EFFICACY OF AKAPULKO (Senna alata) LEAVES AS

ANTHELMINTIC AGAINST Ascaris suum

Undergraduate Thesis
Submitted to the Faculty of the
Department of Medical Technology
College of Nursing
Cavite State University
Indang, Cavite

In partial fulfillment
of the requirements for the degree
Bachelor of Science in Medical Technology

LEILA NICOLE A. PEREZ

ALLAISA JEWEL D. SANTOS
ARAVELLA C. SARMIENTO
LOUISE APRIL T. VARIAS

December 2023
 I

BIOGRAPHICAL DATA

Leila Nicole A. Perez, eldest child of Ronaldo A. Perez and Maria Elena A. Perez,

was born on June 13, 2002 in Sampaloc, Manila and is currently residing at Malabag,

Silang, Cavite. She finished her primary education at Malabag Elementary School on

2014 and her secondary education at Malabag National High School. She graduated

senior high school at Philippine Christian University, Dasmariñas, Cavite in 2020. She

is currently enrolled at Cavite State University Don Severino Delas Alas Campus in

Indang, Cavite where she pursued the degree of Bachelor of Science in Medical

Technology.

Allaisa Jewel D. Santos was born on July 23, 2002, in Cavite City. She is the only

daughter of Edleene D. Santos and Razil B. Santos. Santos’ childhood was marked

by an early fascination with science. She took her primary education at General

Artemio Ricarte Memorial School from the academic year in 2008-2014. She finished

her secondary education in Colegio de San Francisco from academic year

2014-2018, where she graduated with honors. Furthermore, she completed her

Senior High School in San Sebastian College-Recoletos de Cavite from the

academic year 2018-2020, where she graduated with high honors. In 2020, she

enrolled at Cavite State University Don Severino Delas Alas Campus in Indang,

Cavite under the degree of Bachelor of Science in Medical Technology. She is

currently a member of the Philippine Society of Medical Technology Students - CvSU

Chapter and Red Cross Youth - CvSU Chapter.

Aravella C. Sarmiento, the second daughter of Jennifer C. Sarmiento and was born

at 108 Minantok East, Amadeo, Cavite on June 26, 2002. She accomplished
 I

her primary education at Mariano C. Anacay Memorial Elementary School, Amadeo,

Cavite in 2014 and her secondary education at Amadeo National High School, Cavite

in 2018. She graduated in Senior High School at Philippine Christian University,

Dasmarinas, Cavite in 2020, and is currently enrolled at Cavite State University Don

Severino Delas Alas Campus in Indang, Cavite where she pursued the degree of

Bachelor of Science in Medical Technology.

Louise April T. Varias was born at Alfonso, Cavite on April 20, 2002 and the second

daughter of Rowena T. Varias and Nilo F. Varias. She completed her primary

education at Alfonso Central School, Alfonso, Cavite in 2014. While her secondary

education was at Alfonso National High School, Alfonso, Cavite in 2018 where she

was a consistent honor student, and her Senior High School at Saint Gregory

Academy, Indang, Cavite in 2020. She is currently enrolled at Cavite State University

Don Severino Delas Alas Campus in Indang, Cavite where she pursued the degree

of Bachelor of Science in Medical Technology. Louise is not only academically

inclined but also good in other fields such as music, arts, and sciences
 III

ACKNOWLEDGMENT

First of all, the researchers are grateful to the Almighty God for the bestowed

guidance, patronage, and wisdom needed to accomplish this research.

They also like to express their gratefulness to their family, Azurin-Perez,

Domingo-Santos, Corpuz-Sarmiento, and Tan-Varias, for moral and unending

support especially in the financial aspect, for the words of encouragement, love, and

comfort given by their whole family.

With boundless love and appreciation, the researchers would like to extend their

heartfelt gratitude and appreciation for those individuals— Rowell Bernard A. Avilla,

Deonard Euly M. Matanguihan, Justin Carlo G. Concepcion, who in one way or

another made this research study turn into reality.

The researchers would like to extend profound gratitude to the following:

To their adviser, Ma’am Sarah Jane Denia, RMT whose expertise, consistent

guidance, ample time spent in checking the research paper and abundant advice that

helped this research a success. The researchers extend their thanks to Dr. Agnes

Alimboyoguen, PhD and Dr. Evelyn del Mundo, PhD, for their suggestions, critics,

and for helping in making this study possible. In addition, the researchers would like

to express their appreciation to the supportive faculty members of the Department of

Medical Technology, Sir Rhoyette Cuyo, Sir Calixto Sabela, and Ma’am Suzzeth

Autriz, for their parental presence and constant guidance.

To all the workers at the Trece Martires Slaughterhouse who patiently and

passionately spend their time in finding Ascaris suum, the researchers thank them.

Finally, the researchers express their thanks to each other for informative discussions

and brainstorming, for staying despite numerous sleepless nights.

 IV

ABSTRACT

PEREZ, LEILA NICOLE A., SANTOS, ALLAISA JEWEL D., SARMIENTO,

ARAVELLA C., VARIAS, LOUISE APRIL T. IN EFFICACY OF AKAPULKO (Senna

alata) LEAVES AS ANTHELMINTIC AGAINST Ascaris suum. Undergraduate

Thesis. Bachelor of Science in Medical Technology, Cavite State University, Indang,

Cavite. December 2023. Adviser: Mrs. Sarah Jane Denia

This study was conducted at the laboratory of the Natural Product Laboratory,

Research Center and Department of Medical Technology Laboratory, College of

Nursing, Cavite State University, Bancod, Indang, Cavite from March 2023 to

December 2023. It generally aimed to determine the efficacy of akapulko leaf extract

as anthelmintic against Ascaris suum. Specifically, this study aimed to determine the

mortality rate of the Ascaris suum adult worms to akapulko extract in varying

concentrations and significant differences of the akapulko leaf extract to the

commercial anthelmintic drug, Albendazole, against Ascaris suum adult worm.

The freshly collected akapulko leaves were air-dried, cutted into pieces using

scissors, grinded using a blender, and were placed in 4 liters and 2.5 liters of Amber

jar bottle with 95 percent ethanol solvent. The freshly collected Ascaris suum were

placed in a thermos flask, transported using a private car from slaughterhouse to the

Department of Medical Technology Laboratory and transferred into a 500 mL beaker

after rinsing. Completely randomized design was used in assigning the portion of

each extract with Ascaris suum adult worm.

After 12 hours of observation, the experiment showed that the 100 mg/mL, 50

mg/mL, and 25 mg/mL concentration are significantly effective against Ascaris suum

worms. The concentration that exhibited the highest effectiveness of killing worms

was 100 mg/mL. Based on the mortality rate of Ascaris suum worm within the given

concentrations and time intervals, the researchers concluded that 100 mg/mL
 x

concentration of ethanolic leaf extract is the most comparable with Albendazole.

The study recommends that the 100 mg/mL ethanolic leaf extract can be a good

alternative to Albendazole in treating Ascariasis in pigs.

 VI

TABLE OF CONTENTS

Page

BIOGRAPHICAL DATA……………………………………………………………………...i

ACKNOWLEDGMENT………………………………………………………………………ii

ABSTRACT…………………………………………………………………………………..iii

TABLE OF CONTENTS…………………………………………………………………….iv

LIST OF TABLES……………………………………………………………………………v

LIST OF FIGURES…………………………………………………………………………..x

LIST OF APPENDIX FIGURES…………………………………………………………….x

LIST OF APPENDICES…………………………………………………………………….. x

TITLE PAGE…………………………………………………………………………………. x

APPROVAL SHEET………………………………………………………………………… x

INTRODUCTION………………………………………………………………………….. 1

Background of the study……………………………………………………………… 1

Statement of the problem……………………………………………………………….3

Objectives of the study…………………………………………………………………. 4

Hypotheses……………………………………………………………………………….4

Significance of the study…………………………………………………………..…… 5

Time and place of the study………………….…………………………………………6

Scope and limitation of the study……………………………………………………… 6

Definition of Terms…………………………………………….…………………………6

Conceptual framework of the study…………………………………………………… 8

REVIEW OF RELATED LITERATURE 9

Akapulko Leaf……………………………………………………………………………9
 Ascaris suum………………………………………….……………………….…….…10

Phytochemical component………………………………………………………..…..12

Process of Drying…………………………………...………………………………….13

Process of Extraction……………………………..……………………………………15

METHODOLOGY………………………………………………………………………….. 19

Research design…………………………………………….………………………… 19

Research instruments………………………………………………………………… 19

Materials…………………………………..…………………………………………… 20

Methods and procedures…………………………………………………………...… 20

Phytochemical screening…………………………………..………………………… 22

Preparation of treatment……………………………………………………………… 23

Pretesting…………………………………………………………….………………… 24

Application of treatment………………………………………….…………………… 24

Statistical analysis of data………………………………………………………….… 25

Proposed data presentation……………………………………………...………….. 25

Waste management…………………………………………………………………… 26

Ethical considerations………………………….…………………………………….. 26

RESULTS AND DISCUSSION…………………………………………………....………30

Efficacy of akapulko leaves against Ascaris suum……………….………….…30

A. Mean mortality of Ascaris suum per hour…………….…………….…..30

B. Mean mortality of Ascaris suum per treatment…………….…………..31

Mortality rate of A. suum per treatment at different time interval……………..32

SUMMARY, CONCLUSION, RECOMMENDATION…....………….….…….…………35

Summary…………………….……………..……………………….……………. 35

Conclusion………………….…………….……………………………….………36

Recommendation………………….…………….……………………….………37

REFERENCES…………….……………..……………………….………….…….………77
 EFFICACY OF AKAPULKO (Senna alata) LEAVES AS
ANTHELMINTIC AGAINST Ascaris suum

LEILA NICOLE A. PEREZ

ALLAISA JEWEL D. SANTOS
ARAVELLA C. SARMIENTO
LOUISE APRIL T. VARIAS

An undergraduate thesis proposal submitted to the faculty of the Department of

Medical Technology, College of Nursing, Indang, Cavite in partial fulfilment of the
requirements for the degree of Bachelor of Science in Medical Technology with
Contribution No. ___. Prepared under the supervision of Prof. Sarah Jane V. Denia,
RMT, and Dr. Evelyn Del Mundo, PhD.

INTRODUCTION

Akapulko (Senna alata) is a tropical ornamental shrub that thrives across the

low- and medium-altitude areas of the country, including the southern Philippines. It

is a member of the Leguminosae or Fabaceae family and belongs to the invasive

species in Austronesia, making it abundant in different regions in the Philippines. It is

popularly known as "akapulko" in the local language and is one of the ten herbal

medicines recommended by the Department of Health due to extensive research on

its healing activities. Locally, akapulko is frequently used to treat stomach issues,

lung and mouth ailments, and skin conditions in herbal medicine (Oladeji, 2020). The

presence of Chrysophanic acid (chrysophanol); oxymethyl anthraquinone,

aloe-emodin; rhein; cassi axanthone; tannins; saponins; alkaloids; flavonoids were

reported from akapulko leaves. The presence of immense phytochemicals in the

plant can be utilized in the formulation of novel drugs to combat various diseases

(Abraham, 2016).
 2

Ascaris suum, a giant roundworm that causes ascariasis in pigs, is the most

common and widespread parasite of swine (Ballweber, 2022). It has infected a

countless number of feral pigs and domestic pigs especially those that were grown in

backyard operation (Padilla and Ducusin, 2017). Adult worms of Ascaris suum may

appreciably decrease the growth rate of young pigs; in rare cases, the worms may

mechanically obstruct the intestine. Small intestinal stages experience poor feed

conversion and delayed weight growth as a result of nutritional competition with the

host. Accumulation of lymphocytes seen as white spots (called milk spots) under the

capsule and migration of larvae through the liver causes hemorrhage and fibrosis,

leading to condemnation of the liver at slaughter. It can be diagnosed by

demonstrating the usual eggs (golden brown, thick pitted outer wall), by fecal

analysis, or by looking for large worms in feces (MDS Veterinary Manual, 2022).

In the Philippines, the Department of Health (DOH) considered ascariasis as

one of the major helminth infections found in the country with an estimated

prevalence of 34.69% (Deslyper et. al., 2022). Their high infection rates are primarily

due to inaccessibility to clean water, poor sanitation, and hygiene. The nematode

parasite of Ascaris lumbricoides infects the digestive tracts of an estimated 807

million–1.2 billion people in the world. In areas where Ascaris lumbricoides is absent,

ascaris infections in humans are mainly due to its closely related species Ascaris

suum, a pig-derived Ascaris. Cross-infections have been documented in areas where

both parasites occur.

In spite of the type of worm, anthelmintic therapies, drugs that expel parasitic

worms from the body, like albendazole, are the preferred treatments for Ascaris

infections (Centers for Disease Control and Prevention, 2020). Albendazole is used

to treat worm-related illnesses. It prevents the worm from absorbing sugar (glucose)

which causes the worm to lose energy and eventually perish. There are several dose
 3

forms of this medicine available, including tablets, and infections are generally

treated for 1–3 days.

The drugs are effective and appear to have few side effects (National Library

of Medicine, 2019), including fever, chills, sore throat, mouth sores, feeling

light-headed, and seizure or increased pressure inside the skull, severe headaches,

ringing in the ears, dizziness, nausea, vision problems, pain behind the eyes. Severe

side effects of albendazole may include headache, neck stiffness, increased

sensitivity to light, confusion, fever nausea, vomiting, stomach pain, abnormal liver

function tests, dizziness, spinning sensation, or temporary hair loss (Multum, 2022).

Subsequently, the exploration of the potential of medicinal plants has spread

throughout the Philippines because of the high cost and serious to mild or common

side effects of anthelmintic drugs against parasitic infection. With this foundation of

knowledge in hand, the researchers were inspired to learn more about the qualities of

akapulko leaf extract, particularly in terms of its potential to kill parasites like Ascaris

suum.

Statement of the problem

The study was conducted to determine the efficacy of akapulko (Senna alata)

leaves as anthelmintic against Ascaris suum.

Specifically, this answered the following:

1. What are the phytochemical components of akapulko leaf extract?

2. What is the mortality rate of the Ascaris suum adult worms to akapulko

extract in varying concentrations?

 4

3. What are the significant differences of the akapulko leaf extract to the

commercial anthelmintic drug, Albendazole, against Ascaris suum adult

worm?

Objectives

Generally this study aimed to determine the efficacy of akapulko (Senna

alata) leaves as anthelmintic against Ascaris suum. Specifically, it aimed to:

1. determine the phytochemical component of akapulko leaf extract as an

anthelmintic to Ascaris suum.

2. assess the mortality rate of the Ascaris suum adult worms to akapulko leaf

extract in varying concentrations.

3. compare the significant differences of akapulko leaf extract and commercial

anthelmintic drug, Albendazole, against Ascaris suum.

Hypothesis

Based on the given research objectives, the researchers have formulated the

following null hypothesis:

H0 Different concentrations of akapulko (Senna alata) leaf extract had no significant

difference against Ascaris suum.

Significance of the study

The findings of the study aimed to contribute to the following:

Community - The study helped the community understand that akapulko leaf extract

was beneficial for treating Ascaris suum infection in pigs, enhancing animal

productivity and health, reducing the need for synthetic anthelmintics, making
 5

anthelmintics more widely available at more reasonable prices, and promoting

sustainable farming practices.

Department of Medical Technology - This study contributed to the knowledge of

medical technologists in the field of parasitology as it was used as an alternative

anthelmintic to control ascariasis-related diseases.

Pig Production Companies - The study provided knowledge and perceptions to

keep the pig production safe from the risk of infection.

Medical Practitioners (Veterinary Medicine) - The study had the potential to find

viable alternative treatments for parasitic diseases and provided medical practitioners

with more options for treating parasitic illnesses, particularly when standard

treatments were ineffective.

Future Researchers - The study served as an inspiration for future researchers to

innovate and enhance study design, methodology, and analysis in order to solve the

shortcomings and gaps in the current study, which may result in a development of

more effective and efficient approaches to investigating natural compounds as

anthelmintics.

Time and Place of the study

The study was carried out by the researchers at the Cavite State University -

Main Campus, Indang, Cavite, Philippines (Don Severino Delas Alas Campus),

during the Academic Year 2022-2023, second semester until the A.Y. first semester,

2023-2024. In order to obtain the akapulko extract, this study was also carried out in

the Research Center Building, specifically in the Natural Products Laboratory

Additionally, the study used Ascaris suum worms that were gathered at an animal

slaughterhouse in Trece Martires, Cavite and was authenticated in College of

 6

Veterinary Medicine of Cavite State University, and collection of an akapulko plant in

Amadeo, Cavite.

Scope and Limitations

This study was limited on how well the extract of akapulko (Senna alata)

leaves worked against adult Ascaris suum worms. The performance under normal

conditions and processes, such as extraction and phytochemical screening, different

concentration (100 mg/mL, 50 mg/mL, 25 mg/mL, 12.5 mg/mL), and comparison of

the extract to the commercially available anthelmintic, Albendazole, was structurally

observed. The majority of the studies in the paper was carried out and done in the

Biosafety Level 2 Cabinet.

Definition of Terms

The following terms were defined operationally and was used within the

content of the study:

Akapulko. A coarse, erect, branched shrub, 1.5 to 3 m high from which the

extract was taken; leaves are pinnate and 40 to 60 cm long, with orange rachis on

stout branches and contain anthelmintic properties that was used to test the mortality

rate of Ascaris suum.

Ascaris suum. It refers to an intestinal parasite of pigs that can also infect

people; adult females are approximately 20 to 35 cm; while adult males are 15 to 30

cm. In the study, it was used as a dependent variable where the efficacy of akapulko

leaves extract was tested.

Albendazole. It was used as a positive control in the study.

Efficacy of Akapulko. The ability of akapulko leaf extract to kill the adult

worm of Ascaris suum.

 7

Extraction Process

- Drying: Exposing the akapulko leaves to dry air until the moisture

evaporated.

- Extraction. Extracting of akapulko leaves for phytochemical testing.

Normal Saline Solution. It was used as a negative control in the study.

Mortality Rate. A measure of the frequency of occurrence of death in Ascaris

suum during a specified interval, using the formula of: total number of deaths of A.

suum, divided by the total number of A. suum.

Phytochemical. The presence of tannins, saponins, alkaloids, and flavonoids

from akapulko leaf extract that is not nutritive but is biologically active and contributed

to the anthelmintic property of akapulko.

Class AA Slaughterhouse. This refers to the place of the study located in

Trece Martires, Cavite, where the researchers obtained the Ascaris suum.

Conceptual Framework of the Study

The adult Ascaris suum was used as a test organism, while normal saline

solution, albendazole, and temperature are the controlled variables in this

experiment. The akapulko leaves constituted the independent variable of the

experiment. The process consisted of collecting data from the experimentation, doing

statistical analysis and interpretation, and screening for qualitative phytochemicals in

the extract of the akapulko leaves. The extract was tested on Ascaris suum and

compared to albendazole. The intended results include identifying the

phytochemicals present in akapulko leaf extract, evaluating the effectiveness of the

leaves as an anthelmintic against Ascaris suum at various doses, and contrasting the

ability of akapulko leaves to suppress Ascaris suum with albendazole.

 8

Figure 1. Conceptual framework of the study

 REVIEW OF RELATED LITERATURE

This chapter presents a review of related literature and the variables that are

used and relevant to this study.

Akapulko leaves

The scientific name for akapulko is Senna alata that belongs to the

Leguminosae family. There are other names for it that are frequently used, including

candle bush, craw-craw plant, akapulko, ringworm bush, and ringworm plant. This

plant is cultivated in humid and tropic regions of Asia, Africa, Australia, Mexico, South

America, the Caribbean Islands, Polynesia, Hawaii, Melanesia, West Indices,

Philippines, and different areas of India for medicinal purposes (Adelowo, et.al.,

2020).

In the study conducted by Stuart Jr. (2021), akapulko is a rough, upright,

branching shrub that grows 1.5 to 3 meters high. The 40 to 60 centimeter-long,

pinnate leaves have an orange rachis and are attached to strong branches. Each leaf

contains 16–28 leaflets, each measuring 5–15 centimeters long, broad and rounded

at the apex, and ending in a tiny point. The size of the leaflets gradually increases

from the base to the tip of the leaf. Numerous bioactive substances, including

phenolics like rhein, chrysophanol, kaempferol, aloe emodin, and glycosides, are

found in akapulko. Additionally, it contains fatty acids like oleic, palmitic, and linoleic

acids, anthraquinones like alatinone and alatonal, as well as steroids and terpenoids.

The therapeutic capabilities of the plant are influenced by these secondary

metabolites (National Library of Medicine, 2020).

In relation to that, akapulko has diverse medicinal activities which are mainly

related to antidiabetic, antibacterial, antifungal, antioxidant, and anthelmintic

activities. Chrysophanic acid, found in akapulko, is well known for being particularly

successful in treating skin conditions. The plant itself is useful in so many ways.
 10

Although the roots and blossoms are also employed in some formulations with

therapeutic potential, the leaves are the main portion used for herbal purposes. The

leaves serving several uses also includes treatment for cough and expectorant in

bronchitis and asthma. It is also used in treatment for hypertension, stomach

problems, fever, asthma, venereal diseases, and even snake bites. The decoction of

its leaves are even used as a mouthwash in stomatitis (Stuart Jr., 2021).

In addition, Adelowo, et.al. (2020) stated that its leaves with ethanol as its

solvent can be used as astringent, and vermicide. The use of its natural compounds

offers the advantage of exploring new sources of medication while also considering

the safety and sustainability of these resources. These qualities are especially

beneficial in the context of the rise of drug-resistant diseases, where it is essential to

find efficient and secure therapeutic choices.

Overall, the review of akapulko consolidates existing knowledge on the plant

and highlights its potential in various medicinal fields. It serves as a valuable

resource for researchers, healthcare professionals, and individuals interested in

utilizing natural compounds for medicinal purposes.

Ascaris suum

Ascaris suum is found in pigs worldwide. According to the evidence, they are

most certainly genetically independent populations (Ballweber, 2022). Conversely, in

places where Ascaris lumbricoides does not exist, pig-derived Ascaris is the primary

cause of ascarid infections in humans. Cross-infections have also been reported in

regions where both parasites are present. As a result, the pig ascarid is zoonotic and

has been found in humans from different parts of the world, especially individuals

who have had frequent contact with pigs. It has been documented that migratory

larvae cause visceral larva migrans.

According to Midtunn et al. (2017), the soil-transmitted helminth A.

lumbricoides is believed to infect about 1 billion people worldwide and cause

 11

significant morbidity. A. lumbricoides and A. suum, which are both common in pigs

and impair feed consumption and growth performance, are genetically connected

and physically interchangeable. A. suum infection in pigs serves as a great model for

studying host-parasite interactions of A. lumbricoides and offers a chance to

thoroughly examine the host-parasite interaction in the gastrointestinal tract due to

the similarities between host physiology and parasite species. Additionally, it

demonstrates how A. suum strongly controls host immunological and inflammatory

responses to fuel an immune energy stage. Barbosa et al. (2021) claim that A. suum

can infect people, complete the cycle, and produce symptoms comparable to those

of A. lumbricoides, such as coughing, headaches, diarrhea, respiratory discomfort,

and changes in chest x-rays that coincide with larval migration in the lungs.

According to the National Library of Medicine (2023), the average life of the adult A.

suum is one year and the data provide fresh insight into the immune response to this

parasite and support the ability of A. suum to transmit disease.

The surface of the worm is firm and resists deformation. It is covered with a

thick proteinaceous cuticle which plays an important role, in the absence of circular

muscles, in containing the high hydrostatic pressure of the hemocoel. Look for the

characteristic ornamentation of the cuticle, which in this species consists of fine

circumferential ridges. The cuticle of preserved specimens is fragile and rough

handling will cause it to break or peel away. Females reach larger sizes than males.

The posterior end of males is curved ventrally and looks like a shepherd’s crook. Two

tiny copulatory spicules may be visible protruding from the anus on the inside curve

of the crook. The posterior end of females is not noticeably curved. In both sexes,

however, the mouth is terminal at the anterior end but the posterior end has no

terminal opening. Viewed head-on with the help of a hand lens, the mouth can be

seen to be surrounded by three small lips (Richard Fox Lander University, 2019).
 12

As described by Roberts et al. (2018), the criteria for assessing the death of the

worm include changes in vital physiological functions, such as lack of movement,

changes in color or texture, and alterations in internal structures. The effectiveness of

an extract in causing the death of Ascaris suum would be evaluated based on these

observable changes in comparison to control groups.

Phytochemical component

Anthelmintic effects from plants are thought to derive from bioactive

compounds such as alkaloids, polyphenols, and terpenoids (Cowan, 2019). Chicory

contains a wide range of phytochemicals with proposed bioactivity against several

pathologies and infections. The four main groups are hydroxycinnamic acids,

coumarins, flavonoids and sesquiterpene lactones (SL) (Ferioli and D'Antuono,

2022). Several studies have suggested, based on in vitro experiments, that SL could

be responsible for the anti-parasitic activity of chicory (Peña-Espinoza et al., 2018).

The known SL in chicory are lactucin (LAC), 8-deoxylactucin (8-DOL), lactucopicrin

(LACP), 11,13-dihydro-LAC, 11,13-dihydro-8-DOL and 11,13-dihydro-LACP.

According to the Philippine Medicinal Plant (2020), Senna alata, sometimes

referred to as candle bush or ringworm bush, is a widely used medicinal plant that

has been used for hundreds of years to treat fungus infections. A number of chemical

substances, including 2.2% chrysophanic acid (chrysophanol), oxymethyl

anthraquinone, aloe-emodin, rhein, cassiaxanthone, tannins, saponins, alkaloids,

chrysoeriol, kaempferol, quercetin, and different fatty acids, have been found in

Cassia alata.

Tannin, commonly referred to as tannic acid, which are water-soluble polyphenols.

Due to their deterrent effects on food consumption and digestion, tannins were

historically thought to have little nutritional value. Recent studies, however, indicate

that tannins' primary effects may not be on food intake or digestion, but rather on how

well absorbed nutrients are converted into new bodily compounds (NLM, 2021).
 13

According to the National Library of Medicine (2021), Tannins have been shown

to have a range of physiological effects, including the acceleration of blood clotting,

lowering of blood pressure, lowering of serum cholesterol levels, triggering of liver

necrosis, and modulation of immunological responses. The particular effects vary on

the type and dosage of tannins used.

Biological compounds known as saponins are abundant in legume plants and are

distinguished by their capacity to produce soap-like, stable foams in aqueous

solutions. Chemically, a carbohydrate component is joined to a triterpenoid or steroid

backbone to form a saponin. They combine to generate an intricate and varied set of

chemicals (National Library of Medicine, 2020).

According to the clinical studies of National Library of Medicine, these substances

influence the immune system to prevent some malignancies and lower cholesterol

levels while also supporting good health. Saponins have been demonstrated to lower

blood cholesterol levels, lower cancer risk, and control blood sugar response.

Alkaloids are commonly discovered in plants, and certain families of flowering

plants have a higher than average concentration of them. Alkaloids are thought to be

present in about one-fourth of higher plants (angiosperms). It has garnered a lot of

attention in the fields of natural product chemistry and pharmacology due to their

prevalence and diversity (Britannica, 2023).

Process of Extraction

According to the National Library of Medicine (2020), a vital stage in ensuring

the caliber and dependability of research findings is the preparation and screening of

medicinal plants for experimental uses. Before beginning the biological testing, these

stages entail extracting and figuring out the kind and amount of bioactive elements.

Drying:

Initially, choose healthy and desired plant leaves, and gently clean them to

remove any dirt or insects before letting them air dry at 20-25 degrees celsius. Place
 14

the stems in a bundle and hang them upside down in a dim, well-ventilated place.

Until the liquid has evaporated, leave the herbs in a well-ventilated location. Check

for molds or mildew frequently while letting the herbs dry for 1-2 weeks. The leaves

should be separated from the stems once the herbs are totally dry, and they should

be kept in airtight containers in a cool, dark location (Andress, E., et.al., n.d.).

According to Thankaew (2020), by lowering the moisture level, drying

preserves the quality of herbs and prevents the development of microorganisms and

chemical changes that occur during dried storage. Dried herbs can be used in a wide

range of other industries, including the production of cosmetics, toiletries, and

perfumes. Herbs are well-known for being a great source of antioxidants. Depending

on how they are used, dried herbs may or may not have the qualities that are

deemed to be most significant. For instance, the presence of bioactive chemicals in

dried medicinal herbs determines their quality (Ebadi et al., 2015). In recent decades,

numerous herb-drying experiments and approaches have been carried out. Dried

samples were then ground to reduce and homogenise particle size using a

commercial blender (Morris, J., 2019).

Soaking:

According to Cole-Parmer (2020), high-proof alcohols (190 and 200) have

been used for extraction applications. Ethanol is emerging as one of the more

popular solvents, because it is safe for infused edibles and compatible with any type

of container. Ethanol also provides consistent results while being easily recoverable.

Mix plant material with the ethanol. Based on the study of Solvent Extraction Method

of Plants Using Ethanol (2020), submerge the leaves completely and then combine

the ethanol with the plant matter. There should be enough ethanol to completely

cover the material. Put the mixture in an amber bottle and let it sit there for 72 hours.

The ethanol will separate the soluble components of the extract during this soaking

period. Remove all solid materials with a simple filtration step requiring only a
 15

Büchner funnel, filter paper, and a vacuum flask. After the material has been filtered,

a solution of ethanol and extract remains (Solvent Extraction Method of Plants Using

Ethanol, 2020).

Ethanol Extraction:

The extract must then be taken from the ethanol using a rotary evaporator.

Lowering the boiling point of ethanol can hasten evaporation by coupling the rotary

evaporator to a vacuum pump. The resulting oil or extract will be entirely devoid of

ethanol. Additionally, this procedure enables the recovery of ethanol for use in later

extractions (Solvent Extraction Method of Plants Using Ethanol, 2020).

In order to recover phytochemicals from the plant matrix, extraction is crucial.

Studies on the storage of ethanolic extracts in refrigerators indicate that preserving

extracts in a refrigerated setting can improve stability and prolong their shelf life.

Ethanolic extracts are kept intact over time by being kept at a controlled temperature

and receiving little light exposure. "Ethanolic extraction of flavonoids, phenolics, and

antioxidants from Vernonia amygdalina leaf using two-level factorial design" (2020)

describes how the extract was filtered and dried using a rotary evaporator following

extraction. After that, the extract was chilled at 4 °C until additional examination.

Precautions are necessary when handling ethanol due to its flammable

nature. However, ethanol is regarded as being a much safer solvent than alternatives

like hydrocarbon and CO2 extraction. Compared to methods of extracting

hydrocarbons, ethanol has reduced risks of toxicity and explosiveness. (CBG

biotech, 2021)

Powderizing:

As with other powder processing methods, knowledge of particle size and

shape is important process information for L-PBF (Groarke, R., 2021). If the expected

compound is a solid, keep evaporating until a solid or thin film appears (Nichols, L.,

n.d.). The lower limit of layer height chosen for a build is often determined by the D50
 16

of the powder sample with the layer thickness selected not being lower than this. This

will in turn dictate the laser power parameters, in order to ensure melting and partial

remelting of previous layers. Particle shape is important as this is a key factor in how

a powder will pack within the layer or layers and will affect the contact between

powder particles, both in the plane of the build plate, but also vertically through the

build.

Preparation of Plant Extract

As described by Idris et al. (2022), the extraction was done with four solvents;

methanol, ethanol, acetone and water (10:1 v/w), and then the filtrate of ethanol

extract (ETE), acetone extract (ACE) and methanol extract (MEE) were concentrated

with a rotary evaporator (Strike-202 Steroglass, Italy). The filtrate of water extract

(WAE), on the other hand, was chilled at −40 °C and freeze-dried for 72 h. The stock

solutions (extracts) were prepared in concentrations of 0.125, 0.25, 0.5, 1.0, 2.0 and

5.0 mg extract/mL. The available commercial anthelmintic drug, levamisole, was

prepared and used as the positive control.

Albendazole

Anthelmintics are a group of antiparasitic antibiotics that either kill (vermicide)

or expel (vermifuge) infesting helminths. Helminths including tapeworms,

roundworms, and flukes cause helminthiasis. These widespread, frequent illnesses

typically go untreated until they become visible due to the way their clinical

development is. They occur more frequently in hot temperatures, unsanitary

environments, and areas where there are huge water tanks, parasite vectors, and

tainted food and water (Ahire, Lahane, and Deokate, 2020).

This medicine that kills helminths must be specifically harmful to the parasite

and not the host. According to Christman and Ernstmeyer (2020), numerous

anthelmintic drugs inhibit the production of microtubules within the parasite cell,
 17

impairing glucose uptake. Others function by preventing neural transmission inside

the parasite, which starves, paralyzes, and kills the worms.

One class of an anthelmintic drug is albendazole. It inhibits the polymerization

of microtubules by binding to tubulin that causes perturbation of larval intake of

glucose. According to Ahire, Lahane, Deokate (2020), the mature worm is depleted

of glycogen, which reduces the formation of ATP and causes the parasite to become

immobile and eventually die. A broad spectrum antiparasitic, albendazole, a

benzimidazole carbamate (methyl 5-propylthio-1H-benzimidazol-2-yl carbamate) is

used to treat a number of parasites all over the world including Ascaris suum and the

recommended dosage for cattle is 5-10 mg/kg (Junquera, 2022). However, it can

cause some adverse effects in pigs including anorexia, lethargy, and bone marrow

toxicity. (Multum, 2022).

Related Studies on Ascaris suum

In vitro analysis of the efficacy of kamias (Averrhoa bilimbi) fruit extract as an

anthelmintic alternative treatment against Ascaris suum. According to study, the

results showed that the 100 percent concentration of kamias fruit extract killed the

worms the best, followed by the 75 percent and 50 percent concentrations while the

25% concentration was the least effective. The worms treated with Albendazole, a

commonly used anthelmintic treatment, died after three hours of being seen. The

concentrations of kamias fruit extract that were tested were most effective after 12

hours. The greatest efficacy of the kamias fruit extract was found to be 98.31 percent

at 100 percent concentration. The study found that kamias fruit extract could be used

as an alternate treatment to albendazole for Ascaris suum infections, particularly at a

concentration of 100 percent (Linao and Sabela, 2018)

According to Linao, et. al., In the first three treatment hours, a high death rate

was seen at the 100% concentration of kamias fruit extract, in contrast to a relatively
 18

low death rate at the 25% concentration over the same time frame. Moreover, a

significant increase in the death rate was observed at the 12-hour mark for all doses.

This demonstrates how extract efficacy is concentration-dependent, with larger

concentrations being associated with increased mortality rates within predetermined

time frames. Remarkably, the 100% concentration was the best performance,

especially in the first three hours, with the greatest death rate ever reported.

In vitro anthelmintic activity of mani mani plant (Arachis pintoi) against Ascaris

suum in selected slaughterhouses of Cavite. According to this study, it was

discovered that the efficacy of Arachis pintoi stem and leaf extract against Ascaris

suum, a common parasite, was concentration-dependent. They evaluated various

quantities of the extracts in their laboratory investigation and discovered that higher

concentrations resulted in a higher mortality rate of Ascaris suum. When compared to

the leaf extract, the stem extract had a faster and more significant effect, with a 100%

concentration of the stem extract being the most helpful. These findings demonstrate

the potential of Arachis pintois as a natural anthelmintic medication and highlight the

need for additional study on dose optimization and the discovery of active

components. Finally, this research helps to create alternate treatment options for

Ascaris suum infections, notably in slaughterhouses. (Dimakiling and Pangandaman,

2020)
 METHODOLOGY

This chapter discusses the research design, instruments and tools, locale of

the study, sampling procedure, data gathering and statistical tools utilized in the

study. This method was based on the study of In vitro Anthelmintic Activity of

Averrhoa bilimbi Leaf extract against Ascaris suum (2016).

Research Design

This study employed an experimental research design. Sirisilla (2023) claimed

that an experimental research design is a framework of protocols and methods

designed to conduct scientific experimental research with two sets of variables. In

this study, the first set of variables served as a constant for calculating the differences

between the second set. It is used to compare the mortality rate at recorded time

intervals for different concentrations of akapulko leaf extract. Experimental research

is a scientific and methodical approach to research in which the researcher

manipulates one or more variables while controlling and quantifying any change in

other variables.

There were six treatments, and with three replicates each. The following treatments

were applied in the study:

Treatment 1 - Albendazole (positive control)

Treatment 2 - Distilled water (negative control)

Treatment 3 - 100 mg/mL

Treatment 4 - 50 mg/mL

Treatment 5 - 25 mg/mL

Treatment 6 - 12.5 mg/mL

 20

T1R1 T1R2 T1R3

T2R1 T2R2 T2R3

T3R1 T3R2 T3R3

T4R1 T4R2 T4R3

T5R1 T5R2 T5R3

T6R1 T6R2 T6R3

Figure 2. Experimental Layout of the Study

Preparation of akapulko plant

In preparing the akapulko plant, the researchers started by selecting mature, dark

green, healthy leaves from collected akapulko plants. The collected leaves were

placed in the brown paper bag upon transport. The leaves were then washed under

running water to get rid of any dirt or debris, to ensure they are completely dry after

cleaning, the leaves were patted dry with a clean cloth. After that, the leaves were

spreaded out in a single layer on a drying rack and were air dried for twenty-one (21)

days until completely dried in a well-ventilated and shaded area, occasionally turning

them to ensure even drying. Leaves should be allowed to naturally dry in the air
 21

without being exposed directly to the sunlight. After being completely dried and

crunchy, the leaves were cut into small pieces for preparation of grounding. Lastly,

the leaves were grounded finely until it became powder using a normal blender.

Preparation of Ascaris suum

Ascaris suum specimens were obtained from freshly slaughtered pigs at a Class

AA Slaughterhouse in Trece Martires City by the slaughterhouse’s workers, and

placed in a container with NSS to maintain their viability for transport to Cavite State

University using a private vehicle, with efforts made to maintain suitable conditions

including temperature, humidity, and lighting to ensure the well-being of the Ascaris

suum.

Preparation of Materials

The materials that are used in the experiment includes beaker, test tube,

funnel, stirring rod, graduated cylinder, balance, centrifuge, speed vacuum, cheese

cloth, distilled water, Albendazole and a 0.9 percent normal saline solution for

controls. The beakers, funnel, stirring rod, volumetric flask and hot plate were

borrowed from the Natural Products Laboratory in the Research Center Building of

Cavite State University- Indang Cavite and College of Nursing-Medical Technology

Department Laboratory. The 95 percent Alcohol, 0.9 percent normal saline solution,

cheese cloth are then purchased at the REMAN Hospital Medical and Laboratory

Depot, Rizal Avenue, Sta. Cruz, Manila and Medical Depot at Malls of Serin,

Tagaytay, Cavite.
 22

Methods and Procedures

Collection of Plant Material

Freshly harvested mature akapulko (Senna alata) leaves were collected from

Barangay Dagatan, Amadeo, Cavite by the researchers. Sharp scissors, powder free

nitrile gloves, and brown paper bags were used in the collection of akapulko leaves.

According to the Philippine Medicinal Plant. (n.d.), mature and healthy akapulko

leaves are typically dark green and fully grown, with a smooth and firm texture. Each

leaflet must be oval-shaped with a pointed tip and symmetrical on both sides. After

collection of one and a half sacks of fresh akapulko leaves, it was then properly

cleansed with running water to remove particle debris for air drying preparation.

Authentication of Plant Material

One pinnate with sixteen leaflets of fresh akapulko leaves were submitted by

the researchers to the Bureau of Plant and Industry - Malate, Manila for the

identification and authentication of the plant material.

Preparation of Plant Sample

Fresh akapulko leaves were air dried at room temperature (20 to 25 degrees

celsius) for twenty one (21) days using drying racks to allow the air to circulate

around each leaflet and promote even drying. The researchers regularly checked the

leaves to assess the progress. Completely dried leaves must be crispy and crumbly.

The air-dried leaves are then chopped into pieces and powderized using an

electronic blender without any solvent succeedingly.

Preparation of Ethanolic Extract

The powdered Akapulko leaves are then placed in a sealed amber bottle

container with 95 percent ethanol and left at room temperature for at least three days
 23

and set aside in a firm and flat surface to ensure that the soluble substances are

completely soaked. Each 2.5 grams of amber bottle contained 250 grams of

powdered akapulko leaves soaked in 3 Liters of 95 percent analytical grade ethanol.

The combination was filtered using gravity filtration, and the marc (the moist solid

material left over after pressing the leaf in an extraction technique) were pressed.

After standing, it was purified using filtration through a cheesecloth, transferred in a

1000 milliliters beaker.

Rotary Evaporation of Ethanolic Akapulko leaf Extract

The ethanolic extracts are filtered to eliminate the alcohol content of the

extract by exposing the filtrate of the leaves to a rotary evaporator at a temperature

of 75 degrees celsius at 140 RPM for the evaporation of the solvent. The ethanol

extract was collected and stored in a clean beaker at room temperature to let the

remaining ethanol to evaporate to obtain the akapulko leaf extract.

Phytochemical Screening and Analysis

The total of 250 grams of air dried akapulko leaves submitted at the Organic

Chemistry Section of the Department of Science and Technology (DOST) for

ethanolic extract phytochemical screening. The presence of Alkaloids, saponins,

tannins, and flavonoids were examined in the ethanolic leaf extract of akapulko

(Senna alata).

Preparation of Treatment

Four different concentrations of extract solution and one treatment for each

positive control and negative control were prepared and used in the experiment. The

stock solution and served as the first treatment of akapulko extract was prepared with

100 milligrams of akapulko extract dissolved using nss and was diluted with one (1)

liter of distilled water and served as the 100 mg/mL treatment. Secondly, the
 24

treatment of 50 mg/mL was prepared by obtaining the 250 mL from the stock solution

and was then diluted with 500 mililiters of distilled water. Subsequently, the third

treatment concentration of 25 mg/mL was then prepared with 125 milliliters of stock

solution and was diluted with 500 milliliters of distilled water. Lastly, the fourth and

last treatment of 12.5 mg/mL was prepared with 62.5 milligrams from the stock

solution and was diluted with the 500 milliliters. Each treatment has and was divided

into three replicates with a total of 167 milliliters each container.

Furthermore, the concentrations from the stock solution to the following treatment

concentrations that are used in the study were prepared by using the formula:

C1V1 = C2V2

Where: C is the initial concentration

C2 is the final concentration
V1, is the initial volume
V2, is the final volume

Preparation of Control Solution

The 500 milliliters of 0.9 percent Normal Saline Solution (NSS) served as negative

control to maintain the nourishment to support the viability of Ascaris suum worms.

Given a standard anthelmintic medication, 400 mg of Albendazole dissolved in 500

milliliters of distilled water was used as the positive control of the experiment.

Collection of Ascaris suum

Adult Ascaris suum parasites were collected and used in three (3) replicates for

each control and treatment, with three (3) live adult Ascaris suum parasites in each

container. For three trials, mature Ascaris suum worms are randomly assigned to one

of five (5) treatments. This study includes a total of 5 live Ascaris suum adult worms

for pre testing and 170 live Ascaris suum adult worms for actual testing. The
 25

parasites were acquired from a Class AA slaughterhouse in Trece Martires City,

Cavite, with lengths of 20 to 30 millimeters, pinkish tint, alive, and no physical

deformities, it was then validated by Sir Rhoyette A. Cuyo, RMT, of College of

Nursing - Cavite State University, Indang Campus. Adult worms were collected and

stored in a clean plastic jar with normal saline solution at room temperature for

transportation. Adult worms were cleansed with normal saline solution before being

placed in a 500 milliliters beaker of 0.9 percent normal saline solution.

Authentication of Parasite

One (1) freshly collected adult Ascaris suum worm from the slaughterhouse was

submitted to and authenticated by Dr. Adrian Miki C. Macalanda, DVM. of College of

Veterinary and Biomedical Sciences of Cavite State University-Indang, Cavite.

Pre-testing
Pre-testing was done to have initial data of how long the worm would last and still

be able to live outside the host after a certain amount of time. Pre-testing was

performed by the researchers on 5 mature Ascaris suum in a sterile container with a

0.9 percent normal saline solution, the life span of the worms in normal saline

solution were tested in the laboratory of the Department of Medical Technology and

validated by Sir Rhoyette A. Cuyo, RMT, of College of Nursing - Cavite State

University, Indang Campus.

Application of the Treatment

Adult Ascaris suum worms were divided into three (3) groups in each replicate,

whereby, to demonstrate the concentration of the akapulko leaf extracts, a mixture

was prepared using 1 Liter of distilled water and 100 milligrams of akapulko leaf

extract. The mixture is placed in a 500 milliliter beaker with the same volume but

varying concentrations. Treatments involve the use of akapulko leaf extract with

concentrations of 100 mg/mL, 50mg/mL, 25 mg/mL and 12.5 mg/mL. Subsequently,

 26

a total of three mature Ascaris suum are then placed in each equal sized container of

treatments and replicates. As a positive control, 500 milliliters of heated distilled

water dissolved 400 milligrams of albendazole used as the anthelmintic drug. 500

milliliters of 0.9 percent normal saline solution was utilized in the testing process as a

negative control. The researchers checked the viability of the parasite every 3 hours

for 12 hours and the data underwent comprehensive statistical analysis.

Data Gathering

The number of dead and alive were observed and validated for every three (3)

hours in twelve (12) hours. Data are noted according to the number of dead Ascaris

suum in each treatment and according to the time of death in the three hours interval

(3hr, 6hr, 9hr, 12hr). Deaths of worms are recorded after confirming its death by

observing its color and motility. It is also placed in the warm water to ensure its death

and is discarded to prevent its influence on the treatment on remaining live Ascaris

suum. Total of number of dead worms at each observation Total number of worms

examined

𝑇𝑜𝑡𝑎𝑙 𝑜𝑓 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑤𝑜𝑟𝑚𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑜𝑏𝑠𝑒𝑟𝑣𝑎𝑡𝑖𝑜𝑛

% 𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 = 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑤𝑜𝑟𝑚𝑠 𝑒𝑥𝑎𝑚𝑖𝑛𝑒𝑑
𝑥 100

𝑇𝑜𝑡𝑎𝑙 𝑜𝑓 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑤𝑜𝑟𝑚𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑜𝑏𝑠𝑒𝑟𝑣𝑎𝑡𝑖𝑜𝑛

𝐸𝑓𝑓𝑖𝑐𝑎𝑐𝑦 = 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑤𝑜𝑟𝑚𝑠 𝑒𝑥𝑎𝑚𝑖𝑛𝑒𝑑
𝑥100

Statistical Analysis of Data

The number of adult Ascaris suum were thoroughly counted and observed

throughout each observation period. The collected information was calculated and

the result was tabulated. The duration of the parasite-eliminating efficacy of the

akapulko leaf extract was noted by the researchers, and this vital information was

subjected to thorough statistical analysis. Two-way analysis of variance (ANOVA) is

used by the researchers to conduct the statistical evaluation of this study.

 27

Proposed Data Presentation

Table 1 shows the content of the treatment including albendazole as positive

control, normal saline solution as negative control and treatments with varying

concentration of akapulko leaf extract.

TREATMENT NO. TREATMENT CONTENT

Treatment 1 Albendazole (Positive Control)

Treatment 2 Normal Saline Solution(Negative Control)

Treatment 3 100 mg/mL

Treatment 4 50 mg/mL

Treatment 5 25 mg/mL

Treatment 6 12.5 mg/mL

Table 1. The treatment with its corresponding components.

Waste Management and Disposal

All used bottles and containers were disposed of in accordance with strict

safety rules in a sealed waste container inside a biohazardous waste container. The

container had a liner marked with the proper symbol, leak-proof, and designated as a

biohazard. When not in use, it shall be securely sealed. The adult Ascaris suum used

in the study was boiled at 65 degrees celsius and up for 5 minutes in the water for

good precaution to ensure its death. It was then buried at a depth of no less than four

feet in compliance with the correct protocol. Following these rules showed the

commitment of the researchers to moral research practices and safety measures.

Ethical Considerations
 28

In conducting this study, consent from Trece Martires City Class AA

Slaughterhouse was obtained. The data were treated with utmost confidentiality and

was only used exclusively for research purposes. In addition, letter was given to the

Bureau of Plant and Industry, Malate, Manila for authentication of akapulko plant,

College of Veterinary Medicine and Biomedical Sciences for identification of Ascaris

suum adult worms, and Organic Chemistry Section of the Department of Science and

Technology for the screening of phytochemical components of akapulko.

Furthermore, the proper disposal of the used akapulko extract and parasite was

observed to ensure the safety of the researchers as well as the community near the

waste disposal site. Lastly, consent from the research ethics committee was obtained

to comply with a set of ethical guidelines to carry out the study on efficacy of

akapulko leaves as anthelmintic against Ascaris suum.

 29

Figure 2. Flowchart of the Study

 RESULTS AND DISCUSSION

This chapter presents the results, analysis and interpretation of gathered data

of effectiveness of akapulko leaves against Ascaris suum. The akapulko leaf extract

was screened qualitatively to determine the phytochemicals present in the plant. The

akapulko leaf extract was diluted with distilled water to produce a concentration of

100 mg/ml, 50 mg/ml, 25 mg/ml, and 12.5 mg/ml for the in-vitro anthelmintic activity

against Ascaris suum which was observed every 3 hours for 12 hours.

Qualitative Phytochemical Screening of the Plant Extract

Table 1 shows the phytochemicals present in the ethanolic leaf extract of the

akapulko. Preliminary phytochemical analysis revealed the presence of sterols,

triterpenes, flavonoids, alkaloids, saponins, glycosides, and absence of tannins in the

extract. According to the Philippine Medicinal Plant (2020), akapulko has naturally

bioactive substances, including saponins and alkaloids which are known for their

anthelmintic effects, thus suggesting an anthelmintic effect of akapulko leaf extract.

Table 1. Phytochemical constituents of Akapulko leaf extract

Phytochemicals Akapulko Leaf Extract

Sterols +

Triterpenes +

Flavonoids +

Alkaloids +

Saponins +

Glycosides +

Tannins -

*Present = (+); Absent = (-)

 31

Efficacy of akapulko leaves against Ascaris suum

A. Mean mortality of Ascaris suum per hour
Table 2 shows the mean mortality per hour. Note that the treatment 2

(Negative Control) yields 0 mean since no Ascaris suum died in the 12 hour period of

observation. Most deaths occurred under the first 3 hours of the treatment 3 with a

concentration of 100 mg/ml (mean = 2.222). 0 mean death was also recorded on the

12th hour per treatment except treatment 6 with a concentration of 12.5 mg/ml.

According to Linao, et. al., a high death rate was seen at the 100 percent

concentration of kamias fruit extract in the first three hours, in contrast to a relatively

low death rate at the 25 percent concentration over the same time frame. Moreover,

a significant increase in the death rate was observed at the 12-hour mark for all

doses. This demonstrates how extract efficacy is concentration-dependent, with

larger concentrations being associated with increased mortality rates within

predetermined time frames. Remarkably, the 100 percent concentration was the best

performance, especially in the first three hours, with the greatest death rate that has

been reported.

Table 2. Descriptive statistics of mortality of Ascaris suum per hour

Treatment Concentration Hours Mean StDev N

3 hrs 0.333 0.500 9

6 hrs 2.000 0.707 9

T1 Positive Control
9 hrs 0.667 0.707 9

12 hrs 0.000 0.000 9

3 hrs 0.000 0.000 9

6 hrs 0.000 0.000 9

T2 Negative Control
9 hrs 0.000 0.000 9

12 hrs 0.000 0.000 9

 32

3 hrs 2.222 0.667 9

6 hrs 0.778 0.667 9

T3
100 mg/ml 9 hrs 0.000 0.000 9

12 hrs 0.000 0.000 9

3 hrs 1.333 0.500 9

6 hrs 1.667 0.500 9

T4 50 mg/ml
9 hrs 0.000 0.000 9

12 hrs 0.000 0.000 9

3 hrs 0.000 0.000 9

6 hrs 1.333 0.707 9

T5 25 mg/ml
9 hrs 1.667 0.707 9

12 hrs 0.000 0.000 9

3 hrs 0.000 0.000 9

6 hrs 0.000 0.000 9

T6 12.5 mg/ml
9 hrs 0.000 0.000 9

12 hrs 1.333 0.500 9

3 hrs 0.648 0.935 54

6 hrs 0.963 0.931 54

Total
9 hrs 0.389 0.738 54

12 hrs 0.222 0.538 54

Total 0.556 0.844 216

B. Mean mortality of Ascaris suum per treatment

Table 3 shows the mean mortality of Ascaris suum per treatment. The same
mean under treatment 2 (Negative Control) is also observed since no A. suum died in
the 12 hour observation period (mean = 0.000). Similar means were observed under
treatment 1 (positive), treatment 3 (100 mg/ml), treatment 4 (50 mg/ml), and
treatment 5 (25 mg/ml), (mean = 0.750) while the treatment 6 (12.5 mg/ml) treatment
yields the lowest mean mortality rate (mean = 0.333). According to Linao and Sabela
 33

(2018), the results of the study titled “In vitro analysis of the efficacy of kamias
(Averrhoa bilimbi) fruit extract as an anthelmintic alternative treatment against
Ascaris suum”, showed that the 100 percent concentration of kamias fruit extract
killed the worms the best, followed by the 75 percent and 50 percent concentrations
while the 25 percent concentration was the least effective.

Table 3. Descriptive statistics of mortality of Ascaris suum per treatment

Treatment Concentration Mean StDev N
T1 Positive Control 0.750 0.937 36
T2 Negative Control 0.000 0.000 36
T3 100 mg/ml 0.750 1.025 36
T4 50 mg/ml 0.750 0.841 36
T5 25 mg/ml 0.750 0.906 36
T6 12.5 mg/ml 0.333 0.632 36
Total 0.556 0.844 216

Mortality rate of A. suum per treatment at different time interval

Table 4 showed that the p-value of the interaction of the treatment and hours

is 0.000 using Two-Way Analysis of Variance (ANOVA), therefore the null hypothesis

is rejected. This implies that there is a significant interaction between the treatment

and the hours of mortality rate of Ascaris suum, thus, we can conclude that the

mortality rate of A. suum is affected by the interaction of the two factors. The

treatment alone has an effect in the death of A. suum since the p-value is less than

0.05. The same can also be concluded in the hours alone since it also has a

significant effect in the death of A. suum. Looking at the partial eta squared, it can be

concluded that the treatment alone can affect 37 percent of the variation of the

output and the hours alone can affect 35.2% of the variation output which are both

significant at 0.05 level. The interaction of treatment and hours explains 73.7% of the

variation of the mortality rate of A. suum. The R-Squared value implies that 79.7% of

the variation of the mortality rate of A. suum can be explained by the model. The
 34

pairwise comparison also implies that the difference between the mortality rate of

ascaris exists in multiple categories.

Hence, it is to be observed that there is significant difference in the hours as

well as within the treatment. Positive control, 100 mg/ml, 50 mg/ml, and 25 mg/ml

has no significant difference with regards to its ability to kill ascaris where it is to be

noted that 12.5 treatment is the least effective treatment in killing A. suum. With

regards to how fast a treatment kills A. suum, it is to be noted that the first 3 hours

under the treatment of 100 mg/ml is significantly faster with the highest mortality rate

than the rest of the hours per treatment.

Table 4. Mortality rate of A. suum per treatment at different time interval

Mortality rate of A. suum per treatment at different time interval
Dependent Variable: Mortality
Partial
Type III Sum Mean Eta
Source of Squares df Square F Sig. squared
Corrected Model 122.222a 23 5.314 32.795 0.000 0.797
Intercept 66.667 1 66.667 411.429 0.000 0.682
Treatment 18.333 5 3.667 22.629 0.000 0.567
Hours 16.926 3 5.642 34.819 0.000 0.452
Treatment * Hours 86.963 15 5.798 35.779 0.000 0.337
Error 31.111 192 0.162
Total 220.000 216
Corrected Total 153.333 215
a. R Squared = .797 (Adjusted R Squared = .773)
 SUMMARY, CONCLUSION, RECOMMENDATION

Summary

The study entitled “Efficacy of Akapulko (Senna alata) Leaves As Anthelmintic

Against Ascaris suum” was conducted at the laboratory of the Department of Medical

Technology, College of Nursing, Cavite State University, Bancod, Indang, Cavite, in

collaboration with the Natural Products Laboratory at the Research Center from

March 2023 to December 2023. Generally, the study aimed to determine the efficacy

of akapulko (Senna alata) leaves as anthelmintic against Ascaris suum. Specifically,

it aimed to determine the phytochemical component of akapulko leaf extract as an

anthelmintic to Ascaris suum; assess the mortality rate of the Ascaris suum adult

worms to akapulko leaf extract in varying concentrations; compare the significant

differences of akapulko leaf extract and commercial anthelmintic drug, Albendazole,

against Ascaris suum.

The freshly collected mature akapulko leaves were collected from Barangay

Dagatan, Amadeo, Cavite and were air-dried at room temperature (20 degree

Celsius to 25 degree Celsius) for twenty one (21) days using drying racks to allow the

air to circulate around each leaflet and promote even drying. Subsequently, the dried

leaves were pulverized into powder using a blender and soaked in 95 percent

ethanol in an amber bottle for three days. The resulting extract underwent filtration

and rotary evaporation. One hundred eighty Ascaris suum specimens were collected

and transported to the laboratory in a clean gallon with normal saline solution. The

adult worms were then divided into three groups in each replicate, and varying

concentrations of akapulko leaf extract were prepared using distilled water.

Treatments included concentrations of 100 mg/mL, 50 mg/mL, 25 mg/mL, and 12.5

mg/mL. The viability of the parasites was monitored every 3 hours for 12 hours, with

comprehensive statistical analysis applied to the collected data.

 37

Results revealed that after 12 hours of observation, the concentrations of 100

mg/mL, 50 mg/mL, and 25 mg/mL were significantly effective against Ascaris suum

worms, with the 100 mg/mL concentration demonstrating the highest efficacy.

Considering the mortality rate within the specified concentrations and time intervals,

the researchers concluded that the 100 mg/mL concentration of ethanolic leaf extract

closely paralleled the effectiveness of Albendazole.

The study suggests that the 100 mg/mL ethanolic leaf extract could serve as

a viable alternative to Albendazole in the treatment of Ascariasis in pigs.

Conclusion

According to the results of the phytochemical analysis, the chemical

compounds that were determined in the akapulko (Senna alata) ethanolic leaf extract

revealed the presence of sterols, triterpenes, flavonoids, alkaloids, saponins, and

glycosides, which are responsible for the anthelmintic property of the plant.

The efficacy of the akapulko ethanolic leaf extracts was evaluated at various

time intervals (third, sixth, ninth, and twelfth hours), revealing

concentration-dependent effectiveness. After a 12-hour observation, concentrations

of 100 mg/mL, 50 mg/mL, and 25 mg/mL demonstrated significant efficacy against

Ascaris suum worms, with the 100 mg/mL concentration proved to be most effective.

Analysis of the mortality rate indicated that the 100 mg/mL concentration of

ethanolic leaf extract closely resembled the effectiveness of Albendazole. The

efficacy of the extract correlated with concentration, with higher concentrations

leading to increased mortality rates over time. Notably, the 100 mg/mL concentration

exhibited the highest mortality rate within the initial three-hour interval.
 38

In summary, the study highlights the potential of the 100 mg/mL akapulko leaf

extract as a promising alternative to Albendazole in treating Ascariasis in pigs,

emphasizing the concentration-dependent nature of the anthelmintic properties of the

extract.

Recommendations

For further studies, the researchers hereby recommend the following:

1. Future researchers may perform the study in a broader perspective and

involve other solvent substitutes for ethanol in extracting the akapulko leaves.

2. Future investigations should consider increasing the number of adult Ascaris

suum worms in each treatment for more precise observations.

3. Researchers in the future are advised to conduct phytochemical screening on

different parts of the akapulko plant and explore the potential use of its

extracts as anthelmintics against Ascaris suum.

4. It is recommended that future researchers explore the temporary use of

akapulko leaf extract for deworming in pig farming industries.

 39

APPENDIX FIGURES

Appendix Figure 1. Solvents used in the study

Appendix Figure 2. Rotary evaporator used for extraction of akapulko leaves

 40

Appendix Figure 3. Amber jars for soaking and storing of akapulko leaves with 95

percent ethanol

Appendix Figure 4. Slaughter house for collection of Ascaris suum

 41

Appendix Figure 6. Akapulko tree at Amadeo, Cavite

Appendix Figure 7. Procurement of akapulko leaves

 42

Appendix Figure 8. Akapulko Leaves

Appendix Figure 9. Separating of leaves from pinnate

 43

Appendix Figure 10. Air-drying of akapulko leaves

 44

Appendix Figure 11. Cutting and Grinding of dried akapulko leaves

Appendix Figure 12. Weighing the ground akapulko leaves

 45

Appendix Figure 13. Soaking of akapulko leaves in 95 percent ethanol

 46

(a) filter of soaked leaves using cheese cloth,

(b) rotary evaporation of filtered sample,

Appendix Figure 14. Extraction of akapulko leaf ethanolic extract, (a) filter of soaked

leaves using cheese cloth, (b) rotary evaporation of filtered sample,(c) air drying of

extract

Appendix Figure 15. Collected Adult Ascaris suum soaked with 0.9 percent normal

saline solution
 47

Appendix Figure 16. Measuring of Ascaris suum

 48

Appendix Figure 17. Measuring of Extract

 49

Appendix 18. Dilution treatments

(a) Trial 1
 50

(b) Trial 2

(c) Trial 3

Appendix Figure 19. Experimental Setup (a) Trial 1 (b) Trial 2 (c)Trial 3
 51

Appendix Figure 20. Application of Treatments to Ascaris suum

Appendix Figure 21. Screening of Anthelmintic Activity of akapulko extracts after 12

hours of observation
 52

APPENDIX 1

Research Design
 53

APPENDIX 1

Research Design

Figure 1. Procedural manuals of the study.

 54

APPENDIX 2

Sample data sheets

 55

APPENDIX 2

Sample data sheets

Appendix Table 1. Observation of Replicate 1 in Trial 1 of Ascaris suum using

different treatments in varying time.

TREATMENT TIME (HRS)

0 3 6 9 12

T1= 400 mg of albendazole 3 2 1 0 0

(Positive Control)

T2= Normal saline solution 3 3 3 3 3

(Negative Control)

T3= 100 mg/mL 3 2 0 0 0

T4= 50 mg/mL 3 2 0 0 0

T5= 25 mg/mL 3 3 2 0 0

T6= 12.5 mg/mL 3 3 3 3 2

Appendix Table 2. Observation of Replicate 2 in Trial 1 of Ascaris suum using

different treatments in varying time.

TREATMENT TIME (HRS)

0 3 6 9 12

T1= 400 mg of albendazole 3 2 0 0 0

(Positive Control)

T2= Normal saline solution 3 3 3 3 3

(Negative Control)

T3= 100 mg/mL 3 1 0 0 0

T4= 50 mg/mL 3 2 0 0 0

T5= 25 mg/mL 3 3 2 0 0

T6= 12.5 mg/mL 3 3 3 3 2

 56

Appendix Table 3. Observation of Replicate 3 in Trial 1 of Ascaris suum using

different treatments in varying time.

TREATMENT TIME (HRS)

0 3 6 9 12

T1= 400 mg of albendazole 3 2 0 0 0

(Positive Control)

T2= Normal saline solution 3 3 3 3 3

(Negative Control)

T3= 100 mg/mL 3 1 0 0 0

T4= 50 mg/mL 3 2 0 0 0

T5= 25 mg/mL 3 3 3 0 0

T6= 12.5 mg/mL 3 3 3 3 1

Appendix Table 4. Observation of Replicate 1 in Trial 2 of Ascaris suum using

different treatments in varying time.

TREATMENT TIME (HRS)

0 3 6 9 12

T1= 400 mg of albendazole 3 3 0 0 0

(Positive Control)

T2= Normal saline solution 3 3 3 3 3

(Negative Control)

T3= 100 mg/mL 3 1 0 0 0

T4= 50 mg/mL 3 2 0 0 0

T5= 25 mg/mL 3 3 1 0 0

T6= 12.5 mg/mL 3 3 3 3 2

 57

Appendix Table 5. Observation of Replicate 2 in Trial 2 of Ascaris suum using

different treatments in varying time.

TREATMENT TIME (HRS)

0 3 6 9 12

T1= 400 mg of albendazole 3 3 1 0 0

(Positive Control)

T2= Normal saline solution 3 3 3 3 3

(Negative Control)

T3= 100 mg/mL 3 1 0 0 0

T4= 50 mg/mL 3 2 0 0 0

T5= 25 mg/mL 3 3 2 0 0

T6= 12.5 mg/mL 3 3 3 3 2

Appendix Table 6. Observation of Replicate 3 in Trial 2 of Ascaris suum using

different treatments in varying time.

TREATMENT TIME (HRS)

0 3 6 9 12

T1= 400 mg of albendazole 3 3 2 0 0

(Positive Control)

T2= Normal saline solution 3 3 3 3 3

(Negative Control)

T3= 100 mg/mL 3 0 0 0 0

T4= 50 mg/mL 3 1 0 0 0

T5= 25 mg/mL 3 3 1 0 0

T6= 12.5 mg/mL 3 3 3 3 1

 58

Appendix Table 7. Observation of Replicate 1 in Trial 3 of Ascaris suum using

different treatments in varying time.

TREATMENT TIME (HRS)

0 3 6 9 12

T1= 400 mg of albendazole 3 3 1 0 0

(Positive Control)

T2= Normal saline solution 3 3 3 3 3

(Negative Control)

T3= 100 mg/mL 3 1 0 0 0

T4= 50 mg/mL 3 2 0 0 0

T5= 25 mg/mL 3 3 1 0 0

T6= 12.5 mg/mL 3 3 3 3 1

Appendix Table 8. Observation of Replicate 2 in Trial 3 of Ascaris suum using

different treatments in varying time.

TREATMENT TIME (HRS)

0 3 6 9 12

T1= 400 mg of albendazole 3 3 0 0 0

(Positive Control)

T2= Normal saline solution 3 3 3 3 3

(Negative Control)

T3= 100 mg/mL 3 0 0 0 0

T4= 50 mg/mL 3 1 0 0 0

T5= 25 mg/mL 3 3 2 0 0

T6= 12.5 mg/mL 3 3 3 3 2

 59

Appendix Table 9. Observation of Replicate 3 in Trial 3 of Ascaris suum using

different treatments in varying time.

TREATMENT TIME (HRS)

0 3 6 9 12

T1= 400 mg of albendazole 3 3 1 0 0

(Positive Control)

T2= Normal saline solution 3 3 3 3 3

(Negative Control)

T3= 100 mg/mL 3 0 0 0 0

T4= 50 mg/mL 3 1 0 0 0

T5= 25 mg/mL 3 3 1 0 0

T6= 12.5 mg/mL 3 3 3 3 2

Appendix Table 10. Phytochemical constituents of akapulko leaf extract

Phytochemicals Akapulko Leaf Extract

Sterols +

Triterpenes +

Flavonoids +

Alkaloids +

Saponins +

Glycosides +

Tannins -
 60

Appendix Table 11. Post hoc test on treatments

Pairwise Comparisons
Dependent Variable: Death
95% Confidence
Mean Interval for
Difference Std. Differenceb
(I) Treatment (I-J) Error Sig.b Lower Bound
Positive Control Negative .750* 0.09 0.000 0.563
control 5
100 -6.66134E- 0.09 1.000 -0.187
mg/ml 16 5
50 -2.22045E- 0.09 1.000 -0.187
mg/ml 16 5
25 -3.88578E- 0.09 1.000 -0.187
mg/ml 16 5
12.5 .417* 0.09 0.000 0.230
mg/ml 5
Negative Positive -.750* 0.09 0.000 -0.937
Control control 5
100 -.750* 0.09 0.000 -0.937
mg/ml 5
50 -.750* 0.09 0.000 -0.937
mg/ml 5
25 -.750* 0.09 0.000 -0.937
mg/ml 5
12.5 -.333* 0.09 0.001 -0.520
mg/ml 5
100 mg/ml Positive 6.661E-16 0.09 1.000 -0.187
control 5
Negative .750* 0.09 0.000 0.563
control 5
50 2.220E-16 0.09 1.000 -0.187
mg/ml 5
25 2.220E-16 0.09 1.000 -0.187
mg/ml 5
12.5 .417* 0.09 0.000 0.230
mg/ml 5
50 mg/ml Positive 2.220E-16 0.09 1.000 -0.187
control 5
Negative .750* 0.09 0.000 0.563
control 5
100 -2.22045E- 0.09 1.000 -0.187
mg/ml 16 5
25 0.000 0.09 1.000 -0.187
mg/ml 5
12.5 .417* 0.09 0.000 0.230
mg/ml 5
25 mg/ml Positive 3.886E-16 0.09 1.000 -0.187
Control 5
Negative .750* 0.09 0.000 0.563
Control 5
100 -2.22045E- 0.09 1.000 -0.187
mg/ml 16 5
 61

50 0.000 0.09 1.000 -0.187

mg/ml 5
12.5 .417* 0.09 0.000 0.230
mg/ml 5
12.5 mg/ml Positive -.417* 0.09 0.000 -0.604
Control 5
Negative .333* 0.09 0.001 0.146
Control 5
100 -.417* 0.09 0.000 -0.604
mg/ml 5
50 -.417* 0.09 0.000 -0.604
mg/ml 5
25 -.417* 0.09 0.000 -0.604
mg/ml 5
Based on estimated marginal means
*. The mean difference is significant at the .05 level.
b. Adjustment for multiple comparisons: Least Significant Difference (equivalent to no
adjustments).

Appendix Table 12. Post hoc test on hours

Pairwise Comparisons
Dependent Variable: Death
Mean
Differe
nce Std. 95% Confidence
(I) Hours (I-J) Error Sig.b Interval for Differenceb
3 hours 6 hours -.315* 0.077 0.000 -0.468
9 hours .259* 0.077 0.001 0.106
*
12 hours .426 0.077 0.000 0.273
*
6 hours 3 hours .315 0.077 0.000 0.162
*
9 hours .574 0.077 0.000 0.421
12 hours .741* 0.077 0.000 0.588
*
9 hours 3 hours -.259 0.077 0.001 -0.412
*
6 hours -.574 0.077 0.000 -0.727
*
12 hours .167 0.077 0.033 0.014
12 hours 3 hours -.426* 0.077 0.000 -0.579
6 hours -.741* 0.077 0.000 -0.894
*
9 hours -.167 0.077 0.033 -0.319
Based on estimated marginal means
*. The mean difference is significant at the .05 level.
b. Adjustment for multiple comparisons: Least Significant Difference (equivalent to no
adjustments).
 62

APPENDIX 3

Request letters
 63

APPENDIX 3. Request letters

64
65
66
67
68
69
70
 71

APPENDIX 4

Timetable of activities
 72

Appendix 4. Gantt Chart

 73

APPENDIX 5

Budgetary requirements of the study

 74

APPENDIX 5

Budgetary requirements of the study

 75

APPENDIX 5

Budgetary requirements of the study

Budgetary Plan

ITEM DESCRIPTION OF DURATION AMOUNT

ACTIVITIES REQUIRED

Materials and Supplies Materials for September 4000

a. NSS transportation of 2023
b. Gallon parasite and plant
c. Albendazole extraction
d. Ethanol
e. Stainless steel basin
f. Brown paper bag
g. Plant scissor

Token for Slaughter House Parasite Collection August 300

personnel (Pre- testing and 2023-
Actual experiment October
proper) 2023

Printing: 1 reams of 80 gsm March 4000

paper 2023-
a. For title defense and Printing cost December
research proposal 2023
b. For RDE and DMT
forms
c. For final defense

Travel This includes: March 2500

a. Parasite Canvassing Parasite Canvassing 20230-
b. Parasite Confirmation Parasite Collection December
letter Authentication of 2023
c. Parasite Collection Plant and Parasite
d. Parasite Phytochemical
Authentication Testing site
e. Plant Canvassing and
confirmation
f. Plant Collection
g. Plant Authentication

Phytochemical Analysis DOST Plant November 650

Phytochemical 2023
Testing

Authentication of Parasite Consultation fee September 450

2023
 76

Authentication of Plant Consultation September Free

2023

Adviser: Mrs. Sarah Jane Paper consultation March 2, 300

Denia, RMT
2023-
Co-Adviser: Mrs. Evelyn Del
Mundo, PhD December

2023

Technical Critic: Mrs. Agnes Methodology March 2, 300

Alimbuyoguen, PhD consultation 2023-

December

2023

TOTAL: PHP 17, 300

 77

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