Effect of different conditions of subcritical water extraction on antioxidant capacity and amount of phenolic compounds of pomegranate pulp extract

Recently, the role and benefits of antioxidants against many human diseases and food spoilage caused by oxidative corruption has attracted a lot of attention. Antioxidants are vital compounds that are widely used as a parameter for food and bioactive drug compounds. Antioxidants are compounds that inactivate oxygen-free radicals in the human Body. Oxygen-free radicals have a strong tendency to combine with vital molecules in the body, including nucleic acids and proteins. Therefor, free radicals, by combining with these molecules, destroy them and thus cause various diseases such as cancers, cardiovascular disease, inflammatory and Weakening of the immune system (Ayub Nejadgan Jahromi and Hasanpour 1397). Pomegranate pulp is a non-edible part and by-product in the juice industry and is a rich source of polyphenol antioxidants and due to its cure and health benefits, it can be used as a nutritious and useful compound (Kazemizadeh and fadaeeie noghani 1393). There are various methods for extracting bioactive compounds. One of the new methods is extraction with subcritical water. Subcritical water is a region of the condensed phase of water, the temperature of which is between 100°C and 374°C, as well as the pressure that the water stays in the liquid state and does not change the phase (Ramos et al 2002). In the present study, two methods for extraction of subcritical water and maceration and also the effects of subcritical water process variables, including temperature and time, on the extraction of antioxidant and polyphenolic compounds of pomegranate pulp extract were investigated.

In this project, two methods for extraction including subcritical water and maceration were evaluated to determine the antioxidant capacity and phenolic compounds of pomegranate pulp extract. Extract of subcritical water extraction operation was performed by a subcritical water extraction device in a pilot laboratory. Subcritical water extraction variables were two parameters: temperature (120°C,150°C and 180°C) and time (5, 15 and 25 minutes). Volume of the extracts were 75 ml for each treatment (shaddel et al. 1391). In the method of maceration to prepare the extract, mixture pomegranate pulp powder-water were mixed in a dark environment and ambient temperature for 24 hours using a magnetic stirrer, Then, the mixture was centrifuged to separate the solid phase of extract (Mohamadi et al. 2012). In the extracts, the antioxidant capacity was measured using two

the free radical scavenging assay (DPPH) in %RSA (Mohamadi et al. 2012 ) and ferric reducing antioxidant power (FRAP) assay in milligram Fe2+ per gram dry matter (Benzie and Strain 1996), Furthermore, total phenol content by the folin-ciocalteau method in milligram of gallic acid per gram of dry matter (Mohamadi et al. 2012) and flavonoid contents in milligram of quercetin per gram of dry matter (Paul and Bhattacharyya 2015) were evaluated and reported. In this research, statistical analysis was carried out on the effect of temperature and time of extraction by subcritical water on the properties of pomegranate pulp extract from a completely randomized design. (ANOVA) was used to analyze the presence or absence of significant differences between the amounts of different treatments. The results of the analysis of variance method were analyzed at probability level (P<0.01) and averages were compared at probability level (P<0.05). The experiments were carried out in three replications.

The results of the assessment of two methods of maceration and subcritical water showed that the highest extraction was related to the subcritical water method compared to the maceration method. The results of analysis of variance showed that the difference between the values ​​of total phenol, flavonoids, free radical antioxidant and ferric reducing antioxidant for different treatments of pomegranate pulp extract, which was prepared by two methods of subcritical water and maceration, was significant at %1 probability level (P<0.01). The results of the effect of different treatments on the extraction of measured parameters showed that, with increasing temperature and time, the extraction rates of these compounds increased, so that the highest extraction was carried out at treatment at a temperature of 180°C and time of 25 minute. The highest total phenol content was 180°C and time of 25 minute (432.875 mg of gallic acid per gram of dry matter), and the lowest was 120°C and time of 5 minute (205.375 mg / g galic acid per gram of dry matter). In the subcritical water extraction method, with increasing temperature and time in extraction, the amount of flavonoid compounds increased so that the highest amount of flavonoid compounds was related to the treatment of 180°C and time of 25 minute (74.30 mg of quercetin per gram of dry matter) and the lowest amount was related to the treatment of 120°C and time of 5 minute (35.13 mg of quercetin per gram of dry matter). According to the results, the highest amount of antioxidant capacity by DPPH method was related to subcritical water extraction method in 180°C and time of 25 minute (%94.63). According to the results, the highest amount of antioxidant capacity with iron reduction method (FRAP) was related to subcritical water extraction in 180°C and time of 25 minute (1.225 mg of divalent iron per gram of dry matter) and the lowest amount was related to 120°C and time of 5 minute (0.205 mg of divalent iron per gram of dry matter). Increasing the extraction of these compounds by increasing the temperature and pressure in the subcritical water method is described by the fact that water at ordinary temperature and pressure is solvent of polar compounds and non-polar compounds are not soluble in it, But the increase of temperature and pressure reduces the polarity of water and acts as a non-polar solvent, which increases the ability of water to dissolve non-polar compounds. Also, raising the temperature and pressure of water reduces surface tension and viscosity, and thus, during the extraction process, the propagation velocity increases and the mass transfer is facilitated (Brand-Williams et al 1995). In other studies, the results showed that the extracts extracted by subcritical water have significant phenolic and antioxidant compounds than conventional extraction methods and also stated that it was due to increased temperature and pressure during the process, not only the amount of phenolic compounds but also the rate of extraction increased due to increased propagation velocity (Erşan et al 2018; Rangsriwong et al 2009; Mohamadi et al 2012).

According to the results, subcritical water extraction can be used as a suitable method for extraction of polyphenolic and antioxidant compounds of pomegranate pulp. Subcritical water is not only an environmentally friendly technology, but also increases the amount of bioactive compounds extracted compared with the maceration method.