The effect of extraction of probe ultrasound on antioxidant properties of bounded and free phenols of Hyssop (Hyssopus officinalis) extract

Excessive oxidative stress can cause multiple chronic diseases such as diabetes, cardiovascular disease, and cancer and aging-related diseases (Kawabata et al 2015). Phytochemicals reduce cardiovascular disease by decreasing the oxidation of LDL, the absorption and synthesis of cholesterol, blood pressure and increasing the blood clotting time. Also, the potential chemical prevention property of these compounds against cancer is caused by their ability to moderate the epigenetic changes of cancer cells (Mahan et al. 2012). Polyphenols have antioxidant activity, plant polyphenols have different functions and can act as a reducing agent, Phenolic acids and their derivatives form a large group of polyphenols that play an important role in the structure of plants, including the cell wall. (Keshavarz et al 2010). The phenolic acids can be classified as either free or bonded phenolic acids (Renger and Steinhart 2000). Free phenolic compounds in the gastrointestinal system can act against the incidence of colon cancer and other chronic diseases, free phenolic acids can be extracted with solvents such as water, methanol, ethanol and acetone. (Min et al 2012). Bonded phenolic acids are compounds that are insoluble, and are bonded to structural components of cellular wall such as cellulose, hemicellulose (for example arabinoxylans), lignin, pectin and structural proteins (Acosta et al 2014). They cannot be extracted by common techniques which lead to less prediction of total phenolic content from the real extent (Ti and al 2014). Hyssop, scientifically known as Hyssopus officinalis, that belongs to the Lamiaceae family (Ghasemi et al 1392). This plant is used as a flavoring and improving organoleptic properties in the food industry and has aromatic compounds that is used in soaps, cosmetics and perfumes. This plant is rich in volatile oils, flavonoids, tannins, marrubiin. This plant has antimicrobial, laxative and antispasmodic properties and has strong antiviral activity against HIV. It is antibacterial, antifungal and has antioxidant properties. Thus, the study aimed at the separation of the free and bonded phenolic factions and the determination of the phenolic content and antioxidant activity of hyssop plant leaf (Hyssopus officinalis) which partly contributes to the better prediction of the phenolic content and antioxidant activity.

Extraction of the hyssop plant leaf was carried out by using ultrasound probe under 10 and 20 minutes and the intensity of 60 - 100 W and the use of ethanol: water with ratios (80:20) and (50:50) and separating free phenolic fractions from the extractions using methanol and ultrasound for one hour (adjust the pH in the range of 4.5-5.5 by using 6M HCl), Then centrifuged at 4321 g, for 25 min, the supernatant is free phenolic. And then separating the bonded phenolic factions from residues resulting from the extraction of free phenolic by using caustic soda and ultrasound for two hours (adjust the pH in the range of 4.5-5.5 by using 6M HCl) and then centrifuged at 4321 g for 25 min, the supernatant was used as bonded phenolic extract (Kotásková et al 2016). The antioxidant activity was measured by free radical inhibition tests (DPPH) and the evaluation of colorlessness of beta carotene - linoleic acid, and oxidative stability test (OSI).

The results of this study show that hyssop leaf extract which was extracted by ultrasound probe method contains significant amounts of phenolic and flavonoid compounds and there is a direct relationship between these compounds. Therefore, it can be concluded that hyssop leaf extract has antioxidant activity, and the highest amount of phenolic and flavonoid compounds of the extract, free and bonded phenolic compounds are (20-100-80) respectively. According to the determination of antioxidant activity by DPPH test, the maximum inhibition percentage for the obtained extract from treatment is (20-100-80) and the maximum inhibition amount for free and bonded phenols of the treatment is (20-60-80) and the least IC50 of the obtained extract from the treatment is (20-60-80); and is (20-100-80) for free and bonded phenols and the best treatment of free and bonded fractions was shown by the treatment (20-100-80) in the beta - carotene and OSI tests. Thus, there is a direct relationship between phenolic content, free and bonded phenols in beta - carotene and OSI tests. In DPPH radical inhibition, there was no correlation between phenolic content and antioxidant activity, which could be related to other factors (synergistic or antagonistic effect between active compounds, number and location of hydroxyl groups, presence of impurity in the extract, extraction method, etc.). The results also showed that free phenolic fractions had higher phenolic content and better antioxidant activity than bonded phenols. Free and bonded phenols have benefits for health. The released phenolic compounds in food matrix are used as food preservative. In order to use bonded phenolic compounds in food, pharmaceutical and cosmetic industry, some methods should be designed to release and extract them from the field crops and obtained sediments from extraction, which includes a large part of bonded phenols. Also, separation of free phenols can produce bioactive compounds with high antioxidant properties and can be used more efficiently in food and pharmaceutical industry.

Generally, the ratio of solvents, ultrasound intensity and extraction time affect the antioxidant power of hyssop extract. These points indicate the efficiency of ultrasound for recovery of phenolic compounds is mainly due to the cavitation phenomenon. This is due to the emission of ultrasonic waves through a liquid medium which damage the plant wall and improve the solvent penetration and subsequently release the phenolic compounds. In addition, organic solvents are replaced with those which have no toxic effects and consume less energy during the process compared to conventional methods. It also reduces the process time and temperature, which leads it to be useful for extraction of heat - sensitive compounds, such as phenolic compounds. All of these characteristics indicate that ultrasound can be used as a new and alternative process for the use of agricultural and industrial remains and efficient extraction of the phenolic compounds; and it leads to the production of products with high value which have a lot of profit in the industry and finally, it pollutes the environment less than conventional methods.