Investigation of isolation of anthocyanin rich extracts with advanced extraction technologies exhibiting anti-tyrosinase activity

The usage of naturally derived products in the treatment of diseases has a large share in developing countries and keeps its importance as complementary medicine in developed countries. Enlightment of disease development mechanisms and application of the right approach for treatment, guides the production of herbal medicines. The enzyme tyrosinase has been shown to play role in the formation of hyperpigmentation, melanoma and Parkinson's disease. Agents which are able to inhibit tyrosinase activity have been shown to be effective in the treatment of these diseases. While many synthetic and naturally occurring compounds have been shown to possess tyrosinase inhibitory activity, their usage is limited due to safety limitations. For the cosmetics and pharmaceutical industry, the potential of natural compounds for tyrosinase inhibitory activity are of great importance. Advanced extraction technologies which reduce/eliminate organic solvent usage, provide effective and efficient use of resources while utilized for protection of human and environmental health. As many parameters affect extraction processes, advanced extraction systems are open areas for development. While reducing the amount of energy, solvent, time and raw material needs for extraction, development of systems with high efficiency and selectivity has been aimed to obtain bioactive components. In this study, supercritical carbon dioxide, subcritical water and microwave assisted extraction systems, which are defined as advanced extraction technologies, were optimized for the extraction of anthocyanin rich extracts from Morus nigra L. fruits and the tyrosinase inhibitory activities of extracts and fractions were investigated. Quantitative determinations of total phenol, flavonoid and anthocyanin contents were carried out with spectrophotometer and the anthocyanin derivatives cyanidin-3-glycoside, cyanidin-3-rutinoside, pelargonidin-3-glycoside and pelargonidin-3-rutinoside were determined by UPLC-DAD-ESI-MS/MS qualitatively and quantitatively. Active fractions were identified with isolation studies and composition activity relationship was determined. Ethanol:water:TFA (70:29:1) solvent mixture was used for the conventional solvent extraction and IC50 value for tyrosinase inhibitory activity was found as 2.62 mg/mL. Optimum conditions for supercritical carbon dioxide extraction were determined as 60 °C, 200 bar, 8% co-solvent ethanol and IC50 of the extract and isolated active fraction for tyrosinase inhibitory activity was found as 0.344 mg/mL and 10.69 μg/mL, respectively. Optimum conditions for subcritical water extraction were determined as 60 °C and 120 °C, 60 min, 2 ml/min and IC50 of the extracts and isolated active fraction for tyrosinase inhibitory activity was found as 1,84 and 1.71 mg/mL and 86.75 µg/mL, respectively. Optimum conditions for microwave assisted extraction were determined as 500 watt, 35% ethanol, 10 min and IC50 of the extract and isolated active fraction for tyrosinase inhibitory activity was found as 1.44 mg/mL and 166.50 μg/mL, respectively. In the optimized subcritical water and microwave assisted extracts, composition of active fractions were determined with LC-MS/MS as majorly composed of cyanidin-3-glycoside (239,10 mg/g) and cyanidin-3-rutinoside (125,12 mg/g) reaching an anthocyanin amount of 418.22 mg cya-3-gluE/g extract. Raphanus sativus 'Red Meat' root and Brassica oleracea var. capitata f. rubra aerial parts as plant sources rich in anthocyanins, were studied with optimized extraction conditions in order to investigate availability for large scale work and industrial production. The extracts of Brassica oleracea var. capitata f. rubra were determined as suitable for large scale work and production with 31.74 mg GAE/g total phenol, 3,46 mg KE/g total flavonoid and 11,83 mg cya-3-gluE/g total anthocyanin content (IC50: 1.26 mg/mL) values. By using the data obtained from this study, it could be possible to evaluate other plant sources of our flora rich in anthocyanins and contribute to our country's economy using advanced extraction techniques.