Study on the Separation and Preparation of Anthocyanins from Green Mulberry by High Speed Counter Current Chromatography and Their Antioxidant Activity
Mulberry, as the fruit of mulberry trees in the mulberry family, is rich in nutrients and has various effects such as anti-cancer, antioxidant, analgesic and anti-inflammatory, and beauty of black hair. Green mulberries, also known as yellow mulberries, refer to immature mulberries. As a traditional Chinese medicine, green mulberries are easier to pick, transport, and store compared to mature mulberries, and their content of anthocyanins, catechins, and epicatechins is much higher than that of mature mulberries. There have been studies both domestically and internationally on extracting and isolating anthocyanins from various plants such as grape seeds, roses, corn silk, cranberries, cocoa beans, etc. Liu et al. used LSA-10 resin to enrich and purify anthocyanins from hawthorn; Chen Wenliang et al. used membrane separation and filtration to separate oligomeric anthocyanins from grape seeds, resulting in a content of 65% oligomeric anthocyanins, but did not obtain monomer separation components; Glavnik et al. analyzed the anthocyanins in Japanese tiger cane using high-performance thin-layer chromatography and identified monomers, decamers, and B-type anthocyanins from its crude extract. However, this method is difficult to prepare; Sun et al. used semi preparative high-performance liquid chromatography to separate anthocyanins from grape seeds, but the pretreatment was complex and the separation yield was low; This article chooses high-speed counter current chromatography (HSCCC), which has a large separation capacity, can be used for batch preparation and separation, is easy to operate, and has high separation efficiency.
Procyanidins (PC), as a type of polyphenolic compound, are widely present in plants. They are mainly composed of catechins, epicatechins (EC), epigallocatechins (EGC), and epicatechin gallates (ECG), and have strong antioxidant activity. This work aims to use high-speed countercurrent chromatography to separate and purify anthocyanins from green mulberries. High performance liquid chromatography (HPLC) will be used to identify the chemical components of each component and analyze their purity; By examining the scavenging ability of DPPH radicals, ABTS+radicals, and hydroxyl radicals in each component, the strength of their antioxidant capacity can be reflected. This work expands the scope of deep processing and utilization of mulberries, provides direction for the industrial production of anthocyanins in green mulberries, and has certain reference value for the development and utilization of mulberries as medicinal resources.

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Using green mulberry as the raw material, the crude extract was obtained through extraction, defatting, impurity removal, and extraction enrichment under the experimental conditions. The optimal solvent system for HSCCC separation and purification was n-hexane ethyl acetate water (1:20:20, V/V/V). 200mg of green mulberry anthocyanin crude extract was separated into five components by HSCCC, namely F1 (28.5mg), F2 (10.43mg), F3 (6.85mg), F4 (10.65mg), and F5 (13.65 mg). Each component contained catechins, anthocyanin B2, anthocyanin A1, etc. Among them, F4 contained epigallocatechin gallate, with a purity of 95.05%; F5 contains catechins with a purity of 73.75%. The separated components all have certain antioxidant activity. Using Vc as a control, the DPPH free radical scavenging ability is as follows: Vc>F5>F4>F2>F3>F1>crude extract of green mulberry; ABTS+free radical scavenging ability: Vc>F5>F4>F2>F3>F1>crude extract of green mulberry; Hydroxyl radical scavenging ability: F1>F5>Vc>F4>F2>crude extract of green mulberry>F3. HSCCC has a large preparation capacity and no packing material in the chromatographic column. In this experiment, the crude extract can be separated once to obtain five components, and some components have high purity monomer substances and strong antioxidant activity. The research content of the article has opened up new ideas for the production of anthocyanins and provided theoretical support for the study of the medicinal efficacy of anthocyanins in green mulberries.