MAKING AN ADHESIVE PLASTER FROM POMEGRANATE SHELL EXTRACT FOR DIABETES MELLITUS (part3)
MATERİALS AND METHODS
3.1. Materials
3.1.1. Punica Granatum
Pomegranate shells which are used in this study are waste of pomegranate processing, these has been brought from Turkey's Osmaniye province.
3.1.2. Chemicals
Ethanol which is used in the experiment has been provided from Merck. Tetracycline antibiotic has been provided from the Inönü University Molecular Biology and Genetics Department laboratory and the gallic acid standard, orthophosphoric acid and acetonitrile has been provided from the Inönü University Chemistry Department.
3.1.3. Microorganisms source
Escherichia coli, Bacillus subtilis and Candida albicans microorganisms were obtained from the Inönü University Molecular Biology and Genetics Department Laboratory culture collection in order to determine the antibacterial effect of pomegranate shell extract.
3.1.4. Cell source
Mouse (Mus musculus) fibroblast cell (L-929) was obtained from the Inönü University Molecular Biology and Genetics Department Laboratory culture collection in order to determine the epithelialization effects and biocompatibility of adhesive plaster.
3.1.5. HPLC System
The Agilent 1100 series HPLC system located in the Scientific and Technological Research Center of Inönü University has been used.
3.1.6. Electrospinning System
The Electrospinning system located in the Scientific and Technological Research Center of INÖNÜ University has been used.
3.1.7. Spectrophotometer System
The Spectrophotometer system located in the Scientific and Technological Research Center of INÖNÜ University has been used.
3.2. Methods
3.2.1. Preparation of Pomegranate Shells
The pomegranate shells, which were dried and separated from the pomegranate pulp, received dark brown color from the normal red form and has darker spots on it. Since all the pomegranates in our hands were the same species, they were all similar in structure. The shells were shrunk because they were dried also their hardness were enough to broken by hand because of some water in the shells. First, the weights of pomegranate shells has been measured as 1000.93 g with the help of sensitive weighing.
Figure 3. 1. Pomegranate shells before grinding
Then ın order to purify the shells completely from water the pomegranate shells have been kept in the oven at 80 ℃ for 24 hours. After the oven, the size of pomegranate shells has been reduced with the help of pestle and grinded with Retsch brand PM100 grinder at 5 rpm 350 rpm. The ground pomegranate shells has been stored at +4 degrees until use.
Figure 3. 2. Ground pomegranate shells
3.1.1. Extraction of Phenolic Compounds from Pomegranate Shells
At this stage of the experiment, an experiment setup has been created based on the Soxhlet extraction method.
Figure 3. 3. Pomegranate shell extraction process
In this experiment, 35% ethanol has been used as solvent. With the help of a filter paper, the desired extract has been obtained after 24 hours of processing.
3.1.1. Gallic Acid Characterization with HPLC
HPLC has been performed using a reverse phase column through 2-phase gradient system.
The experiment has been carried out as the conditions which were 5% B at the beginning, reached 60% B within 26 minutes. Analysis conditions of the experiment has been identified as; column temperature is 30℃, mobile phase flow rate is 1.2 mL / min and injection volume is 10 μl. After the procedure, observation has been made in 280 nm wavelength with the gallic acid standard which we have.
3.1.1. Investigation of Antibacterial Effect with Disc Diffusion Method
Firstly, Escherichia coli, Bacillus subtilis and Candida albicans microorganisms were reproduced in Luria Bertani (LB) liquid medium overnight at 37 ° C. The solid medium containing LB / Agar has been prepare then autoclaved and poured 20 ml into each petri dish under aseptic conditions and has been wait to solidify. After solidification, 100 μl of each microorganism type has been spread in a petri dish and experiment has been repeat 3 times. Then, 10, 50 and 100 μl pomegranate shell extracts were added to filter papers with a diameter of 6 mm, from pomegranate shell extract dissolved in ethanol with a density of 10 mg / ml, completely dried discs has been become ready to be put into the petri dish. For the positive control, 10 μl of 10 mg / ml tetracycline antibiotic, which was dissolved in water, was dropped on filter paper with a diameter of 6 mm and allowed to dry. Each side of each disc was sterilized under UV light for 30 minutes. Then, the negative control, which is the empty disc (center), the positive control disc with tetracycline (above), the disc containing 1 mg pomegranate extract (right), the disc containing 0.5 mg pomegranate extract (bottom), and the disc containing 0.1 mg pomegranate extract (left) has been put to petri dish under aseptic conditions. Petri dishes has been kept in an incubator at 37 ° C overnight, and the next day, the zone diameters of the region, which did not contain bacteria or fungi (the region where the microorganism died), has been measured in the area where the disc was located.
3.1.2. Making hydrogels into adhesive plaster by spinning method
P(HEMA) – ellagic acid hydrogels are formed into fibers by wet spinning used as a adhesive plaster. Wet spinning requires dissolution in a polymer solvent. The fibers extruded directly into a liquid bath. Wet spinning uses heat to wet the polymer to a viscolity suitable for extrusion through the spinneret. The fibers must be drawn or stretched to orient the polymer to strengthen the fiber. Exruded speed is very important, if done quickly it can create weak spots and micro voids.
3.1.3. Observation of Epithelization Effects and Biocompatibility in Cell Culture Lab
MTT method is one of the widely used enzymatic test methods to evaluate cytotoxic effects. This method is based on the principle that the MTT dye can break up the tetrazolium ring. In this method, MTT is actively absorbed into living cells and the reaction is catalyzed by mitochondrial succinate dehydrogenase and reduced to blue-purple, water-insoluble formazan. Formazan formation occurs only in living cells with active mitochondria. This is accepted as a marker of cell viability and the spectorophotometrically determined value is correlated with the number of viable cells.
