Salting-out Extraction Of Main Active Ingredients From Capsicum Annum L. And Grape (Vitis Vinifera) Seeds

The extraction of natural products from plants is confronted with great challenges, including complex composition in raw material, numerous active ingredients, multiple steps of separation and extraction, and low product yield. For example, capsanthin and capsaicin are usually extracted using conventional organic solvents prior to isolation from the crude extract. As a spicy by-product, capsaicin affects the quality of capsanthin extracted from hot red pepper. Another example is the extraction of total phenolics (TP), total flavonoid (TF), proanthocyanidin (PA) and grape seed oil from grape marc. However, these components are usually extracted individually. Accordingly, this study aims to extract capsanthin and capsaicin from red pepper (Capsicum annum L.) and TP, TF, PA and grape seed oil from grape marc using salting-out extraction (SOE). SOE allows the extraction, concentration, and separation of natural active ingredients with different polarities to be conducted in a single step.Firstly, the phase diagrams and separation behaviors were studied for SOE systems. For aqueous two-phase extraction (ATPE) systems, a linear relationship was obtained between the volume of the top phase and the volume of solvent added. The binodal curves were not sensitive to temperature. The pH affected the phase ratio and the salt concentration of the top phase. For the three-liquid-phase systems (TLPSs), temperature affected the phase ratio, whereas the effect of pH was non-significant.Secondly, ATPE and microwave-assisted ATPE (MAATPE) of capsanthin and capsaicin were studied from red pepper. The capsanthin and capsaicin yields were high when extraction was performed under the aqueous two-phase system (ATPS) of acetone/K2HPO4and ethanol/K2HPO4. The yields of capsanthin and capsaicin were higher with MAATPE than those with ATPE under optimal extraction conditions of32%(w/w) acetone/24%(w/w) K2HPO4and microwave duration of80s. The phase composition and extraction procedures significantly affected the capsanthin and capsaicin yields.Thirdly, the extraction of capsanthin and capsaicin from red pepper was studied using TLPSs of w-hexane/ethanol/f2HPO4and n-hexane/acetone/K2HPO4. Capsanthin was extracted into the top n-hexane-rich phase, whereas capsaicin was mainly distributed in the middle phase. When the mass ratio was1:20at25℃, the capsanthin and capsaicin yields obtained from the10%(w/w) w-hexane/22%(w/w) acetone/25%K2HPO4(w/w) system were 105%and88%of the yields obtained from conventional solvent extraction, respectively. Capsanthin and capsaicin were separated each other in a single step. The color value of capsanthin was improved from129.69to165.83, and the purity of capsaicin was increased by44%using gel column chromatography.The ATPE and MAATPE extraction of TP, TF, and PA from grape seeds were studied. We studied the effect of phase composition on the partitioning behavior of TP, TF, and PA in ATPE. The system comprised32%(w/w) acetone/16%(w/w) ammonium citrate, was used to extract active constituents from different grape seeds and skins. Phenolic compounds were extracted into the top acetone-rich phase using32%(w/w) acetone/16%(w/w) ammonium citrate. The maximum yields of TP, TF, and PA were82.7,52.6, and30.7mg/g, with corresponding recovery rates of97.1%,97.9%, and99.3%, respectively.The yield and antioxidant properties of grape seed extracts (GSEs) obtained using the different extraction techniques were studied. Samples extracted using the different methods were analyzed by HPLC to identify the individual phenolic compounds. The main phenolic compound in grape seed was catechin. The maximum catechin concentration of156mg/100g was obtained using MAATPE. The2,2-diphenyl-1-picrylhydrazyl (DPPH) assays showed different levels of activity. The lowest concentration necessary for the50%inhibition of DPPH was obtained using soxhlet extraction (48.7mg/L), followed by MAATPE (50.3mg/L).Lastly, the three-liquid-phase extraction (TLPE) of active ingredients from grape seeds was studied. The GSEs of grape cv.’Italian Riesling’showed the maximum yield of TP, TF, and PA yields of95.29,60.50, and30.64mg/g, respectively, when the system comprised18%(w/w) n-hexane/32%(w/w) acetone/12%(w/w) K2HPO4, at room temperature. The seed from ’Italian Riesling’ showed lower IC50(60.35mg/L) than those from other varieties, indicating that the seed extracts from ’Italian Riesling’ had higher antioxidant activity. The grape seed oil yield was between17.76and19.49g/100g depending on the grape seed variety. The majority of fatty acids in grape seed were linoleic and oleic acids. Under the SOE system, the TP, TF, PA, and seed oil from grapes were extracted and separated in a single step. SOE required a lower amount of solvent and a shorter extraction time than the conventional solvent extraction method.