A study of nanometre aggregates formation mechanism and antipyretic effect in Bai-Hu-Tang, an ancient Chinese herbal decoction

Bai-Hu-Tang (BHT), a Chinese herbal decoction used as an antipyretic agent, results from the combination of Anemarrhena asphodeloides Bunge, Glycyrrhizae, Japonica rice, and Gypsum. In our previous study, we identified nanoaggregates in BHT. However, the present study aimed to analyze and elucidate the mechanism of nanoaggregate formation and to investigate its antipyretic effect. A BHT decoction extract was split into 15 groups, and in each group, the extract was further separated into two solutions: Nano-phase and Decoction. The physicochemical properties of these solutions, such as particle size, salinity, conductivity, and surface tension were investigated, and analyzed the 15 groups of by transmission electron microscopy (TEM) and fingerprint chromatography. Furthermore, the antipyretic effect of nanoaggregates was evaluated through enzyme-linked immunosorbent assays, HE staining, Western Blot, and Real-time PCR. In the 15 groups, the salinity and conductivity results showed a promoting and stabilizing effect towards the Nano-phase formation. Analysis of the surface tension indicated good solubilization of Radix Glycyrrhizae. The TEM analysis of the BHT separated extracts revealed that only in the presence of Japonica rice the Nano-phase is formed. Sixteen common peaks were identified in the BHT fingerprint chromatogram, and the main chemical components were Neomangiferin, Mangiferin, Liquiritin, and Ammonium glycyrrhizinate. Furthermore, BHT and nanoaggregates from Bai-Hu-Tang (N-BHT) groups did not differ in the main chemical components. Additionally, the N-BHT group had the same antipyretic effect compared with the BHT group. However, the pathological analysis indicated that treatment with N-BHT could ameliorate the lung damage in the rat. At the same time, N-BHT group inhibited expression of several proteins, specifically IL-1β, TRPV4, NF-κB, and TNF-α, which agreed with the Real-time PCR results. We identified the key factors that are involved in the nano-phase formation. Also, by Western blot and Real-time PCR methods, we investigated the N-BHT mechanism of antipyretic action. The discovery of the N-BHT formation would provide a new idea of studying traditional Chinese medicine decoction.