MAKING AN ADHESIVE PLASTER FROM POMEGRANATE SHELL EXTRACT FOR DIABETES MELLITUS (part4)
3. RESULT
3.1. Results of Extraction Process
For the determination of phenolic substance, the extract should be in the most viscous state in the alcohol or hexane phase and should not contain water. A dense consitency extract has been obtained from the pomegranate shells that has been ground to be sent the phenolic sustance characterization stage.
Figure 4.1. Dense pomegranate shell extract obtained by Soxhlet extraction method
3.1. Results of Characterization of Phenolic Compounds
The gallic acid peak value which is obtained in pomegranate extract with HPLC has been compatible with the gallic acid standard we have.
Figure 4. 2. Gallic acid peak value obtained by HPLC
3.1. Results of Antibacterial Effect Determination
Figure 4. 3. Disc diffusion method Escherichia coli effect determination
100 μg Tetracycline Average Zone Diameter = 42.44 ± 4.23 mm
100 μg Extract Average Zone Diameter = 10.66 ± 1.15 mm
500 μg Extract Average Zone Diameter = 18 ± 0 mm
1000 μg Extract Average Zone Diameter = 20 ± 0 mm
Figure 4. 4. Disc diffusion method Bacillus subtilis effect determination
100 μg Tetracycline Average Zone Diameter = 19.66 ± 1.52 mm
100 μg Extract Average Zone Diameter = 6.66 ± 0.57 mm
500 μg Extract Average Zone Diameter = 9.99 ± 1.33 mm
1000 μg Extract Average Zone Diameter = 12.55 ± 2.50 mm
Figure 4. 5. Disc diffusion method Candida albicans effect determination
When the zone diameters has been examined which obtained at the end of the experiment it has been concluded that the antibacterial property increased while the amount of pomegranate shell extract increased, and antifungal effect increase when high dose has been used. It has been understood because of the pomegranate shell extract obtained had a smaller zone diameter, which showed less antimicrobial effects against gram-positive Bacillus subtilis bacteria compared to gra1m-negative Escherichia coli bacteria.
3.1. Result of Spinning making adhesive plaster
In the electo-shooting process, an automatic pump is placed at the back of syringe to control the flow of our polymer solution, mainly containing the delicate extract. It is provided to provide the polymer solution for this autamic sanitary component water. Reducing high pressure to the polymer solution will affect the droplet as the voltage is increased. With the increase of these electrical forces, the drops elongated and took the form of a cone called “Taylor Cone” at the tip of the needle. During the movement toward the grounded collector in the form of the polymer solution jet, which is when the electric field forces reaching the critical value beat the viscoelastic and surface tension forces in the solution. Then, the solvent in the solution was evaporated and the polymer jet solidified to obtain a reticulated layer of fibers with nano-sized diameters on the collecting surface.
3.2. Result of Epithelization Effects and Biocompability
Cytotoxicity of pomegranate sample was determined uzing spectrophotometer device with indirect MTT (3-(4,5-dimethylthiazol-2-YL)-2,5-diphenyltetrazolium bromide) test. The experimental protocol was prepared according to ISO-10993-5 “Biological Evalulation of Medical Devices” dtandards. Mouse (Mus musculus) fibloblast cells (L-929) were used in the study. First, after the sample was washed three times with sterile PBS (Phosphate Buffered Saline, pH 7.4), both sides of the samples were UV sterilized, and then the sample was incubated for 72 hours at 37°C in a DMEM (Dulbecco's Modified Eagle Medium) environment using an incubator containing 5% CO2. For the experiment, the L-929 cell line was pre-enlarged at 37°C in a DMEM environment containing 5% CO2. After the cells reached 80% confluence, 0.25% trypsin-EDTA solution was used to separate the cells from the flask. Then, the cells were centrifuged at 2000 RPM for 5 minutes and planted into 96 well plates, with 104 cells in each well, and incubated for 24 hours under the same conditions. At the end of the incubation period, the media in which the samples were kept for 3 days was replaced with fresh DMEM and an additional 24 hours incubation period was applied for the samples. Then, 90 µL of fresh DMEM media was added to each well of the 96 well plates by removing the medium from the cells. 10 µL of 5 mg/mL MTT solution prepared with sterile PBS was added to Wells and incubated in darkness for 4 hours under the same conditions. Subsequently, the absorbance value of the purple color formed as a result of incubation was measured using the ELISA microplate reader at 550 nm. The medium kept in the incubator for 72 hours was added to the wells of the control groups incubated under the same conditions, and the cells in these wells were considered 100% alive. As a result, cell viability on L-929 cells was calculated compared to the control groups of the pomegranate sample.
Figure 4.6. in vitro Biocompatibility Analysis
3.1. Result
In antibacterial and antifungal studies, it was concluded that the antibacterial property increases as the amount of pomegranate bark extract increases, and that it has an antifungal effect when using a high dose. It was understood that the resulting pomegranate bark extract had less antimicrobial effects against the gram-positive Bacillus subtilis bacterium compared to the gram-negative Escherichia coli bacterium, forming a smaller zone diameter. According to the in vitro biocompatibility results of the hydrogel adhesive plaster obtained by electrospinning method; the electrospin product produced by pomegranate extract increased cell proliferation, which confirms the high biocompatibility of the material. However, it has been concluded that a high number of repetitions of the material produced can be made in order to increase the accuracy of the results of the experiment. In light of these results, we believe that our biocompatible product with antibacterial and epithelialization rate increase properties is a domestic, easily accessible and effective source. According to our results; pomegranate peel extract was determined to be a good antibacterial agent that could be used in various fields, while the results of in vitro biocompatibility analyses were found to be promising for clinical trials.
Accordingly, the stage where obtained the highest phenolic substance from these experiments would be used as a procedure. In the light of previous studies, experiment number 3 was experiment to be the optimum conditions. However, according to the analysis result, the desired phenolic compound could not be obtained in any of these steps. For this reason, Soxhlet extraction method was applied at this stage of the experiment and the desired phenolic substances were successfully obtained.
According to the results of the disk diffusion mehod, pomegranate shell extract has a high antifungal and antibacterial effect. We think that this effect is caused by the high rate of gallic acid that in its content. According to this study, we think that pomegranate shell extract can be used as an antibacterial and antifungal agent, especially infected woulds or medical and biotecnological studies.
