extension. July 19, 2019.
https://extension.unh.edu/blog/2019/07/are-organic-pesticides-safer-my-
garden#:~:text=Many%20organic%20.

“Yield Losses due to Pests.” 2015. AGRIVI. March 26, 2015.

https://www.agrivi.com/blog/yield-losses-due-to-pests/.

31
Mwine, Julius, and Patrick van Damme. 2010. “Evaluation of Selected Pesticidal Plant Extracts
against Major Cabbage Insect Pests in the Field.” Ir.umu.ac.ug, September.
https://doi.org/http://hdl.handle.net/20.500.12280/1254.

Shah, Riaz. 2020. Pesticides and Human Health. Www.intechopen.com. IntechOpen.

https://www.intechopen.com/chapters/73921.

Mkindi, Angela G., Kelvin M. Mtei, Karoli N. Njau, and Patrick A. Ndakidemi. 2015. “The
Potential of Using Indigenous Pesticidal Plants for Insect Pest Control to Small Scale
Farmers in Africa.” American Journal of Plant Sciences 06 (19): 3164–74.
https://doi.org/10.4236/ajps.2015.619308.

Fernandez-Cornejo, Jorge, Richard F. Nehring, Craig Osteen, Seth Wechsler, Andrew Martin,
and Alex Vialou. 2014. “Pesticide Use in U.S. Agriculture: 21 Selected Crops, 1960-
2008.” SSRN Electronic Journal 124. https://doi.org/10.2139/ssrn.2502986.

“Types of Natural Pesticides - Learn about Organic Pesticides for Plants.” n.d. Gardening Know
How. Accessed March 1, 2023.
https://www.gardeningknowhow.com/special/organic/what-are-organic-
pesticides.htm#:~:text=The.

“Preventing Pests in Your Garden: 5 Strategies for Success.” 2016. Savvy Gardening. December
20, 2016. https://savvygardening.com/preventing-pests-in-your-garden/.

“USDA ERS - Pesticide Use Peaked in 1981, Then Trended Downward, Driven by
Technological Innovations and Other Factors.” n.d. Www.ers.usda.gov.
https://www.ers.usda.gov/amber-waves/2014/june/pesticide-use-peaked-in-1981-then-
trended-downward-driven-by-technological-innovations-and-other-factors/.

32
PICTURES

33
34
Curriculum
Vitae

35
 Curriculum Vitae

Student’s Profile

Name: Althea Jasmine T. Arreza

Nickname: Thea

Address: Boniao Village, Telaje, Tandag City

Birthday: May 21, 2007

Parents

Father: Joevarie S. Arreza Sr.

Mother: Gina T. Arreza

Siblings

Brother: Joevarie T. Arreza Jr.

Sister: Riena T. Arreza

Educational background

Elementary: Tandag Pilot Elementary School

Secondary: Jacinto P. Elpa National High School

Achievements:

Consecutive Honor Student

Personal Motto:

“Live fast, die young, be wild, and have fun”.

36
 Curriculum Vitae

Student’s Profile

Name: Hanna Louise U. Acosta

Nickname: Han

Address: Surpoco Village, Rosario, Tandag City

Birthday: November 04, 2006

Parents

Father: Lou Ann L. Acosta

Mother: Florilyn U. Acosta

Educational background

Elementary: Special Science Elementary School

Secondary: Jacinto P. Elpa National High School

Achievements:

Consecutive Honor Student

Personal Motto:

“Live life to the fullest, challenges may come but you can get pass through it”

37
 Curriculum Vitae

Student’s Profile

Name: Aezra V. Moral

Nickname: Aez

Address: Tandurpas, Mabua, Tandag City

Birthday: March 21, 2007

Parents

Father: Azarias L. Moral

Mother: Raselle V. Moral

Sibling

Sister: Anja V. Moral

Educational background
Elementary: Tandag Pilot Elementary School
Secondary: Jacinto P. Elpa National High Scool
Achievements:
Elementary
Consecutive Honor Student
Ukulele Ensemble Awardee
1st place Choral Reading in English
2nd place Buwan ng Wika Quiz Bee
Highschool
Consecutive Honor Student
2nd place Arts Month Photography Contest
1st place Photojournalism School based Press Conference
2nd place Photojournalism Division Press Conference
1st Runner up in School- Based Cheer Dance Competition 2022

Personal motto:
“I am not afraid of death, death is afraid of me.”

38
RESEARCH
JOURNAL

39
February 15, 2022

One of the panelists, sir Joel Cubio recommended us the title “Phytochemical Screening of
Beehive Ginger leaves extract and its insecticidal property” after our previous study was rejected
during the final defense.

February 19, 2022

We decided that instead of focusing on the insecticidal property of beehive ginger it was best to
focus on the agricultural sector so instead we changed it to its pesticidal property and decided to
work on the introduction.

February 26, 2022

We finalized our research study from chapter 1-3 so that it’ll be sent to our research consultant
Sir Venchie Badong.

February 28, 2022

We contacted Sir Badong on our concerns about the study including the cost of the laboratory
analysis and if it was possible for testing.

March 1, 2022

Sir Badong replied that our study was eligible for testing at a cost of 8,000 pesos and sent his
contact details.

March 4, 2022

The payment for the laboratory analysis was sent to Sir Badong and we were told to send a copy
of our study.

March 9, 2022

We conducted a virtual consultation with Sir Badong in terms of his recommendations towards
our study and his contribution on chapter 4 and 5.

March 31, 2022

The revision of our study has started and we slowly start to work on chapter 1 including the
introduction, scope and limitations and the beneficiaries of the study.

May 26, 2022

With our newly revised chapter 1, during face to face classes we were told to present a report
about our study as practice for the final defense once we have received our results from Sir
Badong.

40
June 12, 2022

We have finally revised chapter 2 of our study and is ready to be sent to our research consultant
so that we can finally receive the results.

June 16, 2022

We have sent the revised version of our study to Sir Badong and awaiting further instructions on
his addition to chapter 3-5.

July 18, 2022

After waiting more than a month we finally received the results for our study and as blessed by
the gay gods as we are, our study was a success! Beehive ginger has the potential to become a
pesticide.

August 22, 2022

A brand new chapter begins as we are told that in our last school year here in Elpa High is that
we are to finalize our study and have it book bounded.

September 18, 2022

Since we are done with our study we had it checked by our grade 10 research adviser the
amazing Madam Millan in preparation for our final defense.

October 14, 2022

The creation of our study’s abstract has began and we also received some references regarding
on how to create the research poster.

October 23, 2022

Preparation for the oral exam about results from our study has began as we began to feel anxious
as each passing day pass by.

October 27, 2022

Our oral exam was a success as we definitely answered the questions which we received
adequate scores for. Though, there were some questions we failed to answer but despite it we
paved the way.

October 28, 2022

So we had our revised chapter 1- checked and revision and polishing is still on going as there
were still errors on the abstract and arrangement of our study.

November 15, 2022

41
Everything is finally done and is ready to be printed and checked by Madam Millan.

November 17, 2022

We rushed and printed and submitted it to Madam Millan hoping it’ll finally be approved.

December 7, 2022

We were given numbers as order for the following reporters as practice for the mock defense.

January 3, 2023

The power point presentation for the mock defense is finally finished and are going to have
rehersals virtually.

January 5, 2023

Mock defense is finally over and we served. Although there were comments on how the power
point presentations needed improvements.

February 25, 2023

Research presentation will be finalized since only a few weeks are left till the final deferense
including our final ppt and tarp stand.

March 1, 2023

Another variant is to be checked again of our study including one with plagiarism checked
evidence.

March 23, 2023

We rushed another super final mega ultra-revised version of our study and there were still some
errors and malfunctions on the plagia checker.

March 24, 2023

We managed to submit the research paper with plagia checked including the envelop to be given
to the panelist.

March 30, 2023

We were able to make a tarpaulin for our study and at a cost of 370 pesos and the final power
point presentation is finally finished.

42
March 31, 2023

Final defense was not what we thought it would be but we survived although we weren’t able to
finish the presentation we were able to answer the panelists but at least we were able to win third
place in best in poster.

June 7, 2023

We consulted sir Badong on the conundrum that we had during our defense where we were
questioned as to why the SD on our negative control and beehive extract was the same although
he said that the SD had nothing to do with the efficacy of the plant extract as a pesticide and
solved for it for the right standard deviation.

June 21, 2023

We are days away from book binding our study and now we are finalizing the last pages of our
study.

43
RESEARCH
POSTER

44
45
Plagiarism
Checker

46
47
48
49