CHAPTER 1

INTRODUCTION

Rationale

Cytotoxicity refers to the ability of certain chemicals or mediator cells to destroy living cells.

There are different types of cytotoxicity that will affect different types of cell, generally and

depending on the agent, a cytotoxic agent may be geno toxic (causing DNA damage), mutagenic

(causing gene mutation) or it may be harmful for cell organelles (e.g. cell membrane) or it may

even be teratogenic (Salman S, 2014). In relation to cancer, this will become an alternative

treatment to cancer. Cancer after cardiovascular disease is the second leading cause of death in

the world. Approximately 10 million people are diagnosed with it every year and 6 million die

of it. Treatment options for cancer include chemotherapy, surgery, radiation, and

immunotherapy. The use of any of these treatments depends upon the location, grade, tumour

stage as well as the general state of a person’s health. Chemotherapy and radiation in addition to

cancer cells, destroy normal cells and recently resistance to chemotherapeutic drugs has been

reported in some cases and this has renewed an interest in alternative and herbal methods to

complement conventional treatment. Recent attention has focused on plants which may provide

a good opportunity for complementary cancer treatment. In fact, medicinal plant are more

available, cheaper and possesses less toxicity compared to modern (allopathic) drugs. Biological

components of plants can be important sources for new drugs which may lead to new and better

treatments for cancer, and Muntingia Calabura linn also known as Mansanitas here in the

Philippines is one of those plants.

 Muntingia calabura Linn is a plant that belongs to the Elaeocarpaceae family, commonly

known as cherry tree. This plant has been used as the Peru traditional medicine in reducing the

swell of the prostate gland and alleviating headaches and cold as well as pains associated with

gastric ulcers. Muntingia calabura Linn cytotoxic constituents, these constituents are cytotoxic

chalcones and flavonoids. Chlacones is a unique template that is associated with several

biological activities. Cytotoxicity against tumour cell lines may be the result of the cell cycle,

inhibition of angiogenesis, interference with p53-MDM2 interaction, mitochondrial uncoupling

or induction of apoptosis. Flavonoids are ubiquitous in nature. They are also in food, providing

an essential link between diet and prevention of chronic diseases including cancer. Anticancer

effects of these polyphenols depend on several factors: Their chemical structure and

concentration, and also on the type of cancer.

In this study, the cytotoxicity level of Muntingia calabura Linn leaves will be measured

through counting the dead brine shrimps in every trial.

Statement of the Problem

This study is about using Muntingia calabura Linn or Mansanitas leaves in Brine shrimp

lethality assay aims to identify the cytotoxicity level of mansanitas. Specifically, this study

intends to: 1) Determine the number of Brine shrimps before and after the experiment. 2)

Cultivating the brine shrimp used in the experiment. 3) A stable environment for the whole

experiment set up.

Significance of the Study

The significance of this study is to investigate Muntingia calabura Linn ethanolic extract as

a cytotoxic agent. It also aims to search for a new source of medicine to overcome toxicity
problems in the body cells. This study is beneficial to medical organizations or pharmaceutical

companies because this serves as a precursor to developing medicines. In treating toxins also, it

could be used in treating cancer cells. This is also beneficial to the general public or more

specifically to the cancer patients because current treatments such as chemotherapy is expensive

and using plant as a cytotoxic agent would be cheaper.

CHAPTER 2

REVIEW OF RELATED LITERATURE

Cytotoxicity

The state of being cytotoxic or the quality of being toxic to cell meaning, an agent has a

specific destructive action on certain cells. Researchers and scientists currently have their eyes

on plant derived components as an alternative source to battle diseases including cancer (Tiwari et

al., 2015). Determining the potential toxicity of plant extracts or biologically active compounds

isolated from plants is an essential for the successful development of treatments to diseases

(McGaw et al., 2014). In our study, the leaves of M. calabura were prepared as an ethanol extract

to determine its cytotoxic properties and were then applied to the brine shrimps. Plants found to be

toxic to brine shrimp are likely to be good candidate for anti-cancer research (Ramachandran et al.,

2011).

In the study of Kaneda et al. (1991), the cytotoxic activity of M. calabura was performed

using the roots and were prepared as methanol extract. The extract subjected to the isolation of

bioactive compounds and then tested against BC1 (human breast cancer), HT-1080 (human

fibrosarcoma), Lu1 (human lung cancer), Me12 (human melanoma), Co12 (human colon cancer),

KB (human nasopharyngeal carcinoma), KB-V (vincristine-resistant KB), and P-388 (murine

lymphocytic leukemia) cell lines. Twelve compounds were isolated from the methanol extract of

M. calabura roots, 11 compounds exerted cytotoxicity activity against P-388 cells, 10 compounds

exerted cytotoxicity activity against KB and KB-V cells. As for BC-1, HT-1080, and Lu-1, only

three compounds caused a cytotoxic effect and for the ME-12 cells, only two compounds did not

show any cytotoxic effect. Against Co12, only five compounds did not exert any cytotoxic effect.

The study of Kaneda et al. (1991) is somewhat the same to our study because they also used

Muntingia calabura Linn but they focused on its roots and the type of extract is not the same.

In another study conducted by Chen et al. (2005), 20 bioactive compounds were isolated

from the methanol extract of M. calabura leaves against P-388 (murine lymphocytic leukemia)

and HT-29 cells (human colon cancer). Four compounds were not cytotoxic against both type of

cancer cells and three compounds were not cytotoxic against HT-29 cells. The same as our study,

they used the same part of M. calabura which is the leaves but the type of extract is not the same.

Mansanitas (Muntingia calabura Linn)

Mansanitas is a fast-growing tree reaching 5-10 meters high with spreading branches. Leaves

are hairy, sticky, alternate, distichous, oblong-ovate to broadly oblong-lanceolate, 8 to 13

centimeters long, with toothed margins, pointed apex and inequilateral base, one side rounded and

the other acute. Flowers are about 2 centimeters in diameter, white, extra-axillary, solitary or in

pairs. Sepals are 5, green, reflexed, lanceolate, about 1 centimeter long. Petals are white, obovate,

1 centimeter long, deciduous and spreading. Fruit is a berry, rounded, about 1.5 centimeter in

diameter, red on ripening, smooth, fleshy, sweet and many seeded. In the Philippines, these are

widely scattered as a semi-cultivated tree (Stuart Jr., 2016).

Metabolites found in Mansanitas

Chalcones

Chalcones are pale yellow crystalline ketone, l-2-propen-1-one, C₆H₅CH=CHC(O)·C₆H₅.

Chalcones possess a broad spectrum of biological activities including antioxidative, antibacterial,

antihelmintic, amoebicidal, antiulcer, antiviral, insecticidal, antiprotozoal, anticancer, cytotoxic

and immunosuppressive (Yerragunta et al., 2013). These are considered to be the precursor of

flavonoids and isoflavonoids.

Flavanoids

Flavonoids are the most abundant polyphenols, they are categorized into three main

groups which are: 1) Flavonoids, 2) Isoflavonoids, and 3) Neoflavonoids, with other

subgroupings namely flavones, flavonols, flavanones, flavanonols and anthocyanins. Flavonoids

are beneficial to humans because it provides antioxidants that can be used in various health

issues such as cardiovascular diseases. Flavonoids contains polyphenolic secondary metabolites

with broad-spectrum pharmacological activities including their potential role as anti-cancer

agents (Batra P., Sharma A., 2013).

Mechanism of action of Mansanitas

Antiproliferative activity
 Antiproliferative substance are used to prevent the spread of cells, especially malignant

cells. The study made by Zakaria et al. (2011) used M. calabura leaves and were prepared as

aqueous extract, methanol extract and chloroform extract. The three extract showed

antiproliferative activity against the following: MCF-7 (estrogen-dependent human breast

adenocarcinoma), HeLa (human cervical adenocarcinoma) and K-562 (chronic myelogenous

leukemia).

Antibacterial activity

Antibacterial is anything that destroys bacteria or suppresses their growth or their ability to

reproduce. According to the study of Zakaria et al. (2006), M. calabura leaves has demonstrated

the potential use of M. calabura, especially its aqueous and methanol extracts, as an effective

antibacterial against the infection of some of the bacteria used, particularly C. diphteriae, S.

aureus and P. vulgaris.

Antioxidant activity

An antioxidant is something that prevents or repairs damage that has been done to the

body by oxidation. In the study conducted by Aruna Sindhe et al. (2013),

1.1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and metal chelating ability for

ferrous ions were used to screen the antioxidant property of the ethanol extract and aqueous

extract of M. calabura leaves. Results showed that the ethanolic and aqueous extracts exhibited a

higher antioxidant activity, while the chloroform and petroleum ether extracts was the least.

Anti-inflammatory activity

Anti-inflammatory is a substance that reduces certain signs of inflammatory such as

swelling, tenderness, fever and pain. In the study of Preethi et al. (2012) the anti-inflammatory
activity of M. calabura fruits were evaluated. The fruits were prepared as a methanol extract at

doses of 100, 200, and 300 mg/kg. The methanolic fruit extracts reduced the Carrageenan-induced

edema of the hind paw of adult male Wistar Albino rats in 3 hours. The activity was compared

with that of the standard drug indomethacin. Acute toxicity was investigated and the results

indicated no abnormalities in the behaviour and lethality by the extract up to 1000 mg/kg. These

results indicate the fruit extract of M. calabura possess potent anti-inflammatory activity.

Therefore, these pharmacological results clearly support traditional folkloric application of M.

calabura fruits in the control and/or pain, inflammatory illness as well as an antioxidant agent.

Antinoceptive activity

Antinoceptive describes or relates to any unique factor that increases tolerance for, or

reduces sensitivity to, a dangerous or harmful stimuli, for example, a stimuli that may cause

pain. In the study conducted by Sani et al. (2012), methanol extract of M. calabura leaves

(MEMC) were used to determine its antinociceptive activity and to elucidate the possible

mechanism of antinociception involved. The in vivo chemicals (acetic acid-induced abdominal

constriction and formalin-, capsaicin-, glutamate-, serotonin-induced paw licking test) and

thermal (hot plate test) models of nociception were used to evaluate the extract antinociceptive

activity. The extracts (100, 250, and 500 mg/kg) were administered orally 60 min prior to

subjection to the respective test. The results obtained demonstrated that MEMC produced

significant (P < 0.05) antinociceptive response in all the chemical- and thermal-induced

nociception models, which was reversed after pretreatment with 5 mg/kg naloxone, a

non-selective opioid antagonist.

Anti-ulcer activity
 Anti-ulcer is a substance that prevents, reduce or heal ulcers in the stomach and upper part

of the small intestine. The study of Ibrahim et al. (2012) evaluated the anti-ulcerogenic

properties of the ethanol extract of M. calabura leaves in Sprague-Dawley male rats with

ethanol-induced gastric ulcers. Results showed significant protection of gastric mucosa against

ethanol-induced injury as evidenced by increased mucus production and decrease acidity of

gastric content.

Anti-diabetic activity

Anti-diabetic agent is a substance that helps a person with diabetes control their level of

glucose (sugar) in the blood. The study made by Mohamed Azmathulla Khan et al. (2012)

investigated the in vitro anti--diabetic activity of proteins from M. calabura root. Study

concludes root proteins possess significant antioxidant and antidiabetic activity. In vitro

antidiabetic potential was confirmed through alpha amylase enzyme, alpha glucosidase enzyme

inhibition studies and glucose uptake in yeast cell studies.

Cardioprotective activity

Cardioprotective is a substance that serves to protect the heart or coronary arteries from

injury, disease, or malfunction. In the study of Nivethetha et al. (2009), aqueous extract of M.

calabura leaves (AEMCL) were used. The authors studied the extract ability to attenuate

isoproterenol-induced myocardial infarction in rats. Several parameters (e.g., aspartate

transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH), and creatinine

phosphokinase (CK)) were estimated in both the serum and heart tissues, and the serum uric acid

level was also estimated. From the results obtained, AEMCL caused significant reduction in the

activity of marker enzymes (AST, ALT, CK, and LDH) and the level of uric acid when
compared with the isoproterenol-induced myocardial infarction group. In all parameters

estimated, only 200 and 300 mg/kg AEMCL exerted significant effects.

Cancer

Cancer is the disease caused by an uncontrolled division of abnormal cells in a part of

the body. According to the World Health Organization, cancer is the second leading cause of

death world-wide, and was responsible for 8.8 million cancer deaths in 2015. There are a lot of

ways to treat cancer, one of it is chemotherapy. Chemotherapy is the treatment that uses drugs to

stop the growth of cancer cells, either by killing the cells or by stopping them from dividing.

Chemo drugs kills fast-growing cells like cancer cells however, they can affect normal and

healthy cells that are also fast-growing. Cells that are mostly damaged by chemotherapy are

blood-forming cells in the bone marrow, hair follicles and cells in the mouth and digestive

system. Most common side effects caused by chemotherapy are fatigue, hair loss, anemia,

nausea, etc. The problems of poor selectivity and severe side effects of the available anticancer

drugs, have demanded the need for the development of safer and more effective

chemotherapeutic agents (Ogbole et al., 2017).

Brine Shrimp Lethality Assay

Brine shrimp lethality assay is a simple, high throughput cytotoxicity test of bioactive

chemicals. It is based on the killing ability of test compounds on a simple zoological

organism-Artemia salina or brine shrimp (Harwig J, Scott PM, 1971). The said test is widely

used in medicines especially natural plant extracts. In conducting this test, the nauplii or the

laboratory cultured larvae is exposed for 24 hours to the different concentration of plant extracts.
The number of dead nauplii was counted for the effectiveness of the extract used. This assay is

simple, inexpensive and does not require a lot of test materials.

Scope and Limitations

In this study, studying Mansanitas leaves' cytotoxicity on the possibility in treating cancer.

The extent of this study is that it only used Brine shrimp lethality assay instead of using

laboratory mice, since using animals needs to secure a permit and it needs a longer time. Usually,

the brine shrimp lethality assay consists of exposing the larvae to test samples in saline solution

and lethality is evaluated after 24 hours. The availability of brine shrimp eggs, low cost, and

ease in performing assay, makes brine shrimp lethality assay a very useful bench top method.

Relating is as a cure in humans should not be made, hence it should be taken as a possibility to

become cure. This study used only one part of the plant which is the leaves and one type of

extract which is ethanol extract. The gathering if plants will only be in one location it will be in

Tipolo, Mandaue City to have consistency on the data during its gathering.
 CHAPTER 3

METHODOLOGY

The methodology used are based from the researches of Raja on 2012, Barazi,et.al on 2013

and
I. Collection of plant materials

II. Preparation of plant extract

III. Phytochemical screening

II. Growth of brine shrimp

IV. Cytotoxicity test

V. Analysis of Results

Collection of Plant Materials

The researchers of this study collected the mansanitas leaves in Barangay

Looc, Mandaue City. It was then sent to the Department of Agriculture for authentication to

identify if the plant gathered was really mansanitas.

Growth of Brine Shrimp

The brine shrimps used in this study were bought in Sassy Pets located in Parkmall

Mandaue City. It was bought as an egg only, the researchers were the one who will let it grow

by putting it in a container with one liter of artificial seawater. In making the artificial seawater,

1 liter of distilled water will be added and dissolved in order; having 30g sodium chloride

(Kitchen salt) lined up first, followed with potassium chloride 0.8g, magnesium sulphate 6.6g,

0.5g of sodium hydrogen carbonate and calcium chloride 1.3g (Achour,2016). The container

will be divided into two to separate the hatched ones from the hatching through window screens

and placing one side to the light while the hatching place will be covered with black cloth for the

hatched ones to be attracted to the light and reside there (Hamidi et.al, 2014). pH value will be

monitored to avoid lethality in this area. This will be under observation for 48 hours which will

be subjected under constant aeration

Preparation of Crude Plant Extract

In this study there was only one type of plant extract that was used,

and this is the ethanolic extract. 221.30g were soaked in 1.4 L of 95% of ethanol. The mixture is

then covered with aluminium foil, shaken for 24 hours at 150 rev/min and concentrated using a

rotary evaporator and filtered (Raja, 2012). Consequently evaporated to dryness under the fume

hood.
Phytochemical Screening

The methods used in phytochemical screening was based on a video created by

Barazi published on 2013

1.1 Detection of Alkaloids: Wagner's Test

2mL of the alcoholic extract poured into the test tube will be treated with Wagner's Reagent.

Presence of reddish- brown percipitate indicates the presence of alkaloids

1.2 Detection of Anthroquinone Glycosides Test

2mL of the aqueous extract will be poured into the test tube then will be added with few

drops of FeCl3. Presence of rose red color shows positive result.

1.3 Detection of Carbohydrates: Molish Test

2 ml of plant sample extract will be added with two drops of alcoholic solution of

α-naphthol, shaken and will be added with few drops of concentrated sulphuric acid slowly on

the walls of the test tube .A violet ring indicates the presence of carbohydrate

1.4 Detection of Flavonoids

2mL of acid extract will be poured into two test tubes. The first test tube will be added

with 2mL of distilled water, and 2mL of NaOH in the second test tube. The presence of darker

yellow color indicates positive test.

1.5 Detection of Glycoside: Borntrager's Test

2mL of filtered hydrolysate will be poured into the test tube and will be added with 3mL

of chloroform and shaken, and then 10% ammonia solution was added. Presence of pink color

indicates the presence of glycosides.

1.6 Detection of Phenols: Ferric Chloride Test

50 mg of the extracts was dissolved in 5mL of distilled water, and added with few drops of

5% FeCl3. Presence of a dark green color indicates the presence of phenolic compounds

1.7 Detection of Phytosterols: Salkowski's Test

2mL of alcoholic extract poured into the test tube was added with concentrated H2SO4 on

the walls of it. Presence of red color at the lower layer indicates steroids and yellow at lower

layer indicates triterpenoids.

1.8 Detection of Saponins: Froth Test

2mL of aqueous extract poured into the test tube and was shook. The presence of two to

three centimeters froth that lasts for two to five minutes indicates a positive test.

1.9 Detection of Tannins

2mL of aqueous extract poured into the test tube was added with few drops of FeCl3.

Presence of blue color indicates hydrolyzable tannins and green color indicates condensed

tannins.
Cytotoxicity test

10 nauplii are added to each vial labelled A, B, C and D respectively using pipettes.

This will be added with 0.1 ml of Ethanolic extracts of concentrations of 0, 100, 200 and 300

ppm with dimethyl sulfoxide (DMSO) as its solvent that will be prepared using serial

dillutions. Artificial seawater is then added to the vials reaching its 5 ml mark. The number of

dead nauplii will be counted to get the results of the cytotoxicity test using LC50.

Analysis of Data

The data will be analysed using ANOVA and basing on LC50 by Meyer’s toxicity

index that was published on 1989.