Research Procedure

Gathering of Materials
The Eggplant Peels were gathered by the researchers in various locations in Ormoc City. The
materials were placed in separate containers to ensure an organized and quick preparation for the
Eggplant peels extraction. The researchers cultivated duck eggs for its Chick Chorioallantoic
Membrane Assay to know if the Extract is effective for anti-angiogenesis.
Preparation of Set-ups
The researchers gathered blender, a 1ml of water, a knife, a beaker and a strainer for the
apparatuses in producing the extract of the peel. They also gathered 3 duck eggs, an incubator, a
0.1% Benzalkonium Bromide, a cutter, a dropper, a sterile flexible packing film and a glass slide
to perform Chick Chorioallantoic Membrane (CAM) assay process. For applying the extract on
the incubated eggs, the researchers gathered the incubated eggs, forceps, sterilized filter-paper
disc with the extract of the peelings, normal saline solution, methanol, and acetone.
PROCESS FOR THE EGGPLANT PEELS EXTRACTION (AQUEOUS EXTRACT
METHOD)
Procedure 1. For the Eggplant Peels Extract, the researchers will get fresh healthy eggplant.
Procedure 2. The researchers washed the eggplant thoroughly with running tap water and peel
the eggplant.
Procedure 3. After washing the peels, the researchers weigh 10 grams of eggplant peels and
transferred them into the blender.
Procedure 4. The researchers add 1ml of water in the blender and blend it.
Procedure 5. The researchers mix the pulp well and after mixing, the researchers pour the extract
into the beaker with a strainer.
Procedure 6. And then, the researchers squeeze the remaining peels into the other beaker by
hand. (do not use a cotton sieve to extract).
Procedure 7. Lastly, fill the sterilized filter-paper disc with the extract in the petri dish.
THE PROCESS OF MONITORING EGGPLANT PEELS EXTRACT AS AN ANTI-
ANGIOGENESIS (CHICK CHORIOALLANTOIC MEMBRANE (CAM) ASSAY)
Procedure 1. For monitoring eggplant peels extract as an anti-angiogenesis, the researchers
undertake the CAM assay process.
Procedure 2. The researchers incubated the 3 duck eggs for 3 to 6 days.
Procedure 3. After incubating, disinfect the shell center outside the air sac with 0.1%
Benzalkonium Bromide.
Procedure 4. Buff the marker pen into the shell to highlight a hole in 1 x 1 cm window on the
membrane was sectioned to expose the vascular zone and drill gently over the air sac with a
cutter not to break the shell and the vascular zone was easy to be identified on the CAM.
Procedure 5. Add 2 drops of normal saline to moisten the inner shell membrane near to the CAM
so that the membrane was easy to be separated from CAM.
Procedure 6. And then grasp and raise the membrane by using forceps so that the membrane and
the CAM will be separated unforcedly.
Procedure 7. A 5 mm x 5 mm sterilized filter-paper disks which were used as a carrier for being
directly filled with indicated extract, directly applied and attach to the vascular zone with the
right density of vascular.
Procedure 8. Seal the openings with sterile flexible packing film and incubate the eggs for 2 to 3
days.
Procedure 9. After incubating the eggs, mix methanol and acetone (1:1 in volume) was directly
added to submerge and secure the blood vessels of the experiment zone.
Procedure 10. Grasp and raise the CAM using forceps so that the CAM will be easy to be
separated from the embryo, cut and spread it on the glass slide to view the blood vessels.
Procedure 11. Lastly, the researchers photographed and quantified by counting the number of
blood vessels branch points.