The effect of soaking method on the amount of bioactive compounds and antimicrobial properties of Punica granatum

Medicinal plants are widely used in traditional culture to treat certain illness all over the world and they are becoming popular in many modern societies among costumers as natural preservatives instead of synthetic chemicals in food products. In fact, they are a great source of antioxidants, which can reduce or stop the oxidation reactions, and their benefits against human diseases and food spoilage has attracted food designers and researchers’ attention. (Yang et al., 2016). Punica granatum; non-productive ancient, mystical, unique pomegranate flower borne on a small, long-living tree, is an important medicinal plant in north of Iran. Various studies have announced that has been used to treat wounds, bronchitis, diarrhea, digestive problems, cardiovascular disease, diabetes, dental conditions, bacterial infections, arthritis and obesity due to its astringent, hemostatic and antimicrobial properties. It is also effectively used to help oral inflammation (Gavanji et al., 2014). Secondary metabolites derived from this flower including total poly phenolic and flavonoid compounds such as gallic acid, ursolic acid, triterpenoids, maslinic acid, acetic acid, punicalagin, and anthocyanin are able to inactivating oxygen-free radicals (Jurenka et al., 2008). These secondary compounds can be found in different parts of the plant consists of leaves, trunk skin, roots, fruit peel, pomegranate juice, seeds and flowers (Salahvarzi et al., 2011). Therefore, the extraction of is Punica granatum utilized in pharmacy, cosmetics and food industries. Soaking is one of the methods mostly used to extract plants effective component which is applied by different solvents in different time and temperature.

Formerly, Punica granatum was prepared from Saveh and approved by the herbarium of medicinal plants of the Faculty of Pharmacy, University of Tehran with the scientific name of Punica granatum, pleniflora variety. Thus three soaking variables of solvent type (water and methanol 80%), time (12, 24 and 48 hours) and temperature (20, 35 and 50 °C) were executed for Punica granatum extraction. So 18 treatments were prepared under different conditions of time, temperature and solvent. Total flavonoid by aluminum chloride method, anthocyanin content and IC50 by free radical scavenging activity of Punica granatum extraction were measured. Evaluation of free radical scavenging was conducted with Diphenyl Picrylhydrazyl radical. The IC50 factor, /which demonstrates the amount of required sample to inhibit 50 percent of free radicals, was used. The lower the IC50, the lower concentration with higher antioxidants of extract would prevent oxidation process (Khalili et al., 2015). Confirming its antioxidant properties afterwards, the antimicrobial effect (MIC, MBC) of optimal treatment with two methods of micro dilution broth and diffusion in the well on Staphylococcus aureus, E.coli and Clostridium perfringens were studied. Data were analyzed by one-way variance Duncan test at 95% confidence level by using Minitab 16.

The results of single optimization of independent variables showed the highest flavonoid and anthocyanin content was 8.545 mg/100g during 24h in 35° C using methanol and 6.121 μmol/g during 12h in 50 ° C using water, respectively. The amount of flavonoid and anthocyanin and antioxidant activity enhanced significantly (p≤0.05) over time at higher temperature. Indeed, the solvent has enough time to permeate into plant texture and molecules and the compound can separate from plant matrix and be solved in the solvent. As the increment in temperature decreases the viscosity of solvent and influences the spread rate of it, the mass transfer rate increases. Methanol 80% was more effective to extract antioxidants compared to water. This may be due to solvent polarity since extractability of anthocyanin, flavonoid and phenolic compounds are depended on degree of polarity and ratio of solute and solvent. The polarity of the flavonoid is an important factor to consider; the polarity of the solvent has to be desire for recovery. In addition, the solvent used in the extraction process may have an effect on the bioactivity of the recovered flavonoids. A polar solvent is commonly used for the extraction of isoflavones, flavones, and methylated flavones. In contrast, methanol is sufficient to extract polar flavonoids such as flavonoid glycosides. That’s the reason for ineffectiveness of water to extract total phenols and flavonoids though it can raise the extraction of anthocyanin. Anthocyanin is less stable in higher pH. Methanol raises the pH leading to anthocyanin destruction. The simultaneous optimized condition of Punica granatum extraction to access the most extracted flavonoid and anthocyanin amount and the highest antioxidant properties were predicted during 48h in 50 ° C using methanol solvent with 95/614% acceptability percentage. Under optimum condition 8/6216 mg/g flavonoid, 5/7365 μmol/g anthocyanin and 7.2793 mg/ml IC50 were measured. Based on the results of antimicrobial evaluation of optimized extraction with highest antioxidant activity the highest mean minimum inhibitory concentration(MIC) and the minimum bactericidal concentration (MBC) of Punica granatum against Staphylococcus aureus, E.coli and Clostridium perfringens were observed 468.5 and 1875 μg/ml, 937.5 and 3750 μg/ml , 937/5 and 5000 μg/ml, respectively. The results of the diameter of inhibition zone of Punica granatum extraction showed better antimicrobial effect against Staphylococcus aureus with the diameter of inhibition zone of (13mm) compared to E.coli and Clostridium perfringens. The antimicrobial activity of the Punica granatum extract was more effective against gram positive (S. aureus) compared to Gram-negative (E. coli) bacteria, which are eminent pathogenic microorganisms responsible for food poisoning. A possible explanation for these effects is that Gram-negative bacteria is more resistant. The higher inhibition of Gram-positive bacterium to Gram-negative one is related to structural differences in the cell wall. The Gram-positive bacteria including several layers of peptidoglycans, negatively charged, exhibiting a relatively high level of porosity and permeability. On the other hand, Gram-negative bacteria exposure the cell wall, an additional thoroughly organized outer membrane consisting phospholipids, proteins, lipoproteins and negatively charged lipopolysaccharides, functioning as a hurdle. Consequently, by hydroxyl group of flavonoid and poly phenolic compounds permeating to bacteria cell and bonding their enzymes prevent their metabolism and cause the cell destruction and death.

Thus, by optimizing extraction condition a significant amount of antimicrobial and antioxidant compounds of Punica granatum can be extracted and be used in food and pharmaceutical industry.