Running head: ANGIOSUPPRESSIVE ACTIVITY OF WINGED BEAN 1

Angiosuppressive Activity of Winged Bean (Psophocarpus tetragonolobus) Ethanolic Root Extract

using Chorioallantoic Membrane Assay in Domestic Duck (Anas platyrhynchos domesticus)

Siachua, April Heart A.

Pacita Complex National High School

Running head: ANGIOSUPPRESSIVE ACTIVITY OF WINGED BEAN 2

Angiosuppressive Activity of Winged Bean (Psophocarpus tetragonolobus) Ethanolic Root Extract

Using Chorioallantoic Membrane Assay in Domestic Duck (Anas platyrhynchos domesticus)

Angiogenesis, the formation of new blood vessels, affects the growth of endothelial cells

on the inside wall of blood vessels. It is controlled by chemical signals in the body like

angiogenesis inhibitors. These signals can stimulate both the repair of damaged blood vessels

and the formation of new ones. Normally, blood vessels form only when and where they are

needed as an effect of angiogenesis inhibitors. (National Cancer Institute, 2011)

A blood supply is needed for tumors to grow beyond a few millimeters and tumors give

off chemical signals to stimulate angiogenesis which causes the blood supply. Tumors can also

stimulate normal neighbor cells to produce such signals. The result, the new blood vessels supply

growing tumors with blood, filled with oxygen and nutrients. This allows the cancer cells to raid

nearby tissue and form new colonies of cancer cells.

Scientists are looking into ways to block tumor angiogenesis since tumors cannot spread

or grow beyond a specific size without blood supply. They are studying natural and synthetic

angiogenesis inhibitors or antiangiogenic agents, thinking that these molecules will prevent the

growth of cancer.

The binding of signaling molecules, such as Vascular Endothelial Growth Factor

(VEGF), to receptors of normal endothelial cells, is necessary to angiogenesis since signals

within the endothelial cells are initiated that support the growth and survival of the new blood

vessels when VEGF and other endothelial growth factors bind to the receptors of the endothelial

cells. Angiogenesis inhibitors intrude in the binding of signaling molecules and the receptors on

the surface of normal endothelial cells. For example, bevacizumab, an antibody that recognizes
Running head: ANGIOSUPPRESSIVE ACTIVITY OF WINGED BEAN 3

and binds to VEGF, makes the VEGF unable to activate the VEGF receptor. Other angiogenesis

inhibitors are sorefenib and sunitinib that when they bind to receptors on the endothelial cells,

blocks its activities such as binding to signaling molecules.

In fact, angiogenesis inhibitors are currently being used to treat cancer. Bevacizumab,

used alone for glioblastoma – which is a fast-growing type of central nervous system tumor, was

the first angiogenesis inhibitor that effectively slowed tumor growth and more important,

extended the lives of cancer patients. (National Cancer Institute, 2011)

Angiogenesis inhibitors tend to restrain the growth of blood vessels rather than tumor

cells, which differentiates angiogenesis inhibitors from conventional anticancer drugs.

Angiogenesis inhibitors, when combined with additional therapies like chemotherapy, are most

effective. It has been assumed that these drugs help regularize the blood vessels that supply the

tumor. Angiogenesis inhibitor therapy does not kill tumors but prevent them from growing and

spreading. Therefore, this therapy may need to be administered for a long period of time.

Since it has not been a long time doctors treated patients with angiogenesis

inhibitors, some possible complications of angiogenesis inhibitor remain unknown. (National

Cancer Institute, 2011)

Synthetic angiogenesis inhibitors have been put to test but only few studies have looked

into natural sources of these angiosuppressive agents. Knowing this, the researcher intend to look

into Winged Bean (Psophocarpus tetragonolobus) as a possible source of angiosuppressive agents.

Several studies suggest that winged bean (Psophocarpus tetragonolobus) has antimicrobial, anti-

inflammatory, anti-nociceptive, antioxidant, platelet aggregation inhibitory, hepatoprotective

properties. (Stuart, 2016) Winged Bean (Psophocarpus tetragonolobus) is also said to yield

betulinic acid, specifically on its roots. (Duke, 2007) Considering these facts, this study will explore
Running head: ANGIOSUPPRESSIVE ACTIVITY OF WINGED BEAN 4

the potential angiosuppressive activity of Winged Bean (Psophocarpus tetragonolobus) ethanolic

root extract using chorioallantoic membrane assay in domestic duck (Anas platyrhynchos

domesticus).

Background of the Study

Philippines has diverse plant resources yet only a few of these plants are fully utilized in

search for antiangionetic agents that could prevent the growth and spread of cancer cells. There are

about 10,000 to 11,000 plant species believed to occur in the Philippines and will be more if tropical

forests were fully explored. (Molina, 2012) Over half of this number are endemic. Only around 1,500

plants are used in treatment of various illnesses. If new plant-derived drugs are considered, our

country will improve economically and will be able to make use of other plants. Additionally,

discovery of new plant-derived drugs may give new solutions in the field of cancer therapy.

The plant, Winged Bean (Psophocarpus tetragonolobus) - belongs to a family,

Leguminosae. (Plants For A Future, n.d.) It is commonly known locslly as ‘Sigarilyas’. This

herbaceous perennial plant growing up to 3 to 4 meters. Its green leaves are trifoliate and each

leaflets measure 8 to 15 cm wide. The fruits are elongated and its rectangular pods is usually 15 to 30

cm long and 3 cm wide. Protruding along each corner of the pod are frilly or sometimes smooth

wings. When mature, pods break open and release 5 to 21 round, smooth seeds. The root tubers of

winged bean can be eaten raw or cooked. The size of tuber is affected by climate and it is a plant that

grows well in hot, humid environment and is usually sensitive to frost. The plant does not grow well

in soils with pH less than 5.5 but can tolerate various types of soils. (National Parks Board, 2013)

Several studies about using natural angiogenesis inhibitors have already been conducted.

Herrera, Raga and Ragasa (2013) conducted a research which aimed to explore the angiosuppressive

triterpenoids from Arsidia cf. elleptica (subgenus Tinus) on duck (Anas platyrynchos L.)
Running head: ANGIOSUPPRESSIVE ACTIVITY OF WINGED BEAN 5

chorioallantoic membrane. The research was found effective on different set-ups. Arsidia cf.

elleptica (subgenus Tinus) was also found to have betulinic acid.

Another study evaluated the angiosuppressive activity of Tinospora rumphii Boerl. stem

extract using the chorioallantoic membrane assay in Anas platyrhynchos embryos and had the

same positive result.

Dillenia philippinensis, was also used in a study that explored its angiosuppressive activity

using chorioallantoic membrane assay in Anas platyryncos Linn. embryos. The extract was

partitioned to different concentrations. Nevertheless, each concentration was found to be effective but

highest inhibition of blood vessels was observed at 100%.

Although great progress in the field of cancer treatment has been made, development of

natural antiangiogenic drug for cancer therapy remains difficult. Tumors are responsible for about

85% cancer mortality in humans but tumor growth are dependent on blood vessels. Therefore,

targeting tumor angiogenesis to prevent tumor growth is a possible therapeutic strategy.

Statement of the Problem

The researcher intends to determine the angiosuppressive activity of Winged bean

(Psophocarpus tetragonolobus) ethanolic root extract using chorioallantoic membrane assay in

domestic duck (Anas platyrhynchos domesticus). Specifically, this study aims to achieve the

following goals:

1. Explore the angiosuppressive activity of Winged Bean (Psophocarpus tetragonolobus)

ethanolic root extract; and

2. determine the best solution if the Winged Bean (Psophocarpus tetragonolobus) ethanolic

root extract was partitioned to different concentrations of 25%, 50%, 75% and 100% v/v.
Running head: ANGIOSUPPRESSIVE ACTIVITY OF WINGED BEAN 6

Hypotheses

The research work is anchored on the following null and alternative forms of hypotheses. The

research has two set of hypotheses because the researcher is attempting to answer the goals stated in

the statement of the problem.

SET A: Angiosuppressive Activity of Winged bean (Psophocarpus tetragonolobus) ethanolic root

extract

1.) Ho = Winged bean (Psophocarpus tetragonolobus) ethanolic root extract will have no

angiosuppressive effect on chorioallantoic membrane in Domestic Duck (Anas

platyrhynchos domesticus).

2.) Ha = Winged bean (Psophocarpus tetragonolobus) ethanolic root extract will have an

angiosuppressive effect on chorioallantoic membrane in Domestic Duck (Anas

platyrhynchos domesticus).

SET B: Different Concentrations of the Extract

1.) Ho = Varying concentrations of Winged Bean (Psophocarpus tetragonolobus) ethanolic root

extract will have the same effect on chorioallantoic membrane in Domestic Duck (Anas

platyrhynchos domesticus).

2.) Ha = Varying concentrations of Winged Bean (Psophocarpus tetragonolobus) ethanolic root

extract will have different effects on chorioallantoic membrane in Domestic Duck (Anas

platyrhynchos domesticus).

Significance of the Study

This study may benefit World Health Organization, since the findings may help in the

development of plant-derived cancer drug. Oncologists, especially medical oncologists, may benefit
Running head: ANGIOSUPPRESSIVE ACTIVITY OF WINGED BEAN 7

from this study because it may give them idea to develop natural angiosuppressive agent to be a

possible cure for cancer. It may also help cancer patients who are still struggling to fight for their

condition. Lastly, for the future researchers, this study can serve as their stepping stone if they decide

to investigate similar line of research.

Scope and Limitation

This study focuses on exploring the angiosuppressive activity of Winged Bean

(Psophocarpus tetragonolobus) by making an ethanolic pod extract out of it and testing it on

choriallantoic membrane assay in domestic duck (Anas platyrhynchos domesticus) to see if it

normalized the growth of blood vessels. Winged Bean (Psophocarpus tetragonolobus) will be

collected at the

Materials and Methods

This part describes the methods and procedures which will be used in the preparation of

the product and the experimentation to be conducted for verification purposes about the

angiosuppressive activity of Winged bean (Psophocarpus tetragonolobus).

Research Design

The study will use two-group research design as the setups for this study will use Winged

bean (Psophocarpus tetragonolobus) ethanolic root extract and retinoic acid as negative and positive

control, respectively and Winged bean (Psophocarpus tetragonolobus) ethanolic root extracts in

25%, 50%, 75% and 100% concentrations.

Running head: ANGIOSUPPRESSIVE ACTIVITY OF WINGED BEAN 8

Acquisition of Materials Statistical Analysis Formatted: Font color: Text 1

Formatted: Font color: Text 1
Formatted: Font color: Text 1

Extraction of dried roots Visual Assessment and Formatted: Font color: Text 1
using ethanol Taking Micrographs Formatted: Font color: Text 1
Formatted: Font color: Text 1

Preparation of extract in Formatted: Font color: Text 1

CAM assay Extract Administration
different concentrations Formatted: Font color: Text 1
Formatted: Font color: Text 1

Figure 1 shows the process flow of the methodology of this research

Fresh roots of Winged bean (Psophocarpus tetragonolobus) will be collected in and and

verified in the National Museum. The fresh roots will be rinsed with distilled water and chopped into

smaller pieces. It will be air-dried for four days under direct sunlight. Air-dried roots will be

grounded in a blender and soaked in ethanol for three days. The extracted material will be filtered

with whatman filter paper no. 1. The filtered material will be concentrated by evaporation with rotary

evaporator. The concentrated extract will be partitioned to different concentrations of 25%, 50%,

75% and 100% v/v and will serve as the experimental group. 90% v/v extract and retin A cream, as a

source of retinoic acid, will also be prepared as they will be used as positive and negative control,

respectively. The extracts will be refrigerated at 4°C until needed. Seven fertilized eggs of Anas
Running head: ANGIOSUPPRESSIVE ACTIVITY OF WINGED BEAN 9

platyrhynchos domesticus will be obtained from . At the Institute of Biology, University of the

Philippines, Los Banos, day 0 eggs will be incubated at 37°C with constant humidity. On the 5th

day, the eggs will be candled and inspected for position of embryo and egg viability. On the 7th day

the eggs will be wiped with 70% ethanol and a hole will be made at the end of the egg where the air

space is using a hand held rotary drill. 50μL of each sample will be added to an egg. On the 7th egg,

nothing will be added. The hole will be sealed using sterile parafilm and will be retransferred to the

incubator to continue further incubation for 14 days. Egg viability will be monitored every other day.

After 14 days, the egg will be cracked open, gently placing the embryo along with the yolk on a

petridish that contain saline water. The embryo will be imaged and the image will be transferred to a

computer for image analysis.

Risks and Safety

To minimize, if not, avoid risks and ensure safety during the procedure, the researcher

must be wearing gloves, face mask, goggles and lab coat for protection since caution is needed

when doing the experiment. Used materials should be stored in appropriate containers and kept

out of reach of people who do not know how to properly handle them.

Laboratory equipment are potential hazards when not used properly. The researcher must

be cautious when using them to avoid any harm and damage of property. The researcher must

also be supervised by an expert while performing the experiment to ensure the safety of the

student researcher if any problems arise. After the experiment, the equipment that will used must

be cleaned and stored properly. Disposable items (e.g. gloves, face mask, tissue papers) should

be put in a black bag and disposed properly.

Data Analysis
Running head: ANGIOSUPPRESSIVE ACTIVITY OF WINGED BEAN 10

Analysis of Variance (ANOVA) or F-test will be used to statistically determine the

significant difference among the different concentrations of the Winged bean (Psophocarpus

tetragonolobus) ethanolic root extract between samples in four set-ups and the positive and negative

control. If the null hypothesis is rejected, then Scheffe Test will be used to find which set-up has the

highest inhibition of blood vessels.

Analysis of Variance (ANOVA) or F-Test

Where,

F = Anova Coefficient

MST = Mean sum of squares due to treatment

MSE= Mean sum of squares due to error.

F is based on d.f.1 = (k-1) and d.f.2 = (n-k)

Scheffe Test
Running head: ANGIOSUPPRESSIVE ACTIVITY OF WINGED BEAN 11

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