Citrus fruit skin extract for angiogenesis promotion
An angiogenesis, extract technology, applied in the field of assisting wound healing, treatment such as coronary heart disease, stroke or ulcer need to promote angiogenesis diseases or conditions, grapefruit peel extract
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Embodiment 1
[0054] Embodiment 1: extraction step
[0055] Peel from citrus fruits was pulverized in an electric mixer and 5 volumes of 70% aqueous acetone (acetone / water 7:3 v / v) were added to the material. The mixture was stirred for 2 hours and then left at room temperature overnight. The resulting mixture was filtered on nylon cloth while squeezing the slurry. The solid residue was resuspended in aqueous solvent, stirred for 1 hour, then filtered. The filtrates were combined and acetone was removed by concentration under reduced pressure at 45°C. The remaining aqueous fraction was decanted for collection and processed on a column of non-ionic polystyrene resin. The column was washed with 3 volumes of distilled water, the adsorbed substance was eluted with methanol, and concentrated under reduced pressure. The remaining material was diluted with 1 volume of distilled water and lyophilized to obtain the active extract.
Embodiment 2
[0056] Example 2: Angiogenesis Assay
[0057] After removing fat and perivascular fibrous tissue, the rat aorta was cut into rings with a thickness of about 2 mm. A fibrin gel (0.4 ml) was prepared in one well of a 24-well culture plate by adding coagulation factors to a fibrinogen solution dissolved in MCDB 131 medium. Aortic rings were placed in the center of each hole and covered with another piece of fibrin. Each gel was overlaid with MCDB131 medium (1.5ml) and incubated at 37°C in 3% CO 2 Incubate in an atmosphere of 97% air. Samples to be tested were added to the medium as supplements. Each sample was tested 3 times.
[0058] After about 5 days, the growth of microvessels from the periphery of the ring was detectable. At regular intervals between 5 and 14 days, images of each well were recorded with a digital camera attached to an inverted microscope. The area of microvessel growth relative to the circumference of the ring was measured for each image at each time...
Embodiment 3
[0059] Example 3: Wound Healing
[0060] Two circular incision wounds were created in the back of each rat with a 0.8 cm skin biopsy punch just below the shoulder. One wound is made on each side of the midline. Two doses of the test material were analyzed. For each experiment, 5 male Lewis rats were used. Aliquots (25 μl) of grapefruit peel extract dissolved in saline were topically applied to the left wound of each animal every other day. For one group of rats the concentration was 10 mg / ml and for a second group of rats the concentration was 1 mg / ml. Vehicle was added to the contralateral wound as a control. Photographs of each pair of wounds were taken with a Canon EOS 3000 N camera (F2.8 microlens) and Fuji Professional 400NPH film before each addition. Photographs of each exposure were recorded digitally, and the area of each wound was calculated from these images using NIH Image 1.63 software. The results can be seen in Figures 1 and 2.
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