Averrhoa carambola (balimbing) AND Brassica oleracea (broccoli)

EXTRACT AS ANGIOGENESIS INHIBITORS: AN IN OVO

EVALUATION USING DUCK CHORIOALLANTOIC
MEMBRANE (CAM) ASSAY

This quantitative study is submitted in partial fulfillment of the requirements

in Practical Research II

Katrice Zeann M. Melo

Ashraf Khan N. Santos
Krizztina May L. Songcog
Research Proponents

Leizel F. Mundo
Research Adviser

REGIONAL SCIENCE HIGH SCHOOL-IX

Malasiga, San Roque, Zamboanga City

October 24, 2024

 TABLE OF CONTENTS

INTRODUCTION ……………...………………………………………………...….. 1

Background of the Study ………………………...………………………...… 2

Aims of the Study ………………...……………………………………...……3

Significance of the Study ……………….…..……………………………...... 3

Delimitation of the Study ………..…….…………………………………….. 4

REVIEW OF RELATED LITERATURE ….…………..…………………….…. 5 - 10

REFERENCES ….…………..……………………………………...………….. 11 - 13

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 INTRODUCTION

A. Background of the Study

In the present day, cancer is one of the contributors to high cases of mortality

worldwide, with an estimated 20 million cases and 9.7 million deaths.(World Health

Organization). Cancer refers to a group of diseases that can develop in any organ or

tissue within the body. It occurs when cells grow uncontrollably, leading to rapid

division and spread throughout the body (National Cancer Institute). This

uncontrolled cell growth often results in cancerous tumors or malignant lumps of

tissue, which can invade nearby tissues and form new tumors. A key process in the

growth and spread of tumors is angiogenesis, the formation of new blood vessels.

Angiogenesis can be triggered by releasing a chemical signal called Vascular

Endothelial Growth Factor (VEGF), which is attached to the endothelial cells that

make up the inner lining of blood vessels or simulating near-normal cells to produce

angiogenesis signaling molecules. (Healthline, 2022) Typically, these signals are

regulated to make sure blood vessels form when needed. But, when this balance is

disrupted, it can result in excessive blood vessel growth, contributing to the

development of a disease.

Angiogenesis is essential for tumor development as it supplies oxygen and

nutrients to the cancer cells, and facilitates tumor dissemination to distant organs,

forming groups of cancer cells called metastases. Inhibiting angiogenesis has

emerged as an important strategy in preventing various solid tumors. By cutting off or

reducing the blood supply to tumor regions, angiogenesis inhibitors cause deficiency

in the amount of oxygen being received as well as cause death to tumor tissues,

effectively limiting tumor growth and progression. (National Cancer Institute)

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To combat cancer, multiple treatments have evolved such as Chemotherapy, Hromone

Therapy, Hyperthermia, Radiation Therapy, Stem Cell Transplant, Targeted Therapy,

and more. (National Cancer Institute). One of the approaches that has emerged is the

inhibition of angiogenesis. It is a type of targeted therapy that limits the supply of

blood that the tumor needs to grow and multiply. It works by limiting the formation of

new blood vessels around the tumor, which may involve blocking the attachment of

VEGF to the receptor, preventing the VEGF’s receptor from sending signals and

disrupting the signaling pathways between the cells. (Healthline, 2022)

Following this, using natural compounds as angiogenesis inhibitors has

emerged as some plants may contain bioactive compounds needed to disrupt the

angiogenic process. One of the plants Brassica oleracea (broccoli), is found to have a

huge amount of sulforaphane, a phytochemical that has been said to reduce the risks

of prostate, breast, colon, and oral cancers. Although all cruciferous vegetables

contain anti-cancer properties, broccoli is said to have the most significant amount of

sulforaphane, enhancing the body’s protective enzymes which eliminate

cancer-causing chemicals. (Jed, Fahey, ScD.) Meanwhile, Averrhoa carambola also

known as balimbing is a rich source of natural antioxidant, anti-inflammatory,

anti-infective, and antitumor effects. (Carolino et al., 2005) Flavonoids,

benzoquinone, and glycosides are also found in balimbing, which are known to be

responsible for numerous biological activities. (Kasun, Lakmal, et al.) Both are

promising candidates, thus the study aims to compare the two plants to evaluate their

potential for inhibiting angiogenesis.

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B. Aims of the Study

This study seeks to evaluate the potential of Averrhoa carambola (Balimbing)

and Brassica oleracea (Broccoli) extracts as angiogenesis inhibitors using the in ovo

Duck Chorioallantoic Membrane (CAM) assay. Specifically, it aims to answer the

following questions:

1. Which Balimbing and Broccoli extract concentrations have the most

significant inhibitory effect on blood vessel formation in the duck CAM

assay?

Set-Up A: 0.1 ml Balimbing, 0.1 ml Broccoli

Set-Up B: 0.2 ml Balimbing, 0.2 ml Broccoli

Set-Up C: 0.3ml Balimbing, 0.3 ml Broccoli

Set-Up D: No Balimbing, No Broccoli

2. Is there a significant difference in the average number of blood vessel

junctions seen before and after the treatments?

C. Significance of the Study

This study examines the potential of natural plant extractives to block

angiogenesis, a significant process in cancer and other disease development.

Employing the CAM assay, a standard approach for studying blood vessel formation,

this study aims to validate the anti-angiogenic activities of these two easily accessible

plants. This study may promote a sustainable and lower-cost substitute from synthetic

drugs, contribute to cancer treatment and possibly widen their availability for patients

as well as broaden the knowledge of plant-derived compounds for clinical research.

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D. Delimitation of the Study

This study examines the angiogenesis inhibitory effects of Brassica oleracea

(Broccoli) extracts using the in ovo Duck Chorioallantoic Membrane (CAM) assay. It

aims to identify the most effective concentration for inhibiting blood vessel formation

and compare these effects with a known angiogenesis inhibitor. The scope is limited

to the duck CAM assay within a controlled embryonic environment and does not

explore other models or long-term effects.

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 REVIEW OF RELATED LITERATURE

The Philippines is home to a wide variety of plant resources, but some

scientists feel that these resources are not being completely used in the quest for ways

to disrupt different stages of angiogenesis that are linked to diseases like cancer

(Olarte, 2007). It is well known that a wide range of secondary metabolites with

antimicrobial qualities are incredibly abundant in plants. There are several

anti-microbial medicines made from conventionally used medicinal plants that can be

utilized to treat a wide range of illnesses brought on by microbes. Alkaloids,

flavonoids, tannins, polyphenols, essential oils, phenolics, and polypeptides are some

of the classes of anti-microbial phytochemicals that fall into these categories (Samy

and Gopalakrishnakone, 2010). The compounds that plants contain can be used to

treat or alleviate certain illnesses, which is why they are employed in medicine

(Salmerón-Manzano et al., 2020). Using bioactive substances to block angiogenesis is

one potential use for treating different types of cancer. Angiogenesis plays a crucial

role in the human body from infancy until the onset of serious illnesses including

cancer, heart attacks, and stroke (Karagozar et al., 2020).

In addition to being useful in treating various cancers that arise in various

body areas, broccoli also plays a significant and positive function in the treatment of

other illnesses. Uncontrolled cell division is the end consequence of a multi-step

process called cancer. The primary active component in many cruciferous vegetables,

isothiocyanates, inhibit the development of tumors by either producing reactive

oxygen species or by causing cycle arrest that results in cell apoptosis. Many

isothiocyanates (ITCs), which are well-known types of cancer chemopreventive

agents, are abundant in broccoli sprouts (Ravikumar et al., 2015).

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 The extra-embryonic membrane known as the chick or duck embryo

chorioallantoic membrane (CAM) model is intentionally employed in vivo to

investigate angiogenesis, anti-angiogenesis, or both. In the circumstance of increasing

vessel density surrounding the implant, an angiogenic feedback or reaction takes place

72–96 hours (hr) after stimulation, with the blood vessels radially converging into the

center like spokes in a wheel. On the other hand, when an angiostatic substance is

administered, the vessels on the implant where it is placed become less dense or even

vanish. Several CAM models allow for the quantification of vessels, which may be

used to screen medications derived from organic substances such as plant extracts.

The increasing interest in antiangiogenic therapy for tumors and cancer requires the

advancement of a quantitative angiogenesis assay (Gamallo et al., 2016).

In a study by Magdadaro, Kirstin, et al (2023), the Cananga odorata, from the

Annonaceae or custard apple family, displayed inhibitory activity. Different

concentrations of the plant’s leaves and flower extracts were used to determine the

angiogenic activity on the Chorioallantoic Membrane (CAM) in 12-day-old duck

embryos. The fresh leaves and flowers were collected, dried, and powdered. The

powder was then soaked in 95% ethanol for 48 hours. After incubating with the plant

extracts, the treated eggs were removed, and blood vessel formation was counted and

documented. To figure out the significant changes between the set-ups at p < 0.05, the

Tukey and two-way ANOVA test was used in the statistical analysis. To concentrate

the three different treatments, 100, 200, and 300 ppm, a rotary evaporator was used.

The study's results show that the set-up with 200 ppm and 300 ppm displayed an

anti-angiogenic effect, reducing blood vessel formation .in the CAM of 12-day-old

duck embryos. The two treatments were not quite different from one another.

Furthermore, there was little difference between the leaf and flower extracts at 100

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ppm, because the extracts had lower concentrations of bioactive chemicals. Alkaloids,

flavonoids, glycosides, phenols, saponins, and tannins were found in the set-up that

contained 300 ppm. These are known for their potential to hinder angiogenesis,

causing the strongest inhibitory effect.

A study conducted by Salas, G.M and Totaan, E.V (2015), uses different

Philippine Herbal plant extracts as angiogenesis inhibitors using the Chick

Chorioallantoic Membrane (CAM) assay. The chosen herbal plant extracts were

Carmona retusa, also known as Tsaang-gubat, Pandanus odoratissimus, also known as

Pandan, and Gynura nepalensis, also known as Sabungai. It was discovered that these

plants contain flavonoids, tannins, and alkaloids. Only Pandan contains steroids and

anthraquinones. The three herbal plant extracts caused a different mean number of

branching points than the negative control. The significant difference that the

sabungai extract displayed from the positive control point shows its possible ability to

hinder angiogenesis. However, no difference was found between pandan and

tsaang-gubat when compared to the positive control, demonstrating a similar effect to

the standard treatment. When comparing the three extracts, there was a difference in

the mean number of branching points, showing that each extract had different levels

of inhibiting angiogenesis. Based on the CAM vascularity formula, tsaang-gubat

displayed the highest inhibition, followed by pandan, and then sabungai. These

findings suggest that all three extracts have the ability to hinder angiogenesis, but

further studies are needed to confirm their effectiveness and possible medical

applications, specifically in diseases like cancer where angiogenesis plays a big role.

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 Angiogenesis is the process through which new blood vessels are created from

existing small blood vessels. This important biological process starts when endothelial

cells, which are the specialized cells that line the inside of blood vessels, become

activated. When these endothelial cells are activated, they trigger several changes in

the surrounding environment. One of the first changes is the widening of the vascular

basement membrane, which provides structural support to blood vessels. This

widening is accompanied by an increase in the permeability, or openness, of the

surrounding tissue. The increased permeability allows important molecules to move

more freely through the tissue, which is essential for the formation of new blood

vessels. This initial phase of angiogenesis is crucial because it sets the stage for new

blood vessels to grow and supply necessary nutrients and oxygen to tissues (Mahfam

Moradi & Alireza Foroumadi, 2023).

The regulation of angiogenesis is a complex process that requires a delicate

balance between factors that encourage blood vessel growth, known as proangiogenic

factors, and those that inhibit it, called antiangiogenic factors. Proangiogenic factors

are substances that promote the growth and formation of blood vessels. Some of the

key proangiogenic factors include several types of growth factors. For example,

Vascular Endothelial Growth Factor (VEGF) is one of the most well-known growth

factors that stimulate the growth of new blood vessels. Other important proangiogenic

factors include Fibroblast Growth Factor (FGF), Platelet-Derived Growth Factor

(PDGF), Hepatocyte Growth Factor (HGF), and Epidermal Growth Factor (EGF).

These growth factors signal cells to grow and divide, which is essential for forming

new blood vessels.

In addition to these growth factors, other supportive components also play a

role in angiogenesis. Adhesion proteins are important because they help cells stick

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together and form stable structures. There are also enzymes that break down proteins,

making it easier for cells to move and adapt to new environments. Additionally,

proteins from the extracellular matrix provide support and structure to tissues, helping

to organize the environment around the blood vessels. Transcription factors are also

involved, as they help turn on specific genes that are necessary for cell growth and

division. Finally, various signaling molecules facilitate communication between cells,

allowing them to work together effectively to create new blood vessels (Mahfam

Moradi & Alireza Foroumadi, 2023).

On the other side of this balance, antiangiogenic factors, such as

Thrombospondin (TSP), angiostatin, and endostatin, work to prevent the growth of

unnecessary blood vessels. These factors are essential for maintaining healthy tissue

and preventing the formation of too many blood vessels, which can lead to problems.

In healthy individuals, there is a balance between proangiogenic and antiangiogenic

factors, which helps to control blood vessel growth appropriately. This balance

ensures that blood vessels form only when they are needed, such as during wound

healing or in response to increased tissue demand for oxygen and nutrients.

However, this balance can be disrupted in certain diseases or abnormal

conditions. For instance, in cancer and various chronic illnesses, there may be an

increase in the factors that promote angiogenesis. This increase leads to excessive

blood vessel formation, which can contribute to tumor growth and the development of

chronic conditions. For example, excessive angiogenesis is associated with eye

diseases known as retinopathies, inflammatory joint diseases like arthritis, conditions

affecting the uterus such as endometriosis, and the hardening of arteries

(atherosclerosis). In these conditions, the uncontrolled growth of blood vessels can

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lead to severe health problems and complications (Mahfam Moradi & Alireza

Foroumadi, 2023).

The link between angiogenesis and tumors was first pointed out by the

researcher Judah Folkman in 1971. Folkman described tumors as “hot and bloody,”

indicating that they have a rich supply of blood vessels that support their growth and

survival. This discovery was significant because it highlighted the dependency of

tumors on angiogenesis for their development. Tumors require a constant supply of

nutrients and oxygen, which they obtain through the formation of new blood vessels.

Therefore, understanding how angiogenesis works and the factors that regulate it is

crucial for developing new treatment strategies. These strategies aim to control

abnormal blood vessel growth, particularly in the context of cancer therapy.

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