High-performance liquid chromatography micro-fraction bioactive evaluation combined with countercurrent chromatographic separation of antioxidants from Citrus peel and their tyrosinase inhibition activities.

In the present study, high-performance liquid chromatography micro-fraction bioactive evaluation and high speed countercurrent chromatography were performed on screening, identification and isolation of antioxidants from Citrus peel. Three compounds were screened as antioxidants and tyrosinase inhibitors using 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase activity test, then they were identified as eriocitrin, narirutin and hesperidin. Moreover, the solvent system ethyl acetate-n-butanol-water (6:4:10, v/v/v) was used for separation of ethyl acetate extract of Citrus peel by high speed countercurrent chromatography. In total, 0.45 mg of eriocitrin with 87.10% purity, 2.04 mg of narirutin with 95.19% purity and 1.35 mg of hesperidin with 95.19% purity were obtained from 20 mg of ethyl acetate extract of Citrus peel in a single run and then each component was subjected to 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase inhibition assay. Eriocitrin showed great antioxidant activity (the half-maximum concentration: 3.65 µM) and tyrosinase inhibition activity (the half-maximum concentration: 115.67 µM), while narirutin and hesperidin exhibited moderate activity. Tyrosinase inhibition activity for eriocitrin in vitro was reported for the first time. Furthermore, molecular docking between eriocitrin and mushroom tyrosinase was also studied.

Solvent strength of aqueous phase for two typical biphasic solvent systems in high-speed countercurrent chromatography.

In the present work, influence of solvent strength of aqueous phase for two frequently-used biphasic solvent system in partition coefficient (K) of selected solutes were mainly studied, and a new method for selection of biphasic solvent system was proposed for high-speed countercurrent chromatographic separations. Solvent strength was referred to the typical theory that was deeply investigated in conventional reversed-phase liquid chromatography. Experimental results showed that a linear relationship between log(K) of solutes and apparent content of methanol in biphasic solvent system was found for the biphasic solvent system hexane-ethyl acetate-methanol-water (HEMWat), which was consistent with the relationship between real content and apparent content of methanol in this system. Meanwhile, a quadratic relationship was found between log(K) of solutes and apparent content of methanol in biphasic solvent system chloroform-methanol-water (ChMWat), in which it was found that the relationship between real content and apparent content of methanol in this system was also quadratic. In addition, a visual and simple method was proposed to select a suitable biphasic solvent system for separation of target compounds by high-speed countercurrent chromatography with isocratic elution, which saves a lot of manpower and material resources in order to find a suitable two-solvent system. An optimal biphasic solvent system for isolation of several tested compounds by high-speed countercurrent chromatography was easily obtained using our proposed method.