Running Head: CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.

) LEAF
EXTRACTS AS ANGIOGENESIS INHIBITORS

CALAMANSI (Citrus microcarpa), AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
ANGIOGENESIS INHIBITORS USING CHORIOALLANTOIC MEMBRANE ASSAY
 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
ANGIOGENESIS INHIBITORS

Table of Contents

Abstract . . . . . . . . . . 3

Introduction . . . . . . . . . . 4

Methodology . . . . . . . . . . 9

Results . . . . . . . . . . 12

Discussion. . . . . . . . . . . 15

Conclusion. . . . . . . . . . . 17

Recommendation . . . . . . . . . 17

Acknowledgements . . . . . . . . . 18

Appendices . . . . . . . . . . 19

References . . . . . . . . . . 20

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
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CALAMANSI (Citrus microcarpa), AND GUYABANO (Annona muricata Linn.) LEAF

EXTRACTS AS ANGIOGENESIS INHIBITORS USING CHORIOALLANTOIC
MEMBRANE ASSAY

Adrianne T. Catacutan, Natalie S. Valdez and Aleckzandra M. Tiamzon

Research Adviser: Ariel C. Venida
Bical High School, Mabalacat City, Pampanga

Abstract

Cancer remains to be the most threatening disease to mankind and it becomes an

alarming and serious health concern particularly in the Philippines. Studies have linked

cancer with angiogenesis, a process which allows tumor growth and metastasis through

vascularization. Thus, inhibiting angiogenesis holds a great promise in treating cancers.

Although several anti-cancer therapies such as anti-angiogenic substances exist nowadays,

these remedies are expensive and cause dangerous side effects to patients. Therefore, the

continuous search for less toxic and accessible anti-angiogenesis is highly needed and

encouraged. This study evaluated the anti-angiogenic potential of calamansi (Citrus

microcarpa) and guyabano (Annona muricata L.) ethanolic leaf extracts using duck

Chorioallantoic Membrane Assay and compared the two in terms of their anti-angiogenic

potential. Three groups with 7 replicates were used in the study. Treatment 1 treated with

distilled served as the control group. Treatment 2 was treated with calamansi leaf extract

while treatment 3 was treated with guyabano leaf extract. The study revealed that both plants

are potential anti-angiogenic based on the reduced average numbers of blood vessels formed

in the CAM of the duck embryos as compared to the embryo treated with distilled water

(considered as untreated). It was also revealed that guyabano plant is a more potent anti-

angiogenic as compared to calamansi since embryos treated with its extract registered the

least number of blood vessels and therefore exhibited a greater angiogenesis inhibition. This

study proved that calamansi and guyabano plants have promising anti-angiogenic property

and therefore, hold a great promise in treating cancer.

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
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Introduction

The struggle of searching antidotes for cancer has been progressively escalating over

the years as it is driven by the increasing number of cancer cases worldwide. In the report

released by the International Agency for Research on Cancer (IARC) in September 12 this

year, the global cancer burden is estimated to have risen to 18.1 million new cases and 19.6

million death cases. Of these cases, cancers of the lungs; female breast cancer, colorectal

cancer, prostate cancer and stomach cancer remain to be the top five leading cases worldwide

(IARC, 2018).

In the Philippines, among the different types of cancer; lung cancer, liver cancer,

colorectal cancer, prostate cancer and mouth cancer are the leading types of cancer that cause

death among males while breast cancer, colorectal cancer, lung cancer, cervical cancer, and

liver cancer are the leading types of cancer that cause death in females (World Health

Organization, 2014).

Recent statistics about cancer cases in the Philippines showed that 11 new cases of

cancer in the country are reported daily while 7 adults die of cancer every hour and 8 children

die of cancer everyday (Dizon, 2017). The incidence of cancer in the Philippines remains to

be an alarming and growing national public health concern (Department of Health, 2015).

Thus, the need for continuous and intensive explorations on cancer remedies such as anti-

cancer drug is of high demand.

Previous rigorous studies have correlated cancer with angiogenesis (Gallega & Teves,

2015). Angiogenesis is a normal physiological process in organisms vital for the formation of

new blood vessels, particularly from pre-existing blood vessels (Sun, Heilmann & König,

2015). This process is necessary because it is involved in fetal growth and development,

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
ANGIOGENESIS INHIBITORS

female reproductive cycle as well as in wound healing for tissue repair and restoring of blood

supply after an injury (Gallega & Teves, 2015; Gupta & Zhang, 2005). However, further

studies have linked angiogenesis to tumor growth, metastasis and cancer progression (Hoff &

Machado, 2012). It has been found out that angiogenesis leads to cancer progression because

it allows cancer cells to get enough supply of oxygen and nutrients and help them get rid of

their wastes materials, thus, allowing them to grow, multiply and spread to other parts of the

body (Nishida, Yano, Nishida, Kamura and Kojiro, 2006). Considering these facts, inhibiting

angiogenesis holds a great promise for treating cancer.

Although several anti-angiogenesis therapies to treat cancer have already been

existing nowadays, the search for alternative angiogenesis inhibitors is still being conducted

and remains to be of great interest in order to discover new substances for better remedies

(Verceles & Acebedo, 2013). This is due to the idea that the existing therapies face

challenges which include the cytotoxic side effects of some angiogenesis inhibitors that lead

to an enhanced resistance, invasion and metastasis of cancer cells (Abdalla et al., 2018; Jeong

et al., 2010). Furthermore, the fact is not denied that modern treatments for cancer are highly

expensive making them inaccessible to many less-fortunate patients. Thus, the exploration for

alternative angiogenic inhibitors which are not costly and accessible to all, such of those from

plant sources is needed and highly encouraged (Barluado, Lagang, Gordonas & Bosas, 2013).

World Health Organization (WHO) states that 80 % of the world’s population depend

on plants for medical care (Gurib-Fakim, 2006). The use of plants as alternative sources of

medicine is also a popular practice in the Philippines (Gamallo et al., 2016). Several plants

nowadays, including fruits and vegetables, have become subjects of interest as potential

source of phytochemicals such as phenolic acids that could possibly reduce the risk of certain

types of diseases including cancer (Arts & Hollman, 2005). One consideration of using plants

as sources of alternative medicine is the fact that aside from their properties of being anti-

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
ANGIOGENESIS INHIBITORS

cancer, compounds from plant extracts are organic and less toxic as compared to modern

sources of remedies (Magdalin Joy, Reginal Appavoo & Glad Mohesh, 2016).

Of the many native plants growing in the Philippines, calamansi is one of the most

abundant and widely-grown plants traditionally used as a condiment in many Filipino dishes

and used as a relief for skin itching and mosquito bite (Morte & Acero, 2017). Furthermore,

being known for its richness in Vitamin C, calamansi pulps are usually squeezed to make

fruit beverages which are conventionally taken as home remedy for common coughs and

colds (Philippine Calamansi Association, Inc., 2015). Further studies about the potentials of

calamansi as a medicine revealed that calamansi is not only a good source of Vitamin C

which makes it a powerful antioxidant but also a potential angiogenesis inhibitor due to its

high phenolic acid and tarpenoid contents (Canda & Gasendo-Muaña, 2016; Barluado,

Lagang, Gordonas & Bosas, 2013).

Guyabano (Annona muricata Linn.) is another common plant locally grown in the

Philippines (Gallega & Teves, 2015). Previous studies in the leaf extract of this plant showed

its medicinal uses which include antiparasitic, anticancer, insecticidal, anti-inflammatory,

antibacterial, fungicidal, antioxidant and property (Mithun Pai, Rajesh, Shenoy, Rao, 2016;

Moghadamtousi, et al., 2015; Gallega & Teves, 2015; Kedari & Khan, 2014). Furthermore,

phytochemical analysis conducted on guyabano showed the existence of phenolic compounds

on its extract, a compound that is known to be a natural angiogenesis inhibitor (Sun,

Heilmann & König, 2015; Moghadamtousi, et al., 2015; Gavamukulya, Abou-Ellela,

Wamunyokoli & Ael-Shemy, 2014).

In the Philippines however, only few published studies have been conducted so far

which evaluated the anti-angiogenic potential of calamansi and guyabano and no studies have

been conducted so far that compared the anti-angiogenic potential of these two plants.

Furthermore, studies that were conducted about the anti-angiogenic potential of calamansi

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
ANGIOGENESIS INHIBITORS

utilized its peel extract and only few published studies have been made so far with regards to

its leaf extract’s anti-angiogenic potential.

Another consideration of this study is that traditionally, people only utilize the fruits

of guyabano and calamansi plants but do not usually give much attention to the possible

benefits that the plants’ leaves can offer. Thus, this study aimed to evaluate the potential of

calamansi and guyabano ethanolic leaf extracts as angiogenesis inhibitors and compare the

anti-angiogenesis potential of the two plants using duck Chorioallantoic Membrane (CAM)

Assay.

Chorioallantoic Membrane (CAM) Assay was chosen by the researchers because this

test is considered as an appropriate test widely used to evaluate angiogenesis activity using

plant extracts because of its highly vascularized nature, simplicity and affordability as

compared to animal models (Gamallo, 2016; Lokman, Elder, Ricciardelli & Oehler, 2012).

Moreover, on the guidelines released by National University of Singapore (2016), chick

embryos to be used for scientific purpose which are younger than 15 days old are not

considered as live vertebrates and cannot experience pain; therefore, it is not subject to

Institutional Animal Care and Use Committee (IACUC).

The success of this study is viewed to hold a great promise in the field of cancer

research and in the field of medicine at large in discovering potential remedies for cancer

from natural sources through the help of further intensive research studies that will be

conducted on the same plants used in this study.

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
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Research Questions

This study generally aimed at evaluating the potential of guyabano and calamansi

ethanolic leaf extracts as angiogenesis inhibitors. Specifically, this study sought to answer the

following questions:

1) Which among the three treatments will have greater angiogenesis inhibition based on

the mean number of blood vessels formed?

2) Is there a significant difference between the anti-angiogenic potential of the negative

control, calamansi, and guyabano ethanolic leaf extracts?

3) Which among calamansi and guyabano ethanolic leaf extracts has greater anti-

angiogenic potential based on the mean number of blood vessel formed?

Hypothesis

The study hypothesized that:

1) There is no significant difference between the anti-angiogenic potential of the three

treatments: (T1=distilled water, T2=calamansi ethanolic leaf exrtract and

T3=guyabao ethanolic leaf extract).

2) There is no significant difference between the anti-angiogenic potential of calamansi

and guyabano.

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
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Methodology

Research Design
The study employed the experimental method of research to evaluate the potential of

calamansi (Citrus microcarpa) and guyabano (Annona muricata) ethanolic leaf extracts as

angiogenesis inhibitors using the Duck Chorioallantoic Membrane (CAM) Assay. The design

was selected because it allowed the researchers to test if any manipulation in the independent

variable will have any effects in the dependent variable. The independent variable in this

study is the plant extract used while the dependent variable is the angiogenic activity in the

CAM of the duck embryo. There were three treatments with seven (7) replicates used in the

study: T1-distilled water (negative control), T2- calamansi ethanolic leaf extract and T3-

guyabano ethanolic leaf extract.

Materials & Equipment

The following were the materials used in performing the experiment:
Filter Paper Disc Blender/Pulverizer Ethanol
Filter Paper Alcohol Lamp Plant Extract
Beaker Tripod Distilled Water
Stirring Rod Steam Bath Duck Egg
Containers Incubator
Scalpel Container
Research Procedure

1. Collection of the Plant Leaves

The fresh calamansi (C. microcarpa) leaves and guyabano (A. muricata Linn.)

leaves were collected in Barangay Bical, Mabalacat City, Pampanga on September 14,

2018. The leaves were washed with tap water and distilled water to get rid of

unwanted materials and drained thoroughly. The samples were air-dried for one week

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
ANGIOGENESIS INHIBITORS

in a well-ventilated room without any exposure to solar radiation, and then pulverized

into coarse powder using a blender.

2. Plant Extraction

The leaf ethanolic extract of calamansi (C. microcarpa) and guyabano (A.

muricata Linn.) were gathered after soaking the air-dried powdered leaves with

sufficient amount of 95% ethanol for forty-eight (48) hours at room temperature and

filtered with a clean and fine filter paper. The extracts obtained were then

concentrated using steam bath evaporation.

3. Chorioallantoic Membrane (CAM) Assay

3.1 Incubation of the fertilized Eggs. Twenty-one (21) fertilized duck eggs were

incubated at 37oC with enough relative humidity up to egg development day 10

to prevent them from being dehydrated and damaged.

3.2 Drilling of Holes on the Eggs. On the 10th day, the surface of the eggs was

disinfected with ethyl alcohol then 1cm x 1cm windows were drilled at the broad

end of the shell.

3.3 Abscission of the Eggs. The egg membranes were cut off and plant extracts

were applied directly to the CAM through absorption onto the filter paper disc.

A surgical tape was used to cover the window. The eggs were returned to the

incubator for another two days.

3.4 Visual Assessment of the CAM. CAM was examined after 12 days of

incubation. The windows were reopened for the observation and assessment of

the changes that the treatments have brought to the angiogenesis of the CAM.

The number of branching points of blood vessels was manually counted. The

CAM was also photographed.

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
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Statistical Treatment

The data obtained were recorded, tabulated and analyzed using the One-way Analysis

of Variance (ANOVA) to determine if there is significant difference in the mean count of the

blood vessels’ main branching point in the CAM of the embryos treated with distilled water,

calamansi ethanolic leaf extract and guyabano ethanolic leaf extract. A Post Hoc test using

Tukey HSD was further done in order to determine where the significant difference exists

between the three treatments.

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
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Results
The angiogenic activities of calamansi and guyabano ethanolic leaf extracts were

based on the number of blood vessels branching points formed in the Chorioallantoic

membrane (CAM) of the duck embryos.

Table 1. Angiogenic Activity of the Three Treatments based on the mean number
of blood vessels formed in the CAM
Number of Blood Vessels in the CAM
Treatment
R1 R2 R3 R4 R5 R6 R7 Mean

T1-Distilled Water 78 81 100 82 92 106 90 89.86

T2-Calamansi 56 58 73 65 59 54 53 59.71

T3-Guyabano 49 52 57 57 53 54 46 52.57

Table 1 shows the average number of blood vessels formed in the CAM using the three

treatments: T1-distilled water (control), T2-calamansi ethanolic leaf extract and T3-guyabano

ethanolic leaf extract. Duck embryos treated with distilled water which served as the control

group yielded the highest mean of blood vessel count which is 89.86. It was followed by the

embryos treated with calamansi which is 59.71 while the embryos treated with guyabano

yielded the lowest mean of blood vessel count which is 52.57.

Table 2. Difference in the Anti-angiogenic potential of the Treatments

One-way Analysis of Variance

Source of SS df MS F P-value Sig
Variation
Between Groups 5482.95 2 2741.48 47.18 0.0000 Significant at
Within Groups 1046 18 58.11 5%

Total 6528.95 20

Table 2 shows the analysis of variance among the three treatments. An F-value of 47.18 and a

p-value of 0.0000 were obtained at 5% level of significance.

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
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Table 3. Difference in the Anti-angiogenic potential of each treatment using Post Hoc
Test

Group p-value Remarks

Control vs. Calamansi 0.000 Significant at 5%
Control vs. Guyabano 0.000 Significant at 5%
Calamansi vs. Guyabano 0.214 Not Significant at 5%

Table 3 shows the results of the Post Hoc test. The data reveal that there is a significant
difference between the anti-angiogenic potential of the control treatment and calamansi
extract; and significant difference in the anti-angiogenic potential of control treatment and
guyabano. However, and there is no significant difference between the anti-angiogenic
potential of calamansi and guyabano ethanolic leaf extracts.

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CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
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Figure 1: CAM treated with distilled water (T1)

Figure 2: CAM treated with calamansi leaf extract (T2)

Figure 3: CAM treated with guyabano leaf extract (T3)

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
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Discussion
The angiogenic activities of calamansi and guyabano ethanolic leaf extracts were

based on the number of blood vessels’ branching points formed in the Chorioallantoic

membrane (CAM) of the duck embryos. The fewer blood vessels means the greater degree of

angiogenesis inhibition. The raw data in Table 1 show that embryos treated with calamansi

and guyabano ethanolic leaf extracts showed an inhibition in the angiogenesis activity when

compared to the embryos treated with distilled water (considered as untreated). This means

that both calamansi and guyabano exhibit an anti-angiogenic potential. It was further revealed

that between calamansi and guyabano, guyabano ethanolic leaf extract brought the greatest

angiogenesis inhibition in the CAM of the embryos since the embryos treated with its extract

registered the least number of blood vessels count. Furthermore, Table 2 shows the analysis

of variance among the three treatments. An F-value of 47.18 and a p-value of 0.0000 were

obtained at 5% level of significance. Since the computed p-value is lower than 0.05

(p<0.005), this means that there is a significant difference in the anti-angiogenic potential of

the three treatments. Therefore, the null hypothesis that there is no significant difference

between the anti-angiogenic potential of the three treatments is rejected. Moreover, based on

the results of the post hoc test in Table 3, there is a significant difference between the anti-

angiogenic potential of the control and calamansi (p-value 0.000<0.05); and control and

guyabano (p-value 0.000<0.05). However, there is no significant difference between

calamansi and guyabano (p-value 0.214>0.05). This means that the second null hypothesis is

accepted. This also indicates that calamansi and guyabano ethanolic leaf extracts are

comparable in terms of their anti-angiogenic potential. These findings affirmed the finding of

Gallego and Teves (2015) that guyabano can inhibit blood vessel formation on the CAM of

embryos although the study made use of guyabano supercritical carbon dioxide extract and

also supported by the finding of Barluado, Lagang, Gordonas and Bosas (2013) that

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
ANGIOGENESIS INHIBITORS

calamansi has an antiangiogenic potential although the study utilized its peel ethanolic

extract. The finding that calamansi and guyabano have anti-angiogenic potential is consistent

to finding Canda and Gasendo-Muaña (2016) that calamansi is not only a powerful

antioxidant but also a potential angiogenesis inhibitor due to its high phenolic acid and

tarpenoid contents and also consistent to the finding of Moghadamtousi, et al. (2015) that

guyabano has an anti-cancer property due to the phytochemicals present in it such as phenolic

compounds.

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
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Conclusion

Based on the findings of this study, it can be concluded that calamansi (Citrus

microcarpa) and guyabano (Annona muricata L.) plants are both potential anti-angiogenic

based on the reduced average numbers of blood vessels formed in the CAM of the duck

embryos as compared to the embryo treated with distilled water (considered as untreated).

Furthermore, it can also be concluded from the findings of this study that although the

embryos treated with guyabano extract registered the least number of blood vessels, statistics

revealed that guyabano and calamansi are comparable in terms of their anti-angiogenic

property and therefore appeared to be both important plants with promising anti-cancer

property.

Recommendation

In view of the findings of this study, the researchers recommend that further studies

may be conducted to evaluate the mechanisms on how the phytochemicals in these plants

inhibit vascularization. For the future researchers who wish to conduct the same study, it is

also recommended that a cytoxicity test must be conducted in these plants in order to evaluate

their toxicity level. The researchers also recommend that the future researchers use a positive

control to compare the anti-angiogenic potential of these plants to a commercial anti-

angiogenic chemical such as retinoic acid.

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Acknowledgements
The researchers are grateful to the following individual: Ms. April I. Carbungco, Ms.

Imee M. Dayrit, Mr. Ariel C. Venida and Warren Manalansan for their assistance during the

experimentation process and in the preparation of the papers.

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Appendices

Preparation of the Plant Extracts

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 CALAMANSI (Citrus microcarpa) AND GUYABANO (Annona muricata Linn.) LEAF EXTRACTS AS
ANGIOGENESIS INHIBITORS

CAM Assay Procedures

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ANGIOGENESIS INHIBITORS

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