MAKING AN ADHESIVE PLASTER FROM POMEGRANATE SHELL EXTRACT FOR DIABETES MELLITUS (part2)

1.    GENERAL INFORMATIONS

1.1.Structure of Pomegranate

Pomegranate which is from a family of Punicaceae is a type of red-coloured fruit consisting of hundreds of particles that make up small seeds and body. Pomegranate grows in temperate climates and can withstand temperatures down to -10 degrees. It consists of different parts such as shell, fruit, seed and water and each of them has different phytochemical properties. Approximately 50% of the total weight of the pomegranate is the shell and the other 50% is the fruit. 80% of the fruit portion consists of water and 20% consists of seeds.

Figure 2.1.  Structure of Punica granatum (pomegranate)

1.1.1.    Chemical Composition of Pomegranate

The pomegranate contains about 10% glucose and fructose, as well as complex sugars that have not yet been identified. Also, the polyphenol content varies between 0. 2-1. 0 % (Navindra 2006). It is known while the shell and membrane parts of the pomegranate contain the highest concentration of polyphenol, that the seed part contains the lowest amount of polyphenol (Megan 2010).

1.1.1.    Phytochemistry of Pomegranate

Pomegranate is contained different amounts and qualifications bioactive components such as gallic acid, catechin, flavonoids, ascorbic acid. These components are called phenolic compounds and are effective in the formation of quality properties of food such as taste, smell and colour.

Figure 2. 2.   Classification of phenolic compounds

The main phytochemicals in pomegranate are polyphenols. Pomegranate is especially rich in flavonoids, condensed tannins (proanthocyanidins) and tannins, it also contains high amounts of phenolic acid, sterol, fatty acid, triglyceride and alkaloid. It contains flavonoids such as luteolin, quercetin and kaempferol, especially in the shell part.

1.1.1.    Antimicrobial Effect of Phenolic Compounds

The antimicrobial effect of phenolic compounds has been thought to occur through 2 different mechanisms. These are;

·      The breakdown of the bacterial cell as a result of the reaction of the phenolic compounds with bacterial cell proteins that are found in the cell membrane.

·      Enzymes are inhibited by the reaction of phenolic compounds with proteins which are in the structure of microbial enzymes, and the phenolic compounds react with sulfhydryl groups of microbial proteins so the phenolic toxicity occurs.

·      In previous studies, it has been observed that especially punicalagin, gallic acid and ellagic acid have antimicrobial effects on many different microbiological organisms.

1.2. The Relationship Between Pomegranate, DM and Wound Healing

Diabetes is a chronic disease caused by an increase in the amount of glucose in the blood due to deficiency of insulin secreted from the pancreas. It may occur due to different factors such as environmental, genetics and lifestyle. When DM is not brought under control, it can become a serious threat to the public and individual health by causing an increase in morbidity and mortality rates. Diabetes-induced neuropathy, peripheral vascular diseases and abnormal cellular activities are the main factors preventing diabetic wound healing. Wound healing is a complex process and generally consists of 4 sequential steps, these are; (1) coagulation (2) inflammation (3) construction/ repair (4) maturation. Delayed wound healing and chronic wounds with continuous infection are complications of diabetes this is a serious treatment problem and economic problems of today's medicine. The reason for the effect of DM on wound healing rate is not yet known. But, high blood sugar levels are thought to increase infection by affecting basic events such as cell proliferation, collagen production, fibroblast formation, apoptosis and phagocytosis. In recent years, the use of medicinal plants has increased in the treatment of various diseases worldwide.

It is thought that pomegranate shell, which has been used in China for many years for the treatment of various diseases and against parasites and infections, has a wound-healing effect and increases the level of hydroxyproline in the tissues.

In addition in a study, the polyphenolic content obtained from pomegranate shell extract has been used in the treatment of cutaneous wounds in diabetic rats. As a result of this study, it is thought that pomegranate shell can be used in the treatment of diabetic wound due to the increase in the expression of nitric oxide (NO), hydroxyproline nitric oxide synthase (NOS), VEGF and EGF parameters. In the same study, it has been observed that the rate of epithelialization in wounds increased in the treated group. For this reason, pomegranate is a product that is used more and more frequently in the treatment of diabetes and wounds.

1.3. Soxhlet Extraction Method

Figure 2. 3.  Soxhlet extraction apparatus

Soxhlet extraction method is commonly used for many types of biological and environmental samples. Generally, Soxhlet extraction apparatus is consist of a condenser, siphon, and porous thimble (sample holder). A Soxhlet extraction method involves the series of steps, Firstly, the sample is put in filter paper and placed in the thimble.

Next, fresh solvent vapors and pass through the thimble. When the liquid reaches the outpouring level of the thimble, a siphon aspirates the solution and the liquids falls back into the distillation flask.

The separation of solute and solvent occurs in the distillation flask. After that solute is left in the distillation flask but fresh solvent vapours back into the solid bed of samples. This process is repeated until extraction becomes complete.

1.1. HPLC

High pressure liquid chromatography (HPLC) is a commonly used separation technique. Thanks to this method, it is possible to separate, identify and quantify the components in any mixture. Today, HPLC is used in many fields such as chemistry, biochemistry, biotechnology, pharmacology, medical chemistry, agriculture and chemical engineering. HPLC apparatus generally consists of the mobile phase, pump, injector, column, detector and recorder parts also the stationary phase consists of porous particles in mm. In this method, the liquid mobile phase is mainly used to separate the components in the mixture. The components in the mixture are first dissolved in the solvent and then passed through the chromatography column under high pressure. HPLC offers many advantages. These are;

·      Matrices with a very small particle size are used.

·      Sample requirement is low.

·      Thanks to the continuous flow detector, small quantities can be determined.

·      It has fast analysis possibility and high separation power.

1.2. Disc Diffusion Method

Antimicrobial tests are tests that are performed to determine the effect of an antimicrobial agent against a specific bacterial species in in-vitro conditions. 2 main methods are used in antibiotic susceptibility tests these are "diffusion" and "dilution". Disc diffusion method is among the diffusion tests and is the most commonly used method in laboratories. This test is also called agar diffusion test or Kirby-Bauer test. It is a very cheap and simple application.

The basic logic of this test is; after antibiotics are saturated on paper discs it is diffused into the medium (agar) where the organism which is sensitivity to be investigated is inoculated. While these discs dissolve over time and diffuse into the agar, microorganisms begin to multiply in the medium. After the incubation period, which is usually 24 hours at 35℃, no growth is observed around the disc. The more resistant the microorganism is to the drug, the wider the inhibition zone around the disc occurs. In order not to overlap the inhibition zones that will occur during this process, there must be 22mm distance between the disks and 14mm distance from the petri edge.

1.1. Electrospinning

Electropinning is a simple and versatile method of preparing micro or nanopolymer fibes. In the methpds, high voltage electricity is applied to the solution and a collector that allows the solution to exit the header forming a jet. Electrospinning is affected by various parameters such as temperature, pressure, operating voltage, viscosity and flow rate. The presence of polymers can create variability in these parameter. Viscosity is an important parameter for electrospinning, it is related to how mixed the polmer molecule chain is in the solution. 

Figure 2. 5. Electospinning device

The electospinning device is a device for production of nanofiber networks from polymer solutions with high voltage and high electric field by electro-magnifacturing method. The required high voltage, high voltage power is boiled and supplied to the approriate polymer solution in the range of 10-40 kV. The electric field created by the high voltage creates a nanofiber membrane surface by creating life in nanometer orders by thinning the charged polymer solution by spinning against metal surface with the opposite pole or grounded with the opposite pole.

1.1. Epithelialization Effects and Biocompatibility

MTT methods is one of the widely used enzimatic test methods to evaluate cytotoxic effects. This methods 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 is living cells with active mitochondria. This is regarded as a marker of cell viability, and the spectrophotometrically determined value is associated with the number of viable cells.