Establishment of Polydopamine-Modified HK-2 Cell Membrane Chromatography and Screening of Active Components from Plantago asiatica L.

Cell membrane chromatography (CMC) has been widely recognized as a highly efficient technique for in vitro screening of active compounds. Nevertheless, conventional CMC approaches suffer from a restricted repertoire of cell membrane proteins, making them susceptible to oversaturation. Moreover, the binding mechanism between silica gel and proteins primarily relies on intermolecular hydrogen bonding, which is inherently unstable and somewhat hampers the advancement of CMC. Consequently, this investigation aimed to establish a novel CMC column that could augment protein loading, enhance detection throughput, and bolster binding affinity through the introduction of covalent bonding with proteins. This study utilizes polydopamine (PDA)-coated silica gel, which is formed through the self-polymerization of dopamine (DA), as the carrier for the CMC column filler. The objective is to construct the HK-2/SiO2-PDA/CMC model to screen potential therapeutic drugs for gout. To compare the quantity and characteristics of Human Kidney-2 (HK-2) cell membrane proteins immobilized on SiO2-PDA and silica gel, the proteins were immobilized on both surfaces. The results indicate that SiO2-PDA has a notably greater affinity for membrane proteins compared to silica gel, resulting in a significant improvement in detection efficiency. Furthermore, a screening method utilizing HK-2/SiO2-PDA/CMC was utilized to identify seven potential anti-gout compounds derived from Plantago asiatica L. (PAL). The effectiveness of these compounds was further validated using an in vitro cell model of uric acid (UA) reabsorption. In conclusion, this study successfully developed and implemented a novel CMC filler, which has practical implications in the field.

A Strategy for Identification and Structural Characterization of Compounds from Plantago asiatica L. by Liquid Chromatography-Mass Spectrometry Combined with Ion Mobility Spectrometry.

Plantago asiatica L. (PAL) as a medicinal and edible plant is rich in chemical compounds, which makes the systematic and comprehensive characterization of its components challenging. In this study, an integrated strategy based on three-dimensional separation including AB-8 macroporous resin column chromatography, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF MS), and ultra-high performance liquid chromatography-mass spectrometry with ion-mobility spectrometry (UHPLC-IM-MS) was established and used to separate and identify the structures of compounds from PAL. The extracts of PAL were firstly separated into three parts by AB-8 macroporous resin and further separated and identified by UHPLC-Q-TOF MS and UHPLC-IM-MS, respectively. Additionally, UHPLC-IM-MS was used to identify isomers and coeluting compounds, so that the product ions appearing at the same retention time (RT)can clearly distinguish where the parent ion belongs by their different drift times. UNIFI software was used for data processing and structure identification. A total of 86 compounds, including triterpenes, iridoids, phenylethanoid glycosides, guanidine derivatives, organic acids, and fatty acids, were identified by using MS information and fragment ion information provided by UHPLC-Q-TOF MS and UHPLC-IM-MS. In particular, a pair of isoforms of plantagoside from PAL were detected and identified by UHPLC-IM-MS combined with the theoretical calculation method for the first time. In conclusion, the AB-8 macroporous resin column chromatography can separate the main compounds of PAL and enrich the trace compounds. Combining UHPLC-IM-MS and UHPLC-Q-TOF MS can obtain not only more fragments but also their unique drift times and RT, which is more conducive to the identification of complex systems, especially isomers. This proposed strategy can provide an effective method to separate and identify chemical components, and distinguish isomers in the complex system of traditional Chinese medicine (TCM).

Comparative pharmacokinetics of Ding-Zhi-Xiao-Wan preparation and its single herbs in rats by using a putative multiple-reaction monitoring UPLC-MS/MS method.

INTRODUCTION: The formula of Chinese medicine, Ding-Zhi-Xiao-Wan (DZXW), has the distinct feature of compatibility therapy, which is attributed to the interactions of multi-herbs. However, the quantification problem caused by the absence of pure reference standards is a bottleneck to clarify the compatibility advantages from the perspective of pharmacokinetics (PKs). OBJECTIVE: This study aimed to develop a putative multiple-reaction monitor (PMRM) strategy for exploring the comparative PKs of DZXW and its single herbs. METHODS: First, precursor ion and tandem mass spectrometry (MS/MS) chromatograms were obtained via ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight MS (UHPLC-Q-TOF-MS) under different collision energy (CE) values. Then, the two most abundance ions in the MS/MS chromatograms were chosen as product ions, and CE values were selected according to the abundance of the product ion peaks. Next, a PMRM strategy consisting of optimal MRM parameters was constructed. Finally, the established PMRM parameters were imported to UHPLC coupled with triple quadrupole MS (UHPLC-TQ-MS) for quantification. RESULTS: The strategy was exemplified by the comparative PK study of DZXW and its single herbs. This strategy could extend the PK scopes of multi-components. The quantitative results displayed substantial variations in PK parameters between DZXW and its single herbs. CONCLUSION: The PK parameters indicated that the DZXW formula could increase the exposure levels of most ingredients and reduce the maximum concentration (Cmax ) of Radix Polygala, indicating that herb compatibility could produce synergistic effects and diminish possible toxic effects. This study provides a viable orientation for the compatibility investigation of traditional Chinese medicine.