Rapid screening and identification of active ingredients in licorice extract interacting with V3 loop region of HIV-1 gp120 using ACE and CE-MS

• A new screening method of HIV-1 inhibitor from natural extracts without isolation.
• The CE-MS method of seeking active ingredients in licorice extract is developed.
• We identified the structure of main ingredients from licorice active mixture.
• The Kb of compounds isolated from licorice extract with R15K are calculated by ACE.
• Glycyrrhizin could be HIV-1 entry inhibitor through binding to gp120-V3 loop.

The binding of envelope protein gp120 to glycosphingolipids is very important during the human immunodeficiency virus entering into the host cell. This step occurs in the V3 loop region in particularly. The conserved core sequence of V3 loop in gp120 was named R15K. Anti-HIV drug targeting to R15K would avoid the drug-resistance caused by HIV-1 genetic diversity. Here, for the first time, affinity capillary electrophoresis (ACE) and capillary electrophoresis-mass spectrometry (CE-MS) were used for establishing a simple, rapid and effective method of screening the licorice extract for biological activity (anti-HIV), which avoided the complicated isolation and purification process. R15K, 3′-sialyllactose (the positive control), and d-galactose (the negative control) were used for the development and validation of ACE method. After the interaction between licorice extract and R15K was confirmed by ACE, the relative active ingredients were isolated by SPE and their structures were determined by CE-ESI-MS online. In this research, two mixtures from licorice extract were found to be active. Furthermore, glycyrrhizin and licorice saponin G2 were verified as the main ingredients that significantly interacted with R15K via CE-MS and LC–MS. The results of quantitative assays showed that the active mixture contained glycyrrhizin of 74.23% and licorice saponin G2 of 9.52%. Calculated by Scatchard analysis method, glycyrrhizin/R15K complex had the highest binding constant (1.69 ± 0.08) × 107 L/mol among 27 compounds isolated from licorice extract. The anti-HIV activity of glycyrrhizin was further confirmed by bioactive experiment of cellular level. This strategy might provide a high throughput screening and identifying platform for seeking HIV-1 inhibitors in natural products.

Here, the interaction between licorice extracts and R15K was investigated by ACE for the first time. CE-ESI-MS was performed to identify the structures of the active ingredients.Figure optionsDownload as PowerPoint slide