Preparative isolation of maltol glycoside from Dianthus superbus and its anti-inflammatory activity in vitro.

Dianthus superbus is a traditional Chinese medicine that is commonly utilized as a treatment for inflammation, pain, and immunological conditions. In this study, an anti-inflammatory maltol glycoside derived from Dianthus superbus was isolated for the first time via medium and high-pressure liquid chromatography, and at the same time, the in vitro anti-inflammatory activity of this maltol glycoside was preliminarily explored. Initially, crude samples of Dianthus superbus were preprocessed via MCI GEL® CHP20P and Spherical C18 medium-pressure chromatography, under the guidance of evaluation of in vitro anti-inflammatory activity. Fr44 was found to be the target fraction, and it was further isolated via two-dimensional reversed-phase/hydrophilic interaction liquid chromatography, yielding > 95% pure and was identified as tunicoside B. MTT assay, nitric oxide and nitric oxide synthase were used to evaluate the effects of tunicoside B on murine macrophage Raw264.7 by nitric oxide synthase assay kit, molecular docking, and western blotting. The results showed that tunicoside B did not affect the viability of cells and exhibited significant anti-inflammatory activity. As far as we know, this is the first report of tunicoside B from Dianthus superbus and the first study on the anti-inflammatory activity of tunicoside B. More importantly, the approach established in this study is expected to provide a theoretical basis for the separation and pharmacological activity study of maltol glycosides from other natural products.

The applicability of high-speed counter-current chromatography for preparative separation of biosynthesis products: Glycosylation products as example.

Biosynthesis is a promising way to manufacture desired products, however, the purification of its final products is a tough work due to the huge amount of reaction matrix. Liquid stationary phase of high-speed counter-current chromatography could easily avoid the commonly disadvantages that occurred in traditional column chromatography in the field of biosynthesized products purification. This characteristic makes high-speed counter-current chromatography particularly applicable for final products separation in biosynthesis. In this study, the glycosylation products of Silybin B by one-pot glycosylation were successfully purified by high-speed counter-current chromatography to show the applicability of high-speed counter-current chromatography for preparative separation of biosynthesis products. An optimized n-hexane/ethyl acetate/methanol/water (2:5:2:3, v/v/v/v) system was applied in this study. As a result, four Silybin B glycosylation products, including 7 mg of Silybin B-5-O-ß-D-glucoside (SG-1), 12 mg of Silybin B-3-O-ß-D-glucoside (SG-2), 10 mg of Silybin B-7-O-ß-D-glucoside (SG-3), and 24 mg of Silybin B-20-O-ß-D-glucoside (SG-4), were simultaneously separated from 200 mg of glycosylation crude products, with the purity of 89.3, 95.2, 96.4, and 97.5%, respectively. Their structures were identified by spectroscopic analysis.

The applicability of pH-zone-refining counter-current chromatography for preparative separation of biosynthesis products: Glycosylation products as example.

Biosynthesis is a research hot-spot in recent years, however, the purification of its final products is a tough work. Liquid stationary phase and large-scale separation ability of PZRCCC could easily avoid the commonly disadvantages occurred in traditional column chromatography. These characteristics makes PZRCCC particularly applicable for final products separation in biosynthesis. In this study, the glycosylation products of ellagic acid by one-pot glycosylation were successfully purified by PZRCCC to show the applicability of PZRCCC for preparative separation of biosynthesis products. An optimized ethyl acetate/n-buthanol/water (3:3:5, v/v/v) system was applied in this study, where 5 mM trifluoroacetic acid (TFA) as the retainer and 30 mM triethylamine (TEA) as the eluter were added. As a result, four ellagic acid glycosylation products, including 51 mg of ellagic acid-4, 3'-O-ß-D-diglucoside (EG-1), 24 mg of ellagic acid-4, 4'-O-ß-D-diglucoside (EG-2), 11 mg of ellagic acid-4-O-ß-D-glucosyl (1→2)-ß-D-glucoside (EG-3) and 64 mg of ellagic acid-4-O-ß-D-glucoside (EG-4) were simultaneously separated from 500 mg of glycosylation crude products, with the purity of 93.3%, 91.2%, 89.4% and 95.5%, respectively. Their structures were identified by spectroscopic analysis.