Cytoxicity of Lemon Grass (Cymbopogon citratus) Extract

Cytoxicity of Lemon Grass (Cymbopogon citratus) Extract
Though Brine Shrimp Bioassay



A Research Paper
Presented to the
Department of Science and Technology
(Special Science Curriculum)
Iligan City National High School
Iligan City

__________________

In Fulfillment of the Requirements
In Research II
_________________

Mrs. Annallee Q. Aron
Adviser
__________________


By:
Adanza, Faith Stephanie E.
Bala, Clendelyn Jane D.
Redublado, Marjorie S.


March , 2008

ABSTRACT
Nowadays, there are many plants found in our environment, some of it is used in our daily living such as spices. But yet, we don’t know if these plants are bioactive. Determining plants bioactivity can lead to discoveries of the other uses of this plant, especially in the field of medicine.
This research study aims to determine the bioactivity of lemon grass extract through brine shrimp bioassay.
Lemon grass is commonly use as spices in the Philippines and is also known in our dialect us “Tanglad”.
Brine shrimp bioassay uses brine shrimps to be able to determine how bioactive a certain plant is.
The brine shrimps are hatched for two days and after that, preparations of the test solutions are made. The researchers prepared three test-solutions namely 1000-ppm, 100-ppm, 10-ppm. After getting the number of mortality on different concentrations and the data is tabulated in table form and in a plotted in a graph. The researchers use ANOVA statistical test in determining the significant difference between the experimental set-up and the control. The researchers also use the table in finding the LC50 of experimental set-up.
Results from ANOVA statistical test shows that there is a significant difference between the experimental set-up and the control. Results from the LC50 table shows that the number of concentration is directly proportional to the number of mortality, and LC50=52.596.
From the result of the ANOVA statistical test and LC50, it was then concluded that lemon grass is bioactive.

APPROVAL SHEET

The research proposal entitled “Cytoxicity of Lemon Grass Extract Though Brine Shrimp Bioassay”, prepared by Clendelyn Jane D. Bala, Marjorie S. Redublado, Faith Stephanie Adanza, in fulfillment of the requirements in Research II have been examined and recommended for acceptance and approval.

Annallee Q. Aron
Research II Adviser



Marjorie Y. Dumaug Aimee R. Pareño Annalle Q. Aron
Panel Member Panel Member Panel Member

This research proposal is hereby approved in fulfillment of the requirements for the subject Research II.

Nida H. Gumera
Science Dept. Head (Day)


ACKNOWLEDGEMENT

The researchers express their thanks and sincere praises to the Most Merciful and Most Loving God for the guidance and strength which they needed to overcome all the obstacles in their life. Without God’s help and love, this research work would have not been possible.

They also express their thanks and sincere gratitude to the following whose contribution have made the completion of this research paper:
Mrs. Annallee Aron, their research adviser, whose intellectual guidance and sound advices made possible to complete this research paper.
Ms. Victoria Cordova, for letting them use the computer laboratory for the completion of their research.
Panel members, whose comments and suggestions made changes in some parts of this research paper.
Ms. Desimie Gumabat, for allowing the researcher to continue their unfinished study, together with her partner.
To the brine shrimp team (Anamel, Alyzza and Ginuel) for the sleepless nights they’ve shared together. For the friendship and conversations they have been through.
There classmates, Maxtein ’08, for the laughter, kindness and companionship throughout the high school years.
Finally, the researchers express their deepest gratitude to their family for their support (financially and emotionally), enduring love, encouragement, and understanding to finish the research paper.
To all persons who have contributed to this paper, thank you very much.

Clenz, Faith, Marj

TABLE OF CONTENT

Title Page ………………………………………..………………………
Abstract ………………………………………..………………………
Approval Sheet ………………………………..………………………
Acknowledgement ………………………………..………………………
Table of Contents …………………………………………..……………

CHAPTER I- Introduction
Background of the Study …………………………………..……
Statement of the Problem …………………………………..……
Significance of the Study ……………………………..…………
Scope and Delimitation ……………………………..…………

CHAPTER II – Review of Related Literature and Studies
Review of Related Study ………………………………………..
Conceptual Framework ………………………………………..
Null Hypothesis ………………………………………..
Definition of Terms ………………………………………..

CHAPTER III – Methodology
Materials ………………………………………..
Procedure ………………………………………..
Data Gathering Procedure ………………………………………..
Statistical Used ………………………………………..
Instruments Used ………………………………………..
CHAPTER IV- Results and Discussions
Table1 ……………………………………………………..…
Chart 1 ……………………………………………………..…

Table 2 ……………………………………………………..…
Chart 2 ……………………………………………………..…
Decision ……………………………………………………..…
CHAPTER IV – Summary, Conclusion and Recommendation
Summary ……………………………………………………..…
Conclusion ……………………………………………………..…
Recommendation ……………………………………………..…
BIBLIOGRAPHY ……………………………………………..…
APPENDICES ……………………………………………………..…

CHAPTER I
INTRODUCTION

Background of the Study

Tanglad or Lemongrass extract (Cymbopogon citratus) s a fragrant grass that grows in abundant clumps just about anywhere in the tropics. It possesses a larger bulb near the base stalks and it is here that the lemon or citrus flavoring is most often obtained ( though we Filipinos use the entire leaf for stuffing ).Lemongrass is used as a base for curries, soups, stews and it is also used to flavor salads, as part of marinades and as stuffing material. It can be infused into hot water for a simple herbal infusion and it now flavors desserts as well (http:/www.marketmanila.com/archives/tanglad-lemongrass).
Just over a centimeter in size, the adult brine shrimp Artemia is an extremely well-known animal because of its importance as a food source for fish and crustaceans raised in home aquariums, aquaculture systems and in laboratories. One can buy brine shrimp at practically any pet display. It looks like a powdery brown substance but in reality the substance is thousands of cysts—eggs surrounded by protective cases. When added to water, these cysts will hatch into shrimp nauplii within a few hours (http://seagrant.gso.uri.edu/factsheets/931brine.html).
Brine shrimp are food source not only for humans but also for aquatic life, since they are found naturally in about 250 locations around the world. Brine shrimp have been used as a “bench top bioassay” for the discovery and purification of bioactive natural products and they are an excellent choice for elementary toxicity investigations of consumer products (http://scholar.google.com/q=importance of brine shrimp bioassay=scholart).
Bioassay is shorthand commonly used term for biological assay and is a type of scientific experiment. Bioassays are typically conducted to measure the effects of a substance on a living organism. It may be qualitative or quantitative. Qualitative bioassays are used for assessing the physical effects of a substance that may not be quantified, such as abnormal development or deformity. Quantitative bioassays involve estimation of the concentration or potency of a substance by measurement of the biological response that it produces. Quantitative bioassays are typically analyzed using the methods of biostatistics. Bioassays are essential in the development of new drugs and monitoring pollutants in the environment (Microsoft Corporation © 1993-2003).

Statement of the Problem
This study aims to answer the following questions:
1.) Is there any significant difference in terms of brine shrimp lethality with the lemongrass extract and its control?
2.) Is there any significant difference in terms of toxicity in different concentrations?

Significance of the Study
This research study is about identifying toxicity of Lemongrass (Cymbopogon citratus) extract through Brine Shrimp Bioassay having different concentrations.
Brine shrimp have been used as “bench top bioassay” for the discovery and purification of bioactive natural products and they are excellent choice for elementary toxicity investigations of consumer products.
This could help future researches who will be conducting studies relating to lemongrass to decide if lemongrass would fit to their study.

Scope and Delimitation
This research study was conducted at Iligan City National High School, ESEP building last February 23, 2008. The focus of the study is to test the toxicity of lemongrass extract in order to find out how bioactive lemongrass is. The extraction was conducted at MSU-IIT (Chemistry Laboratory of College, Science and Mathematics) last December 20, 2007.

CHAPTER II
REVIEW OF RELATED STUDY AND LITERATURE

Related Study
“A brine Shrimp Bioassay for measuring Toxicity and Remediation of Chemicals”
This article describes a laboratory project that uses a bioassay on brine shrimp to measure LC50 values or a variety of insecticides. This project was the center piece of a five-week unit on toxicology and chemical remediation. The 22 students in the class were first-year. Two 3-hour lab periods are required, the first for toxicity testing, and the second for remediation and retesting. Any chemical that can be dissolved or dispersed into water can be studied in this bioassay; plant extracts or other natural products. Although the bioassay is simple enough to use with student who have had little lab experience, it could also be integrated into an environmental, organic, medical or biochemistry lab course.
This set of labs helps student how toxicity is measured and how harmful molecules can be decomposed to harmless products.
The bioassay and remediation procedures described in the supplementary material could be used as straight and remediation procedures described in the supplementary material could be used as straight labs as a multi-week project. In such project, students were given a letter from the owner of a dog-grooming salon, who was worried that the insecticide products she uses in the salon are leaching into a nearby stream. She asks the students to test the flea products to determine their toxicity in aquatic life, and to devise some method that could use to make the wastewater less toxic before she pours it down the drain.
The students were then divided into groups of 3 or 4 and given an insecticide product. They measured the LC50 value of their insecticide o brine shrimp; all seven groups were able to complete this lab in three-hour lab period. Over the next two-weeks, the groups researched the chemicals present in their insecticide product. In the second lab period, the remediation procedures were carried out and the remediate material was assayed on brine shrimp (Marya.Lieberman, 1994).

Conceptual Framework
Independent Variable Dependent Variable

Null Hypothesis
1. There is no significant difference in terms of brine shrimp lethality with the lemongrass extract and its control.
2. There is no significant difference in terms of toxicity in different concentration.

Definition of Terms
1. Artificial Seawater –can be attained by dissolving 40g of salt in 100ml of water. Also used as substitute for pure seawater
2. Brine Shrimp –very tiny crustaceans that are easy to breed and maintain for long period of time and are commonly used as food for marine fish
3. Bioassay –a test used to evaluate the relative potency of a chemical or mixture of chemicals by comparing its effect on a living with the effect of a standard preparation on the same organism
4. Compressor –a mechanical devise that increases the pressure of gas
5. Cyst –are sac-like lesion containing liquid material consisting of white blood cells, dead cells, and bacteria
6. Extract –to obtain from a substance by chemical or mechanical action, as by pressure, distillation, or evaporation
7. Lemongrass -is a genus of about 55 species of grasses, native to warm temperate and tropical regions of the old world and Ocennia. It is a tall perennial grass; common names include lemongrass, lemon grass, barbed wire grass, silky heads citronella grass, fever grass or Hierba Luisa
8. Ppm –a unit of measure used to express the number of parts of substance contained within a million parts of a liquid, solid, or gas
9. Nauplii –a newly hatched brine shrimp
10. Yeast -any of a number of microscopic, one-celled fungi important for their ability to ferment carbohydrates in various substances

CHAPTER III
METHODOLOGY

Materials
20 Test tubes Lemon grass
Jar Brine Shrimp
Syringe Yeast
500mL Distilled water Graduated Cylinder
20 grams Salt Paper Disc

Procedure
I. Gathering and of Lemon grass
Lemon grasses were gathered in Tambo, Iligan City.
II. Preparation of Lemon grass Extract
The gathered lemon grass was cut for about ½ inch. Then, it was air-dried for about 3 hours. Then, the air-dried lemongrass was soaked to 900mL of ethanol. Then the soaked lemon grass were sent to MSU-IIT College of Science and Mathematics for the extraction.
III. Brine Shrimp Assay (BSA)
The brine shrimp assay (BSA) was employed to determine the toxicity and general bioactivity of lemon grass.
a. Hatching of Brine Shrimp
Brine shrimp eggs were made to hatch into a jar. The jar is divided into two compartments; half of the compartment is wrapped around to prevent light. Into this jar, artificial sea water is prepared by dissolving 20grams of ordinary table salt in 500mL distilled water. Brine shrimp cysts are then placed and allowed to hatch. The tank should not be moved after adding the eggs to prevent the eggs from getting to other compartment. The sea water is supplemented with yeast to serve as food for the shrimp. After 48 hours of incubation at room temperature, the active brine shrimp nauplii are ready for transfer to the test solution.
b. Preparation of Test Solution of Lemongrass Extract
i. Prepare paper disc of about 0.6cm in diameter.
ii. Calibrate twenty (20) test tubes to 5.0mL volume with a 5.0-mL disposable syringe.
iii. Prepare a stock solution of 50,000-ppm by dissolving 50grams of lemongrass extract in 1.0mL of the artificial seawater.
iv. First, prepare test-tubes of 1,000-ppm test solutions. Transfer 0.1mL of the 50,000-ppm stock solution to each of four test tubes (label a1, a2, a3, a4). Test tube a1, a2, a3 should contain the prepared paper disc and was not air dried.
v. Next, prepare test-tubes of 100-ppm test solutions. Transfer 0.5mL of the 1,000-ppm stock solution to each of four test tubes (label b1, b2, b3, b4). Test tube b1, b2, b3 should contain the prepared paper disc and was not air dried.
vi. Then, prepare test-tubes of 10-ppm test solutions. Transfer 0.5mL of the 100-ppm stock solution to each of four test tubes (label c1, c2, c3, c4). Test tube c1, c2, c3 should contain the prepared paper disc and was not air dried.
IV. Bioassay
Add about 5ml of seawater to each of the test tubes to air-dried extract. Using a syringe, ten (10) brine shrimp nauplii are transferred with seawater from the lighted compartment of the tank into each of the test tubes containing the air-dried samples. Then, the volumes of the test solutions are adjusted to 5ml with artificial seawater.
Data Gathering Procedure
After 24 hours, the survivors are counted and the number of mortality is recorded for each test solution and reported as the average of three replicates. The percent mortality in test solution is calculated as:
Number of mortality
% mortality = ----------------------------- x 100
Number of test shrimp

Statistical Test Used
The statistical test used in this study is ANOVA.

Instruments Used
A. Compressor

CHAPTER IV
RESULTS AND DISCUSSION
In this chapter, discussion about the results will be the focus of the research showing the bioactivity of “Lemongrass” through brine shrimp bioassay.

Table 1
Concentration Test tube 1 Test tube 2 Test tube 3 Average
1000-ppm 9 8 10 9
100-ppm 6 8 7 7
10-ppm 4 2 5 3.7
Ethanol 10 10 10 10
A. Seawater 0 0 0 0
The table shows the number of the brine shrimp mortality in different concentration. It is shown here that 1000-ppm concentration have the highest number of mortality in terms of the three set-up.
Chart 1

Ld50= 52.596
The chart shows the comparison of mortality of different test tubes in different concentration. It shows that the higher the concentration, the higher the mortality. And base on ld50 result, it shows that the experiment shows bioactivity.
Table 2
Concentration Test tube 1 Test tube 2 Test tube 3 Average
1000-ppm 90% 80% 100% 90%
100-ppm 60% 80% 70% 70%
10-ppm 40% 20% 50% 37%
Ethanol 100% 100% 100% 100%
A. Seawater 0% 0% 0% 0%
The table shows the percentage of the brine shrimp mortality in different concentration. It is shown here that 1000-ppm concentration have the highest number of mortality in terms of the three set-up.
Chart 2

Ld50= 50.576
The chart shows the comparison of mortality of different test tubes in different concentration in percentage. It shows that the higher the concentration, the higher the mortality. And base on ld50 result, it shows that the experiment shows bioactivity.

Anova: Two-Factor Replication

SUMMARY Count Sum Average Variance
Row 1 4 36 9 0.666667
Row 2 4 28 7 0.666667
Row 3 4 14.7 3.675 1.555833
Row 4 4 40 10 0

Column 1 4 29 7.25 7.583333
Column 2 4 28 7 12
Column 3 4 32 8 6
Column 4 4 29.7 7.425 7.7225
Decision
ANOVA
Source of Variation SS df MS F P-value F crit
1.15E-
Rows 93.41688 3 31.13896 43.11134 0.5 3.862548
Columns 2.166875 3 0.722292 1 0.4362 3.862548
Error 6.500625 9 0.22292

Total 102.0844 15

Base on the statistical test ANOVA, it shows that Ho is rejected. Therefore H1 is accepted, it is because there is significant difference in terms of mortality of brine shrimp in the experimental and control. And there is also significant difference of mortality in different con concentration.

The researchers have observed that as the concentration of the crude extract of lemon grass increases, the number of mortality also increases.

CHAPTER V
SUMMARY, CONCLUSION, RECOMMENDATION

Summary
This study was conducted at Iligan City National High School, ESEP building, last February 23, 2008 It is all about finding out the bioactivity of lemongrass extract though brine shrimp bioassay. After conducting the bioassay, it was found out that lemongrass has bioactive compound but its bioactivity depends upon the concentrations of the test solution.
The table for mortality of brine shrimp extract shows that lemongrass is more bioactive at higher concentration.

Conclusion
Based on the LC50 and ANOVA statistical test, it shows that there is significant difference between the control and the experimental set-up with different concentrations.
Therefore it was concluded that lemon grass is more bioactive. And the concentration of the lemon grass extract is directly proportional to the mortality of the brine shrimps.

Recommendation
It is highly recommended by the researchers that lemon grass can be used as a raw material for making product that can be used in studies involving bioactivities.
The researchers also recommend the future researchers to use lemongrass in making product such as anti-fungal ointment.
It is also suggested to the future researchers to conduct other form of bioassay to further determine the bioactivity of lemongrass and to conduct study in other different concentration.

BIBLIOGRAPHY

Books
Tim Johnson, CRC Ethanobotany Desk Reference, N.Y. New York, Newday Publishing House, 1993, p.193.

Dictionary
Webster’s Third New International Dictionary, 1995, page 633.

Encyclopedia (Online)
“Bioassay”, Encarta Encyclopedia, Microsoft Corporation, 1993-2003, all rights reserved.

Internet
http://www.seagrant.gso.uri.edu
http://www.scholar.google.com
http://www.sciencedirect.com
http://www.marketmanila.com
http://www.nd.edu