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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
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
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.
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,
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,
Angiogenesis inhibitors tend to restrain the growth of blood vessels rather than tumor
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
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
Several studies suggest that winged bean (Psophocarpus tetragonolobus) has antimicrobial, anti-
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
root extract using chorioallantoic membrane assay in domestic duck (Anas platyrhynchos
domesticus).
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.
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.
Another study evaluated the angiosuppressive activity of Tinospora rumphii Boerl. stem
extract using the chorioallantoic membrane assay in Anas platyrhynchos embryos and had the
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
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,
domestic duck (Anas platyrhynchos domesticus). Specifically, this study aims to achieve the
following goals:
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.
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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
extract
1.) Ho = Winged bean (Psophocarpus tetragonolobus) ethanolic root extract will have no
platyrhynchos domesticus).
2.) Ha = Winged bean (Psophocarpus tetragonolobus) ethanolic root extract will have an
platyrhynchos domesticus).
extract will have the same effect on chorioallantoic membrane in Domestic Duck (Anas
platyrhynchos domesticus).
extract will have different effects on chorioallantoic membrane in Domestic Duck (Anas
platyrhynchos domesticus).
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
normalized the growth of blood vessels. Winged Bean (Psophocarpus tetragonolobus) will be
collected at the
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
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
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
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
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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
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
Data Analysis
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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
Where,
F = Anova Coefficient
Scheffe Test
Running head: ANGIOSUPPRESSIVE ACTIVITY OF WINGED BEAN 11
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