Centella asiatica

De La Salle University - Dasmariñas

BIOLOGY PROGRAM

EFFECTS OF Centella asiatica L. (GOTU KOLA)

TO THE EMBRYONIC DEVELOPMENT OF

Danio rerio (ZEBRAFISH)

An Undergraduate Research Presented to the

Faculty of the Biological Sciences Department

College of Science and Computer Studies


Dasmariñas City

In Partial Fulfillment of the Requirements

for the Degree of Bachelor of Science Major in Human Biology

Precious Ann O. Padilla

Harold L. Maglalang

March 2013

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BIOLOGY PROGRAM

CHAPTER 1

INTRODUCTION

1.1 Background of the Study

The Zebrafish (Danio rerio), since the 1800’s, have been used as excellent

systems for studying vertebrate embryogenesis. It provides several opportunities

to examine cellular processes in its early development. (Detrich, III et al., 1999) It

has become the Drosophila of vertebrate developmental biology (Kane, 1999) for

it has fewer cell number than most of the studied vertebrate species in terms of

embryo development (Westerfield, et al, 1999).

1.2 Conceptual Framework

The epitome of the study is an attempt to determine the effects of Centella

asiatica L. to the formation of the heart viscera of Danio rerio (Zebrafish), study

is as follows:

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1.3 Statement of the Problem (or Objectives of the Study)

The study aims to determine the effects of Gotu kola(Centella asiatica) to the

changes in the rate of embryonic development and changes in the physical

appearance of Zebrafish (Danio rerio).

1. To determine the effects of Centella asiatica L. to the embryonic

development of Danio rerio (Zebrafish).

2. To determine the morbidity of eggs during the administration of the

concentrate in the Danio rerio (Zebrafish).

1.4 Hypotheses

The following hypotheses were used to accept or reject the study on the effects of

Centella asiatica to the embryonic development of Danio rerio:

1. There are no significant differences on the effects different

concentrations of Centella asiatica extract to the embryonic

development of Danio rerio.

2. There is no significant difference on the effects between the Danio

rerio that is chorionated and with treatment of the extract and the

normal chorionated and untreated one.

1.5 Scope and Limitations

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The study will solely focus on the effects of Centella asiatica L. to the

embryonic development of Danio rerio (Zebrafish). The experiment should be

undertaken in a laboratory setting. The time for breeding, extraction of eggs, and

the staging series shall be more or less expected to be completed in 5 months.

This study is solely for the purpose of understanding more about the effects of the

administered concentrate in the embryo development and the quest for finding

new ways to develop effective methods of enhancing growth.

1.6 Significance of the Study

This study aims to determine the effect of Centella asiatica L. (Gotu kola)

to the embryonic development of Danio rerio (Zebrafish) and the morbidity of

eggs in that matter. This study will also assess Centella asiatica’s effectiveness as

a growth enhancer. This will also help other researchers in their quest to

understand more about the embryonic development of Danio rerio (Zebrafish)

and the use of Centella asiatica (Gotu Kola) as an herbal drug.

1.7 Definition of Terms

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CHAPTER 2

REVIEW OF RELATED LITERATURE

Centella asiatica L. is a colonal, perennial herbaceous creeper belonging

to the family Umbelliferae (Apiceae) which is found throughout India growing in

moist places up to an altitude of 1800 m. It is found in most tropical and

subtropical countries growing in swampy areas. (Gohil et-al, 2010)

It has a small fan-shaped green leaves with white or light purple to pink or

white flowers and it bears small oval fruit. The whole plant is used for medicinal

purposes. It is widely used as a blood purifier as well as for treating high blood

pressure, for memory enhancement and promoting longevity.

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CHAPTER 3

METHODOLOGY

3.1 Research Design

The study is descriptive and experimental at the same time. Its primary

aim is to determine the rate of development and describe the changes that will

manifest on the treated Danio rerio specimen. The staging series developed by

Kimmel, et al. will be used to observe the specimens in growth.

3.2 Research Setting

The experiment will be done in the laboratory setting and will be performed at the

Biological Research Laboratory at De La Salle University – Dasmariñas. The

entire research procedure is estimated to be done in about 10 weeks depending on

the results and other factors that might hinder the completion of the research such

as: contamination of the embryo water, death of the embryo, and academic

responsibilities.

3.3 Research Procedure

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BIOLOGY PROGRAM

Detailed standard procedures for the experiment can be found on the Appendices

of this proposal.

3.3.1. Acquisition of plants

The plant that will be used in this experiment is Gotu kola or commonly

known in the Philippines as Takip-kohol. It can be gathered from Baguio City,

Philippines according to Dalluay and Panem. Other sources of the plant will still

be researched in order to minimize cost. (2012) All of the plant parts will be used

in the extraction process (see Appendices for Extraction process).

3.3.2. Acquisition and care of the Zebrafish (Danio rerio)

Zebrafish and its feed will be bought in accredited pet stores. It will be kept in a

glass aquarium fitted with a filter with occasional water maintenance for the fish

to breed. Zebrafish will be kept in the glass aquariums with the ratio of 1 male: 2

females and in 28oC. In order to breed efficiently, adult fishes shall be fed 1-2

times a day with a variety of food (see appendix for list). Also sanitary procedures

listed in the Appendices of this proposal will be observed to ensure optimum

breeding performance and to avoid diseases.

3.3.3. Egg Harvesting

Embryos can be harvested by siphoning them from the bottom of the tank once

the eggs of the fish have been fertilized. With the use of a glass tubing, medium

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mesh nylon net and a piece of rubber or tygon tubing, sweep the bottom of the

aquarium. Using 10% Hank’s saline(recipes for embryo, egg, and fish water in the

appendices) the harvested embryo should be rinsed to help prevent molds.

3.3.4. Subjection to extract and Microscopy

Siphoned embryos will be subjected to differing concentrations of 0.05%, 0.10%,

0.15% and 0.20% of the Centella asiatica extract in order to test the efficacy of

the extract. Staging series will be used to record the observations.

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APPENDIX A

The Methodology of the study required the following:

Pharmacology – Toxicology (Bernas et al. 2003)

Plant Extraction

1. Centella asiatica plant sample gathering

2. Plants should be cleansed and harvested to avoid contamination.

3. Plants should be dried in an oven at 60-80o C for one to two days.

4. Plant samples should then be crushed into small particles and grinded with

the use of mortar and pestle.

5. Powdered plant, weighed should be soaked into 95% ethanol for 2-3 days

in preparation for ROTAVAP.

6. Mixture will then be run into ROTAVAP.

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APPENDIX B

Zebrafish (Danio rerio) Acquisition, Care, and Water recipes

(from Monte Westerfield’s Zebrafish book)

Danio rerio samples shall be acquired through pet stores or breeding labs.

Sample specimens shall be contained in a 10 gallon fish tank with instant

ocean salts in deionized or distilled water for efficient and successful breeding. To

ensure successful breeding, water should be maintained at about 28 degrees

Celsius. (Note: Adults can live in tap water.) Only 25 fishes with ratio of 2

females: 1 male should be in the tank.

Adult breeding fish should be fed 1-2 times per day with variety of

manually ground moist trout pellets, dry flake food, or adult brine shrimp and

must be finished within 5 minutes every feeding session. Multiple light feeding

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routine can also be established in order to make the food be utilized more by the

fish.

Place single layer of marbles to keep fish from eating newly spawned eggs

that shall be used for observation.

Egg Harvesting

To siphon eggs, use a plastic or glass tube covered with a piece of tygon

tubing to protect it from breaking. Using medium-mesh nylon net at the end of the

tubing, start suction by mouth and sweep the sides of the tank. After siphoning,

invert the net over a petri dish filled with 10% Hank’s saline solution. The

embryos will then fall off the net when they come in contact with the solution. To

prevent from molds, rinse embryos several times with 10% Hank’s saline

solution. ( For Hank’s solution and Embryo water mixtures see below.)

Egg water, Hank’s stock solution, and embryo water mixtures

Egg water is used for in vitro fertilization and raising young embryos.

Stock salts: 40 g "Instant Ocean" Sea Salts added to 1 L distilled water

Egg water = 1.5 ml stock salts added to 1 L distilled water = 60 μg/ml final

concentration.

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Embryo medium: Don't confuse with "egg water" above. Used in handling

dechorionated embryos and storing young embryos in dishes. This is basically

10% Hank's with full strength calcium and magnesium.

Embryo medium

1.0 ml Hank's Stock #1



95.9 ml dd H2O


1.0 ml fresh Hank's Stock #6

Use about 10 drops 1 M NaOH to Ph 7.2

Hank's solutions: Hank's solutions can be made from stock solutions (kept

refrigerated, they will last for several months). A premix of the salts can be stored

in the refrigerator for several weeks. Sodium bicarbonate does not store well, so it

is made up fresh each time Hank's solution is made.

Full Strength Hank's

0.137 M NaCl

5.4 mM KCl

0.25 mM Na2H PO4

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0.44 mM KH2 PO4

1.3 mM CaCl2

1.0 mM Mg SO4

4.2 mM NaH CO3

Hank's Stock Solutions

Stock #1

8.0 g NaCl

0.4 g KCl

in 100 ml ddH2O

Stock #2

0.358 g Na2HPO4 Anhydrous

0.60 g KH2PO4

in 100 ml ddH2O

Stock #4

0.72 g CaCl2

in 50 ml H2O

Stock #5

1.23 g MgSO4-7H2O

in 50 ml ddH2O

Stock #6

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0.35 g NaHCo3

10.0 mls dd H2Oz

Hank's Premix - Combine the following in order:

10.0 ml Solution #1

1.0 ml Solution #2

1.0 ml Solution #4

86.0 ml ddH2O

1.0 ml Solution #5

Store Hank's Premix in the refrigerator along with the Hank's solutions.

Final Hank's

9.9 ml Hank's Premix

0.1 ml fresh Stock #6

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CITED REFERENCES

Detrich, III, HW, Westerfield M, Zon L. 1999. The Zebrafish (Danio rerio)

Biology. Volume I. Pp. xviii, 6,

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Centella asiatica