TERATOGENIC PROPERTY OF EUPHORBIA HIRTA LEAF EXTRACT USING

ZEBRAFISH ASSAY

Submitted to:

Mr. Ephraim Villacrusis

In partial fulfillment of the requirements in

Research

Submitted by:

De Vera, Mchael Jireh L.

Diosana, Danielle Jonnar E.

Celebre, Trina Franchezca L.

Cruz, Isabelle Regine B.

Llana, Inah U.

G 10 - Ferrel

July 15, 2017

Pasig City Science High School

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 TABLE OF CONTENTS

Chapter I: Introduction

A. Background of the study

B. Statement of the problem

C. Hypothesis

D. Scope and Delimitation

E. Significance of the study

Chapter II: Review of Related Literature

Chapter III: Research Methodology

A. Gathering of Materials

B. Experimentation

C. Data collecting

D. Methodological Framework

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 CHAPTER 1

INTRODUCTION

A. Background of the Study

Euphorbia hirta, which is commonly known as “tawa-tawa”, is primarily

used in the Philippines and Asia. Also it is traditionally medicine, like asthma,

diarrhea, dysentery and coughs. Part of diet uses for dysentery. In Mauritius, It

is used to treat respiratory tract infection, vomiting, fever, bronchitis,

pulmonary disorders, asthma, coughs, diarrhea and dysentery.

Teratogen is any agent that can disturb the development of an embryo or

fetus. Teratogens may cause a birth defect in the child. Or a teratogen may halt

the pregnancy outright. The classes of teratogens include radiation, maternal

infections, chemicals, and drugs. Exposure to the teratogen affects the fetus or

embryo in a variety of ways, such as the duration of exposure, the amount of

teratogenic substance, and the stage of development the embryo or fetus is in

during the exposure. Teratogens may affect the embryo or fetus in a number of

ways, causing physical malformations, problems in the behavioral or emotional

development of the child, and decreased intellectual quotient (IQ) in the child.

Additionally, teratogens may also affect pregnancies and cause complications

such as preterm labors, spontaneous abortions, or miscarriages.

B. Statement of the Problem

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 Many women and mothers have undergone a birth defect and

malformation in their fetuses during their pregnancy. This was a big problem

in their child as they grow up. In this researcher’s study, they picked

Euphorbia hirta leaves to study its teratogenic property. Since there are many

people, especially those pregnant, were using Tawa-Tawa (Euphorbia hirta) as

their medicine in their specific diseases. This study aims to know or determine

on how Euphorbia hirta will affect the fetuses inside their mother’s womb. This

study will also specifically tell its phytochemicals using phytochemical

analysis, its toxicity level using Brine Shrimp Lethality Assay, and its

teratogenic properties using the Zebrafish assay. The testing of its teratogenic

properties will specifically identify the problem in heartbeats, in sensory

organs, the malformations in body and the number of melanocytes produced.

C. Hypothesis

Based on the formulated problems, the researchers ended up with three (3)

hypothesis which consist of one (1) general and two (2) specific hypothesis:

null:

1. There will be no teratogenic property in Euphorbia hirta leaf extract.

2. There’s no amount of toxicity level I the Euphorbia hirta leaf extract.

3. There will be no significant difference in number of heartbeats, and no

teratogenic effect in sensory organs

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Alternative:

1. There is a possibility of having Teratogenic Property in Euphorbia hirta

Leaf Extract

2. There will be an amount of

a. 2mg/ml

b. 5mg/ml

c. 10mg/ml

3. There is a significant difference in the number of heartbeats and will

form teratogenic effect in sensory organs

D. Scope and Delimitation

The scope of this study is to test if the teratogenic property of Euphorbia

hirta leaf extract using zebrafish assay. Researchers will limit the study in

using Euphorbia hirta leaf extract. The researcher will also determine its

toxicity using brine shrimp lethality assay with the help of phytochemical

analysis. The researchers scope on how Euphorbia hirta leaf extract will affect

the birth in embryos using zebrafish (Danio rerio). On the other hand, the

study is limited on using brine shrimp lethality assay in testing its toxicity level

and zebrafish assay as to test its teratogenicity.

E. Significance of the Study

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Obstetrician who specializes in births to know further about teratogenic

properties of Euphorbia hirta leaf extract when using as a medicine

Pregnant woman may have enough knowledge about the teratogenic property

of Euphorbia hirta therefore to lessen the intake of Euphorbia hirta as a

medicine.

Future Researchers who wish to continue this further study about the

teratogenicity of Euphorbia hirta leaf extract

CHAPTER II

Review of Related Literature

Euphorbia hirta

Tawa-tawa is an epiphytic orchid which is variable in size. Pseudo bulbs

are stout, ovoid or fusiform, terete, and 12 to 20 centimeters long. Leaves are

oblanceolate, oblong, and 20 to 40 centimeters long. Racemes with the

peduncle are 0.6 to 1.5 meters long, spreading, and nodding or pendulous.

Flowers are numerous, each about 5 to 8 centimeters across. Sepals and petals

are obovate-oblong, yellowish-green, and covered with large, reddish-brown

blotches. Lip is three-lobed, and yellow with purple nerves. Column is

incurved. Anther is orbicular and hairy, the pollinia subglobose.

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- Studies have isolated gallic acid, quercetin, triacontane, cetyl alcohol,

phytosterol, phytosterolin (phytosterol glucoside); jambulol, melissic, and a

mixture of acids consisting chiefly of palmitic, oleic, and linoleic acid.

- Phytochemicals screenings have yielded alkaloids, essential oil, phenols,

sterol, flavones and fatty acids.

- Yields flavonoids: euphorbianin, leucocyanidol, camphol, quercitrin and

quercitrol.

- Study has suggested that some of the constituents of the plant are similar to

those of the jambul (Syzygium cumini) seeds.

- Dried leaves yielded a moisture content of 9.70%, protein 13.5% ±0.15, fat

1.13% ±0.06, ash 3.13% ±0.06, crude fiber 3.57% ±0.06, and carbohydrate

69.5% ±0.20. Vitamin content showed ascorbic acid 26.1 mg/100g, thiamine

0.60, riboflavin 1.20, and niacin 0.70.

- Phytochemical screening of extracts yielded the presence of carbohydrates,

lipids, proteins, flavonoids, alkaloids, saponins resins, steroids, acidic

compounds, tannins, glycosides, phenols and terpenoids. (see study below)

- Study of aerial parts (leaves and stems) revealed saponin, sterol, terpene,

alkaloids, polyphenols, tannins, flavonoids and especially mucllage.

Physiochemical study yielded a moisture content of 7.73% ± 0.00%, total ash

7.48% ± 0.03%, sulfuric ash 9.05% ± 0.01%, hydrochloric acid insoluble ash of

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0.8% ± 0.02%. Mineral analysis yielded magnesium, potassium, calcium, zinc,

and traces of chrome.

Teratogen

Teratogen is any agent that can disturb the development of an embryo or

fetus. Teratogens may cause a birth defect in the child. Or a teratogen may halt

the pregnancy outright. The classes of teratogens include radiation, maternal

infections, chemicals, and drugs. Exposure to the teratogen affects the fetus or

embryo in a variety of ways, such as the duration of exposure, the amount of

teratogenic substance, and the stage of development the embryo or fetus is in

during the exposure. Teratogens may affect the embryo or fetus in a number of

ways, causing physical malformations, problems in the behavioral or emotional

development of the child, and decreased intellectual quotient (IQ) in the child.

Additionally, teratogens may also affect pregnancies and cause complications

such as preterm labors, spontaneous abortions, or miscarriages.

Zebrafish

Zebrafishes are vertebrate organisms that are of growing interest for

preclinical drug discovery applications. Zebrafish embryos develop most of the

major organ systems present in mammals, including the cardiovascular,

nervous and digestive systems, in less than one week. Additional

characteristics that make them advantageous for compound screening are their

small size, transparency and ability to absorb compounds through the water.

Furthermore, gene function analysis with antisense technology is now routine

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procedure. Thus, it is relatively simple to assess whether compounds or gene

knockdowns cause toxic effects in zebrafish. Assays are being developed to

exploit the unique characteristics of zebrafish for pharmacological toxicology.

This review discusses assays that may be used to assess in vivo toxicity and

provides examples of compounds known to be toxic to humans that have been

demonstrated to function similarly in zebrafish. The zebrafish embryo

toxicity/teratogenicity assay is described as a useful alternative screening

model to evaluate the effect of drugs on embryofoetal development. Fertilized

eggs were exposed to different concentrations of 15 compounds with

teratogenic (8) and non-teratogenic (7) potential until 96h post-fertilization

when 28 morphological endpoints and the level of compound uptake was

assessed. The majority of drugs testing positive in mammals was also positive

in zebrafish (75% sensitivity), while a relative high number of false positives

were noted (43% specificity). Compound uptake determination appears useful

for clarifying classifications as teratogenic or potential overdose although assay

sensitivity could be improved to 71% if the exposure threshold, previously

suggested as ∼50ng/larvae, is reconsidered. The zebrafish assay shows some

potential, though limited in its current form, as a screening tool for

developmental toxicity within Janssen drug development. Further assay

refinement with respect to endpoints and body burden threshold is required.

Phytochemicals

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 Phytochemicals are non-nutritive plant chemicals that have protective or

disease preventive properties. They are non-essential nutrients, meaning that

they are not required by the human body for sustaining life. It is well known

that plant produce these chemicals to protect them but recent research

demonstrate that they can also protect humans against diseases. There are

more than thousand known phytochemicals. Some of the well-known

phytochemicals are lycopene in tomatoes, isoflavones in soy and flavanoids in

fruits. Phytochemicals can be defined, in the strictest sense, as chemicals

produced by plants. However, the term is generally used to describe chemicals

from plants that may affect health, but are not essential nutrients. While there

is ample evidence to support the health benefits of diets rich in fruit,

vegetables, legumes, whole grains, and nuts, evidence that these effects are

due to specific nutrients or phytochemicals is limited. Because plant-based

foods are complex mixtures of bioactive compounds, information on the

potential health effects of individual phytochemicals is linked to information on

the health effects of foods that contain those phytochemicals.

There are many phytochemicals and each works differently. These are

some possible actions:

Antioxidant

Hormonal action

Stimulation of enzymes

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 Interference with DNA replication

Anti-bacterial effect

Physical action

Brine shrimp lethality assay

A method, utilizing brine shrimp (Artemia salina Leach), is proposed as a

simple bioassay for natural product research. The procedure determines LC

(50) values in microg/ml of active compounds and extracts in the brine

medium. Activities of a broad range of known active compounds are manifested

as toxicity to the shrimp. Screening results with seed extracts of 41 species of

Euphorbiaceae were compared with 9KB and 9PS cytotoxicities. The method is

rapid, reliable, inexpensive, and convenient as an in-house general bioassay

tool Brine shrimp lethality bioassay 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-brine shrimp (Artemia salina).[1] This assay

was first proposed by Michael et al., and further developed by several groups.

[2,3,4] The brine shrimp lethality bioassay is widely used in the evaluation of

toxicity of heavy metals, pesticides, medicines especially natural plant extracts

and etc.[5,6] It's a preliminary toxicity screen for further experiments on

mammalian animal models.

One important aspect of this assay is that the solvent used in this assay

may give false positive signals due to the toxicity of the solvent itself. It has

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been well-known that some organic solvent and detergents have high

cytotoxcity in vivo. A systematic study on how high concentration of solvent

affects the results from brine shrimp lethality bioassay and guidance for

maximum working concentration of solvents is needed. The article published in

this issue “Interference from ordinarily used solvents in the outcomes of A.

salina Lethality Test” fills this gap very well. In this study, the authors tested

the solvent toxicity effect on brine shrimp experimentally. This is a very

significant study. The authors concluded that dimethyl sulfoxide (DMSO) is a

safer solvent in brine shrimp lethality bioassay and the maximum working

concentration of solvents such as DMSO, methanol and Tween 20 was

suggested.

LC50

LC50 is a standard measure of the toxicity of the surrounding medium

that will kill half of the sample population of a specific test-animal in a

specified period through exposure via inhalation (respiration). LC50 is

measured in micrograms (or milligrams) of the material per liter, or parts per

million (ppm), of air or water; lower the amount, more toxic the material. Used

in the comparison of toxicities, LC50 values cannot be directly extrapolated

from one species to the other or to humans. Also called median lethal

concentration or population critical concentration 50. Written also as LC50. LC

stands for "Lethal Concentration". LC values usually refer to the concentration

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of a chemical in air but in environmental studies it can also mean the

concentration of a chemical in water.

According to the OECD (Organisation for Economic Cooperation and

Development) Guidelines for the Testing of Chemicals, a traditional experiment

involves groups of animals exposed to a concentration (or series of

concentrations) for a set period of time (usually 4 hours). The animals are

clinically observed for up to 14 days.

The concentration of the chemical in air that kills 50% of the test

animals during the observation period is the LC50 value. Other durations of

exposure (versus the traditional 4 hours) may apply depending on specific laws

An LC50 is the lethal concentration required to kill 50% of the

population. The LC50 is a measure, e.g. in mg/l, of the concentration of the

toxin whereas a dose is a more general term (need not be a concentration but

may be a specific temperature, etc.). These bioassays involve subjecting several

replicate groups of individuals to a range of concentrations (or doses) of a toxic

compound and measuring the mortality after a defined time interval, e.g. 24

hours, 1 week, 1 month, etc. The data are then plotted and the LC50 is

interpolated from the graph.

TOXICITY TEST OF EUPHORBIA HIRTA

Brine shrimp lethality assay was used to calculate the median lethal

concentration (LC50) of E. hirta (for leaves, stems, flowers and roots)

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methanolic extracts at concentrations from 100 to 0.07 mg/ml. The LC50

values of 1.589, 1.420, 0.206 and 0.0827 mg/ml were obtained for stems,

leaves, flowers and roots, respectively. Potassium dichromate (the positive

control) had LC50 value of 0.00758 mg/ml. The acute oral toxicity study of the

leaf extract resulted in one third mortality and mild behavioral changes among

the treated mice. No significant statistical differences found between body

weight, relative (%) and absolute (g) organ weights of treated and untreated

groups (P> 0.05). Gross and microscopic examination of the vital organ tissues

revealed no differences between control and treated mice. All the tissues

appeared normal.

CHAPTER III

METHODOLOGY

A. Gathering of materials

Drying and pulverizing of Euphorbia hirta leaves

- Drying the Euphorbia hirta leaves under the heat of the sun for

approximately three days and pulverizing it with mortar and pestle or using an

electronic blender.

Ethanol Extraction

- The pulverized Euphorbia hirta will undergo the ethanol extraction which

will be the process of extracting the fragrant compounds straight from dry raw

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materials and to impure oils or concrete from it. The extracted ethanol from dry

raw materials will be then called as tinctures whereas ethanol washes are done

for purifying oils and concretes which are called absolutes. The impure oil

substances and some oils are combined with 95% of ethanol.

Rotary Evaporation

- Rotary evaporation is the process of reducing the volume of a solvent by

distributing it as a thin film across the interior of a vessel at elevated

temperature and reduced pressure. It is the process of separating the solvent

from many organic, inorganic, and polymeric materials.

B. Experimentation

Phytochemical Analysis

- Phytochemical analysis is non-nutritive plant chemicals that have

protective or disease preventive properties. It can be defined, in the strictest

sense, as chemicals produced by plants. However, the term is generally used to

describe chemicals from plants that may affect health, but are not essential

nutrients.

Toxicity Test Using Brine Shrimp Lethality Assay

- A method, utilizing brine shrimp , is proposed as a simple bioassay for

natural product research. The procedure determines LC50 (is the lethal

concentration required to kill 50% of the population) values in microg/ml of

active compounds and extracts in the brine medium.

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Zebrafish Teratogenicity Testing

- After having the brine shrimp lethality assay we will test the teratogenic

properties on zebrafish. The process where the teratogenic properties of

Euphorbia hirta will be injected in the embryos of the Zebrafish.

C. Data Collecting

Collecting of Active Compounds in Euphorbia hirta

- After the experiment, specifically the phytochemical analysis, the

researchers will then collect the active compounds and determine them to be

test its toxicity level by lc50 by using brine shrimp lethality assay

Linear Regression

- Linear regression is the most basic type of regression and commonly

used predictive analysis. The researchers will use this kind of regression to

identify the least concentration 50 (lc50) of the active compounds given by the

phytochemical analysis.

ANOVA Table

- Analysis of variance (ANOVA) is an analysis tool used in statistics that

splits the aggregate variability found inside a data set into two parts:

systematic factors and random factors. The systematic factors have a

statistical influence on the given data set, but the random factors do

not. Analysts use the analysis of the variance test to determine the

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 result independent variables have on the dependent variable amid a

regression study. Researchers will use analysis of variance because it

will help determine the dependent variable in the study.

D. Methodological framework

III. Data
I. Gathering of materials II. Experimentation
Collecting

Drying and pulverizing Phytochemical Collecting of active

Of Euphorbia hirta leaves Analysis Compounds in
Euphorbia
hirta
Toxicity Test using
Brine Shrimp
Ethanol Extraction Lethality Assay
Linear Regression
To identify the LC50
Testing teratogenic
Rotary Evaporation Properties on
Zebrafish assay
ANOVA Table

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