THE ANGIOGENIC PROPERTY OF COLLAGEN FROM promoting blood vessel formation to restore the

EGGSHELL MEMBRANE OF CHICKEN USING EX-OVO

CHORIO-ALLANTOIC normal circulation of blood in the arteries.
MEMBRANE ASSAY OF DUCK EGGS (8 secs)
Collagen is a fibrous protein which is dominant in
INTRODUCTION (2:50 mins) the connective tissue of animals. It has a wide
range of application in the food, pharmaceutical
and cosmetic industries. The primary sources of
Ischemic heart disease, also called coronary
collagen are bovine and swine skins and bones.
artery disease, is a disease characterized by
However, extraction of collagen from these sources
reduced blood supply to the heart. It develops
was restricted after the outbreaks of mad cow
when cholesterol particles in the blood begin to
disease and foot and mouth infection. Safe
accumulate on the walls of the arteries that supply
alternative source of collagen have to be explored.
blood to the heart. Eventually, deposits called
And according to Long, et al. (2004), collagen
plaque may form, which narrows the arteries and
extracted from eggshell membrane of chicken is a
eventually blocks the flow of the blood. This
safe alternative because it has low autoimmune
decrease in blood reduces the amount of oxygen
and allergic reaction and high in biosafety. Collagen
supplied to the heart causing chest pain or heart
also plays a vital role in giving structural support
attack.
and functions on the, growth of tissues and
regeneration.
According to World Health Organization, ischemic
heart disease and stroke are the world’s biggest
Eggshell is an abundant waste that can be collected
killers, accounting for a combined 17.9 million
aplenty anywhere. It is a good source of collagen.
deaths in 2016.
According to A.M. King Ori (2011), “presence of
hydroxyproline in hydrolysates of membrane layers
Treatment for ischemic heart disease involves
of eggshell suggests the presence of collagen”.
lifestyle changes and, if necessary, drugs and
certain medical procedures. In present time,
This study utilized eggshell membrane as a source
cholesterol-modifying medications, beta- blockers,
of collagen to at least lessen the waste associated
calcium channel blockers, ACE inhibitors and
with eggshells wisely.
angiotensin II receptor blockers are group of drugs
used to treat ischemic heart disease. However,
This study determined the angiogenic property of
these agents can cause serious side effects such
collagen from eggshell membrane of chicken using
as angioedema, rebound hypertension, myopathy
ex-ovo chorioallantoic membrane assay of duck
and many more. Procedures to improve blood flow
eggs.
such as angioplasty, stent placement and coronary
bypass surgery may also be performed. Heparin
also induces proliferation of blood vessels and has OBJECTIVES (15 secs)
a mechanism directly to the heparin binding
sequence. 1. To extract collagen from eggshell membrane
of chicken.
Angiogenesis is the process wherein new blood 2. To perform different tests for the
vessels form and grow on pre-existing vasculature, identification of collagen and its properties.
allowing the delivery of oxygen and nutrients to the 3. To determine the angiogenic property of
body’s tissues. According to Zhang et al. (2013), collagen from the eggshell membrane of
therapeutic angiogenesis is said to be effective in chicken and compare it to the standard drug,
treating or preventing ischemic heart disease by Heparin.
 METHODOLOGY (2:20) The temperature of the autoclave was adjusted to
37.5C.
COLLECTION, SEPARATION, PRE-
TREATMENT, EXTRACTION
On the third day, all of the contents of the eggs were
transferred in the sterilized plastic container.
The eggshells were collected at Rizal Poultry and
Livestock, Inc. Then, washed with distilled water to
The eggs were further incubated until the chorio-
remove the dirt and particle size was reduced using
allantoic membrane is fully developed.
a blender. 5% EDTA was used to separate the
membrane from the shell. Then, eggshell
On day 8 of incubation, under sterile conditions
membranes were subjected to a pre- treatment
within a laminar flow hood, a 1 millimeter sterilized
process using 10% NaCl to remove non-
filter paper disks were soaked with used substance
collagenous proteins. Collagen was extracted from
individually and dried before implanting on the
the eggshell membranes obtained from the pre-
CAM.
treatment process using 0.5M acetic acid.
yielding a final concentration of: 3milligram per
PERCENTAGE YIELD
milliliter, 5milligram per milliliter 10milligram per
milliliter, 10 milligram of Heparin in 1milliliter, 1
The percentage yield was computed using the
milliliter of Normal Saline Solution
formula weight of the collagen after extraction
divided by weight of eggshell membrane before
The purpose was to make sure the used substance
extraction multiplied by 100
was concentrated and contained only on the
chosen area of interest.
PHYSICAL AND CHEMICAL EVALUATION
After locating an area containing a fine vessel
For the physical evaluation of the extracted
networks between large blood vessels. The disk
collagen, organoleptic evaluation was performed.
was lifted individually with fine end forceps and
For the chemical evaluation, tests for proteins were
implanted onto the CAM.
performed by dissolving 1milligram of extract to
0.45 molar of Sodium Hydroxide.
Samples were incubated for another 48 hours.
MORPHOLOGICAL EVALUATION
After 48 hours, the samples were removed from
the incubator and mounted on the
Scanning electron microscopy coupled with energy
stereomicroscope for evaluation.
dispersive x-ray spectroscopy was performed in the
morphological analysis of the extracted collagen.
QUANTIFICATION (15 secs)
INSTRUMENTAL EVALUATION
Quantification of angiogenesis was accomplished
by counting the blood vessels near and beneath
Fourier transform infrared spectroscopy, High
the filter paper disc applied.
performance liquid chromatography, and
Differential scanning calorimetry were performed.
Only blood vessels that branched out near and
beneath the confined region of the filter paper disc
CAM ASSAY- Native duck eggs locally known as
were tallied. Pre-existing large-vessels were not
“balut” were bought from a poultry in Pateros. The
counted. Only newly formed thin branches were
eggs were incubated for 72 hours.
counted.
SUMMARY OF FINDINGS (2:30
secs)
PERCENTAGE YIELD
The percentage yield of the extracted collagen is
14.48%
PHYSICAL EVALUATION
The extracted collagen is a white powder with a
fine texture and an acrid odor.
CHEMICAL EVALUATION
The extracted collagen produced a positive result in
the tests: Ninhydrin Test, Biuret Test and
Xanthoproteic Test. Negative results were seen on
other protein tests because according to Amino
Acids Sequence Analysis On Collagen, Sorana et.
al. 2007, Collagen usually does not contain amino
acids such as Tryptophan, Cysteine and Tyrosine
Therefore, the extracted collagen is characterized
only by chemical groups essential for collagen.
MORPHOLOGICAL EVALUATION
The scanning electron microscopic images showed
that both collagen scaffolds of Rat tail collagen and
extracted collagen spontaneously have macro
porous frameworks. Therefore, the images proved
the similarity of the two in microscopic level.
INSTRUMENTAL EVALUATION
FOURIER TRANSFORM INFRARED
SPECTROSCOPY
Fourier transform infrared spectrum of eggshell
membrane collagen showed that the main
absorption bands are AMIDE A (stop,direct),
AMIDE B (stop,direct), AMIDE I (stop,direct),
AMIDE II(stop,direct), and AMIDE III (stop,direct).
The absorption bands are based on Yu-Hong et. al
‘Characterization of Collagen’ 2008
Therefore, The fourier transform infrared spectrum
of eggshell membrane collagen proved the
presence of essential functional groups of collagen.
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
The confirmation of purity of the extracted collagen
was established by using retention time
comparison with the standard, type I collagen.
The detector showed 1.829 and 1.803 retention
time for the standard collagen and extracted
collagen, respectively.
Therefore, The consistency of the retention time at
concentration of 0.5 gram per liter proved the
existence of collagen and its properties in the
extracted collagen.
DIFFERENTIAL SCANNING CALORIMETRY
Differential Scanning Calorimetry thermogram
showed that thermal denaturation is 106.85°C
indicating that there is a high stability of the
structure maintained by the hydrogen bond
Thermogram indicates that the eggshell membrane
collagen retained its intermolecular cross links after
the extraction process.
Therefore, The eggshell membrane collagen may
serve as an attractive alternative to mammalian
collagen for biomedical and pharmaceutical
applications owing its greater thermal denaturation
than other mammalian collagen available in the
market.
CAM ASSAY
1: Chorio-allantoic membrane assay was used in
determining the angiogenic property of the eggshell
membrane collagen, and these are the number of 6. To conduct a toxicity test on different doses to
collaterals counted in each controls. be administered to the eggs.

2: To further visualize the differences per control,

these are their representatives who have the
greatest number of collaterals.

3: 3 milligrams per milliliter and 10 milligrams per

milliliter showed no significant difference with
Heparin while 5milligram per milliliter showed the
most significant difference compared with Heparin.

CONCLUSION (22 secs)

The characteristics of collagen extracted from
eggshell membrane were determined and proved
to be comparable with the standard collagen.

There is a significant difference in the angiogenic

activity of eggshell membrane collagen among the
three control group. At concentration of 5 milligram
per milliliter, eggshell membrane collagen exhibited
the most angiogenic activity greater than the
positive control, Heparin.

RECOMMENDATION (30 secs)

1. To perform the use of ultrasound which proved
to increase the yield and reduce the extraction
time without damaging the quality of collagen.

2. To further use nylon mesh and histogram

equalization of green channel image for easier
quantification of the collateral vessels.

3. To add more instrumental test to determine the

specific type of collagen such as SDS-PAGE

4. To find different source of collagen

5. To make a formulation of the eggshell

membrane collagen extract