Anthelmenthic Property of Ai Leaf Extract - Forprinting

Iloilo Doctor’s College
College of Medical Laboratory Science

West Avenue Molo, Iloilo City
ABSTRACT
Soil-transmitted helminths (STHs) have traditionally been managed through the
administration of anthelmintics, which are medications designed to eliminate parasitic
worms. Despite these efforts, the emergence of resistant strains of these helminths has
necessitated the exploration of alternative control strategies. Traditionally, elders are
using Azadirachta indica (Neem) leaf extract as an organic medicine because of its known
properties. This plant has been documented to exhibit insecticidal, pesticidal, and
agrochemical properties. This study investigates the anthelmintic properties of
Azadirachta indica (Neem) leaf extract against hookworm larvae to explore its potential
as a local anthelmintic treatment. We aimed to determine the minimum concentration
required to immobilize hookworms and the time needed for effective immobilization.
Neem leaf extracts were prepared at concentrations of 10 mg/ml, 20 mg/ml, 30 mg/ml,
40 mg/ml, 50 mg/ml, 60 mg/ml, and 70 mg/ml and exposed to hookworm larvae for 8,
12, and 24 hours. Using a test tube culture method with positive stool samples and
freshly prepared Azadirachta indica (Neem) leaf extract, which is then turned into
powder, the study measures larval mobility across treatments. Microscopic examinations
assessed the larvae's mobility. Results indicated that at 8 hours, no concentration
immobilized the larvae effectively. At 12 hours, significant immobilization occurred at 50
mg/ml, 60 mg/ml, and 70 mg/ml, though not complete. By 24 hours, concentrations of
60 mg/ml and 70 mg/ml successfully immobilized the larvae. Statistical analysis revealed
significant differences in mobility related to both concentration (F(18,42)=106.928,
p=0.000) and exposure time (F(2,18)=7.216, p=0.005), with increased concentration
and longer exposure correlating with reduced mobility. The results suggest that
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Azadirachta indica (Neem) leaf extract has significant anthelmintic properties, with higher
concentrations and longer exposure times enhancing its effectiveness against hookworm
larvae. This research emphasizes the potential of Azadirachta indica (Neem) leaf extract
as a possible alternative for local anthelmintic treatments.
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Anthelminthic Property of Azadirachta indica (Neem) Leaf Extract against

Hookworm
A Research Paper Presented to the Faculty of the College of Medical Laboratory Science
Iloilo Doctors` College
West Avenue, Molo, Iloilo City
In Partial Fulfillment of the Requirements for the subject Introduction to Research
By
Edmar Saligumba
Angel Dumip-ig
Crystel Jade Delos Reyes
Kuh Angel Tinguban
Annah Maria Noble
Rey Ann Concepcion
December 2023
3
Acknowledgements
The researchers would like to extend their deepest gratitude and appreciation to the
following person who never ceased and have extended their valuable guidance, assistance and
support for the completion of their study.
We extend our heartfelt thanks to everyone who supported us in completing this research.
First, we are deeply grateful to our research adviser, sir Wandro Thomas, for his expert
guidance and encouragement. His support was essential in navigating the challenges of this
study and ensuring its success.
We also wish to thank our Dean, Maria Eden Patriarca for her valuable guidance and
support throughout our research. Her assistance was crucial in shaping our study and achieving
our goals.
Our sincere appreciation goes to the research director, Anna Liza Macalalag, for providing
the necessary materials and equipment. Their support was vital in carrying out the experiments
and completing our research.
We are profoundly thankful to those who generously donated their stool samples. Their
contributions were crucial to our research and greatly appreciated.
We also want to express our gratitude to our parents for their constant support and
encouragement. Their belief in us helped us through the entire research process.
Finally, we give thanks to God for providing us with the strength, wisdom, and
perseverance needed to complete this study.
Thank you all for your invaluable help and support.
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CHAPTER I
Introduction to the Study
Chapter 1 is divided into five parts: (1) Background and Theoretical Framework of the
Study (2) Statement of the Problem (3) Definition of Terms (4) Delimitation of the Study; and (5)
Significance of the Study.
Part One, Background and Conceptual Framework of the Study, explains the reason for the
choice of the problem and presents the Paradigm of the Research Study.
Part Two, Statement of the Problem, states the purpose of the study and the
specific question that the study seeks to answer.
Part Three, Definition of Terms, gives the conceptual and operational meaning of important
words used in the study.
Part Four, Delimitation of the Study, sets the precise limits in the conduct of the study.
Part Five, Significance of the Study, provides the rationale why the research
study is conducted.
5
Background of the study
An estimated 576-740 million individuals worldwide suffer from hookworm infection.
Although hookworms were historically common in the US, especially in the Southeast, infections
have significantly decreased as living conditions have improved. Ascaris, whipworm, and
hookworm are examples of soil-transmitted helminths, or parasitic worms. Collectively, they carry
a significant portion of the global disease burden. Among the most prevalent roundworms in
humans, hookworm is a soil-transmitted helminth (STH). Necator americanus and Ancylostoma
duodenale are nematode parasites that cause infection. The use of human waste as fertilizer or
the discharge of human waste onto soil are common sites for hookworm infections. Hookworms
resides in the small intestine. If the infected person defecates outside (near bushes, in a garden,
or field) or if the feces of an infected person are used as fertilizer, eggs are deposited on soil.
They can then mature and hatch, releasing larvae (immature worms). The larvae mature into a
form that can penetrate human skin. The most common way to be infected with hookworm is to
walk barefoot on contaminated soil. It is also possible to spread one type of hookworm by
ingesting its larvae. (Global Health, Division of Parasitic Diseases and Malaria).
Climate and soil structure are crucial determinants of hookworm prevalence, as the parasite
thrives in tropical and subtropical zones, where moisture and temperature are ideal for larval
development outside the host. The differential distribution of different hookworm species is not
absolute, and mixed infections often occur in individual. An estimated 438.9 million people (95%
confidence interval: 406.3 to 480.2 million) were infected with hookworm in 2010, with the
greatest concentration of hookworm infections in South Asia, Southeast Asia, and followed by
sub-Saharan Africa. (Loukas, et. Al., 2016)
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Soil transmitted helminths are a major public health problem in the Philippines, particularly
among school-aged children; these infected children may suffer from profound physical deficits,
including anemia and malnutrition, stunted growth, reduced fitness, and cognitive delays. Most
protozoan and helminth parasitic infections, prevalence peaks in childhood. However, the age
specific epidemiology of hookworm infection is different: although prevalence is high among
children, it increases until it plateaus in adulthood (Ross. et.al., 2017).
The two major medications used to treat hookworm infections are albendazole and
mebendazole. Data indicates an albendazole single-dose of 400 mg over a mebendazole single-
dose of 500 mg. Three consecutive daily doses of either drug demonstrate superior cure and egg
reduction rates, but it is less convenient for mass treatment campaigns. Alternatively, a 3-day
regimen of 100 mg twice daily, mebendazole, is suitable for stable uncomplicated cases. Another
option is to take pyrantel pamoate 11 mg/kg (maximum 1 g) orally every day for three days.
In accordance with a systematic meta-analysis, single-dose oral albendazole, mebendazole,
and pyrantel pamoate were 72%, 15%, and 31% effective against hookworm infections. The
efficacy of treatment varies depending on age groups, regional distribution, and the severity of
the infection. Both mebendazole and albendazole are usually safe with few transient side effects
such as dizziness, headache, and abdominal upset. Albendazole, however, can cause a sharp,
persistent headache, blurred vision, yellowing of the eyes and skin, intense stomach or
abdominal pain, dark urine, unusual fatigue, easy bruising and bleeding, fever, chills, persistent
sore throat, signs of kidney problems, altered urine volume, disorientation, and stiff neck.
(WebMD LLC, 2023). Drug resistance is a concern when the same medication is used repeatedly,
even while the exact cause is unknown. Pyrantel pamoate and levamisole are alternative
treatments, but neither has equal efficacy with albendazole (GhodeifandJain,2023)
7
To prevent hookworm infections, elders have traditionally introduced neem trees as an
alternative herbal remedy. The neem tree, Azadirachta indica (A. Juss), has been utilized for ages
as a traditional medicine for a wide range of human conditions. Neem is a widely acknowledged
broad-spectrum insecticide and fertilizer with uses in agriculture and other fields. At present, the
broad antibacterial actions of Azadirachta indica Research in the domains of dentistry, food
safety, virology, mycology, and parasitology are being conducted on indica. According to the
aforementioned study topics, a summary of some of the most recent studies demonstrates neem's
potential as a previously undiscovered source of innovative medicines. Furthermore, neem
extracts and chemicals are shown to have the ability to combat biofilm-forming and drug-
resistant organisms, two major pathogen groups for which there are few available treatments.
Also covered are the most recent data on the phytochemistry and safety of products made from
neem. While there is an increasing amount of compelling data to back up the usage of
Azadirachta indica as an antimicrobial, additional studies are clearly needed to determine the
specific mechanisms of action. The investigation of plant-based products as medicines has
historically been overtaken by standard antibiotics, even if herbal medicine continues to be
popular worldwide. But as the demand for novel antimicrobial compounds has grown, many
researchers have increasingly widened the scope of their searches to encompass novel sources
found in plants and other environments. Indeed, mainstream medicine is increasingly receptive
to the use of plant-derived drugs, especially those to which antimicrobial resistance is more
difficult or unlikely to d e v e l o p (WylieandMerrell,2022).
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Statement of the Problem
This study aims to determine the anthelminthic property of Azadirachta indica (Neem) leaf
extract against hookworm, help promote effective local anthelminthic medicine to community.
Specifically, it seeks to answer the following:
1. What is t h e effect of Azadirachta indica (Neem) leaf extract to hookworm after
exposed in different amount in grams?
2. How much is the amount needed to immobilize the hookworm?
3. How much time is consumed to immobilize the hookworm?
Objectives
The main objective of this research is to determine the significant effect of Azadirachta indica (Neem)
leaf extract as anthelminthic against hookworm. In terms of amount (in grams) and time duration
1. To measure the mobility rate of hookworm in different amount (in grams)
2. To measure the effectivity of Azadirachta indica (Neem) leaf extract in a given time (8 hours, 12
hours, and 24 hours).
3. To determine the effectiveness of Azadirachta indica (Neem) leaf extract in different amount (in
grams).
4. To determine whether the difference in amount of Azadirachta indica (Neem) leaf extract are
statistically significant.
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Hypothesis
In view of the preceding problems, the proposed null hypothesis is:
The mobility rate, concentration, and time duration have no significant effect on the
Anthelminthic property of Azadirachta indica leaf extract.
10
Theoretical Framework
Amount (in grams)

used
Azadirachta Effect on
indica (Neem) Hookworm
Leaf Extract Duration of Mobility
Exposure
Figure 1: The Research Paradigm

Positive control: Albendazole (70 mg)
Negative control: Distilled water
This study provides a framework based on research of Kudom et al. (2011) stated the
efficient dose of antimicrobial extract property of Azadirachta indica (Neem), our independent
variable. The microscopic examination performed as our intervening variable based in the study
of Abu Hawsah et al. (2023), showed the general changes in organism used upon responding in
Azadirachta indica (Neem) leaves extract with its corresponding time. In our study, these
approaches influence the mobility rate of hookworm with the corresponding time applied upon
determining the anthelminthic property of Azadirachta indica (Neem) leaves extract.
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Conceptual Framework
INDEPENDENT VARIABLE INTERVENING VARIABLE DEPENDENT VARIABLE
Different Remarks with regards to

the Average Hookworm Mobility
Rate
Azadirachta Different amount (grams)
Azadirachta indica (Neem) leaf Hookworm
indica (Neem)
extract mobility
leaf extract
Different duration for each amount
(grams) of Azadirachta indica
(Neem) leaf extract
Figure 1.1: The Research Paradigm
As shown in Figure 1, the conceptual paradigm shows the anthelmintic effects of Azadirachta
indica (Neem) leaf extract on Hookworm.
The main concern of this study is to assess the effects of Azadirachta indica (Neem) leaf
extract on Hookworm mobility. The conceptual paradigm explains the process by which the
researchers determine the anthelmintic effect Azadirachta indica (Neem) leaf on Hookworm.
Azadirachta indica (Neem) leaf is the independent variable, whereas Hookworm mobility is the
dependent variable. From the results of this study, the researchers were able to determine the
anthelmintic effects of Azadirachta indica (Neem) leaf extract on Hookworm. The researchers
utilized amounts (in grams) of Azadirachta indica (Neem) leaf during different durations (8 hours,
12 hours, 24 hours) and grade them with different remarks base on the average mobility rate.
These intervening variables serve as a link between the independent and dependent variables
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Operational Framework
INDEPENDENT VARIABLE INTERVENING VARIABLE DEPENDENT VARIABLE
Different Remarks with regards

to the Average Hookworm
Mobility Rate (100.00-75.00
Not effective, 74.99-50.00 Less
Effective, 49.99-25.00 Effective,
24.99-0.00 Very effective)
Azadirachta • Different amount (10 mg, 20 Anthelmintic
indica (Neem) leaf mg 30 mg, 40 mg, 50 mg, 60 effect on
extract mg, 70 mg) of Azadirachta Hookworm
indica (Neem) leaf extract
• Different duration (8 hours, 12
hours 24 hours) for each amount
(grams) of Azadirachta indica
(Neem) leaf extract
Figure 1.2: The Research Paradigm
As shown in Figure 2, the operational paradigm illustrates the measured effects of
Azadirachta indica (Neem) leaf extract on Hookworm mobility.
The operational paradigm shows the process by which the researchers measure the
anthelmintic effects of Azadirachta indica (Neem) leaf extract on Hookworm. Azadirachta indica
(Neem) leaf on Hookworm. Azadirachta indica (Neem) leaf is the independent variable, whereas
Hookworm mobility is the dependent variable. From the results of this study, the researchers
were able to determine the anthelmintic effects of Azadirachta indica (Neem) leaf extract on
Hookworm. From the results of this study, the researchers were able to determine the
anthelmintic effects of Azadirachta indica (Neem) leaf extract on Hookworm. The researchers will
utilize amounts (10 mg, 20 mg 30 mg, 40 mg, 50 mg, 60 mg, 70 mg) of Azadirachta indica
(Neem) leaf during different durations (8 hours, 12 hours, 24 hours). A total of three tests were
conducted and observed for every concentration and duration. Different Remarks with regards
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to the Average Hookworm Mobility Rate (100.00-75.00 Not effective, 74.99-50.00 Less Effective,
49.99-25.00 Effective, 24.99-0.00 Very effective) is used. These intervening variables serve as a
link between the independent and dependent variables.
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Definition of Terms
The following terms are conceptually and operationally explained to aid in understanding
the study.
Anthelminthic. Expelling or destroying parasitic worms especially of the intestine
(Meriam-Webster, 2023).
In this study, anthelminthic is the activity of the Azadirachta indica (Neem) against
hookworm.
Azadirachta indica (Neem). Fast-growing tree of the mahogany family (Meliaceae),
valued as a medicinal plant, as a source of organic pesticides, and for its timber (Petruzzello, 2023).
In this study, Azadirachta indica (Neem) is used as the main local plant to study its
anthelminthic property to immobilize hookworm.
Efficiency. Refers to the ability to achieve an end goal with little to no waste, effort, or
energy (Banton, 2023)
In this study, the efficiency of Azadirachta indica (Neem) leaves extract are being
determined as the anthelminthic against Hookworm.
Harada Mori. Method of incubating fecal material on a filter paper strip in a test tube
containing water (cover one-third of the length of the paper strip) for the purpose of culturing
and recovering nematode larvae (GenScript, 2022)
This method will be the kind of culture that this study will perform to attain the larvae form
of Hookworm needed for the experiment.
Hookworm. Any of several parasitic nematode worms (family Ancylostomatidae) that
have strong buccal hooks or plates for attaching to the host's intestinal lining and that include
serious bloodsucking pests (Meriam-Webster, 2023).
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In this study, hookworm is the parasite to be eliminated as the main subject to test
the efficacy of Azadirachta indica (Neem) leaf extract.
Immobilize. To stop something or someone from moving (Cambridge Dictionary, 2023)
In this study, immobilize refers to the a c t i o n of various treatments of Azadirachta
indica leaf extract on the hookworm's ability to move.
Mobilize. To put into movement or circulation (Meriam Webster, 2024)
In this study, mobilize refers to the action of hookworm upon exposure with different
treatments of Azadirachta indica leaf extract.
Pulverized. To press or crush something until it becomes powder or a soft mass
(Cambridge Academic Content Dictionary, 2024).
In this study, it is an extraction that we will perform to attain the powdered form of
Azadirachta indica (Neem).
Soil-transmitted helminths (STH). Refer to the intestinal worms infecting humans that are
transmitted through contaminated soil (Centers for Disease Control and Prevention, 2022).
The study employs the mode of transmission taken by the Hookworm.
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Scope and Limitation of the Study
This study focuses on the anthelminthic property of Neem Azadirachta indica (Neem)leaf
extract at varying concentrations and time durations. It will employ an experimental research
design. Azadirachta indica (Neem) tree leaves were obtained at Dongsol, Pototan, Iloilo. The
quantitative approach used in this is based on the extraction of Azadirachta indica (Neem) leaves
extract by pulverizing. This will be done at Iloilo Doctors’ College, West Timawa, Iloilo,
Philippines.
Azadirachta indica (Neem) tree leaves extract was obtained through pulverizing by mortar
and pestle. A total of three (3) set ups was prepared based on different treatments namely,
Treatment A: 21 test tubes with 10, 20, 30, 40, 50, 60, 70 mg/ml of leaf extract with time of
exposure, Treatment B: 21 test tubes with mg 10, 20, 30, 40, 50, 60, 70 mg/ml of leaf extract
with a time of exposure of 12 hours, and Treatment C: 21 test tubes with 10, 20, 30, 40, 50, 60,
70 mg/ml of leaf extract with a time of exposure of 24 hours. All treatments were triplicated.
The roots, stem, and flowers of Azadirachta indica (Neem) tree were not utilized in this
study. The anthelminthic property of Azadirachta indica (Neem) leaves extract was introduced
only to hookworm. Microscopic examination for each concentration will be performed within the
given time (8 hours, 12 hours, and 24 hours).
If the conducted cultures of Hookworm are still microscopically mobile after the last
duration of exposure, we, the researchers, will continue to monitor the effects of each test until
parasites are immobilized.
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CHAPTER II
Review of Related and Literature Studies
This chapter presents the Review of Related Literature about the topic, the fact and writing
in this chapter will give the researchers and the readers a wider background and information
about the study and thus, give them direction and motivation that would a good progress toward
the goal of this study.
This chapter is composed of (1) Efficacy of Azadirachta indica as an organic plant (2)
Mobility rate of Hookworm upon exposure to Azadirachta indica leaf extract (3) Related studies.
Part One, Efficacy of Azadirachta indica as an organic plant
Part Two, Mobility rate of Helminth upon exposure to Azadirachta indica leaf extract
Part Three, Related studies
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Efficacy of Azadirachta indica (Neem) as an organic plant
Azadirachta indica (Neem), which belongs to the family Meliaceae, is one of the most
versatile medicinal plants, with a broad spectrum of agricultural and medicinal applications
(Anyaehie, 2009; Britto & Gracelin, 2011). This plant has been reported to exert insecticidal,
pesticidal, and agrochemical properties. In addition, the active constituents of neem and its
derivatives are applied in alternative and modern therapy, such as the treatment of diverse
infectious, metabolic, and cancer diseases (Brahmachari, 2004; Ezz-Din et al., 2011; Dkhil et al.,
2012; Gotep et al., 2016; Mohamed et al., 2021; Ishaq et al., 2022). Previous studies
demonstrated the various multi-targeted biological activities of neem, such as hypoglycemic (Murty
et al., 1978), anti-ulcer (Pillai & Santhakumari, 1984), anti-inflammatory (van der Nat et al., 1991),
antimalarial (MacKinnon et al., 1997), chemo preventive (Tepsuwan et al., 2002),
chemotherapeutic
(Paliwal et al., 2005), and antibacterial (Thakurta et al., 2007) properties, as well as an
antioxidant (Yanpallewar et al., 2003), and cardioprotective (Peer et al., 2008) effects. Previous
studies were recorded for the evaluation of anthelmintic effect of neem leaves on Haemonchus
contortus in goats (Radhakrishnan et al., 2007; Rahman et al., 2011; Sakti et al., 2018), and
bovine strongylosis (Jamra et al., 2015).
Azadirachta indica (Neem) is a tropical evergreen with a great deal of versatility.
Originating in India as well as Burma, it has been imported to Africa, the South America,
Australia, and the Middle East. It works well in semi-arid circumstances and flourishes even in the
dirtiest areas with rainfall as little as 450 millimeters (18 inches) a year and temperatures as high
as 120°F (50°C). It could develop into 200 years old, 50 feet (15 meters) tall. The length of time
of the Neem tree is said to be found anywhere 150–300 years in the lifespan range.
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It produces tiny, white flowers with a very nice, jasmine-like aroma. The fruit that it can eat
is roughly long, with white kernels, about 3/4 of an inch (2 cm). In most cases, a neem tree
starts fruiting around three to five years old and is capable of producing 50 kg (110 pounds) of
ripe fruit every year. The pinnate leaves smell like garlic and taste extremely unpleasant (Haque
Rabiu et al).
Over 135 substances have been identified from various neem components, and
numerous reviews have additionally published works regarding the chemical and structural these
compounds' variety. The most significant part of the neem is the neem leaf which has various
applications.
Neem leaves' physio-chemical characteristics contribute to the preservation of general health as
it undergoes additional processing and are utilized to make a variety of medications. Because
organic trees guarantee the preservation of the ecosystem and lessen contamination from
synthetic and environmental pollutants, neem leaves are typically only collected from these
trees.
The identified compounds in different crude extracts from A. indica are hydrocarbons,
terpenoids, phenolic, alkaloids and their derivatives and are biologically active molecules (Kar et
al. 2007; Cock et al. 2009; Lucantoni et al. 2010; Hossain et al. 2013). Aqueous extract of A.
indica leaves and flowers showed anthelmintic activity by inhibiting mitochondrial enzymes of S.
digitata (Banu et al. 1992). Alcoholic and aqueous extracts of flowers of A. indica was tested in
vitro for their antifilarial activity against whole worm, nerve muscle preparation and microfilariae
of S. cervi (Mishra et al. 2005c). Recently reactive oxygen species mediated apoptosis of S.
cervi has also been demonstrated by polyphenol rich ethanolic leaves extract of A. indica.
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Neem extracts possess anti-diabetic, antibacterial and anti-viral properties. The tree stem,
root and bark possess astringent, tonic, and anti-periodic properties. The bark is beneficial in
malarial fever and useful in cutaneous diseases. It is used for external applications in skin
diseases. It possesses antidandruff, antibacterial, antiviral and fungicidal properties. Neem
aqueous leaf extract significantly (P<0.05) prevented changes in the serum levels of bilirubin,
protein, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase.
Similarly, it significantly prevented the histological changes as compared to the group receiving
anti-tubercular drugs. It also significantly reversed the biochemical and histological changes. So,
Neem aqueous leaf extract significantly prevents and reverses the hepatotoxic damage induced
by anti-tubercular drugs in rats.
Traditionally, individuals experiencing discomfort received baths made of raw neem. Poor
people are dependent on several plants’ preparations for the treatment of various ailments in
their traditional system. The use of traditional medicine in getting rid of worm infections is
widespread across the world. It is an undeniable fact that in today’s world, herbal medicine plays
a vital role in health care of large sections of the population, particularly in developing countries,
where they often bridge the gap between the availability and demand for modern medicines. In
many parts of India, especially those inhabited by tribal populations, there persists a rich folklore
regarding the vermicidal and vermifugal properties of many plants. This plant has been reported
to exert insecticidal, pesticidal, and agrochemical properties. In addition, the active constituents
of neem and its derivatives are applied in alternative and modern therapy, such as the treatment
of diverse infectious, metabolic, and cancer diseases (Brahmachari, 2004; Ezz-Din et al., 2011;
Dkhil et al., 2012; Gotep et al., 2016; Mohamed et al., 2021; Ishaq et al., 2022). The present
study has been designed to evaluate the potential role of A. indica leaf extracts as an
21
anticoccidial agent against Eimeria papillata, as well as its in vitro anthelmintic activity. \
Moreover, Rabiu and Subhasish (2011) reported the effect of aqueous extract of neem leaves on
the adult earthworm, roundworm, and tapworms. It contains several biologically active
constituents such as azadirachtin, meliantriol and salanin (Naganishi 1975; Lavie et al. 1967;
Shin-Foon 1984). Of these constituents, azadirachtin has been demonstrated to have
antimicrobial and antifungal properties (Kudom et al. 2011; Ramesh et al. 2011).
Mobility rate of Helminth upon exposure to Azadirachta indica (Neem) leaf extract
Coccidiosis is considered the most important intestinal disease caused by apicomplexan
protozoan parasites belonging to the genus Eimeria (family Eimeriidae) which spends its life cycle
invasive multiplication within the intestinal tract of many species of farm and domestic animals
(Allen & Fetterer, 2002; Mehlhorn, 2014; Lai et al., 2018). Most of the research programs for
parasite control are focused on the use of anti-parasitic drugs. However, indiscriminate and
longtime use of these drugs has led to the emergence of drug resistance and adverse side effects
(Abbas et al., 2011; Dkhil, 2013). Gastrointestinal helminths also affect farming systems
worldwide (Alzahrani et al., 2016). These parasitic infections disrupt nutrient uptake for animals,
resulting in reduced body weight and increased susceptibility to secondary infections
(LópezOsorio et al., 2020)
Search for alternatives to anticoccidial drugs to treat and control coccidiosis is an important
field of study. Several studies used plant extracts as antiparasitic agents with minimum side
effects different in vitro and in vivo studies (Aljedaie & Al-Malki, 2020; Yousaf et al., 2021; Qaid
et al., 2022). This study assessed the potential role of Azadirachta indica leaf extract (AILE) as
22
anthelmintic and anticoccidial effectors. Several studies have reported the anthelmintic role of
AILE (Radhakrishnan et al., 2007; Priscilla et al., 2014; Jamra et al., 2015). Herein, earthworms
were used as a model for the anthelmintic activity of AILE. In the current in vitro study, 200
mg/mL concentration of AILE produced a significant anthelmintic activity that is comparable with
the conventional anthelmintic agent, mebendazole. This finding is in line with Adjorlolo et al.
(2016) and Salma et al. (2021) confirmed the efficacy of AILE due to the presence of active
constituents, i.e., alkaloids, phenolics, flavonoids, saponins, tannins. Roy et al. (2010) reported
that alkaloids may act on the central nervous system to cause paralysis of the parasite and
death.
Gastrointestinal (GI) parasitic infections are one of the major constraints for profitable
dairy industry in tropical and subtropical countries. The economic impact of GI parasites in
livestock industry includes both mortality and morbidity losses in terms of sub-optimum
production of meat, milk, enhanced susceptibility to diseases, losses resulting from condemnation
of carcasses, cost of drugs and veterinary aids (Rajakaruna and Warnakulasooriya 2011). The
control of GI nematodes is mainly based on the use of chemical anthelmintics (Waller 1987) but
these are generally expensive and further not frequently available to farmers residing in rural
areas. In this regard, Azadirachta indica, contains several biologically active constituents such as
azadirachtin, meliantriol and salanin (Naganishi 1975; Lavie et al. 1967; Shin-Foon 1984). Of
these constituents, azadirachtin has been demonstrated to have antimicrobial and antifungal
properties (Kudom et al. 2011; Ramesh et al. 2011). For these various reasons, interest in
the screening of medicinal plants for their anthelmintic activity remains of great scientific interest
(Akhtar et al. 2000).
23
Higher concentration of extract produced paralytic effect much earlier, and the time taken
for death was shorter for all types of worms. The aqueous extract of Neem exhibited
anthelmintic activity in dose-dependent manner showing maximum efficacy at 40 mg/ ml
concentration for all three types of worms. Our plant extract exhibited more potent activity at
lowest concentration (10 mg/ml) against (roundworm) Ascaridia galli. Anthelmintic activity of
the extract was compared with the standard drug Piperazine citrate (Table.1). From the above
results, we can conclude that Neem, which is used traditionally to treat intestinal worm
infections, exihibited significant anthelmintic activity. (Haque Rabiu et al., 2011).
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Related Studies
Azadirachta indica (Neem)which belongs to the family Meliaceae, is one of the most
versatile medicinal plants, with a broad spectrum of agricultural and medicinal applications
(Anyaehie, 2009; Britto & Gracelin, 2011). This plant has been reported to exert insecticidal,
pesticidal, and agrochemical properties. It is used in traditional and modern therapies for various
diseases, including infectious, metabolic, and cancer-related ones (Brahmachari, 2004; Ezz-Din et
al., 2011; Dkhil et al.,2012; Gotepetal.,2016; Mohamedet al.,2021; Ishaq et al.,2022) The
aqueous extract of A. indica leaves and flowers showed anthelmintic activity by inhibiting
mitochondrial enzymes (malate dehydrogenase and malic enzyme) of S. digitata (Banu et al.
1992)
Coccidiosis is a significant intestinal disease caused by apicomplexan protozoan parasites,
which disrupt nutrient uptake in animals (Allen & Fetterer, 2002; Mehlhorn, 2014; Lai et al., 2018)
The study investigated Neem (Azadirachta indica) leaf extract's potential as an anthelmintic and
anticoccidial agent, using earthworms as a model. In vitro studies showed AILE's efficacy,
comparable to mebendazole, due to its active constituents, including azadirachtin, meliantriol, and
salanin, which have antimicrobial and antifungal properties.
A study found that (Azadirachta indica) extract, when used as an anthelmintic agent,
showed comparable activity to mebendazole against live adult A. caliginosa worms. The most
effective dose was 200 mg/kg, with paralysis and death times of 9.328 ± 2.183 and 10.024 ±
1.542 min, respectively. (Abu Hawsah et al., 2023).
“Therapeutic Potentials of Neem Against Malaria Parasite,” study found that controlling
malaria vectors in their larval stage was effective in reducing transmission, but it lost popularity
due to synthetic insecticides and bed nets. The study assessed the effectiveness of neem leaf
25
and seed extracts in combating malaria parasites. The findings suggest neem seeds or leaves
show promise, but further research is needed to confirm their antimalarial potency (Eribe
Jonathan et al., 2023).
“Anthelmintic effect of betel nut (Areca catechu) and neem Azadirachta indica (Neem)
extract against liver fluke (Fasciola spp.),” stated that neem extracts can effectively control Fasciola
spp., a liver fluke disease that affects cattle and buffalo in the Philippines. (Yamson et al., 2023)
Effectiveness of Azadirachta indica (Neem) Leaves and Flubendazole treatment against
Diplectanum in Sea Bass Dicentrarchus labrax. Monogenetic trematodes are a common parasitic
problem causing significant economic losses. Neem Azadirachta indica is a promising medicinal
plant with antimicrobial, immunostimulant, appetite stimulation, anti-inflammatory, and
antiparasitic properties. (Aly et al. in 2022) 22 sheep with gastrointestinal parasite history involved
in study on the in vitro anthelmintic activity of neem Azadirachta indica (Neem) leaves against
sheep strongyle larvae. The results showed that neem Azadirachta indica (Neem) leaves showed
significant anthelmintic efficacy against L3 larvae, with 93% and 83% of the L3 mortality rates
observed post-treatment. The study highlights the therapeutic benefits of neem Azadirachta indica
(Neem) leaves in treating sheep parasitic diseases. (Nurul Haiunnisa et al., 2016).
The investigated Antifilarial effect of Neem Azadirachta indica (Neem) leaves extract in
different solvents on Setaria cervi microfilariae used four extracts prepared with different solvents,
and their mortality rates were recorded. All solvent extracts showed significant anthelmintic
activity, with methanol and ethanol extracts showing the highest mortality rate. The dose-dependent
effects of ethanol and chloroform were analyzed, suggesting the extracts may be used as
phytopharmacological agents in the future (Sharba Kaunsar et al., 2016)
26
Neem Azadirachta indica (Neem) leaf powder has marked anthelminthic properties, even
though it is slow acting. This study supports previous research on the anthelminthic potential of
Neem Azadirachta indica (Neem) leaves in controlling gastrointestinal parasites, particularly
strongyle infection. The anthelminthic property of neem is likely due to the presence of an active
alkaloid, azadirachtin, which interferes with the parasite's central nervous system and blocks the
calcium channel, expulsion of parasites from the host body. Further studies are needed to
determine active components, lethal dose, administration route, and parasite species or
developmental stages (Nirmala Jamra et al., 2014)
The study antimalarial activities of two fractions of neem Azadirachta indica (Neem) leaf
extract (IRDN-A and IRDN-B) compared to chloroquine in in-vitro assays against Plasmodium
falciparum found that the neem-leaf fractions lysed 50% and 100% of developing and mature
gametocytes, respectively. If found safe and effective in vivo, these fractions could form the basis
of new antimalarial drugs that not only cure chloroquine-sensitive and chloroquine-resistant
malaria but also significantly reduce transmission (Benelli et al., (2013)
An in vitro anthelmintic activity of Neem Azadirachta indica (Neem) leaves assay using
adult earthworms, similar to human intestinal round worm parasites stated that the extract
showed significant anthelmintic activity at a concentration of 40 mg/ml, indicating its vermicidal
activity and effectiveness as an anthelmintic. The study used piperazine citrate as a reference
standard (Haque Rabiu et al., 2011)
An in vitro study on the anthelmintic efficacy of medicinal plants from Northeast India
against hookworms tested the crude extracts of these plants on an experimental hookworm
model, Ancylostoma ceylanicum. The parasites were exposed to different concentrations of the
plant- derived components and compared with a reference nematocidal drug, mebendazole.
27
The results showed that the plant-derived components had a dose-dependent onset of paralysis,
with the root peel extract of F. vestita causing paralysis faster than mebendazole. The
phytochemicals of the test plants suggested a vermifugal role, though less effective than in soft-
bodied flukes and tapeworm parasites (Lyndem et al., 2008)
28
Conceptual Literature
A study by Dr. Rajeevet Sharma et al., in a year of 2005, entitled “Dry Fruits and Medicinal
Plants,” stated that Neem, also known as Margosa tree, is an antiseptic in nature. It is a blood
and air purifier. It helps in eliminating digestive problems, cough, cold, fever, and other physical
ailments.
The tree's entire body, including its rind, leaves, wood, flowers, and fruits, has significant
therapeutic and medicinal qualities. Neem has been used in the production of several allopathic,
Ayurvedic, Unani and Allopathic Drug. The use of bark of neem purifies the blood. It is digesting
and reduces wind, bile, and phlegm-related ailments. Neem oil is an excellent antibacterial and a
highly helpful tool for energy transfer. The oil extracted from its ripe, yellow flowers known as
‘Margosa oil’ is very beneficial for curing arthritis, leprosy, leukoderma, and other skin diseases.
Neem is an insecticide, which destroys and kills germs and insects. Dry neem leaves kept in the
cupboard or boxes protect clothes, books etc., from worms and other insects. Neem’s tender
twigs used as toothbrushes are very beneficial to keep gums and teeth strong and shining and
free from bad odor and pyorrhea. Its wood is used for making furniture. Neem tree is a natural
air purifier, which wards off foul gases and gives oxygen. Its medical properties include headache,
ear trouble, dental troubles, cough, constipation, vomiting, digestive problem, especially diarrhea,
by heating the inner bark of Neem tree on Iron Tava: grinding it nicely when burnt to be
pulverized.
In the study by Irma Remo Castro et al., in a year of 2006, entitled “A Guide to Families of
Common Flowering Plants in the Philippines,” stated about the Meliacaea, family name of
Azadirachta indica, its characteristic and physical property including its fruit, flowers, trunk etc.
29
The family Meliacaea includes some food plants such as Lansium domesticum or “lansones,” and
Sandoricum koetjape or “santol.” Some are ornamental such as Azadirachta indica or neem tree,
and Melia azidirach or “paraiso” or pride-of-India, both known to possess insecticidal properties.
30
CHAPTER III
Methodology
Chapter three consists of four parts (1) Research Design, (2) Instrumentations, (3) Data
Gathering Procedure, (4) Data Analysis Procedure.
Part One, Research Design, indicates the framework of research methods and techniques
chosen by the researchers.
Part Two, Instrumentations, refers to the measuring tools that the researchers may use to
collect, measure, and analyze data.
Part Three, Data Gathering Procedure, indicates the methodical process of collecting,
gathering and analyzing the gathered data.
Part Four, Data Analysis Procedure
This study discusses a thorough procedure for obtaining Azadirachta indica (Neem) Leaf
Extract and evaluating its anthelmintic effects against Hookworm larvae. The process involves
fresh leaf extraction and pulverization of Azadirachta indica (Neem) Leaf followed by a test tube
culture method (Harada Mori) to ensure growth of Hookworm larvae. The preceeding materials
will then be utilized for Anthelmintic property testing based on different Azadirachta indica
(Neem) Leaf concentration.
31
Research Design
This study intends to determine the significant effect of Azadirachta indica (Neem) leaf
extract as anthelmintic against Hookworm in terms of concentration and time duration.
This research follows a quantitative approach which will rely on the extraction of
Azadirachta indica (Neem) leaf extract by pulverization. Powdered material will be prepared in
milligrams to get the amount of leaf extract (10, 20, 30, 40, 50, 60, and 70 mg/mL). Harada mori
must then be performed for culturing Hookworm larvae. The researchers would afterwards do a
microscopic viewing for growth before the addition of powdered extract of specific concentration
to test anthelmintic property. Another microscopic examination will be performed together with
the counting of mobile and immobile hookworm per slides to confirm the effectiveness of each
extract. Finally, data will be gathered and analyzed statistically.
Research instruments
The primary research instrument is the Azadirachta indica (Neem) leaf extract that is to be
tested for its Anthelmintic property againts Hookworm larvae. The extract of different
concentrations is given importance as they become the starting off point to record the effectivity
on the Hookworm sample. In addition, bright field microscope is the key instrument used in
observing and recording data by microscopic examination. The entire procedure is preceded by
Azadirachta indica (neem) leaf extraction.
Azadirachta indica (neem) leaf extraction:
• Fresh mature Azadirachta indica (neem) leaf, to be utilized as anthelmintic extract.
• Use blender to pulverize Azadirachta indica (neem) dried leaves
• A weighing scale will be used to measure 500g of powdered Azadirachta indica (neem) dried leaves
32
• Used 1000ml of triple distilled water to soak Azadirachta indica (neem) dried leaves for 24 hours.
• Double filter the extract using muslin cloth and Watman No.5
• Evaporate the extract using water bath to produce powdered Azadirachta indica (neem) dried leaves
Harada Mori Method
• Stool Positive of Hookworm egg sample to be utilized in Harada mori procedure.
• A total of 65 test tubes for Hookworm culture
• Pipette 7ml of distilled water in 65 test tubes
• Use applicator stick to strip positive stool in a 13×200 nm filter paper
• Cover the test tubes with aluminum foil
• Place at room temperature (24°C to 28°C) for 7 to 10 days.
Observation and Measurement
• Microscopic observation: count for the number of mobile and immobile Hookworm.
• Data recording sheets
Data Collection and Procedure
Procedure
Gathering of materials
The Azadirachta indica (Neem) leaves will be gathered in Dongsol Ilaya, Pototan Iloilo. Positive
stool bought in Lanit Jaro, Iloilo City. Other materials will be provided in the laboratory.
1.1 Azadirachta indica (Neem) leaf extraction (liquid)
Freshly collected mature Azadirachta indica (Neem) leaf samples are washed gently with tap
33
water and placed in natural sunlight to initiate the drying process. The leaves are then shredded using a
blender into small parts amounting to 500 grams then weighed. One liter of double distilled water is added
to the shredded leaves, mixed until moist and left to macerate for 24 hours. Placing the leaves on a Muslin
cloth, the researchers squeezed on the cloth until liquid from the leaves are extracted. The extract is then
double filtered with Whatman filter paper, and the resulting liquid is evaporated into powdered form using
an evaporating dish on water bath set-up. Store in refrigerator until use.
1.2 Harada Mori or Test tube culture method
We started this procedure by placing a 7 mL distilled water on a 20 mL test tube. (A total of 63
test tubes will be used for culturing). Using an applicator stick, spread a thin film of feces on the middle
third of a 13x200 nm strip filter paper (one side only). Next, the researchers will insert the smeared filter
paper strip into the test tube with the un-smeared portion touching the water near the bottom of the tube.
Ensure about 1 cm distance of the filter paper from the bottom of the tube. Finally, cover the tube loosely
with an aluminum foil to allow air to get through and place the tube in a dark place at room temperature
(24°C to 28°C) for 7 to 10 days.
34
Data analysis
Microscopic examination is the statistical tool that will be used to determine the
anthelmintic properties of Azadirachta indica (Neem) leaf extract. This procedure will be performed
during the following durations (8 hours, 12 hours, and 24 hours) requiring 21 test tubes in each
treatment containing 10,20,30,40,50,60,70 mg/ml. The study’s results will be derived from the
statistical tool to test whether the extract will showcase the immobilized Hookworm larvae as well
as determine whether the difference in concentration and duration are statistically significant.
35
CHAPTER IV
Results and Discussion
This chapter presents the results, analysis, and interpretation of data gathered. The data
represented in tabular form in accordance with specific questions post in the statement of the problem.
This study is to determine the anthelminthic property of Azadirachta indica (Neem) leaf extract against the
Hookworm. The mobility rate of Hookworm was measured by counting through microscopic observation.
This parasite was chosen as it is one of the most common intestinal parasites among children.
This table shows the total number of mobile and immobile Hookworm counted in 8 hours of exposure in
different concentrations
Time of Exposure (8 hours)

TRIAL 1 TRIAL 2 TRIAL 3
TREATMENT A Mobile Non Non Mobil Non
mobile Mobile mobile e mobile
10 mg/ml 5 0 6 0 10 0
20 mg/ml 6 0 8 0 9 0
30 mg/ml 6 0 10 0 8 0
40 mg/ml 5 0 7 0 8 0
50 mg/ml 4 0 6 0 9 0
60mg/ml 5 0 9 0 6 0
70 mg/ml 5 0 8 0 8 0
In trial 1, all treatments (10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml 50 mg/ml, 60 mg/ml, and 70
mg/ml) shows a total number of Hookworms (10mg/ml=5, 20mg/ml=6, 30mg/ml=6, 40mg/ml=5,
50mg/ml=4, 60mg/ml=5, 70mg/ml=5) that demonstrates mobility, and a 0 number of Hookworms are
non-mobile. In trial 2, all treatments (10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml 50 mg/ml, 60 mg/ml,
and 70 mg/ml) shows a total number of hookworms (10mg/ml=6, 20mg/ml=8, 30mg/ml=10,
40mg/ml=7, 50mg/ml=6, 60mg/ml=9, 70mg/ml=8) that demonstrates mobility, and a 0 number of
Hookworms are still non-mobile. Finally, In trial 3, all treatments (10 mg/ml, 20 mg/ml, 30 mg/ml, 40
mg/ml 50 mg/ml, 60 mg/ml, and 70 mg/ml) shows a total number of Hookworms (10mg/ml=10,
36
20mg/ml=9, 30mg/ml=8, 40mg/ml=8, 50mg/ml=9, 60mg/ml=6, 70mg/ml=8) that demonstrates mobility,
and a 0 number of Hookworms are still non-mobile. In all the treatments (10 mg/ml, 20 mg/ml, 30 mg/ml,
40 mg/ml 50 mg/ml, 60 mg/ml, and 70 mg/ml) from trial 1, 2, and 3, Hookworm larvae were observed
demonstrating a mobile movement. The researchers concluded that the amount of time given is not
sufficient to immobilize the hookworm larvae. Therefore, the research proceeded to perform the same
process using the tubes containing Azadirachta indica (Neem) powder for 12 hours.
This table shows the total number of mobile and immobile Hookworm counted in 12 hours of
exposure in different concentrations

TREATMENT B Mobile Non Mobile Non Mobil Non
mobile mobile e mobile
10 mg/ml 5 0 10 0 13 0
20 mg/ml 7 0 9 0 9 0
30 mg/ml 6 0 10 0 11 0
40 mg/ml 6 0 10 0 11 0
50 mg/ml 6 2 8 2 10 1
60mg/ml 5 1 10 1 11 2
70 mg/ml 5 2 6 1 9 3
In trial 1, all treatments (10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml 50 mg/ml, 60 mg/ml, and 70
mg/ml) shows a total number of Hookworms (10mg/ml=5, 20mg/ml=7, 30mg/ml=6, 40mg/ml=6,
50mg/ml=6, 60mg/ml=5, 70mg/ml=5) that demonstrates mobility, and a 0 number of Hookworms are
non-mobile in concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml and 40 mg/ml. For the remaining
concentrations (50 mg/ml, 60mg/ml, and 70 mg/ml), the initial efficacy of Azadirachta indica (Neem) leaf
extract has occur. As indicated in trial 1 from the table, treatment 50 mg/ml, 60mg/ml, and 70 mg/ml
shows a total number of Hookworm (50 mg/ml=2, 60mg/ml=1, and 70 mg/ml=2) that demonstrate
37
non- mobile. In trial 2, all treatments (10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml 50 mg/ml, 60 mg/ml, and
70 mg/ml) shows a total number of Hookworms (10mg/ml=10, 20mg/ml=9, 30mg/ml=10, 40mg/ml=10,
50mg/ml=8, 60mg/ml=10, 70mg/ml=6) that demonstrates mobility, and a 0 number of hookworms are
non-mobile in concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml and 40 mg/ml. For treatment 50 mg/ml,
60mg/ml, and 70 mg/ml, shows a total number of Hookworms (50 mg/ml=2, 60mg/ml=1, and 70
mg/ml=1) that demonstrate non-mobile. Finally, in trial 3, all treatments (10 mg/ml, 20 mg/ml, 30 mg/ml,
40 mg/ml 50 mg/ml, 60 mg/ml, and 70 mg/ml) shows a total number of Hookworms (10mg/ml=13,
20mg/ml=9, 30mg/ml=11, 40mg/ml=11, 50mg/ml=10, 60mg/ml=11, 70mg/ml=9) demonstrates mobility,
and a 0 number of Hookworms are non-mobile in concentration of 10 mg/ml, 20 mg/ml, 30 mg/ml and 40
mg/ml, and the same remaining concentration (50 mg/ml=1, 60mg/ml=2, and 70 mg/ml=3) demonstrates
non mobile. In 10 mg/ml, 20 mg/ml, 30 mg/ml and 40 mg/ml, the Hookworm larvae were observed
demonstrating a mobile movement. Significant changes were seen during the last three tubes containing 50
mg/ml, 60 mg/ml and 70 mg/ml where some of the larvae show mobility and some are non-mobile. The
researchers concluded that the amount of time given is not sufficient to completely immobilize the
Hookworm larvae. Therefore, the research proceeded to perform the same process using the tubes
containing Azadirachta indica (Neem) powder for 24 hours.
38
This table shows the total number of mobile and immobile Hookworm counted in 8 hours of exposure in
different concentrations

TREATMENT C Mobile Non Mobile Non Mobile Non
mobile mobile mobile
10 mg/ml 7 0 8 0 12 0
20 mg/ml 6 0 10 0 10 0
30 mg/ml 5 1 9 1 10 3
40 mg/ml 2 3 5 5 7 6
50 mg/ml 1 3 0 8 0 7
60mg/ml 0 6 0 6 0 8
70 mg/ml 0 7 0 9 0 11
During the 24-hour mark, the Hookworm larvae in tubes containing 10 mg/ml, 20 mg/ml and 30
mg/ml, 40 mg/ml, and 50 mg/ml of treatment demonstrates mobility of Hookworm, and a complete 0
number of Hookworms are non-mobile in 60mg/ml and 70mg/ml treatment in trial 1, however, in trial 2
and 3, changes have shown in treatment 50mg/ml. In trial 1, treatments (10 mg/ml, 20 mg/ml, 30 mg/ml,
40 mg/ml, and 50 mg/ml) shows a total number of Hookworms (10mg/ml=7, 20mg/ml=6, 30mg/ml=5,
40mg/ml=2, and 50mg/ml=1) that demonstrates mobility, and a total number of Hookworms
(10mg/ml=0, 20mg/ml=0, 30mg/ml=1, 40mg/ml=3, 50mg/ml=3, 60mg/ml=6, and 70mg/ml=7) indicates
non-mobile. The Hookworm larvae were successfully immobilized in the 60 mg/ml and 70 mg/ml
treatment. The data specify that a 0 number of Hookworms are all completely non-mobile (60mg/ml=0
and 70mg/ml=0). In trial 2, treatments (10 mg/ml, 20 mg/ml, 30 mg/ml, and 40 mg/ml) shows a total
number of Hookworms (10mg/ml=8, 20mg/ml=10, 30mg/ml=9, and 40mg/ml=5) that demonstrates
mobility, and a total number of Hookworms (10mg/ml=0, 20mg/ml=0, 30mg/ml=1, 40mg/ml=5,
50mg/ml=8, 60mg/ml=6, and 70mg/ml=9) indicates non-mobile. The Hookworm larvae were successfully
immobilized in 50mg/ml, 60 mg/ml, and 70 mg/ml treatment. The data specify that a 0 number of
39
Hookworms are all completely non-mobile (50mg/ml=0, 60mg/ml=0, and 70mg/ml=0). In trial 3,
treatments (10 mg/ml, 20 mg/ml, 30 mg/ml, and 40 mg/ml) shows a total number of Hookworms
(10mg/ml=12, 20mg/ml=10, 30mg/ml=10, 40mg/ml=7) that demonstrates mobility, and a total number
of Hookworms (10mg/ml=0, 20mg/ml=0, 30mg/ml=3, 40mg/ml=6, 50mg/ml=7, 60mg/ml=8,
70mg/ml=11) indicates non-mobile. The Hookworm larvae were successfully immobilized still in
treatments of 50mg/ml, 60 mg/ml, and 70 mg/ml. The data specify that a 0 number of Hookworms are all
completely non mobile (50mg/ml=0, 60mg/ml=0, 70mg/ml=0). The researchers concluded that the
amount of time given (24 hours) is sufficient to immobilize the hookworm larvae given the treatment
should contain 60 mg/ml 0r 70 mg/ml of Azadirachta indica (Neem) powder
40
Discussion
Table 1. shows the mobility rate of hookworm in different amount of Azadirachta indica (Neem)
leaf extract regardless of time duration.
At 10 mg/ml and 20 mg/ml amount Azadirachta indica (Neem) leaf extract, hookworms have
mobility rate (M= 100.000, SD=0.000) which is the highest mobility rate of hookworm in all concentration
and rated as Not effective. 30mg/ml and 40mg/ml amount Azadirachta indica (Neem) leaf extract,
hookworms have mobility rate (M= 94.4729, SD=8.91197 and M= 82.6496, SD=26.26985 respectively)
which are also rated as Not effective. 50mg/ml, 60mg/ml and 70mg/ml amount Azadirachta indica
(Neem) leaf extract, hookworms have mobility rate (M= 63.4343, SD=42.86493), (M= 62.0953,
SD=46.99231) and (M= 59.1270, SD=45.56586) respectively, which are rated as Not Less effective.
Table 1. Mobility rate of hookworm in different amount of Azadirachta indica (Neem) Leaf
Extract
Mobile
Concentration Mean N Std. Deviation Remarks
10.00 100.0000 9 .00000 Not Effective
20.00 100.0000 9 .00000 Not Effective
30.00 94.4729 9 8.91197 Not Effective
40.00 82.6496 9 26.26985 Not Effective
50.00 63.4343 9 42.86493 Less Effective
60.00 62.0953 9 46.99231 Less Effective
70.00 59.1270 9 45.56586 Less Effective
Total 80.2542 63 34.44305
Legend:
100.00 – 75.00 Not effective, 74.99 – 50.00 Less Effective, 49.99 – 25.00 Effective, 24.99
– 0.00 Very effective
41
Table 2, shows the mobility rate of hookworm in different amount of Azadirachta indica (Neem)
Leaf Extract when grouped according to time duration with their specific concentration.
At 8.00 hours all concentration 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml
and 70 mg/ml amount Azadirachta indica (Neem) leaf extract, hookworms have mobility rate (M=
100.000, SD=0.000) rated as Not effective.
At 12.00 hours all concentration 10 mg/ml, 20 mg/ml, 30 mg/ml, and 40 mg/ml of amount
Azadirachta indica (Neem) leaf extract, hookworms have mobility rate (M= 100.000, SD=0.000) while 50
mg/ml, 60 mg/ml and 70 mg/ml hookworms have mobility rate (M= 81.9697 SD=8.13539), (M= 86.2859
SD=4.05476), and (M= 77.3810 SD=7.43452) respectively, also rated as Not effective.
At 24.00 hours 10 mg/ml, and 20 mg/ml amount of Azadirachta indica (Neem) leaf extract,
hookworms have mobility rate (M= 100.000, SD=0.000), 30 mg/ml of amount Azadirachta indica (Neem)
leaf extract, hookworms have mobility rate (M= 83.4188 SD=6.53888) rated all concentrations as Not
effective. 40 mg/ml of amount Azadirachta indica (Neem) leaf extract, hookworms have mobility rate (M=
47.9487 SD=7.14736) rated as Effective. 50 mg/ml of amount Azadirachta indica (Neem) leaf extract,
hookworms have mobility rate (M= 8.3333 SD=14.43376), 60 mg/ml and 70 mg/ml amount of
Azadirachta indica (Neem) leaf extract, hookworms have mobility rate (M= .0000 SD=.0000) are rated as
Very effective.
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Table 2. Mobility rate of hookworm in different amount of Azadirachta indica (Neem)

Leaf Extract when grouped according to time duration with their specific concentration.
Hours Concentration Mean N Std. Deviation Remarks
8.00 10.00 100.0000 3 .00000 Not Effective

20.00 100.0000 3 .00000 Not Effective
30.00 100.0000 3 .00000 Not Effective
40.00 100.0000 3 .00000 Not Effective
50.00 100.0000 3 .00000 Not Effective
60.00 100.0000 3 .00000 Not Effective
70.00 100.0000 3 .00000 Not Effective
Total 100.0000 21 .00000
12.00 10.00 100.0000 3 .00000 Not Effective
20.00 100.0000 3 .00000 Not Effective
30.00 100.0000 3 .00000 Not Effective
40.00 100.0000 3 .00000 Not Effective
50.00 81.9697 3 8.13539 Not Effective
60.00 86.2859 3 4.05476 Not Effective
70.00 77.3810 3 7.43452 Not Effective
Total 92.2338 21 10.20679
24.00 10.00 100.0000 3 .00000 Not Effective
20.00 100.0000 3 .00000 Not Effective
30.00 83.4188 3 6.53888 Not Effective
40.00 47.9487 3 7.14736 Effective
50.00 8.3333 3 14.43376 Very Effective
60.00 .0000 3 .00000 Very Effective
70.00 .0000 3 .00000 Very Effective
Total 48.5287 21 44.23251
Legend:
100.00 – 75.00 Not effective, 74.99 – 50.00 Less Effective, 49.99 – 25.00 Effective, 24.99 –
43
Table 3, Results shows the difference in mobility in different amount of Azadirachta indica
(Neem) leaf extract and duration.
The effect on mobility of hookworm in different amount of Azadirachta indica (Neem) leaf
extract and in different duration, F(18,42)= 106.928, p=0.000. A probability of 0.000 is less than the
level of significance which is 0.05. Therefore, there is a significant difference in in amount of
Azadirachta indica (Neem) leaf extract and duration: The longer the duration with different
concentration has lower mobility of hookworm. It also shows the result that the effect of duration on
mobility of hookworm, F(2,18)= 7.216, p=0.005. A probability of 0.005 is less than the level of
significance which is 0.05. Therefore, there is also a significant difference in the mobility of
hookworm in terms of the duration: The longer the duration, the lower mobility of hookworm.
Table 3. The difference in mobility in different amount of Azadirachta indica (Neem) leaf
extract and duration.
Type III Sum Mean
Source of Squares df Square F Sig.
Intercept Hypothesis 405766.041 1 405766.041 181.084 .000
Error 40333.721 18 2240.762
Concentration (Hours) Hypothesis 40333.721 18 2240.762 106.928 .000
Error 880.146 42 20.956
Hours Hypothesis 32338.218 2 16169.109 7.216 .005
Error 40333.721 18 2240.762
a. MS(Disinfectant(Concentration))
b. MS(Error)
Moreover, In Table 3 displayed were Pearson’s r test of correlation result to determine whether
there is a significant correlation between the amount of Azadirachta indica (Neem) leaf
extract(concertation), and time duration (Hours) with the mobility rate of hookworm.
Result shows that the amount of Azadirachta indica (Neem) leaf extract(concertation), and time
44
duration (Hours), at 5% level of significance with all p-value equal to 0.00, there are significant
relationship between the amount of Azadirachta indica (Neem) leaf extract(concentration), and time
duration (Hours) with respect to the mobility rate of hookworm. In fact, negative values of Pearson’s r
on concentration and duration means that the increase of amount of concentration and duration
decreases the mobility rate of the hookworm. The magnitude of 0.480 on concentration means that the
relationship between concentration and mobility rate is fair. While the magnitude between duration and
mobility rate has 0.660 the relationship between two variables is moderate. The same result when using
non-parametric test.
Table 4. Relationship in mobility in different amount of Azadirachta indica (Neem) leaf extract
and different duration.
Mobility
Concentration Pearson Correlation -.480**
Sig. (2-tailed) .000
N 63
Hours Pearson Correlation -.660**
Sig. (2-tailed) .000
N 63
45
CHAPTER V
Summary
This study investigated the anthelminthic effects of Azadirachta indica (Neem) leaf extract on
hookworm larvae, exploring various concentrations (10 to 70 mg/mL) and exposure times (8, 12, and 24
hours). The experiment found that after 8 hours, larvae remained highly mobile at all concentrations,
indicating insufficient time for immobilization. Extending the exposure to 12 hours resulted in notable
immobilization at higher concentrations (50, 60, and 70 mg/mL), but larvae at lower concentrations (10,
20, 30, and 40 mg/mL) still moved moderately. Further extending the exposure to 24 hours
demonstrated that only the 70 mg/mL concentration effectively immobilized hookworm larvae
completely. The results indicate that Neem leaf extract possesses significant anthelminthic properties,
with higher concentrations and longer exposure times yielding better immobilization outcomes. These
findings suggest Neem leaf extract could be a valuable alternative for treating hookworm infections,
particularly in resource-limited settings. Future research should focus on understanding the exact
mechanisms of Neem’s anthelminthic action, evaluating its safety profile, and testing its effectiveness in
real-world conditions. Overall, this study supports the potential of Neem leaf extract as a promising
anthelminthic agent.
Conclusions
This study provides data that shows strong evidence of the anthelminthic property of Azadirachta
indica (Neem) leaf extract against the parasitic larvae form of Hookworm. The immobility of Hookworm to
the concentration with the given time of exposure to Azadirachta indica (Neem) leaf extract signifies its
anthelminthic property. The results of this study indicate that the effectiveness of Azadirachta indica
(Neem) leaf extract varies to the amount of concentration and time of exposure: the higher the amount of
concentration, the greater its effectiveness will be and the longer the time of exposure, the higher the rate
of immobility. These variables signify and underscore the potential remarks of anthelminthic property of
Azadirachta indica (Neem) leaf extract. The selective impact of Azadirachta indica (Neem) leaf extract
46
highlights the importance of choosing the right amount varying to time duration for intestinal parasite to
achieve the best health results.
Recommendations
Based on the findings and conclusion made from the experiment conducted, the following recommendations
were given by the researchers.
I. Future researchers could explore the anthelmintic properties of Azadirachta indica (neem) leaf
extract by investigating its effects on other types of parasites.
II. Researchers are recommended to conduct further studies to evaluate the effectiveness of neem leaf
extract against various stages of hookworm development, including eggs, larvae, and adults, to
comprehensively understand its anthelmintic potential.
III. Researchers can furtherly assess the safety profile of neem leaf extract in various doses to ensure it is
non-toxic and safe for potential therapeutic use
IV. Future researchers could further investigate the potential for hookworm resistance to neem leaf.
V. Future researchers could explore the different methods of preparing neem leaf extracts, such as using
various solvents and extraction techniques, to enhance the concentration of active anthelmintic
compounds.
VI. Researchers can explore different concentrations and doses of neem leaf extracts to determine the most
effective dosage for anthelmintic activity.
VII. Researchers could further investigate how neem leaf extracts affect hookworm egg production and
viability.
47
REFERENCES
(Das G, 2014). Anthelmintic efficacy of crude neem (Azadirachta indica) leaf powder against bovine
strongylosis.
(Rabiu Haque, 2011). Investigation of in Vitro Anthelmintic activity of Azadirachta indica Leaves.
(National Library Of Medicine, 2016). In vitro evaluation of antifilarial effect of Azadirachta indica leaves
extract in different solvents on the microfilariae of Setaria cervi.
(Mingala, 2019). Anthelmintic effect of betel nut (Areca catechu) and neem (Azadirachta indica) extract
against liver fluke (Fasciola spp).
(Udeinya, 2006). Fractions of an antimalarial neem-leaf extract have activities superior to chloroquine and
are gametocytocidal.
(N. H. Suhaimi, 2006). In vitro anthelmintic activity of neem leaves (Azadirachta indica) chloroform extract
against the third-stage larvae of strongyles from sheep.
(L.M. Lyndem, 2008). Anthelmintic Efficacy of Medicinal Plants from Northeast India against Hookworms: an
In Vitro Study on Ancylostoma Ceylanicum.
(E. Jonathan,2023). Therapeutic Potentials of Neem Against Malaria Parasite: A Review.
(Abu Hawsah, 2023). In vitro studies for the antiparasitic activities of Azadirachta indica extract.
(S. M.Aly, 2022). Comparative Studies on The Efficiency of Neem Leaves Azadirachta indica and
Flubendazole Treatment Against Diplectanum in Sea Bass Dicentrarchus labrax.
(Ross, 2017). Risk factors for human helminthiases in rural Philippines.
(Wylie and Merrell ,2022). The Antimicrobial Potential of the Neem Tree Azadirachta indica.
(WebMD, 2004). Albendazole - Uses, Side Effects, and More.
(Adam, et al., 2018) Centers for disease control and prevention. About Soil-transmitted helminthscenter.
(J. Bethony, 2016). Hookworm infection.
48
Appendix A. Gant Chart
ACTIVITIES Date Started Date Completed
Submission of Methodology to Technical adviser for 3/ 15/2024 3/19/2024
approval
Gathering of materials needed 3/18/2024 3/21/2024
Experimentation/ Data Collection 4/16/2024 4/26/2024
Data Analysis 5/2/2024 7/3/2024
Report writing 7/10/2024 7/28/2024
Submission of manuscript 9/14/2024 9/28/2024
Presentation of research output
49
Appendix B. Letters
PLANT VERIFICATION
50
51
52
Letter for Laboratory
53
54

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Iloilo Doctor’s College

College of Medical Laboratory Science

Soil-transmitted helminths (STHs) have traditionally been managed through the

administration of anthelmintics, which are medications designed to eliminate parasitic

necessitated the exploration of alternative control strategies. Traditionally, elders are

agrochemical properties. This study investigates the anthelmintic properties of

as a local anthelmintic treatment. We aimed to determine the minimum concentration

Neem leaf extracts were prepared at concentrations of 10 mg/ml, 20 mg/ml, 30 mg/ml,

assessed the larvae's mobility. Results indicated that at 8 hours, no concentration

immobilized the larvae effectively. At 12 hours, significant immobilization occurred at 50

mg/ml, 60 mg/ml, and 70 mg/ml, though not complete. By 24 hours, concentrations of

significant differences in mobility related to both concentration (F(18,42)=106.928,

p=0.000) and exposure time (F(2,18)=7.216, p=0.005), with increased concentration

as a possible alternative for local anthelmintic treatments.

Anthelminthic Property of Azadirachta indica (Neem) Leaf Extract against

Iloilo Doctors` College

West Avenue, Molo, Iloilo City

In Partial Fulfillment of the Requirements for the subject Introduction to Research

Crystel Jade Delos Reyes

Kuh Angel Tinguban

Annah Maria Noble

Rey Ann Concepcion

support for the completion of their study.

study and ensuring its success.

and completing our research.

contributions were crucial to our research and greatly appreciated.

encouragement. Their belief in us helped us through the entire research process.

perseverance needed to complete this study.

Thank you all for your invaluable help and support.

Introduction to the Study

Significance of the Study.

specific question that the study seeks to answer.

words used in the study.

Background of the study

An estimated 576-740 million individuals worldwide suffer from hookworm infection.

humans, hookworm is a soil-transmitted helminth (STH). Necator americanus and Ancylostoma

sub-Saharan Africa. (Loukas, et. Al., 2016)

specific epidemiology of hookworm infection is different: although prevalence is high among

children, it increases until it plateaus in adulthood (Ross. et.al., 2017).

mebendazole. Data indicates an albendazole single-dose of 400 mg over a mebendazole single-

In accordance with a systematic meta-analysis, single-dose oral albendazole, mebendazole,

treatments, but neither has equal efficacy with albendazole (GhodeifandJain,2023)

To prevent hookworm infections, elders have traditionally introduced neem trees as an

potential as a previously undiscovered source of innovative medicines. Furthermore, neem

specific mechanisms of action. The investigation of plant-based products as medicines has

historically been overtaken by standard antibiotics, even if herbal medicine continues to be

difficult or unlikely to d e v e l o p (WylieandMerrell,2022).

Statement of the Problem

Specifically, it seeks to answer the following:

1. What is t h e effect of Azadirachta indica (Neem) leaf extract to hookworm after

exposed in different amount in grams?

2. How much is the amount needed to immobilize the hookworm?

3. How much time is consumed to immobilize the hookworm?

1. To measure the mobility rate of hookworm in different amount (in grams)

hours, and 24 hours).

In view of the preceding problems, the proposed null hypothesis is:

Anthelminthic property of Azadirachta indica leaf extract.

Amount (in grams)

Figure 1: The Research Paradigm