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, Michael Jireh L.

Diosana, Jonnar Danielle E.

Celebre, Trina Franchezca L.

Cruz, Isabelle Regine B.

Llana, Inah U.

G 10 - Ferrel

Pasig City Science High School

 :ABSTRACT (WHOLE PAPER SUMMARY)

CHAPTER 1: AAYUSIN

CHAPTER 2: LOL

CHAPTER 3: SPECIFY

CHAPTER 4: PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

(YUNG PRESENTATION YUNG SA TABLE AND PHYTO, ANALYSIS BUKAS AND

INTERPRETATION)

CHAPTER 5: SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

(SUMMARY YUNG MGA AVERAGE NG MGA TEST, CONCLUSION YUNG

PARANG RESULTS DIN KASAMA HYPOTHESIS, RECOMMENDATIONS ALAM

NYO NA YON)

:ACKNOWLEDGEMENT

: BIBLIOGRAPHY (SOURCES AND BOOKS MISMO, YUNG PAGGAWA NYA IS

YUNG PINATEST SATIN NI SIR ALMEN DATI)

 TABLE OF CONTENTS

Chapter I: Introduction………………………………...........................................3

A. Background of the study…………………………………………………………3

B. Statement of the problem………………………………………………………..4

C. Hypothesis…………………………………………………………………………...4

D. Scope and Delimitation…………………………………………………………...5

E. Significance of the study………………………………………………………….6

Chapter II: Review of Related Literature……………………………………………….6

Chapter III: Research Methodology…………………………………………………….14

A. Gathering of Materials……………………………………………………………..14

B. Experimentation…………………………………………………………………….15

C. Data collecting……………………………………………………………………….16

D. Methodological Framework……………………………………………………….17
 CHAPTER 1

INTRODUCTION

A. Background of the Study

-brief description and uses of tawa-tawa (with citations every sentence)

- brief history of pregnancy in Ph (with citations kung meron??)

- relate tawa tawa as a problem to pregnancy (sariling gawa)

B. Statement of the Problem

This study aims to determine the teratogenic property of Euphorbia hirta

leaf ethanolic crude extract using Zebrafish assay. Specifically, this study aims

to:

1. To know the chemicals in E. hirta leaf extract using phytochemical analysis.

2. To measure its toxicity level using Brine Shrimp Lethality Assay with the

following measurements:

a. 100ug/mL b. 50ug/mL c. 25ug/mL d. 12.5ug/mL

3. To determine if there will be any malformation on

Zebrafish when injected with Euphorbia hirta Leaf Extract

a. Mean length
 b. Number of melanocytes

c. Rate of heartbeat per minute

d. Mortality rate

(REVISION PLS!!!)

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 the mean length, number of

melanocytes, rate of heartbeat per minute and mortality rate

Alternative:

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

Extract

2. The amount to get the lethal concentration 50 (LC50) of Euphorbia hirta leaf

extract using brine shrimp is 25ug/ml.

3. There is a significant difference in the mean length, number of melanocytes,

rate of heartbeat per minute and mortality rate

(AYUSIN??????)

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. (AAYUSIN PA BA????)

E. Significance of the Study

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 (OKAY NA TO, DAMI LANG

REDUNDANT NA WORDS)

CHAPTER II

Review of Related Literature

ANG LT NETO HAHAHAHAH

AYUSIN NATIN, WITH CITATIONS EVERY SENTENCE (SA LIB TAYO

MAGHANAP!!!)

-TAWA TAWA

-BRINE SHRIMP AND TOXICITY LEVEL

-ZEBRAFISH AND TERATOGENS

CHAPTER III

METHODOLOGY

The researchers went to Bureau of Plant Industry in Malate, Manila for

the plant identification and certification of the Euphorbia hirta leaves.

A. Gathering of materials

Drying and pulverizing of Euphorbia hirta leaves

 The researchers pulverized the 1 kg of Euphorbia hirta leaves sun dried

for three days using mortar and pestle and/or electronic blender.

Ethanol Extraction

The researchers mixed the pulverized Euphorbia hirta leaves to a 95%

ethanol. This will undergo the ethanol extraction which will be the process of

extracting the fragrant compounds straight from dry raw 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. The mixed solutions is then left

for 3 days in the laboratory. The researchers extracted and filtered the

pulverized leaves to ethanol, leaving pure ethanol extract and is put into a

bottle.

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.

The researchers went to University of the Philippines-Institute of

Chemistry for the rotary evaporation of the Euphorbia hirta leaf ethanol extract.

The extract is chuchu at 400 Celsius.

B. Experimentation

Phytochemical Analysis

The researchers went to University of the Philippines-Institute of

Chemistry for phytochemical analysis. Phytochemical screening based on

Hasborne (1984), Edeoga et al. (2005), and Onwukaerne et al. (20017) with

slight modifications.

 TANNINS

 SAPONINS

 TERPENOIDS

 FLAVONOIDS

 CARDIAC GLYCOSIDES (Killiani-Keller test)

 PHENOLIC COMPOUNDS

 ALKALOIDS

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.

The researchers went to University of the Philippines-Institute of Biology

for the assistant of the laboratory. They made a solution for brine shrimp
lethality test to determine the Euphorbia hirta leaf extract’s toxicity level using

the following procedures:

1. The researchers measured 10mg of the extract and mixed with 1mL of

DMSO (Dimethyl sulfoxide) and 9mL of improvised seawater for the solution of

1mg/mL extract.

2. The researchers get 2.4mL in the solution and mixed with 21.6mL of

improvised seawater for the solution of 100microg/mL. Then they get 10mL of

it and mixed again with 10mL seawater for the solution 50microg/mL. Then

they repeated the procedure of getting 10mL from the previous solution and

added 10mL distilled water to half the solution, making 25microg/mL and

12.5microg/mL of solutions.

3. They mixed a solution of 3mL chuchu and 7mL seawater for positive control.

Then they mixed 100micrograms of DMSO (Dimethyl sulfoxide) for the negative

control.

4. The researchers put the solutions in a small chuchu, 4 samples for each

solutions, and put atleast 10 brine shrimps to each sample and to be observed

after 24 hours.

5. The researchers will count the number of brine shrimp survived and dead to

determine its LC50.

Zebrafish Teratogenicity Test

 The zebrafish assay is used to test the teratogens of the Euphorbia hirta

leaf extract. The researchers went to University of the Philippines-Institute of

Biology for the laboratory assistance and made the following procedures of the

assay:

1. The researchers get 10mg of Euphorbia hirta leaf extract and mixed with

1mL DMSO (Dimethyl sulfoxide) and 9mL distilled water.

2. The researchers get 2.4mL of the solution and mixed with 21.6mL

distilled water for the solution of 100microg/mL. The researchers the

get 10mL of it and mixed with 10mL distilled water for 50microg/mL

solution. Then they repeated the procedure of getting 10mL from the

previous solution and added 10mL distilled water to half the solution,

making 25microg/mL and 12.5microg/mL of solutions.

3. The researchers put the solutions of 12 samples each in a small chuchu

and put 1 egg of zebrafish in each chuchu.

4. The zebrafish is left and observed after 24 hours for 1pdf (0), and

observe their survival rate, after 3 days for 3pdf to get their hatch rate

and heartbeat rate, and 5 days for 5pdf to get their mean length.

(HINDI PA KUMPLETO ITO PLUS KAILANGAN DAW SA NARINIG KO,

KAILANGAN KUMPLETO PERO WAG IBIGAY LAHAT (PARANG MGA

KUMPLETONG IMPORTANTE GANUN)