Republic of the Philippines

Department of Education
Region III
Schools Division of Nueva Ecija
Bartolome Sangalang National High School
Barawid St., St. John District, Guimba, Nueva Ecija

Embryo-toxic and Teratogenic Activity of Snake Grass (Clinacanthus nutans) Leaf Extracts in
Developing Embryos of Zebrafish (Danio rerio)

An Investigatory Project Presented to

The Panel of Oral Examiners
In Partial Fulfilment of the Requirement in Research II

by

Mylene Kaye Dianne G. Ramos

Johanna Mae L. Gamis
Eduardo L. Sanidad
Jasper F. Fernandez
Dreddfil S. Padre
Mark Aaron E. Dubal

Arjanne J. Villamarzo
Research Adviser

March 2019
 RESEARCH PLAN/PROJECT SUMMARY

Embryo-toxic and Teratogenic Activity of Snake Grass (Clinacanthus nutans) Leaf Extracts in
Developing Embryos of Zebrafish (Danio rerio)

Rationale

Cancer is a large family of diseases that involves abnormal cell growth with the potential to invade

or spread to other parts of the body. According to World Health Organization (WHO), cancer is the second

leading cause of death globally. There are many types of cancer such as breast cancer, Liver cancer, lung

cancer, color cancer, cervix cancer and cancer of the blood (leukemia). Cancer is caused by changing

(mutations) to the DNA within cells. The DNA inside a cell is package into a large number of individual

genes, each of which contains a set of instructions telling the cell what functions to perform, as well as

how to grow and divide. Errors in the instructions can cause the cell to stop its normal function and may

allow a cell to become cancerous. In 2016, Philippines has the highest breast cancer prevalence and also

one of the countries worldwide with the highest prevalence rates of cancer.

Today’s medicinal world is dynamic in searching for effective medicines to cure or prevent the

proliferation of virulent diseases affecting the human race. Despite the dynamism of science however, the

world is still in trouble, as these diseases, such as cancer. Though nowadays, there are health professionals

that can offer chemotherapies and surgeries to cancer patients, there are patients who are in low and

middle income families who could not afford medication. So now, herbal medicines are getting popular

as an alternative way in treating diseases. Many plants considered to be an alternative medicine that can

be used to treat cancer and one of those herbal plants is Snake Grass (Clinacanthus nutans). (Romagosa

et. al, 2016)

Snake grass (Clinacanthus nutans) is a natural herb that is part of Acanthaceae family which can

grow from 20 to 50 centimeters. Leaves are little bit long and pointed in the tip. Flowers are commonly
white and red with color yellow on the tip. The plant has already been used in different studies like anti-

oxidant, cytotoxic antimicrobial, etc.

Teratogens are agents that cause abnormalities in the growth and development of an organism.

However, teratogens are anticancer agents and vice versa. (Dulay et. al, 2017)

Zebrafish embryo as animal model is a very reliable and important tool due to its very rapid

developmental processes, high fecundity, transparency, easy maintenance in the laboratory accessible to

experimental manipulation and similarity to the embryonic development of higher forms of vertebrates.

(Romagosa et. al, 2016)

This study aims to assess the embryo-toxic and teratogenic activity of Sabah snake grass

(Clinacanthus nutans) leaves extract in developing embryos of zebrafish (Danio rerio) and provide pieces

of evidence that the plant can contribute in the medical field by further studies.
Statement of the problem

This study entitled “Embryo-toxic and Teratogenic Activity of Snake Grass (Clinacanthus nutans)

Leaf Extracts in Developing Embryos of Zebrafish (Danio rerio)” aims to determine the following

objectives:

1. Determine the mean percentage of the mortality of zebrafish after 12, 24, 36, and 48 hours of

exposure in different concentrations of (Clinacanthus nutans) leaf extracts.

2. Find out the percentage hatchability and growth retardation if zebrafish treated with the different

concentrations of Clinacantus nutans at 48 hours of treatment application.

3. Describe the percentage mortality, hatchability and growth retardation of embryos treated with

the different concentrations of Clinacantus nutans at 48 hours of post-treatment applications.

Hypotheses

This study will be conducted to validate the following hypothesis:

1. There is a significant difference in the mean percentage of the mortality of zebrafish after hours

of exposure in different hours of exposure to Clinacanthus nutans leaf extracts.

2. There is a significant difference between the percentage hatchability and growth retardation of

zebrafish treated with the different concentrations of Clinacanthus nutans after 48 hours of

applications.

3. There is a significant effect between the percentage mortality, hatchability and growth

retardation of embryos treated with the different concentrations of Clinacanthus nutans

after hours of application.

1
Significance of the study

Society

Society will develop their knowledge about the benefits of Clinacanthus nutans that can

be used soon as an anti-cancer.

Medical Workers

The findings of this study will help the medical workers in expanding their knowledge on

developing products that will help treat cancer.

Teachers

This study will benefit the teachers, especially the research teachers. Through this study,

teachers will have more knowledge to guide their students.

Students

This will help the students to have knowledge about the plant’s components that can be

used in different studies.

Researchers

This study will aid other researchers to develop and improve new findings in treating

cancer with the use of Clinacanthus nutans.

2
Scope and Delimitation

This study aims to determine the Embryo-toxic and Teratogenic activity of Sabah Snake

Grass (Clinacanthus nutans) in developing embryos of zebrafish. It will be conducted from June

2018 to February 2019 at Bartolome Sangalang National High School.

Engineering Goals

This study aims to prove that Sabah Snake Grass (Clinacanthus nutans) leaves has an

embryo-toxic and teratogenic activity and address to the community that this plant can cause

delayed growth, low-success hatchability and mortality in teratogenic assay that signifies that it

can be used in anti-cancer activities. Besides, the result of this research study will become useful

for the pharmaceuticals to produce anti-cancer drugs and also for the cancer patient to treat

their disease using low-cost Sabah Snake Grass (Clinacanthus nutans).

Expected Outcomes

The Sabah Snake Grass (Clinacanthus nutans) leaves extract will cause mortality, growth

retardation, low-success hatchability and abnormalities to the zebrafish (Danio rerio) embryo and

will therefore prove the teratogenic property of the plant. The success of the study will provide

awareness for members of the community in discovering alternative ways to cure cancer,

knowledge for medical workers about the benefits that Sabah Snake Grass (Clinacanthus nutans)

could give---a potential additive in anti-cancer drugs, and will definitely help cancer patients in

finding alternative ways in curing cancer.

3
 CHAPTER I

INTRODUCTION

Cancer is a large family of diseases that involves abnormal cell growth with the potential

to invade or spread to other parts of the body. According to World Health Organization (WHO),

cancer is the second leading cause of death globally. There are many types of cancer such as

breast cancer, Liver cancer, lung cancer, color cancer, cervix cancer and cancer of the blood

(leukemia). Cancer is caused by changing (mutations) to the DNA within cells. The DNA inside a

cell is package into a large number of individual genes, each of which contains a set of instructions

telling the cell what functions to perform, as well as how to grow and divide. Errors in the

instructions can cause the cell to stop its normal function and may allow a cell to become

cancerous. In 2016, Philippines has the highest breast cancer prevalence and also one of the

countries worldwide with the highest prevalence rates of cancer.

Today’s medicinal world is dynamic in searching for effective medicines to cure or prevent

the proliferation of virulent diseases affecting the human race. Despite the dynamism of science

however, the world is still in trouble, as these diseases, such as cancer. Though nowadays, there

are health professionals that can offer chemotherapies and surgeries to cancer patients, there

are patients who are in low and middle income families who could not afford medication. So now,

herbal medicines are getting popular as an alternative way in treating diseases. Many plants

considered to be an alternative medicine that can be used to treat cancer and one of those herbal

plants is Snake Grass (Clinacanthus nutans). (Romagosa et. al, 2016)

 Snake grass (Clinacanthus nutans) is a natural herb that is part of Acanthaceae family

which can grow from 20 to 50 centimeters. Leaves are little bit long and pointed in the tip.

Flowers are commonly white and red with color yellow on the tip. The plant has already been

used in different studies like anti-oxidant, cytotoxic antimicrobial, etc. Snake Grass are used in

native countries as an herbal plant. This is a rare type of grass that has believed to heal health

problems, but it is not proven yet by professionals. This is one of the possible cancer-curing plants

that have been lately being tested by the Oncology Team (a group of Scientists who specializes

in cancer). (Modi, 2013). Extracts of 6-month old snake grass exhibited a significant In-vitro anti-

cancer activity against HeLa cancer cells, thus, early harvesting of snake grass may yield increased

concentrations of potent anti-oxidant compounds. (Ghasemzadeh et. al, 2014)

“Teratology is the aspect of embryology involved with the study of abnormal

development and a teratogen is any agent which overtly causes the production of a particular

congenital defect.” (Tolentino et. al, 2016). Teratogenicity assay is evaluated using zebra fish

embryos. Teratogen are those substances that is responsible for the formation of anatomical

abnormalities or defects of embryos. Zebrafish embryo is a very reliable and important tool due

to its very rapid developmental processes, high fecundity, transparency, easy maintenance in the

laboratory, accessible to experimental manipulation and similarity to the embryonic

development of higher forms of vertebrates as an animal model.(Romagosa et. al, 2016).

According to Dulay et. al, (2014), “Teratogenicity assay using zebrafish embryos as animal

model is a very reliable and important tool in establishing whether certain compounds or food

materials could cause deformity to the new being. This is also used in various toxicological

research such as environmental toxicity dealing with the harmful effects of chemical pollutants,

5
safety assessment of new pharmaceutical product, and evaluation of new toxic compounds with

potential anticancer properties.”

Zebrafish embryo as animal model is a very reliable and important tool due to its very

rapid developmental processes, high fecundity, transparency, easy maintenance in the

laboratory accessible to experimental manipulation and similarity to the embryonic development

of higher forms of vertebrates. (Romagosa et. al, 2016) Based on the periodicals, zebrafish

embryo model in environmental risk assessment of SETAC, the use of fish embryos is not

regulated, Fish embryos are commonly used as a model for determining toxicity and studies

aimed at understanding toxic mechanism and the indicaton of possible adverse and long-term

effects.

This study aims to assess the embryo-toxic and teratogenic activity of Sabah snake grass

(Clinacanthus nutans) leaves extract in developing embryos of zebrafish (Danio rerio) and provide

pieces of evidence that the plant can contribute in the medical field by further studies.

6
Statement of the problem

This study entitled “Embryo-toxic and Teratogenic Activity of Snake Grass (Clinacanthus nutans)

Leaf Extracts in Developing Embryos of Zebrafish (Danio rerio)” aims to determine the following

objectives:

1. How may the mortality rate of zebrafish embryo be affected by:

1.1 Time of exposure to Clinacanthus nutans leaf extracts ; and

1.2 Concentrations of Clinacanthus nutans leaf extracts

2. How may the percentage of hatchability and growth retardation of zebrafish embryo be affected

when treated with different concentrations of Clinacanthus nutans leaf extract after 48 hours

of treatment application?

3. Is there significant difference between the effect of different concentrations of C. nutans to the

mortality, hatchability and growth retardation?

Hypotheses

This study will be conducted to validate the following hypothesis:

1. Ha: There is a significant difference between the effects of different concentrations and time

exposure of Clinacanthus nutans leaf extract to mortality rate after 48 hours of application.

2. Ha: There is a significant difference between the percentage hatchability and growth

retardation of zebrafish treated with the different concentrations of Clinacanthus nutans after

48 hours of applications.

3. Ha: There is a significant effect between the percentage mortality, hatchability and growth

retardation of embryos treated with the different concentrations of Clinacanthus nutans after

48 hours of application.

7
Scope and Delimitation

This study aims to determine the Embryo-toxic and Teratogenic activity of Sabah Snake

Grass (Clinacanthus nutans) in developing embryos of zebrafish. It will be conducted from June

2018 to February 2019 at Bartolome Sangalang National High School.

8
 CHAPTER II

Methodology

Collection of Sabah Snake Grass Leaves

Extraction of Sabah Snake Grass

Spawning of Zebrafish

Embryo-toxicity and Teratogenicity Assay

Statistical Analysis

Figure 1. Methods Flowchart

Collection of the Leaves of Sabah Snake Grass

The researchers will use Sabah Snake Grass leaves which will be collected in Guimba,

Nueva Ecija and were washed to eliminate dirt and dust. The leaves were out into small pieces

and air-dried for 3-5 days prior to extraction.

Extraction of Sabah Snake Grass Leaves

By following the protocol of Eguchi et al. the functional components of the air-dried snake

grass leaves were acquired by following the hot water extraction. The 20g snake grass leaves

sample was extracted at 80-90o C in 600mL hot water in a water bath for 2 hours.

Spawning of Zebrafish

For the spawning of zebrafish, matured male and female zebrafish were present at 1:2

ratio, the researchers used a non-treated stock of tap water in a glass aquarium with oxygen

saturation. At a 12-hour day and night light regime where the condition was 26 ± 1o C. The

zebrafish were fed twice a day and excess food was removed daily to ensure optimum water

quality. The glass aquarium was covered with a black plastic for 12 hours for the zebrafish to

spawn. The fish were confined in a plastic mesh to spawn and fertilize; following the procedure

of Dulay et al. After the segmentation phase ( 12-24 and 26-48 hours of post fertilization),

embryos were collected and placed in a watch glass to examine the uniformity and normal

condition of embryos.

10
Embryo-toxicity and Teratogenicity Assay

The assay that this study followed was established by Dulay et al. Embryos were exposed

to the different concentrations ( 10,000 ppm, 1000 ppm, 100 ppm, 10 ppm, 1 ppm and 0% ) of

each extracts. Embryo water served as the control in the 12-well ELISA plate. Four embryos were

assayed per treatment and each treatment was replicated three times. The plates were

incubated at 26o C ± 1 o C. Toxicity and teratogenicity were examined using a compound

microscope every after 12 hours of treatment exposure, mortality, hatchability and delayed

growths were recorded. Death was defined as coagulated embryos and as no visual heartbeat.

Statistical Analysis

Data were analyzed using Analyss of Variance (ANOVA) compared using T-test at 5% level

of significance.

11
 CHAPTER III

RESULTS AND DISCUSSION

In the embryotoxicity and teratogenicity test, zebrafish, which is a new vertebrate model was

used as an experimental animal model. The toxic and teratogenic effects of the extracts of C. nutans was

assessed using zebrafish embryos. The extract was dilluted into varying concentrations prior to the

exposure of the embryos at the segmentation phase. The mortality, hatchability, and delayed growth were

recorded.

Toxic Effects of C. nutans leaves:

Embryos with no visual heartbeat and coagulated embryos are considered toxic effects of a

certain compound being tested. The results of the recorded mortality after 12, 24, 36 and 48 hours

exposure in different extract concentrations of snake grass are shown in Table 1. In all periods of

observation, the lethal effect of the extracts of C. nutans was dependent on the time of exposure and

dose. At 24 hours of exposure, 8.33% mortality was recorded to the embryo at 10000ppm concentration.

However, after a longer period of exposure, the developmental processes of embryos were affected.

Table 1. Mean percentage mortality of zebrafish embryos after 12, 24, 36 and 48 hours of exposure in

different concentrations of the extract.

Treatment Concentration Mortality (%)

(ppm) 12 hours 24 hours 36 hours 48 hours
T1 10000 0.00 8.83 66.67 100.00

T2 1000 0.00 8.83 25.00 41.67

T3 100 0.00 0.00 0.00 8.83

T4 10 0.00 0.00 0.00 0.00

T5 1 0.00 0.00 0.00 0.00

 Embryos exposed at 10000ppm concentration showed 66.67% mortality after 36 hours and 100%

mortality after 48 hours of exposure. Some embryos exposed to 1000ppm extract showed 8.33%

mortality after 24 hours, 25% mortality after 36 hours and 100% mortality after 48 hours. While other

embryos treated with 100ppm extract resulted in a mortality rate of 8.33% after 48 hours. No mortality

was observed in those embryos with 10ppm or lower concentrations. These results suggested that a

prolonged exposure of embryos at higher concentation (10000ppm) of extract increased the mortality

rate of zebrafish embryos.

Hatchability of Embryos:

Hatchability takes place between 48-72 hours depending on the thickness of chorion and

muscular activity of the embryos. The hatchability of embryos treated with the different concentrations

of the extracts of C. nutans after 48 hours is presented in Table 2. A 100% hatchability rates were recorded

in the control embryos and 1ppm extract concentration. Embryos at 10ppm extract concentration

resulted in a hatchability rate of 75%.

Table 2. Percentage hatchability of zebrafish treated with the different concentrations of the extract at

48 hours post treatment application.

Treatment Concentration Hatchability (%) after 48 hours

(ppm) R1 R2 R3 Mean
T1 10000 0 0 0 00.00

T2 1000 0 0 0 00.00

T3 100 0 50 50 33.33

T4 10 50 75 100 75.00

T5 1 100 100 100 100.00

T6 0 100 100 100 100.00

13
The lowest hatchability was noted to embryos at 10000ppm and 1000ppm extract concentration

which resulted to a low-success hatchability of 0%. The present results clearly indicate that

exposure of embryos to C. nutans could cause the decreased on the hatching rate of embryos.

Growth Retardation as Teratogenic Effect:

The embryos exposed to the varying concentrations of C. nutans extracts were evaluated

based on the morphological endpoints established by Nagel. The growth retardation is the most

teratogenic effect of C. nutans leaf extracts. This is obvious to all extract-treated embryos showed

by 100% growth retardation at 10000ppm and 1000ppm extract concentration. Embryos at

100ppm extract concentration resulted in a delayed development percentage of 66.67%.

Table 3. Percentage delayed growth of embryos treated with the different concentrations of

Treatment Concentration Delayed Development (%) after 48 hours

(ppm) R1 R2 R3 Mean
T1 10000 100 100 100 100.00

T2 1000 100 100 100 100.00

T3 100 100 50 50 66.67

T4 10 50 25 0 25.00

T5 1 0 0 0 00.00

T6 0 0 0 0 00.00

The lowest percentage growth was noted for embryos exposed at 10ppm concentration.

While embryos at 1ppm extract concentration resulted as a no delayed growth in embryo water.

Growth retardation could be explained by the hindered enzymatic activities which play a

significant role in the developmental processes of embryo.

14
 CHAPTER IV
SUMMARY, CONCLUSION AND RECOMMENDATION

Summary of Findings

This study was conducted to determine the mean percentage of the mortality,

hatchability and growth retardation of zebrafish embryos treated by the different concentrations

of C. nutans leaves extract at 48-hours post-treatment application. Different concentrations of C.

nutans leaves extract was used to prove that it contains teratogenic property, and therefore

effective as anti-cancer. Several descriptive and experimental methods were conducted in this

study which was deemed to be the most appropriate in obtaining the needed information.

On the basis of the analysis of data, the following results were determined:

1) The results suggested that the mortality rate of zebrafish embryos increased through a

prolonged exposure of embryos of higher concentrations (10000 ppm) of extract.

2) The present results clearly indicate that the hatching rate of embryos may decrease by

longer exposure of embryos to higher concentrations of C. nutans extract.

3) The growth retardation of embryos may increase by adding higher concentrations of C.

nutans leave extract.

Conclusion

This study focused on te embryotoxicity and teratogenic activity of C. nutans leaves

extract using zebrafish (Danio rerio) embryo. The following conclusions were drawn:
 1) Varying concentations of C. nutans leaves extract affects the mortality and hatchability of

embryos. Therefore, there is no significant difference in the mean percentage of mortality

of hatchability of zebrafish treated with exposure in different concentrations.

2) The teratogenic effect of the C. nutans leaves extract to zebrafish embryos was growth

retardation. Therefore, there is significant difference between the percentage growth

retardation of embryos treated with the different concentrations of Clinacanthus nutans

at 48 hours post treatment applications

3) Higher concentrations of C. nutans leaves extract exhibit teratogenic and toxic effect in

the developing embryos of zebrafish which suggests that these extracts contain

teratogens that could develop anti-cancer drug.

Recommendations

Based on the observations regarding this study, the researchers recommend the

following:

1) Try using higher concentrations of C. nutans leaves extract and longer exposure of

zebrafish embryos to extract.

2) Other parts of the plant may also be used as anti-cancer and other studies as it may

contain antioxidant, anti-inflammatory, antimicrobial, antifungal, antiviral properties and

others.

3) C. nutans leaves may be used to produce anti-cancer drug.

16