Toxicity and Phytochemical Screening of Muntingia Calabura (Aratilis) Leaf Extract - DATA

HOLY TRINITY COLLEGE
Toxicity and Phytochemical Screening of Muntingia calabura

(Aratilis) Leaf Extract
A Quantitative Research Paper

Presented to the Faculty of Senior High School Program
Holy Trinity College of General Santos City
In Partial Fulfillment of
the Requirements for the Senior High School
Science, Technology, Engineering, and Mathematics
Lynnch Data
Gideon C. Akieh
Christel Maree A. Boyboy
Shady Nash A. Kamenza
Al Jaylone A. Balt
April 2021
i
S E N I O R H I G H S C H O O L E D U C A T I O N
Holy TrinityCollegeof General SantosCity

Fiscal Daproza Avenue, General Santos City
Basic Education Department – Senior High School Program
APPROVAL SHEET
This research entitled TOXICITY AND PHYTOCHEMICAL SCREENING OF

Muntingia calabura (Aratilis) LEAF EXTRACT prepared and submitted by LYNNCH
DATA, GIDEON C. AKIEH, CHRISTEL MAREE A. BOYBOY, SHADY NASH A.
KAMENZA, and AL JAYLONE A. BALT, has been examined and is hereby
recommended for acceptance and approval.
ANDREVE JOHN L. REBUCIAS, LPT.

Adviser
________________________________________________________________
PANEL OF EXAMINERS
JUDY ANN H. VERONILLA, LPT.

_____________________________
Chairman
JAYBOY M. SARTORIO JAMES BERNHARDT P. PERPETUA, LPT.

____________________________ _______________________
Examiner Examiner
________________________________________________________________
Accepted and approved in partial fulfilment of the requirements for the subject
Inquisitive Investigation and Immersion.
MARIA GIOVANNA G. ABECIA, EdD.

High School Principal
ii
ACKNOWLEDGEMENT
This study would not be possible without the people who helped and supported the
researchers. The researchers would like to express their deepest gratitude and sincere
appreciation to the following:
To Mr. Andreve John L. Rebucias, their research adviser for his unending
support, guidance and patience all throughout the conduct of this study.
To Ms. Judy Ann H. Veronilla for granting them permission to use the
alternatives including the materials for the conduct of the experiment.
To the Chemistry Student Assistant, Mc Lin G. Data, with his expertise in the
field and for giving his time and assistance in the conduct of the study.
To the Data Family for their effort in helping the researchers in getting the
Muntingia calabura Leaf.
To their parents, for the love, care and support needed to the completion of this
study. Your effort is so much appreciated.
To the panelists, Judy Ann H. Veronilla, Jayboy M. Sartorio, and James

Bernhardt P. Perpetua, for their time and effort in validating the study.
To all the people who have become part of the study, for their support in the
completion of this study.
To Almighty God, whom all the brilliance and honor are offered for the blessing of
strength, patience, grace and possibility for He has given the needed things, be it
material or not, for the completion of this study. Through Him, anything is possible.
- The Researchers
iii
ABSTRACT
This experimental study determined the secondary metabolites present and the
toxicity of M. calabura Leaf Extract. For the brine shrimp lethality assay, five (5)
treatments were used with three (3) replicates. The treatments were Brine Solution
(Negative Control), Methanol (Positive Control), 1ug/mL fruit and leaf concentration,
10ug/mL leaf concentration and 100ug/mL leaf concentration for treatments one (1) to
five (5), respectively.
The result of the phytochemical screening was obtained from a related study, it
revealed the presence of secondary metabolites in the plant extract. The leaf extract is
positive for Carbohydrates, Alkaloids, Flavonoids, Glycosides, Phenols, Saponins,
Steroids, Sterols, and Tannins. However, no Carotenoids were detected. The brine
shrimp lethality assay using the probit analysis and linear regression method showed
that the M. calabura leaf extract is toxic. Result shows that the leaf has a great value of
0.0165 ug/mL in the LC50 computation. The difference in the mortality rates of Artemia
salina (Brine Shrimp) nauplii were also determined, and with a significant difference
shown in One-Way Analysis of Variance (ANOVA).
The Toxicity and the presence of Secondary Metabolites on Muntingia calabura

(Aratilis) Leaf Extract were determined to generate information about its toxic properties
for pharmaceutical industries to adequately evaluate the health risks prior for medical
use and its potential as a source of effective drug.
Keywords: Muntingia calabura, phytochemical screening, secondary metabolites,

Artemia salina, toxicity
iv
TABLE OF CONTENTS
Preliminaries
Title Page ........................................................................................................................ i
Approval Sheet ............................................................................................................... ii
Acknowledgement .......................................................................................................... iii
Abstract ......................................................................................................................... iv
List of Figures ............................................................................................................... vii
List of Tables ................................................................................................................ viii
1 THE PROBLEM AND ITS SETTING

Introduction | Background of the Study................................................................ 1
Statement of the Problem ................................................................................... 2
Hypothesis……………………………………………………………………………….3
Scope and Delimitation………………………………………………………………....3
Significance of Study .......................................................................................... 3
2 REVIEW OF RELATED LITERATURE AND STUDIES

Related Literature .............................................................................................. 5
Related Studies ................................................................................................ 12
Theoretical Framework ..................................................................................... 15
Conceptual Framework ................................................................................................15
Definition of Terms… .................................................................................................... 16
3 METHODOLOGY
Research Design ........................................................................................................... 18
Materials and Instruments… ....................................................................................... 19
Chemicals ......................................................................................................... 19
Preparation of Aqueous Extract......................................................................... 19
Preparation of Concentrated Extract .................................................................. 19
Serial Dilution…............................................................................................................. 19
Phytochemical Screening……………………………………………………………..20
Brine Shrimp Lethality Assay ............................................................................ 21
• Preparation of the Simulated Sea Water .................................... 21
v
• Hatching of Brine Shrimp Eggs and Toxicity Test ......................21
Data Gathering Procedure ............................................................................... 22

Statistical Tool .................................................................................................22
Ethical Consideration….............................................................................................. 23
4 PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA

Phytochemical Screening of M. calabura Leaf ................................................. 24
Toxicity of M. calabura ..................................................................................... 25
Mortality Rate of A. salina ................................................................................ 26
5 SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

Summary of Findings ....................................................................................... 28
Phytochemical Screening..................................................................................28
Toxicity of Muntingia calabura (Aratilis) ............................................................ 28
Conclusion……………………………………………………………………………..28
Recommendations…………………………………………………………………….29
REFERENCES ............................................................................................................. 30
APPENDICES
A. Certification
• Statistician ................................................................................. 36
• Grammarian ............................................................................... 37
C. Research Layout .......................................................................................... 38

D. Statistical Analysis… ............................................................................................... 39
E. Budget Allocation .......................................................................................... 42
F. Documentation ............................................................................................. 43
CURRICULUM VITAÉ.................................................................................................. 45
vi
LIST OF FIGURES
FIGURE PAGE
1
1 Paradigm of the Study: The IV-DV Model of the Toxicity and
Phytochemical Screening of Muntingia calabura (Aratilis) 16
Leaf Extract
2 Research Design: Toxicity and Phytochemical Screening of 18

M. calabura
3 Brine Shrimp Lethality Procedure of M. calabura Leaf Extract 22
vii
LIST OF TABLES
TABLE PAGE
1 Secondary Metabolites Present in M. calabura 13

Leaf Extract
2 Phytochemical Screening of M. calabura Leaf 24

Extract
3 Mean Number of Dead Nauplii after 24-hour 25

exposure to different concentrations of M.
calabura
4 LC50 value of M. calabura 26
5 Difference between the mortality rates of Artemia 26

salina (Brine Shrimp) nauplii
6 Summary Table for One-way ANOVA of A. salina 27

Mortality Rate
viii
CHAPTER I
THE PROBLEM AND ITS SETTING
Introduction
The corona virus disease outbreak commenced a global menace. As a result, the
development of new medicinal compounds with wide diverse chemical structures and
novel bioactivities that would help aid the resistance of harmful microorganisms has
become a top priority. However, throughout history, plants have been used for medicinal
purposes long before its actual recorded history. It can be traced back to 3000 B.C. as
documented in ancient Chinese and Egyptian papyrus writings. However, it was only
during the 19th century when scientists started to extract and modify the active
ingredients, and the toxicity of its constituents from the plants. Later on, chemists started
making their own version of plant compounds which resulted in the decline of herbal
medicine use with its identified phytochemical constituents (Tapsell et al., 2006).
During recent years, the cost of medical drugs has been rapidly increasing, the
toxicity of plants was identified to consider its benefits and risks in therapeutic
treatments. It also took a huge toll to those who live in poverty. The Philippines is
classified as a lower-middle income country. About 22.6% of the population live on less
than US $1/day, and 45.0% live on less than US $2/day (United Nations Development
Program, 2009). Majority of Filipinos have to pay out from their own pockets for their
own medicines, but with its high cost, about 30% of the population do not have access to
these necessities (World Health Organization, 2004). Under the Republic Act (RA) 8423
or Traditional and Alternative Medicine Act of 2007 by the former Sec. Juan M. Clavier
states that the use of plants as alternative medicine is encouraged by the Philippine
Government.
In the study of Mahmood et al. (2014) entitled “Muntingia calabura: A review of its
traditional uses, chemical properties, and pharmacological observations,” where it
showed the isolation of bioactive compounds done from the leaves of M. calabura,
collected in Purus, Peru, in October 1997. As per the study, the Leaf extract of M.
calabura possesses remarkable medicinal value such as, cytotoxic and anti-
inflammatory activities. This study justifies the constant need for discoveries of plants
that will help aid and contribute to pharmaceutical industries as a potential source of an
effective drug.
1
The result of this study determined the effectiveness of Muntingia calabura

(Aratilis) Plant Leaf Extract using different tests such as Phytochemical Screening and
Toxicity Testing. The secondary metabolites present were identified. There has also
been a comparison on the significant difference in the mortality rate of Artemia salina
(Brine Shrimp) nauplii. This would be of help in medical industries to discover treatments
using a new abundant alternative.
Statement of the Problem:
The key purpose of this analysis aimed to evaluate the Secondary Metabolites
present in M. calabura Leaf Extract that are accountable for its Toxicity. Toxicity of the
plant extract were determined to generate information about its toxic properties to
evaluate the health risks prior for medical use and its potential as a source of an
effective drug. Specifically, the purpose of the analysis is to address the following
questions:
1. Are the following secondary metabolites present in Muntingia calabura (Aratilis)

Leaf:
1.1 Alkaloids;
1.2 Carotenoids;
1.3 Flavonoids;
1.4 Glycosides;
1.5 Phenols;
1.6 Saponins;
1.7 Sterols;
1.8 Tannins?
2. What is the toxicity level (LC50) of M. calabura leaf in the mortality rate of Artemia
salina (Brine Shrimp) nauplii?
3. Is there a significant difference in the mortality rate of Artemia salina (Brine

Shrimp) nauplii?
2
Hypothesis
1. The LC50 of Muntingia calabura (Aratilis) Leaf Extract is greater than

100ug/mL.
2. There is no significant difference in the mortality rates of Artemia salina (Brine
Shrimp) nauplii.
Scope and Delimitation
This study aimed to determine the Phytochemical Constituents and toxicity of

Muntingia calabura (Aratilis) Leaf Extract. The experimentation of this study was
conducted at Brgy. Lagao, General Santos City during the second semester of the
school year 2020-2021.
The M. calabura leaves used in the study were collected from Barangay Lagao,
General Santos City. The Crude ethanolic extract was collected through evaporation.
The Phytochemicals present was based on a related study, and Brine Shrimp Lethality
Assay was tested on the leaf part of M. calabura, to test for the toxicity and secondary
metabolites present.
The study was limited to the use of M. calabura leaves, it did not investigate the
properties of the other parts of the Aratilis tree such as the barks, fruits, roots, or flowers.
The result from a related study for the phytochemical screening only covered the
identification of the presence or absence of its secondary metabolites.
Significance of the Study
This study aimed to identify the Secondary Metabolites present in M. calabura

Leaf Extract that are accountable for its Toxicity. The Toxicity of the plant extract was
determined in order to consider its safety and its potential as a source of an effective
drug. Furthermore, this study will be of benefit to the following:
Pharmaceutical Industries. This study will provide an additional information in

regards with the Toxic Property of M. calabura Leaf Extract, as well as the Secondary
Metabolites Present.
Community. This study will impart new knowledge on people about the benefits
of M. calabura Plant, its Toxicity and the Secondary Metabolites present in it.
3
Students. This study will give them deeper knowledge with regards to the
benefits of the Muntingia calabura (Aratilis) Plant, such as the Secondary Metabolites
present that are accountable for its Toxicity.
Researchers. The results from the conducted research will give them ideas in
conducting further studies about the phytochemical constituents and toxicity of Muntingia
species, and to the other plants found in their locality. Moreover, this study will test their
patience, perseverance and understanding.
4
Chapter II
REVIEW OF RELATED LITERATURE AND RELATED STUDIES
This chapter presents the relevant works, literature, and studies of local and
foreign authors for the purpose of establishing a deeper and better understanding of the
topic and the problem and as basis of the methods and claims used in the study.
RELATED LITERATURE
The Use of Herbal Medicine
Botanical medicine or most commonly known as Herbal medicine refers to the

use of any parts of the plant, either bark, stem, roots, leaf, seeds, fruit, or berries as cure
for ailments. The use of Botanical medicine can be dated back in ancient Egypt where
record shows the use of garlic and juniper as early as 1700 B.C. A comprehensive
philosophy of herbal medicine was developed by the Greeks during the 100 B.C. and
was adapted by the Romans where they contributed to the theories of medicine creating
a vast knowledge of medical practices some of which we are still practicing today
(Gelayee et al., 2017).
Botanical medicine is widely used in the country. The reason as to why is

because Herbal medicine is inexpensive, abundant and has no side effects since it is all
natural. Though Herbal medicine is a natural product it is often mistaken as completely
safe. Herbal medicine contains active ingredients that may react badly to your body.
Traditional medicine encompasses a wide range of approaches. Herbs are currently
used to treat chronic and acute illnesses, as well as a variety of diseases and
complications, including cardiovascular disease, inflammation, and boosting the immune
system (Galor & Benzie, 2011).
Muntingia Species
Muntingia calabura L. (Muntingiaceae) is a tree native to tropical America, it is

known as Jamaica Cherry in the U.S.A. and calabura in Brazil, where it occurs as an
exotic species (Souza & Lozenzi, 2005).
Muntingia calabura L. (Aratilis), a shrub introduced to Southeast Asia from

Tropical America. It is also known locally as Jamaica cherry, belonging to
5
Elaeocarpaceae family. It is often seen growing as roadside trees and is also used as an
air pollution tolerance indicator. It is an annual plant, and flowers throughout the year. Its
leaves are distinctively lanceolate in shape, with margins irregularly serrate and fruits are
berries which turn red on maturation and are sweet in taste (Invasive Species
Compendium, 2019).
Muntingia calabura (Aratilis)
Aratilis, from the family of Muntingiaceae, stands 4 to 10 meters with hairs in a

radiating pattern. It has a soft and smooth appearance, and its leaves are large with a
heart-shaped base and is ranging from 10 to 25 centimeters. The flowers of M. calabura
are greenish yellow with fruit capsules that are millimeters away with each other. This
plant is commonly found in thickets and secondary forests in a low and medium altitudes
all over the Philippines. The Bark, Leaves, and Flowers of Aratilis are used to treat some
diseases like dandruff, chest pains, and fever. It is also considered as a source of fuel
wood (Stuart, 2016).
Muntingia calabura is known throughout the world as ‘‘Jamaican cherry,’’ in

Malaysia, particularly among the Malay, it is known as ‘‘kerukup siam’’. Being the sole
species within the genus Muntingia, it is native to southern Mexico, tropical South
America, Central America, the Greater Antilles, Trinidad, and St. Vincent. It is also
widely cultivated in warm areas in India and Southeast Asia such as Malaysia,
Indonesia, and the Philippines. In Malaysia and the Philippines, the Aratilis tree is
commonly cultivated as a roadside tree (Morton, 1987; Sani et al., 2012).
Muntingia calabura as Traditional Medicine
According to Sarojini & Mounika (2018), the barks of Muntingia calabura are
commonly used as an antiseptic and to help diminish the swelling in lower extremities.
The leaves of M. calabura are either boiled or steeped in water, they are also used to
reduce gastric ulcer, and to alleviate headache. Moreover, the boiled barks can be used
as a wash to reduce swelling in some areas of lower extremities. In the Philippines, the
flowers are also used to treat headaches, incipient cold, as tranquillizers, and
antispasmodics. Other than its medicinal uses, its fruits, which are commonly eaten
fresh, are frequently cooked in tarts or made into jam, while the leaf infusions are
consumed as a tea-like beverage.
6
According to Zakaria et al. (2008), pharmaceutical remedies have been

developed along with unwanted side effects. To combat with the situation, people looked
for an alternative method to treat their disease without any side effects. Muntingia
calabura have recently gained limited traditional uses throughout the world with record in
particularly Peru, Colombia, Mexico, Vietnam and Philippines. The flowers and barks
have antiseptic activity and boiled barks can be used as a wash to reduce swelling in the
lower extremities.
Phytochemicals
Phytochemicals are chemicals that can be found in plant which has preventive
properties in diseases. Phytochemicals are not required in sustaining life in human body.
They are non-essential nutrients. In the past research, phytochemicals are known to
produce chemicals to employ a defense mechanism against competitors, but recent
studies said that they can also prevent diseases in human body. Phytochemicals are so
many that each works in different actions; it has antioxidant activity that protects our
cells, hormonal action to help reduce menopausal symptoms and osteoporosis,
stimulation enzymes which help reduce the risk of breast cancer, interference with DNA
replication to prevent multiplication of cancer cells, antibacterial effects, and physical
action to prevent the adhesion of pathogens to human cell wall. Phytochemicals are
naturally present in many foods such as in whole grains, fruits, beans, herbs and
vegetables. There are some known phytochemicals like lycopene in tomatoes,
isoflavones in soy and flavonoids in fruits (Phytochemicals, n.d).
Alkaloids
Alkaloids are naturally composed of organic nitrogen, hydrogen and carbon.

Alkaloids are basic, they are single molecules, and it means they can absorb acid or
hydrogen ions. They react with acids to form salts just like other alkalis. They have a ring
structure with one nitrogen atom. They also have bitter taste. Alkaloids have different
physiological effects both in humans and other animals. Most alkaloids can be found in
plants and they can be also found in animals and fungi. There are also alkaloids that is
lethal to humans (Марія, n.d.).
Alkaloids are useful in the field of medicine. Mostly known alkaloids are quinine,
morphine, ephedrine, and atrophine. Quinine is known for being powerful in treating
7
disease like malaria. Morphine is best known for painkiller; they are mostly used in
surgery. It prevents the nerve cells to receive the message from the body about the pain.
Ephedrine functions as blood-vessel constrictors. It reliefs the uncomfortable common
colds. Atrophine is used to treat digestive symptoms. It slows down muscles and it
prevents the messages from being sent from the brain to muscle. Although alkaloids are
very useful in medicine, they can be also lethal to humans if overused (Kaur, 2015).
Carotenoids
Carotenoids are colorful plant pigments which play an important role if extracted.
They are found in fruits and vegetables like bell peppers, tomatoes, carrots, broccoli,
mangoes and etc. They can be spot in red, orange, yellow and green hues in fruits and
vegetables. Carotenoids are very beneficial to human's health. In order for the body to
absorb carotenoids, they must be consumed with a fat (Nagarajan et al., 2017).
Carotenoids help plants in absorbing light energy in photosynthesis. They act as

an antioxidant in the human body. They help to prevent cancer, like breast and prostate
cancer, for they have strong cancer-fighting properties. Fruits and vegetables containing
carotenoids can help people achieve a younger look. Carotenoids prevent
cardiovascular diseases and also have anti-inflammatory and immune system benefits.
Carotenoids can be converted into Vitamin A which is responsible for protecting eyesight
(Varzakas & Zakynthinos, 2016).
Flavonoids
Flavonoids are a group of polyphenolic compounds, diverse in chemical structure

and characteristics, found ubiquitously in plants. There is an increasing interest in the
studies related to flavonoids from dietary sources, due to growing evidence of the
versatile health benefits of flavonoids including anti-inflammatory, antioxidant,
antiproliferative and anticancer activity. Based on a published literature, this study
reviews current developments in flavonoids in food, with a focus on mechanism aspects,
where it can provide guidance for researchers conducting further studies and industries
in developing practical health agents (Xiao et al., 2011).
Flavonoids are best known for its anti-inflammatory and antioxidant properties,
as well as the ability to protect the cardiovascular and nervous systems. Since they aid
in the detoxification of potentially tissue-damaging molecules, though not always, their
8
consumption has been linked to a lower risk of some cancers, such as lung and breast
cancers. Flavonoids are quite remarkable group of phytonutrients that falls into the
chemical category of polyphenols. Flavonoids are highly bioactive and play a wide
variety of different roles in the health of plants, animals, and human health (What Are
Flavonoids?, 2020).
Glycosides
Glycosides are commonly found in plants; they have an intense bitter taste.
These are colorless, crystalline carbon, hydrogen and oxygen-containing water-soluble
phytochemical constituents, found in the cell sap. Glycosides contain a carbohydrate
and a non-carbohydrate part (Hamuel, 2012). The bitter taste acts on gustatory nerves,
which results in increased flow of saliva and gastric juices. Chemically, lactone groups
can be found on those bitter principles which can be a triterpenoids or lactones. Some of
the bitter principles are either used as astringents due to the presence of tannic acid, as
antiprotozoan, or to reduce thyroxin and metabolism. This includes cardiac glycosides
(acts on the heart), anthracene glycosides (purgative and for treatment of skin diseases),
chalcone glycoside (anticancer), amarogentin, gentiopicrin, andrographolide, ailanthone
and polygalin. Sarker and Nahar (2012) said that plants containing cyanogenic glycoside
can be fatal to living things.
Phenols
Phenols are a class of chemical compounds in which a hydroxyl group is

attached to an aromatic hydrocarbon group. It is produced by plants and other micro-
organisms but can also be synthesize industrially. In appearance pure phenols is a
colorless crystalline solid which has a sweet and tarry odor. Phenols has good
penetrating power into organic matter which is why it is often used as a disinfectant that
is actively effective against a wide range of micro-organisms including some fungi and
viruses, though phenols are only mildly effective against spores. It also has anesthetic
and antibacterial properties which makes it useful as an antiseptic but only to cauterize
infected areas because of its irritant and corrosive properties, as well as its potential
systemic properties (National Center for Biotechnology Information, 2014).
Phenols are also used in the preparation for cosmetics including sunscreens,
hair dye, and skin lightening products. It is also used in the production of drugs, weed
9
killers, and synthetic resins. Oral ingestion and skin exposure to concentrated solution of
phenol may cause chemical burn systemic toxicity, manifested primarily by CNS and
cardiovascular effects leading to death (Lin, 2016).
Saponins
Saponin is a toxic compound that has foaming characteristics. It also has

glucosides, steroids and terpenoid. The taste of saponin is bitter. Saponins are mainly
from Quijallasaponaria and Yucca schidigera extract. Saponins has the foaming ability
because of the combination of hydrophobic (fat-soluble) sapogenin and a hydrophilic
(water-soluble) sugar part. There are saponins that are toxic and called sapotoxin.
Saponins are phytochemical and mostly found in beans, vegetables and herbs; Peas,
soybeans and other herbs are best known for saponins, those who have foaming
properties such as soap wort, soap root, soap berry, and soap bark. There are many
health benefits in Saponin. There are studies that show the beneficial effect of saponin
such as in cancer, blood cholesterol levels, bone health and immune-stimulating activity.
The antimicrobial properties of saponins show that it is particularly against fungi, bacteria
and protozoa.
Saponins have a large role in medicinal field since it has hypolipidemic and
anticancer activity. It is also needed for the activities of cardiac glycosides. It has two
main types, diosgenin and hecogenin (Sarker & Njahar, 2007).
Steroids
Steroids are used to treat a variety of conditions in which the body’s defense
system malfunctions and causes tissue damage. Steroids are used as the main
treatment for certain inflammatory conditions, such as systemic vasculitis (inflammation
of blood vessels) and myositis (inflammation of muscle). They may also be used
selectively to treat inflammatory conditions such as rheumatoid arthritis, lupus, Sjögren’s
syndrome, or gout.
Steroids are used by prescription to treat certain types of medical disorders.

They are used to treat boys and men who don’t produce enough male hormones for
formal development. For example, they are given to me who have testes removed
because of testicular cancer and in treatments for impaired growth. They are also used
to treat some forms of anemia (a condition in which a person has too few red blood
10
cells), certain types of cancers, autoimmune diseases, certain skin diseases, and
osteoporosis (the loss of bone, which often occurs as people age) (“Steroids,” 2009).
Tannins
Tannin is a type of biomolecule, (as opposed to modern synthetic tannin), is an

astringent, bitter plant polyphenolic compound that either bonds and precipitates or
shrinks proteins and various other organic compounds including amino acids and
alkaloids. Tannins can be present in the leaves, bark, and fruits, and are thought to
protect the plant against infection and herbivory (“Phenolic Compound Biochemistry,”
n.d).
Tannin is used in leather manufacture and dyeing. They are also used in the
clarification of wine and beer, as a constituent to reduce viscosity of drilling mud for oil
wells, and in boiler water to prevent scale formation. Because of its styptic and
astringent properties, tannin has been used to treat tonsillitis, pharyngitis, hemorrhoids,
and skin eruptions; it has been administered internally to check diarrhea and intestinal
bleeding and as an antidote for metallic, alkaloidal, and glycosidic poisons, with which it
forms insoluble precipitates. Soluble in water, tannins form dark blue or dark green
solutions with iron salts, a property utilized in the manufacture of ink.
Serial Dilution
Serial dilution is used in pharmaceutical laboratories to obtain the necessary

concentration of chemicals and compounds as it is more efficient than independent
dilutions. Also, to prevent having to pipette very small volumes to produce a dilution of a
solution, serial dilutions are frequently used (Sapkota, 2020).
According to Sapkota (2020), serial dilution is the method of diluting a sample in

a sequence of standard volumes. In Biology, the degree of dilution is calculated based
on the approximate concentration of cells/organisms in a sample. Thus, to prevent
having to pipette very small volumes, and in order to produce a dilution of a solution,
serial dilutions are widely used.
Brine Shrimp
Brine Shrimps are mostly found in inland salt waters. Artemia, the only genus in
the family Artemiidae, has changed little externally since the Triassic period. The first
11
historical record of the existence of Artemia dates back to the first half of the 10th
century AD from Urmia Lake, Iran, by Iranian geographer as "aquatic dog", although the
first unambiguous record is the report and drawings made by Schlösser in 1757 of
animals from Lymington, England Brine shrimp usually occur in huge numbers and can
be seen in vast windblown lines in the Great Salt Lake. Their absence from the sea has
been explained by their vulnerability to be attacked by predators and the absence of the
latter in their inland saline habitat (Maszczyk & Wurtsbaugh, 2017).
Although brine shrimps are considered to be members of a single genus,

Artemis, and possibly a single species, there are several varieties. Generally, they have
stalked, compound eyes and tapered bodies with a trunk that bears 11 pairs of leaf like
legs. Females have a brood pouch from which active young are liberated under
favorable conditions (The Columbia Encyclopedia, 2017). Otherwise, eggs are laid
partheno genetically (unfertilized by sperm) or fertilized and can either hatch immediately
or be dried and remain viable for many years. These eggs are remarkably resistant to
adverse environmental conditions, which is why they can be hatched so easily in
saltwater and used for fish food; adult brine shrimp are also used as food in aquariums
and are generally sold frozen. Brine shrimps are classified in the phylum Arthropoda,
subphylum Crustacea, class Branchiopoda, order Anostraca (Brine Shrimp Cycle, 2017).
RELATED STUDIES
Foreign Studies
An attempt by (Mahmood et al., 2014), in the study entitled “Muntingia calabura:
A review of its traditional uses, chemical properties, and pharmacological observations,”
helped in isolating the bioactive compounds done from the leaves of M. calabura,
collected in Purus, Peru, in October 1997. The MEMCL was first partitioned into water,
petroleum ether (PEE), and ethyl acetate (EAE). Only the EAE partition was further
subjected to the isolation procedures, which led to the identification of 25 compounds,
and the identification of 12 flavonoids from the methanol extract.
A study by Singh et al. (2017), entitled “Phytochemical Analysis of Muntingia
calabura Extracts Possessing Anti-Microbial and Anti-Fouling Activities,” showed that the
phytochemical screening of M. calabura extracts revealed the presence of sterols,
flavonoids, alkaloids, and tannins. This activity can be developed if active components
are synthesized, and appropriate dosages are determined using in vivo studies for
12
efficient administration of the results. The study also concluded the presence of a huge
amount of lectin activity, if purified, it can be used for drug targeted therapy depending
on its carbohydrate specificity.
An experimentation done by Fitri et al. (2017), was conducted to identify the

secondary metabolites present in M. calabura Plant Extract. It revealed various
secondary metabolites present namely, Tannin and Saponins. After identifying each
active compound on the cherry leaf (Muntingia calabura), sirih leaf (Piper betle Linn) and
beluntas leaf, they discovered that one of the factors affecting the results of which
secondary metabolites are present were the kind of leaf, the type of plant, plant location,
local climate, genetics of the plant, and the weather when the research was performed.
In the study conducted by Krishnaveni & Dhanalakshmi (2014) entitled

“Qualitative and Quantitative Study of Phytochemicals in Muntingia calabura L. Leaf and
Fruit.” It states that carbohydrates, glycosides, tannin, phenolic compounds, proteins,
and amino acids showed intense reactions among the phytochemicals examined, while
the rest of the phytochemicals showed moderate reactions. The figure below shows the
Secondary Metabolites present in M. calabura Leaf Extract.
Table 1.
Secondary Metabolites Present in M. calabura Leaf Extract
Secondary Metabolites Leaf Extract

Test for Carbohydrates
a) Molisch’s test +++
b) Fehlings test +++
c) Benedict’s test +++
Test for Alkaloids
a) Wagners test ++
b) Hagers test ++
Test for Steroids
a) Libermann-Burchard test +++
Test for Sterols
a) Salwoski test ++
Test for Glycosides
a) Legal test +++
b) Baljet test +++
Test for Saponins
a) Saponin test ++
13
Test for Flavonoids

a) Shinoda test +
b) Zinc hydrochloride test ++
Test for Tannins
a) Ferric chloride test +++
b) Potassium dichromate test ++
Test for Phenols
a) Gelatin test +
Test for Carotenoids –
(+) Slight changes, (++) Moderate, (+++) Stronger reactions, (–) Absence
Local Studies
In the study of Barcelo (2015) entitled “Phytochemical Screening and Antioxidant

Activity of Edible Wild Fruits in Benguet, Cordillera Administrative Region, Philippines,”
the secondary metabolites present in the fruit of Muntingia calabura were identified.
Additionally, the study determined the antioxidant activity of the plant. Based on the
findings of the study, the following are the phytochemicals present in the fruit: Alkaloids,
Steroid, Glycosides, Saponins, Flavonoids, Polyphenols, and Tannins.
In the study entitled “Chemical constituents of Muntingia calabura L.” of Ragasa

et al. (2015) which reports on the non-polar constituents from the fruit of M. calabura, the
plant was reported to exhibit diverse biological activities. Moreover, there were also
studies on the isolation of non-polar components of the plant. Previous literature on M.
calabura reported mainly on the isolation of chalcones and flavonoids which exhibited
cytotoxic and anticancer properties.
In University of the Philippines, Diliman, Quezon City, a study entitled “Bioactive

metabolite profiles and antimicrobial activity of ethanolic extracts from Muntingia
calabura L. leaves and stems” of Buhian et al. (2016) states that antimicrobial activity
were exhibited by the leaf and stem extracts against Pseudomonas aeruginosa (P.
aeruginosa), Salmonella typhimurium, Staphylococcus aureus (S. aureus), Bacillus
subtilis, and Candida albicans (C. albicans), and also with minimal activity against
Escherichia coli. The extracts showed the highest activity against P. aeruginosa, C.
albicans, and S. aureus based on the MIC. The Phytochemical screening of the plant
extract revealed the presence of sterols, flavonoids, alkaloids, saponins, glycosides and
tannins in the leaf extract; however, no triterpenes were detected. In the stem extract of
14
the plant, triterpenes were detected along with large amounts of flavonoids, saponins,
glycosides and tannins. Alkaloids and sterols were absent in the stem extract. As a
conclusion, M. calabura leaf and stem ethanol extracts are considered to as a potential
source of antibacterial agents against P. aeruginosa and S. aureus. This is the first to
report the high degree of antifungal activity of M. calabura ethanolic extract, especially
against C. albicans.
Theoretical Framework
This study is anchored on the “Ecological Crop Rotation Theory of Antibiotic Use”
of Venter et al. (2016). The theory proposed that if we regularly adjust our "to-go"
antibiotic in the ICU, we will decrease the production of resistance because the selection
pressure for bacteria to establish resistance to a particular antibiotic will be reduced as
organisms become exposed to constantly changing antimicrobials.
The increased response of the microbial richness scores to plant rotations

formed over longer periods which is not reflected in the diversity scores. This illustrates
the susceptibility of next generation sequences of plants; this study is a justification of
plant series as an effective alternative that can be used for the development of an
effective drug.
Conceptual Framework
This study is anchored in the research conducted by Krishnaveni &

Dhanalakshmi (2014) entitled “Qualitative and Quantitative Study of Phytochemicals in
Muntingia calabura L. Leaf and Fruit.” It states that carbohydrates, glycosides, tannin,
phenolic compounds, proteins, and amino acids showed intense reactions among the
phytochemicals examined, while the rest of the phytochemicals showed moderate
reactions. According to the study of Nshimo et al. (2008) entitled “Cytotoxic Constituents
of Muntingia calabura Leaves and Stems Collected in Thailand,” the result revealed that
the cytotoxic flavonoids were extracted from Thai-grown Muntingia calabura leaves and
stems. The compounds that were found proved to be effective against a variety of
human murine cell lines and can combat against certain diseases. The inactive
compounds were also isolated.
15
Toxicity and
Muntingia calabura
Phytochemical
(Aratilis) Leaf Extract
Screening
Figure 1. Paradigm of the Study: The IV-DV Model of the Toxicity and
Phytochemical Screening of Muntingia calabura (Aratilis) Leaf
Extract
The diagram of the conceptual framework, as presented in Figure 1, denotes the

causal relationship between the independent variable as Muntingia calabura (Aratilis)
Leaf Extract, and the dependent variables as Phytochemical Screening and its Toxicity.
The arrow shows the effects between the two variables, in which the independent
variable will affect the dependent variable.
Definition of Terms
These are some of the terms that are defined conceptually and operationally to
provide clarity and better understanding.
Bioassay. Conceptually, it refers to the test method employed in measuring the

response of living animal or plant tissue to the toxicity of chemical contaminants
(Jeyanathan & Ghani, 2016). Operationally, this will be the test that will be used to
determine the Toxicity of the Muntingia calabura (Aratilis) Leaf Extract.
Brine Shrimp. Conceptually, it refers to the genus of aquatic crustaceans that

can be found in Salt Lake and marshes and used as living, frozen, or dried food in
aquarium (Brine Shrimp, 2021). In the study, it will be used to determine the toxicity of
M. calabura.
Brine Shrimp Lethality Assay. Conceptually, it refers to the general bioassay

system using Brine Shrimp nauplii (Cock & Ruebhart, 2009). In this study, it is the
procedure conducted to screen for the biological activity of M. calabura expressed as
Median Lethal Concentration.
16
Toxicity. Conceptually, it refers to the quality of being toxic (Sesli Sözlük -

toxicity, n.d.). Operationally, it is the ability of M. calabura to cause mortality of the brine
shrimp.
Leaf. Conceptually, It is one of the flat and typically green parts of a plant that
grows from a stem or twig (PNC Grow Up Great - Spring Lessons, n.d.). In the study,
this will be the part of the Muntingia calabura (Aratilis) Plant that will be used to
determine the secondary metabolites present and Its Toxicity.
Median Lethal Concentration. Conceptually, it refers to the average

concentration of a chemical or mixture in air as a gas, vapor, mist, or dust capable of
annihilating test animals exposed by inhalation under specific conditions (Segen’s
Medical Dictionary, 2011). Operationally, it refers to the statistically derived
concentration of the leaf extract of M. calabura expected to kill 50% of the brine shrimp
nauplii.
Muntingia calabura (Aratilis). Conceptually, it refers to an evergreen tree

growing to 9 m (29ft) by 12 m (39ft) at a fast rate, with leaves that are hairy and sticky,
also, a round and sweet berry fruit, giving a reddish appearance (Muntingia Calabura -
Useful Tropical Plants, n.d.). This plant will be used in the study for determining the
secondary metabolites present and Its Toxicity.
Serial Dilution. Conceptually, it refers to the method of dilution in which a

solution is diluted in a step-by-step manner with a dilution factor (Sapkota, 2020). In this
study, it was used to obtain the optimal plant extract concentration from a higher
concentration.
Probit Analysis. Conceptually, it refers to the parametric procedure that relies

on linear regression following transformation of toxicity data (Golan & Follett, 2017).
Operationally, It refers to the statistical tool used to obtain the LC50 of the leaf of M.
calabura.
Secondary Metabolites. Conceptually, it refers to the organic compounds that

are not directly involved in the normal growth, development or reproduction of an
organism (Li et al., 2016). Operationally, these are the substances that were determined
in the Phytochemical Screening and identified its presence in the M. calabura Leaf
Extract.
17
Chapter III
METHODOLOGY
This chapter gives the details of progressive development of the research that
will be conducted in a step-by-step manner. It includes the research design, materials
and instruments and the experiments and general procedures which will cover all the
methods that was conducted in this study.
Research Design
This study used Quantitative research method with the specific usage of an
Experimental design shown in Figure 2 which also presents the variables, treatments
and replicates used in the study. The Independent variable in the study is the Muntingia
calabura leaf extract while the dependent variables are Phytochemical Screening and its
Toxicity. For the Brine Shrimp Lethality Assay, Treatment 1 (T1) – Brine Solution
(Negative Control), Treatment 2 (T2) – Methanol (Positive Control), Treatment 3 (T3) –
1ug/mL Plant Concentration, Treatment 4 (T4) – 10ug/mL Plant Concentration and
Treatment 5 (T5) – 100ug/mL Plant Concentration.
Figure 2. Research Design: Toxicity and Phytochemical

Screening of M. calabura
18
General Procedure:
Materials and Instruments
The Muntingia calabura leaf was collected from Barangay Lagao, General
Santos City. Crude Plant Extract was collected through evaporation. The materials (e.g.,
weighing scale, funnel and beaker) used for the experimental procedure was collected
and borrowed from Barangay Lagao, General Santos City.
Chemicals
The chemicals and the reagents (e.g., ethanol, and methanol) used for the
Toxicity experimental procedure were obtained from RTE General Merchandise and
Amesco Drug Store.
Preparation of Aqueous Extract
Two hundred fifty grams (250g) of freshly washed M. calabura leaf was
completely soaked in 1.5 L of 95% ethyl alcohol for 48 hours and was filtered using a
Whatman No.1 Filter Paper. The crude ethanolic extract was concentrated through
evaporation, resulting to a thick, viscous and syrupy extract which was used for the tests
involved in the study.
Preparation of Concentrated Extracts
The solvent used in the extraction process was disposed by evaporation until the
syrupy extract was obtained. Serial dilution was then used to obtain the desired
concentration of 1ug/mL, 10ug/mL, and 100ug/mL.
Serial Dilution
The M. calabura leaf extract was taken in three vials, each with 9 mL of distilled
water. 1 ml of properly mixed sample is drawn and was added to the first vial to make
the total volume of 10 ml. This yields an initial dilution of 10^-1. The dilution was
thoroughly mixed. 1 ml of the mixture from the 10^-1 (1ug/mL) dilution was poured into
the second tube. The gross dilution factor in the second tube is now 10^-2 (10ug/mL).
The remaining tube is then processed in the same way, with 1 ml from the previous tube
added to the next 9 ml diluents. As three tubes are used, the final dilution for the M.
calabura leaf extract was 10^-3 (100ug/mL).
19
Phytochemical Screening
Test for Alkaloids (Mayer’s Test)

One (1) mL of the stock solution was placed in a test tube and treated
with Mayer’s reagent (Potassium Mercuric Iodide). The formation of a yellow-
colored precipitate indicated the presence of alkaloids.
Test for Carotenoids

One (1) mL of plant extract was mixed with 5mL of chloroform in a test
tube. It needs to be shaken vigorously. The resulting mixture was filtered, and
85% sulfuric acid was added. A blue color at the interface indicated the presence
of carotenoids (Raja, 2012).
Test for Flavonoids (Alkaline Reagent Test)
In a test tube, 1 mL of the stock solution was treated with a few drops of
sodium hydroxide solution. Formation of an intense yellow color, which becomes
colorless on addition of dilute acid, indicated the presence of flavonoids.
Test for Glycosides (KellarKillani Test)
In a test tube, two (2) mL of the aqueous extract with glacial acetic acid,
one drop of 5% FeCl3 and concentrated H2SO4 were prepared. Reddish brown
color at the junction of the two liquids and a bluish-green upper layer indicated a
positive result.
Test for Sterols (Salkowski’s Test)
In a test tube, 1 mL of the stock solution was treated with chloroform and
filtered. The filtrates were treated with few drops of Conc. Sulphuric acid, shaken
and allowed to stand. Appearance of golden yellow color indicated the presence
of triterpenes.
Test for Phenols (Ferric Chloride Test)
The ferric chloride test was used. In a test tube, 1 mL of the stock solution
was treated with three to four drops of ferric chloride solution. Formation of bluish
black color indicated the presence of phenols.
20
Test for Saponins (Froth Test)
In a graduated cylinder, one (1) mL of the stock solution was diluted with
20 mL of distilled water and was shaken for 15 minutes. Formation of one (1) cm
layer of foam indicated the presence of saponins.
Test for Tannins (Gelatin Test)
One (1) mL of the extract and 1% gelatin solution containing sodium

chloride were prepared in a test tube. Formation of white precipitate indicated the
presence of tannins.
Brine Shrimp Lethality Assay
The toxicity of M. calabura was tested using the Brine Shrimp Lethality Assay
from the methods of (Guevarra, 2005) with some modifications. The M. calabura leaf
extract was prepared prior to the bioassay. The different concentrations (1ug/mL,
10ug/mL, and 100ug/mL) were transferred to the vials using a dropper. An artificial
seawater was added to bring the volume of the water to 5mL.
Preparation of the Simulated Sea Water
For the hatching of eggs, an aquarium was separated into two sections,
one for the dim and the other for the illuminated part. The divider was punctured
with 3mm hole. It was then filled with an artificial sea water by dissolving 228g of
rock salt in 6L of distilled water.
Hatching of Brine Shrimp Eggs and Toxicity Test
The brine shrimp eggs were placed into the dim section. The hatched
nauplii were taken after 72 hours. Ten (10) nauplii were then transferred to the
vials using a dropper. A yeast suspension was added into each solution which
served as the food for the nauplii. The vials containing the nauplii were kept
under illumination for 24 hours. The number of dead nauplii were counted and
the percent death were determined.
21
Figure 3. Brine Shrimp Lethality Procedure of M. calabura Leaf Extract
Data Gathering Procedure
The gathering of data of the Phytochemical Screening was obtained from the
study of Krishnaveni & Dhanalakshmi (2014). Brine Shrimp Lethality Assay was done
after the conduct of each trial, wherein 5 treatments [Treatment 1 (Brine Solution),
Treatment 2 (Methanol), Treatment 3 (1ug/mL), Treatment 4 (10ug/mL), Treatment 5
(100ug/mL)] with 3 replicates each were employed. The researchers determined the
Median Lethal Concentration of the leaf extract, and the significant difference in the
mortality rate of Artemia salina (Brine Shrimp) nauplii.
Statistical Tool
The researchers used One-way Analysis of Variance (ANOVA) in determining

the significant difference in the mortality rate of Artemia salina (Brine Shrimp). For the
22
Toxicity, the results were analyzed using Probit Analysis as described by Finney. The
median lethal concentration (LC50) was determined using Linear Regression Analysis.
Ethical Consideration
In this experimental study, no participants were used. The researchers ensured

that the data results will have potential benefits in the society, and that no manipulation
of the gathered data was done during the conduct of the experiment. There are no
fabrications of data committed throughout the study, the researchers paraphrased and
used citations to avoid plagiarism. The experiment justified the claim and showed the
consistency of the results. It reveals that multiple justification, rather than single
justification, is suitable to make scientifically acceptable claims. When conducting the
experiment, equipment and materials were handled with care, the researchers ensured
their safety and that no harm was instilled to any members. The researchers also asked
for assistance from the experts in the field.
23
Chapter IV
PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA
This chapter presents, analyzes and interprets the data gathered in the study
through phytochemical screening and using the different treatments used in toxicity on
the leaf aqueous extract of M. calabura. The data gathered in this chapter were
subjected to analyze the relationship between the variables under investigation.
Phytochemical Screening of M. calabura Leaf
From the results gathered based on the study of Krishnaveni and Dhanalakshmi,
(2014), Table 1 shows the secondary metabolites present in the leaf of M. calabura.
Carbohydrates, Alkaloids, Flavonoids, Glycosides, Phenols, Saponins, Steroids, Sterols,
and Tannins were present. Additionally, Carotenoids were not detected on M. calabura
leaf based on the related study presented. This only means that the leaf has secondary
metabolites present.
Table 2.
Phytochemical Screening of M. calabura Leaf Extract by
(Krishnaveni & Dhanalakshmi, 2014).
Secondary Metabolites Leaf Extract
Carbohydrates +++
Alklaloids ++
Steroids +++
Sterols ++
Glycosides +++
Saponins ++
Flavonoids ++
Tannins +++
Phenols +
Carotenoids –
(+) Slight changes, (++) Moderate, (+++) Stronger reactions, (–) Absence
24
According to many previous studies, these phytochemicals has varied functions

and applications. Carbohydrates are the key source of nutrition for both animals and
plants. Plants produce carbohydrates using energy from sunlight (photosynthesis), they
store carbohydrates as starch, which are long polysaccharide strings (“Metabolism of
Carbohydrates,” 2021). Alkaloids are single molecules; they can absorb acid or
hydrogen ions. They have a ring structure with one nitrogen atom (‘Alkaloids,” n.d).
Flavonoids are found in most plant material which serves as an anti-cancer, anti-allergic,
antioxidant, anti-inflammatory and antiviral (“Flavonoids,” n.d). Glycosides are commonly
found in plants, they contain a carbohydrate and a non-carbohydrate part (Kar, 2007;
Firn, 2010). Phenols have antioxidant property and lowers the risk of having
cardiovascular diseases and cancer (“Types of Phytochemicals,” n.d). Saponins have
toxic effects which is noted to its hemolytic property, binds cholesterol which results to
the property of anti-cancer (“Saponins,” n.d). Steroids are used by prescription to treat
certain types of medical disorders, such as inflammation of blood vessels (“Steroids,”
2009). Sterols act on lowering the cholesterol levels (Griffin, 2005). Tannin is a bitter
plant polyphenolic compound that bonds, precipitates, or shrinks proteins and various
other organic compounds including amino acids and alkaloids. However, no Carotenoids
were detected (“Phenolic Compound Biochemistry,” n.d).
Toxicity of M. calabura Leaf

The results gathered were subjected to Probit Analysis and Linear Regression
Method. It indicated that the plant extracts are toxic based on the number of the dead
brine shrimp nauplii after 24 hours which is shown on Table 2.
Table 3.
Mean Number of Dead Nauplii after 24 hour-exposure to different concentrations of

Muntingia calabura
Muntingia T3 (1ug/mL) T4 (10ug/mL) T5 (100ug/mL)
calabura
Leaf 8.33 7.33 5.33
Table 3 shows the LC50 values of M. calabura leaf. The leaf has a great value of
0.0165 ug/mL in the LC50 computation. It is believed that the toxicity of M. calabura is
due to the presence of Saponins.
25
Saponins are toxic compounds that has foaming characteristics. It is a complex

compound that has a wide range of health benefits such as anti-cancer. (Francis et. al,
2002). It binds cholesterol which results in the formation of spores in the cell membrane
which leads to cell lysis (“Saponins”, n.d).
Table 4.
LC50 Value of M. calabura
M. calabura LC50 (ug/mL)
Leaf 0.0165
Brine shrimp lethality assay was first proposed by Michael, et al. and was
enhanced by other scientists. It is now widely used in the evaluation on the toxicity of
heavy metals, plant extracts etc. (Wu, 2014). Median Lethal Concentration is the dose
that is deadly to animals where the plant extract and chemical was administered to
under controlled conditions. According to Gupta et al. (1985), the LC50 value that is
greater than 100ug/mL is considered to be non-toxic which means that the M. calabura
leaf (0.0165ug/mL) is toxic. This is because the lower the value of the LC50, the more
toxic a substance is. This could be used as an anti-tumor and to diseases caused by
bacteria with antibiotic resistance if further study will be conducted and when it is already
safe for human intake.
Mortality Rate of A. salina
From the gathered data, table 4 shows the difference in the mortality rate of Artemia
salina (Brine Shrimp) nauplii.
Table 5.
Difference in the mortality rate of Artemia salina (Brine Shrimp) nauplii
Replicates Treatments
T1 T2 T3 T4 T5
(-) Control (+) Control 1ug/mL 10ug/mL 100ug/mL
R1 0.00 3.00 8.00 7.00 5.00
R2 0.00 2.00 8.00 8.00 5.00
R3 0.00 2.00 9.00 7.00 6.00
Mean 0.00 2.33 8.33 7.33 5.33
26
The results revealed that the M. calabura leaf extract has an effect to the
mortality rate of A. salina. The Treatment 3 (T3) – 1ug/mL Concentration of M. calabura
Leaf Extract had the highest mortality rate from all the treatments, with a mortality rate of
(8.33). It was followed by Treatment 4 (T4) – 10ug/mL Plant Concentration of M.
calabura Leaf Extract with a mortality rate of (7.33). Treatment 5 (T5) – 100ug/mL Plant
Concentration of M. calabura Leaf Extract with a mortality rate of (5.33), Treatment 2 (T2)
– Methanol (Positive Control) had an effect to the mortality rate of A. salina with a
mortality rate of (2.33), and Treatment 1 (T1) – Negative Control (Distilled Water) which
did not have an effect to the mortality rate of A. salina (0.00).
For the difference in the mortality rate of Artemia salina (Brine Shrimp) nauplii,
the data were subjected to One-way Analysis of Variance to further analyze the data
gathered. Table 5 shows the summary for one-way ANOVA of the mortality rate of
Artemia salina (Brine Shrimp) nauplii. The computed F-value (135.60) is greater than the
tabulated F-value (3.48) at 0.05 level of significance. This implies that the null hypothesis
is rejected (One-Way ANOVA Fcomp= 135.60 > Ftab = 3.48, α = 0.05). As such, there is a
significant difference in the mortality rate of Artemia salina (Brine Shrimp) nauplii.
According to the study of Ramos et al. (2009), Artemia salina (Brine Shrimp)
nauplii is helpful in evaluating the toxicity of Muntingia calabura plant extract. The toxicity
of Muntingia calabura to A. salina varied from low to high, which also depends on the
part of the plant used in the assay. Thus, this highlights the potential of M. calabura for
new pharmacological studies.
Table 6.
Summary Table for One-Way ANOVA of A. salina Mortality Rate
Source of Degree of Sum of Mean of Computed Tabulated

Variation freedom squares Squares F F
Between groups 4 144.67 36.17
135.60 3.48
Within groups 10 2.67 0.27
27
Chapter V
SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATIONS
This chapter presents the summary of findings, conclusion made from the study
and the recommendations given by researchers.
Summary of Findings
This study investigated the Secondary Metabolites present in M. calabura Leaf
Extract. In the procedure, the Plant Extract was assessed through Phytochemical
Screening from the result in a related study, and Toxicity Testing. The researchers
followed the standard procedures in testing the M. calabura Leaf Extract.
Phytochemical Screening
The M. calabura leaf extract based on the study of Krishnaveni & Dhanalakshmi
(2014), was positive for Carbohydrates, Alkaloids, Flavonoids, Glycosides, Phenols,
Saponins, Steroids, Sterols, and Tannins. However, no Carotenoids were detected.
These would account for the toxic property of the leaf extract.
Toxicity of Muntingia calabura (Aratilis)

The Toxicity level of M. calabura leaf extract in terms of LC50 is 0.0165ug/mL.
This indicates that the crude ethanolic extract from M. calabura leaf is toxic, the lower
the LC50, the more toxic it is. This can be also traced back to the present secondary
metabolites. This suggests that the M. calabura can be used for future medical
applications after further research.
Conclusions
The following conclusions were formulated based on the findings of the study.
1. The leaf extract was positive for the presence of Carbohydrates, Alkaloids,
Flavonoids, Glycosides, Phenols, Saponins, Steroids, Sterols, and Tannins. However,
no Carotenoids were detected.
2. The toxicity level against the Artemia salina (Brine Shrimp) nauplii using LC50 of the
leaf extract is 0.0165ug/mL.
3. The difference in the mortality rate of A. salina for Treatment 1 – Negative Control
(Distilled Water) is 0.00, Treatment 2 – Positive Control (Methanol) is 2.33, Treatment
3 – 1ug/mL Concentration of M. calabura Leaf Extract is 8.33, Treatment 4 – 10ug/mL
28
Concentration of M. calabura Leaf Extract is 7.33, and Treatment 5 – 100ug/mL

Concentration of M. calabura Leaf Extract is 5.33.
4. Based on the results, the null hypothesis (Ho) that there is no significant difference in
the mortality rates of Artemia salina (Brine Shrimp) nauplii was rejected.
Recommendations
This study has analyzed and showed the critical importance of Muntingia
calabura (Aratilis) Tree and its Leaves. The researchers discovered that it could produce
plentiful results. In relation to the conclusions, further research and recommendations
are hereby recommended:
1. Conduct a toxicity screening and observe it for a longer time rather than observing it
for 24 hours.
2. Formulate an ointment for the M. calabura Plant Extract and test it for its safety and
biological assays.
3. Investigate on the mutagenic activity of the M. calabura Plant Extract.
4. Conduct an antibacterial screening on Muntingia calabura (Aratilis) Plant Extract.
29
REFERENCES
Barcelo, R. (2015). eJBio. Phytochemical Screening and Antioxidant Activity of Edible

Wild Fruits in Benguet, Cordillera Administrative Region, Philippines, 11(3), 1–10.
https://ejbio.imedpub.com/phytochemical-screening-and-antioxidant-activity-of-
edible-wild-fruits-in-benguet-cordillera-administrative-region-philippines.pdf
Britannica. (2016). Alkaloid. Retrieved October 1, 2017 from

https://www.britannica.com/science/alkaloid
Britannica. (2016). Tannins. Retrieved October 1, 2017 from

https://www.britannica.com/topic/tannin.
Buhian, W. P. C., Rubio, R. O., Valle, D. L., & Puzon, J. J. M. (2016, June). Bioactive
metabolite profiles and antimicrobial activity of ethanolic extracts from Muntingia
calabura L. leaves and stems. ResearchGate.
https://www.researchgate.net/publication/303953129_Bioactive_metabolite_profil
es_and_antimicrobial_activity_of_ethanolic_extracts_from_Muntingia_calabura_
L_leaves_and_stems
Cock, I. E., & Ruebhart, D. R. (2009, January). Comparison of the brine shrimp nauplii
bioassay and the ToxScreen-II Test for the detection of toxicity associated with
Aloe vera (Aloe barbadensis Miller) leaf extract. Research Gate.
https://www.researchgate.net/publication/38310407_Comparison_of_the_brine_s
hrimp_nauplii_bioassay_and_the_ToxScreen-
II_Test_for_the_detection_of_toxicity_associated_with_Aloe_vera_Aloe_barbade
nsis_Miller_leaf_extract
Fitri, G. D., Tistiana, H., & Radiati, L. E. (2017, August). Review Study on Antibacterial
Activity of Cherry Leaf (Muntingia Calabura) Against Staphylococcus Spp. and
Salmonella Spp. the Most Causing Disease in Livestock. Neliti.
https://www.neliti.com/publications/98810/review-study-on-antibacterial-activity-
of-cherry-leaf-muntingia-calabura-against
Galor, S. W., & Benzie, I. F. (2011). Herbal Medicine: Biomolecular and Clinical Aspects.
NCBI Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK92773/
Gelayee, A. D., Mekonnen, G. B., Atnafe, S. A., Birarra, M. K., & Asrie, A. B. (2017,
August 22). Herbal Medicines: Personal Use, Knowledge, Attitude, Dispensing
Practice, and the Barriers among Community Pharmacists in Gondar, Northwest
Ethiopia. Hindawi. https://www.hindawi.com/journals/ecam/2017/6480142/
Golan, R. B., & Follett, P. A. (2017). Probit Analysis - an overview | ScienceDirect
Topics. ScienceDirect. https://www.sciencedirect.com/topics/agricultural-and-
biological-sciences/probit-analysis
Hampshire, R. G. (2009). United Nations Development Programme (UNDP). Human

Development Report 2009 Overcoming Barriers: Human Mobility and
Development, 1–229. http://hdr.undp.org
Hamuel, J. D. (2012, March). Phytochemicals: Extraction Methods, Basic Structures and

Mode of Action as Potential Chemotherapeutic Agents. ResearchGate.
30
https://www.researchgate.net/publication/221929467_Phytochemicals_Extraction
_Methods_Basic_Structures_and_Mode_of_Action_as_Potential_Chemotherape
utic_Agents
Hassali, H. A., Adam, Z., Saaya, F. M., Zainob, M., Hanif, A., & Aziz, A. (2015, March).
PHYTOCHEMICAL EVALUATION AND CYTOTOXIC ACTIVITIES OF LEAF
EXTRACTS OF MUNTINGIA CALABURA. ResearchGate.
https://www.researchgate.net/publication/280684691_PHYTOCHEMICAL_EVAL
UATION_AND_CYTOTOXIC_ACTIVITIES_OF_LEAF_EXTRACTS_OF_MUNTI
NGIA_CALABURA
Herbal Medicine. (2017). Retrieved September 27, 2017 from

https://www.unh.edu/health/ohep/complementaryalternative-health-
practices/herbal-medicine.
Herbal medicine. (n.d.). Retrieved September 27, 2017 from

http://www.umm.edu/health/medical/altmed/treatment/herbal-medicine.
Infoplease. (2017, January 24). Brine shrimp.

https://www.infoplease.com/encyclopedia/ecology/animals/invertebrates/brine-
shrimp
Jeyanathan, L., & Ghani, M. (2016, May 10). Analysis of Toxicity Effects of Triton X-100
on Tetrahymena ThermophiliaSpecies. Course Hero.
https://www.coursehero.com/file/21017829/Lab-Experiment-1/
Kaur, R. (2015, January). Alkaloids – Important Therapeutic Secondary Metabolites of

Plants origin. Research Gate.
https://www.researchgate.net/publication/319748653_Alkaloids_-
_Important_Therapeutic_Secondary_Metabolites_of_Plants_origin
King, A., & Young, G. (n.d.). Characteristics and occurrence of phenolic phytochemicals.
PubMed. Retrieved April 10, 2021, from
https://pubmed.ncbi.nlm.nih.gov/9972191/#:%7E:text=Phenolic%20phytochemica
ls%20are%20the%20largest,phenols%20and%20the%20most%20studied.
Krishnaveni, M., & Dhanalakshmi, R. (2014, September). QUALITATIVE AND

QUANTITATIVE STUDY OF PHYTOCHEMICALS IN MUNTINGIA CALABURA
L. LEAF AND FRUIT. ResearchGate.
https://www.researchgate.net/publication/265198290_QUALITATIVE_AND_QUA
NTITATIVE_STUDY_OF_PHYTOCHEMICALS_IN_MUNTINGIA_CALABURA_L
_LEAF_AND_FRUIT
Lavilla, C., & Villazorda, G. (2015, August). PHYTOCHEMICAL, BRINE SHRIMP

LETHALITY ASSAY, ANTIBACTERIAL AND ANTIOXIDANT ACTIVITY
SCREENING OF NYPAFRUTICANS ( " NIPA " ) LEAF EXTRACTS.
ResearchGate.
https://www.researchgate.net/publication/283278107_PHYTOCHEMICAL_BRIN
E_SHRIMP_LETHALITY_ASSAY_ANTIBACTERIAL_AND_ANTIOXIDANT_ACT
IVITY_SCREENING_OF_NYPAFRUTICANS_NIPA_LEAF_EXTRACTS
31
Li, S., Wang, P., Yuan, W., Su, Z., & Bullard, S. H. (2016). Endocidal Regulation of
Secondary Metabolites in the Producing Organisms. PubMed Central (PMC).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937345/
Libretexts. (2021, January 3). 2.6.1: Metabolism of Carbohydrates. Biology LibreTexts.

https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Bou
ndless)/2%3A_Chemistry/2.6%3A_Energy/2.6.1%3A_Metabolism_of_Carbohydr
ates#:%7E:text=Plants%20build%20carbohydrates%20using%20light,carbohydr
ates%20as%20the%20molecule%20glycogen.
Lin, D., Xiao, M., Zhao, J., Li, Z., Xing, B., Li, X., Kong, M., Li, L., Zhang, Q., Liu, Y.,
Cheng, H., Qin, W., Wu, H., & Chen, S. (2016, October 1). An Overview of Plant
Phenolic Compounds and Their Importance in Human Nutrition and Management
of Type 2 Diabetes. PubMed Central (PMC).
List of phytochemicals. (n.d.). Phytochemicals. Retrieved April 10, 2021, from

https://www.phytochemicals.info/phytochemicals.php
Loh, C. M., Mamphweli, S., Meyer, E., & Okoh, A. (2018, April 1). Antibiotic Use in
Agriculture and Its Consequential Resistance in Environmental Sources:
Potential Public Health Implications. PubMed Central (PMC).
M. (2021, January 14). Brine Shrimp: Life Cycle, Benefits & DIY Hatchery. Shrimp and
Snail Breeder. https://aquariumbreeder.com/brine-shrimp-life-cycle-benefits-diy-
hatchery/
Mahmood, N. D., Nasir, N. L. M., Rofiee, M. S., Tohid, S. F. M., Ching, S. M., Teh, L. K.,
Salleh, M. Z., & Zakaria, Z. A. (2014, July 28). Muntingia calabura: A review of its
traditional uses, chemical properties, and pharmacological observations. Taylor &
Francis. https://www.tandfonline.com/doi/full/10.3109/13880209.2014.908397
Mamedov, N. A., & Egamberdieva, D. (2019, February 12). Phytochemical Constituents

and Pharmacological Effects of Licorice: A. SpringerLink.
https://link.springer.com/chapter/10.1007/978-3-030-04408-
4_1?error=cookies_not_supported&code=fcd60b8f-17f5-47a3-beab-
f40467026188
Марія, М. (n.d.). TEXTBOOK OF PHARMACOGNOSY AND PHYTOCHEMISTRY.

ACADEMIA. Retrieved April 10, 2021, from
https://www.academia.edu/41974113/TEXTBOOK_OF_PHARMACOGNOSY_A
ND_PHYTOCHEMISTRY
Maszczyk, P., & Wurtsbaugh, W. (2017, May 20). Brine shrimp grazing and fecal
production increase sedimentation to the deep brine layer (monimolimnion) of
Great Salt Lake, Utah. Hydrobiologia.
https://link.springer.com/article/10.1007/s10750-017-3235-
32
Muntingia calabura - Useful Tropical Plants. (n.d.). Useful Tropical Plants. Retrieved
April 10, 2021, from
http://tropical.theferns.info/viewtropical.php?id=Muntingia+calabura
Nagarajan, J., Ramanan, R. N., Raghunandan, M. E., Galanakis, C. M., &

Krishnamurthy, N. P. (2017, January 1). Carotenoids. ScienceDirect.
https://www.sciencedirect.com/science/article/pii/B9780128052570000089
NHS website. (2020, January 17). Steroids. Nhs.Uk.

https://www.nhs.uk/conditions/steroids/
Nshimo, C. M., Pezzuto, J. M., Kinghorn, A. D., & Fansworth, N. R. (2008, September).
Cytotoxic Constituents of Muntingia calabura Leaves and Stems Collected in
Thailand. ResearchGate.
https://www.researchgate.net/publication/232048099_Cytotoxic_Constituents_of
_Muntingia_calabura_Leaves_and_Stems_Collected_in_Thailand
Phenol. (n.d.). PubChem. Retrieved April 10, 2021, from

https://pubchem.ncbi.nlm.nih.gov/compound/Phenol
Phytochemicals. (n.d). Saponins. Retrieved October 14, 2017 from

http://www.phytochemicals.info/phytochemicals/saponins.php.
Plant Sterols and Blood Cholesterol â€ • Scientific substantiation of a health claim

related to plant sterols and lower/reduced blood cholesterol and reduced risk of
(coronary) heart disease pursuant to Article 14 of Regulation (EC) No 1924/2006.
(2008, August 1). EFSA.
https://efsa.onlinelibrary.wiley.com/doi/pdf/10.2903/j.efsa.2008.781
PNC Grow Up Great - Spring Lessons. (n.d.). GROW UP GREAT. Retrieved April 10,
2021, from https://www.pnc.com/en/about-pnc/corporate-responsibility/grow-up-
great/lesson-center/spring/parts-of-a-plant.html
Ragasa, C., Tan, M. C., Chiong, I., & Shen, C. (2015, May). Chemical Constituents of
Muntingia calabura. ResearchGate.
https://www.researchgate.net/publication/275963205_Chemical_Constituents_of
_Muntingia_calabura
Ramos, D. R. S., Rodrigues, A. B. L., Farias, A. L. F., Simões, R. C., Pinheiro, M. T.,
Ferreira, R. M. D. A., Barbosa, L. M. C., Souto, R. N. P., Fernandes, J. B.,
Santos, L. D. S., & Almeida, S. S. M. D. S. D. (2017, January 1). Chemical
Composition and In Vitro Antioxidant, Cytotoxic, Antimicrobial, and Larvicidal
Activities of the Essential Oil of Muntingia calabura L. (Aratilis). Hindawi.
https://www.hindawi.com/journals/tswj/2017/4927214/
Sapkota, A. (2020, May 27). Serial dilution- definition, formula, calculator, procedure,
uses. Microbe Notes. https://microbenotes.com/serial-dilution/
Sarker, S., & Nahar, L. (2012, May). Chemistry for Pharmacy Students: General,
Organic and Natural Product Chemistry. ResearchGate.
33
https://www.researchgate.net/publication/225188868_Chemistry_for_Pharmacy_
Students_General_Organic_and_Natural_Product_Chemistry
Sarojini, S., & Mounika, B. (2018, January). Muntingia calabura (Jamaica Cherry): An
Overview. ResearchGate.
https://www.researchgate.net/publication/328739887_Muntingia_Calabura_Jama
ica_Cherry_An_Overview
Segen’s Medical Dictionary. (2011). median lethal concentration.

TheFreeDictionary.Com. https://medical-
dictionary.thefreedictionary.com/median+lethal+concentration#:%7E:text=median
%20lethal%20concentration%20The%20average%20concentration%20of%20a,
mg%2Fm3.%20Segen%27s%20Medical%20Dictionary.%20%C2%A9%202012
%20Farlex%2C%20Inc.
Sesli Sözlük - toxicity. (n.d.). Sesli Sözlük. Retrieved April 10, 2021, from
https://www.seslisozluk.net/toxicity-nedir-ne-
demek/#:%7E:text=The%20quality%20of%20being%20toxic%20The%20extent%
2C%20quality%2C,toxicity%20generally%20refers%20to%20intake%20of%20ex
cessive%20dosages
Singh, R., Iye, S., Prasad, S., Deshmukh, N., Gupta, U., Zanja, A., & Patil, S. (2017,
June). Phytochemical Analysis of Muntingia calabura Extracts Possessing Anti-
Microbial and Anti-Fouling Activities. ResearchGate.
https://www.researchgate.net/publication/318284975_Phytochemical_Analysis_o
f_Muntingia_calabura_Extracts_Possessing_Anti-Microbial_and_Anti-
Fouling_Activities
Souza, & Lozenzi. (2005). Muntingia calabura (Jamaica cherry). CABI Invasive Species
Compendium. https://www.cabi.org/isc/datasheet/35164
Stuart, G. (2016, January). Aratiles Muntingia calabura Linn. CHERRY TREE. Philippine
Medicinal Plants. http://stuartxchange.com/Aratiles
Study. (n.d). What is an Alkaloid.Retrieved October 1, 2017 from

http://study.com/academy/lesson/what-is-an-alkaloid-definition-examples.html.
Tapsell, L., Hemphill, I., Cobiac, L., & Patch, C. S. (2006, August). Health benefits of
herbs and spices, the past, the present. ResearchGate.
https://www.researchgate.net/publication/284309133_Health_benefits_of_herbs_
and_spices_the_past_the_present
Tripathi, G., & Shukla, S. P. (1988, June 1). Toxicity bioassay of technical and
commercial formulations of carbaryl to the freshwater catfish, Clarias batrachus.
ScienceDirect.
https://www.sciencedirect.com/science/article/abs/pii/0147651388900826
Varzakas, T. H., & Zakynthinos, G. (2016, March). Carotenoids: From Plants to Food
Industry. Research Gate.
34
https://www.researchgate.net/publication/297032265_Carotenoids_From_Plants
_to_Food_Industry
Venter, Z. S., Jacobs, K., & Hawkins, H. (2016, May). The impact of crop rotation on soil
microbial diversity: A meta-analysis. ResearchGate.
https://www.researchgate.net/publication/301791200_The_impact_of_crop_rotati
on_on_soil_microbial_diversity_A_meta-analysis
Vermerris, W., & Nicholson, R. (n.d.). Phenolic Compound Biochemistry. Phenolic

Compound Biochemistry. Retrieved April 10, 2021, from
https://link.springer.com/content/pdf/bfm%3A978-1-4020-5164-
7%2F1.pdfy?error=cookies_not_supported&code=98c52b24-1a51-4a76-bc61-
4c4d3647d606
What are Flavonoids? (2020, October 5). DJAB. https://www.drjosephahrens.com/what-

are-flavonoids
World Health Organization. (2004). Equitable access to essential medicines: a

framework for collective action. Institutional Repository for Information Sharing.
https://apps.who.int/iris/handle/10665/68571
Wu, L. B., Shhadi, M. Y., Gregorio, G., Matthus, E., Becker, M., & Frei, M. (2014).
Genetic and physiological analysis of tolerance to acute iron toxicity in rice.
PubMed Central (PMC). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4052628/
Xiao, Z., Peng, Z., Peng, M. J., Yan, W. B., Ouyang, Y. Z., & Zhu, H. L. (2011,
February). Flavonoids health benefits and their molecular mechanism. PubMed.
https://pubmed.ncbi.nlm.nih.gov/21222576/
35
APPENDIX A
CERTIFICATIONS
This to certify that the undersigned has closely examined the research study of
LYNNCH DATA, GIDEON C. AKIEH, CHRISTEL MAREE A. BOYBOY, SHADY NASH
A. KAMENZA, and AL JAYLONE A. BALT entitled TOXICITY AND
PHYTOCHEMICAL SCREENING OF Muntingia calabura (Aratilis) LEAF EXTRACT,
as to the content, organization, mechanism and format for improvement.
This certification is granted upon the request of the researchers and this is issued
at Basic Education Department, Senior High School Program of Holy Trinity College of
General Santos City.
JOHALIBRASSIM MAMALINTA LU
Statistician
36
APPENDIX B
CERTIFICATIONS
This to certify that the undersigned has closely examined the research study of
LYNNCH DATA, GIDEON C. AKIEH, CHRISTEL MAREE A. BOYBOY, SHADY NASH
A. KAMENZA, and AL JAYLONE A. BALT entitled TOXICITY AND
PHYTOCHEMICAL SCREENING OF Muntingia calabura (Aratilis) LEAF EXTRACT,
as to the content, organization, mechanism and format for improvement.
This certification is granted upon the request of the researchers and this is issued
at Basic Education Department, Senior High School Program of Holy Trinity College of
General Santos City.
ANDREVE JOHN L. REBUCIAS, LPT

Grammarian
37
APPENDIX C
RESEARCH LAYOUT
Brine Shrimp Lethality Assay of M. calabura Leaf
T3R1 T2R3 T4R1 T5R3 T3R3
Complete Randomized Design
Legend:
Rn = Replicates
T1 = Brine Solution
T2 = Methanol
T3 = 1ug/ml Leaf Extract Concentration
T4 = 10ug/mL Leaf Extract Concentration
T5 = 100ug/mL Leaf Extract Concentration
38
APPENDIX D
STATISTCAL ANALYSIS
Toxicity
Probit Calculation of M. calabura Leaf Extract
Concentration Total no. No. of Dead Corrected

(ug/mL) of Nauplii % Mortality Mortality Probit
Nauplii
0 10 2.33 23.03 0.00 -
1 10 8.33 83.03 78.00 5.77
10 10 7.33 73.03 65.00 5.39
100 10 5.33 53.03 39.00 4.72
Abbot’s formula for control mortality of M. calabura Leaf Extract
Corrected % Mortality = % 𝑀𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦−𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑥 100

100−𝑐𝑜𝑛𝑡𝑟𝑜𝑙
23.03 − 23.03
= 𝑥 100
100−23.03
=0

83.03− 23.03
= 𝑥 100
100−23.03
= 78

73.03 − 23.03
= 𝑥 100
100−23.03
= 65

39
53.03 − 23.03
= 𝑥 100
100−23.03
= 39
Regression Method Table of M. calabura Leaf Extract
x y ̅)
(x-𝒙 ̅ )2
(x-𝒙 ̅
y-𝒚 ̅ )2
(y-𝒚 ̅)(y-𝒚
(x-𝒙 ̅)
1 5.77 -1 1 0.48 0.2304 -0.48
2 5.39 0 0 0.1 0.01 0.00
3 4.72 1 1 -0.57 0.3249 -0.57
̅
𝒙=2 ̅=5.29
𝒚 ∑(x-𝒙
̅)2=2 ∑(y-𝒚
̅)2=0.5653 ∑[(x-𝒙
̅)(y-𝒚
̅)]=-1.05
̅)(𝑦−𝒚
∑[(𝑥−𝒙 ̅)]
b1 = ̅)2)
b0 = y – b1x
∑[(𝑥−𝒙
−1.05
b1 = b0 =5.29 – (-0.525) (2)
2
b1 = −0.525 b0 =5.29 – (-1.05)
b0 = 6.34
y = b0 + bx ; where y = probit of 5
5.0 = 6.34+ (-0.525) x
5.0 – 6.34 = -0.525 x
−1.34 −0.525𝑥
−0.525
= −0.525
-2.55 = x
LC50 = antilog (-2.55)
LC50 = 0.0165 ug/mL
40
One-way ANOVA
Replicates Treatment 1 Treatment 2 Treatment 3 Treatment 4 Treatment 5
1 0 3 8 7 5
2 0 2 8 8 5
3 0 2 9 7 6
SUM 0 7 25 22 16
MEAN 0 2.33 8.33 7.33 5.33
Anova: Single Factor
SUMMARY
Groups Count Sum Mean Variance
Treatment 1 3 0 0 0
Treatment 2 3 7 2.333333 0.333333
Treatment 3 3 25 8.333333 0.333333
Treatment 4 3 22 7.333333 0.333333
Treatment 5 3 16 5.333333 0.333333
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 144.6667 4 36.16667 135.60 1.15E-08 3.47805

Within Groups 2.666667 10 0.266667
41
APPENDIX E
BUDGET ALLOCATION
These are all the material and other expenses gathered by the researchers.
Expenses Quantity Amount Total Price
Aquarium 1 Php700 Php700

Aerator 1 Php300 Php300
Brine Shrimp Eggs 15g Php120 Php120
Distilled Water 7L Php80 Php80
Rock Salt 228g Php30 Php30
Foil 1 Php100 Php100
Ethanol 1.5L Php200 Php200
Disposable Pipette 3 Php30 Php90
Vials 15 Php25 Php375
Whatman Filter Paper 10 Php450 Php450
Nitrile Gloves 2 Php60 Php120
Tricycle Fare Php120 Php120
TOTAL ₱2,685
42
APPENDIX F
DOCUMENTATION
43
44
CURRICULUM VITAE
PERSONAL BACKGROUND
Name: Lynnch Data

Age: 18
Birthday: March 08, 2003
Birthplace: General Santos City
Email Address: datalynnch@gmail.com
Address: 10 Royeca Subd., Brgy. Lagao, General Santos City
SKILLS
• Attention to Detail
• Analytical Skills
• Writing
• Leadership Skills
EDUCATIONAL BACKGROUND
Secondary: Holy Trinity College of General Santos City | Senior High School
2019 – 2021
Secondary: Holy Trinity College of General Santos City | Junior High School
2015 – 2019
Elementary: Dadiangas West Central Elementary School
Magsaysay Avenue, General Santos City
2009 – 2015
45
CURRICULUM VITAE
PERSONAL BACKGROUND
Name: Gideon C. Akieh

Age: 20
Birthday: February 19, 2001
Birthplace: Lagos
Email Address: gideonakieh2@gmail.com
Address: Blk. 7 Lilac Street, Brgy. City Heights, General Santos City
SKILLS
• Boxing
• Soccer
• Drawing
• Basketball
2019 – 2021
Secondary: Lagacy High School | Junior High School
No. 16, Victoria Island
2015 – 2019
Elementary: Lagacy High School
No. 16, Victoria Island
2009 – 2015
46
CURRICULUM VITAE
PERSONAL BACKGROUND
Name: Christel Maree A. Boyboy

Age: 18
Birthday: December 24, 2002
Birthplace: Quezon City, Manila
Email Address: cmab1224@gmail.com
Address: Purok 7, Glamang, Polomolok, South Cotabato
SKILLS
• Making organic pesticides

• Playing outdoor sports
• Speech making
• Playing instruments
2019 – 2021
Secondary: Nicolas B. Barreras National High School | Junior High School
Purok 3, Glamang, Polomolok, South Cotabato
2015 – 2019
Elementary: Glamang Elementary School
Purok 4, Glamang, Polomolok, South Cotabato
2009 – 2015
47
CURRICULUM VITAE
PERSONAL BACKGROUND
Name: Shady Nash A. Kamenza

Age: 18
Birthday: September 04, 2002
Email Address: shadynash.02@gmail.com
Address: Doña Soledad Subd., Brgy. Labangal, General Santos City
SKILLS
• Cooking
• Assembling motor parts
• Billiard
• Driving long trips
2019 – 2021
Secondary: Holy Trinity College of General Santos City | Junior High School
2015 – 2019
Elementary: Saint Salvador Del Mundo Montessori School
Doña Soledad Subd. Brgy. Labangal, General Santos City
2009 – 2015
48
CURRICULUM VITAE
PERSONAL BACKGROUND
Name: Al Jaylone A. Balt

Age: 17
Birthday: June 17, 2003
Email Address: jayajay0912@gmail.com
Address: Brgy. Bula, Alunan St., General Santos City
SKILLS
• Cooking
• Badminton
2019 – 2021
Secondary: Ireneo L. Santiago National High School of Metro Dadiangas | High School
Niyog Street, General Santos City

2015 – 2019
Elementary: Dadiangas East Elementary School
Laurel Avenue, General Santos City
2009 – 2015
49

Toxicity and Phytochemical Screening of Muntingia Calabura (Aratilis) Leaf Extract - DATA

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Toxicity and Phytochemical Screening of Muntingia Calabura (Aratilis) Leaf Extract - DATA

Uploaded by

Copyright:

Available Formats

HOLY TRINITY COLLEGE

Toxicity and Phytochemical Screening of Muntingia calabura

A Quantitative Research Paper

Holy TrinityCollegeof General SantosCity

This research entitled TOXICITY AND PHYTOCHEMICAL SCREENING OF

ANDREVE JOHN L. REBUCIAS, LPT.

JUDY ANN H. VERONILLA, LPT.

JAYBOY M. SARTORIO JAMES BERNHARDT P. PERPETUA, LPT.

MARIA GIOVANNA G. ABECIA, EdD.

To the panelists, Judy Ann H. Veronilla, Jayboy M. Sartorio, and James

The Toxicity and the presence of Secondary Metabolites on Muntingia calabura

Keywords: Muntingia calabura, phytochemical screening, secondary metabolites,

1 THE PROBLEM AND ITS SETTING

2 REVIEW OF RELATED LITERATURE AND STUDIES

• Hatching of Brine Shrimp Eggs and Toxicity Test ......................21

Data Gathering Procedure ............................................................................... 22

4 PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA

5 SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

C. Research Layout .......................................................................................... 38

2 Research Design: Toxicity and Phytochemical Screening of 18

3 Brine Shrimp Lethality Procedure of M. calabura Leaf Extract 22

1 Secondary Metabolites Present in M. calabura 13

2 Phytochemical Screening of M. calabura Leaf 24

3 Mean Number of Dead Nauplii after 24-hour 25

4 LC50 value of M. calabura 26

5 Difference between the mortality rates of Artemia 26

6 Summary Table for One-way ANOVA of A. salina 27

The result of this study determined the effectiveness of Muntingia calabura

Statement of the Problem:

1. Are the following secondary metabolites present in Muntingia calabura (Aratilis)

3. Is there a significant difference in the mortality rate of Artemia salina (Brine

1. The LC50 of Muntingia calabura (Aratilis) Leaf Extract is greater than

Scope and Delimitation

This study aimed to determine the Phytochemical Constituents and toxicity of

Significance of the Study

This study aimed to identify the Secondary Metabolites present in M. calabura

Pharmaceutical Industries. This study will provide an additional information in

REVIEW OF RELATED LITERATURE AND RELATED STUDIES

The Use of Herbal Medicine

Botanical medicine or most commonly known as Herbal medicine refers to the

Botanical medicine is widely used in the country. The reason as to why is

Muntingia calabura L. (Muntingiaceae) is a tree native to tropical America, it is

Muntingia calabura L. (Aratilis), a shrub introduced to Southeast Asia from

Muntingia calabura (Aratilis)

Aratilis, from the family of Muntingiaceae, stands 4 to 10 meters with hairs in a

Muntingia calabura is known throughout the world as ‘‘Jamaican cherry,’’ in

Muntingia calabura as Traditional Medicine

According to Zakaria et al. (2008), pharmaceutical remedies have been

Alkaloids are naturally composed of organic nitrogen, hydrogen and carbon.

Carotenoids help plants in absorbing light energy in photosynthesis. They act as

Flavonoids are a group of polyphenolic compounds, diverse in chemical structure

Phenols are a class of chemical compounds in which a hydroxyl group is

Saponin is a toxic compound that has foaming characteristics. It also has

Steroids are used by prescription to treat certain types of medical disorders.

Tannin is a type of biomolecule, (as opposed to modern synthetic tannin), is an

Serial dilution is used in pharmaceutical laboratories to obtain the necessary

According to Sapkota (2020), serial dilution is the method of diluting a sample in

Although brine shrimps are considered to be members of a single genus,

An experimentation done by Fitri et al. (2017), was conducted to identify the

In the study conducted by Krishnaveni & Dhanalakshmi (2014) entitled

Secondary Metabolites Leaf Extract

Test for Flavonoids

In the study of Barcelo (2015) entitled “Phytochemical Screening and Antioxidant