Rapid screening of lipid-lowering active ingredients in rhubarb using chemically modified L02 liver cell membrane biocompatible materials
The screening of active ingredients in traditional Chinese medicine mainly relies on activity tracking, with monomer components as the screening objects, which has the disadvantages of large workload, cumbersome operation, and low efficiency. Compared with traditional activity tracking methods, cell membrane chromatography (CMC) is a rapidly developing biological affinity chromatography technique in recent years, which has the characteristics of specific recognition and efficient separation. The basic principle of CMC is to bind the cell membrane to the surface of a silica gel carrier to form a stationary phase. The cell membrane receptor can specifically bind to drug molecules, and the interaction between drug molecules and cell membrane receptors can be studied using chromatographic analysis. CMC combines the advantages of chromatographic technology, cellular and molecular biology, and receptor pharmacology to achieve approximately dynamic simulation of the action process of active pharmaceutical ingredients in vivo within the chromatographic column; Compared to commonly used adsorbents such as ion exchange resins and macroporous adsorption resins, CMC has the advantage of combining chromatographic separation with activity screening. It is easy to operate, fast and stable, and has high sensitivity, making it particularly suitable for screening active ingredients in complex systems such as traditional Chinese medicine.
When preparing cell membrane chromatography, it can be divided into physical adsorption and chemical modification based on the different binding forms of cell membrane and silica gel carrier. Physical adsorption cell membrane biological affinity chromatography is prepared by utilizing the fusion effect of the cell membrane itself and the adsorption effect of silicon hydroxyl groups on the surface of silica gel; This type of force is relatively weak, causing the cell membrane to easily detach from the carrier, resulting in a short lifespan of the chromatographic column. Chemical modification of cell membrane for biological affinity chromatography involves covalent bonding between silica gel and cell membrane; The silanol group of silica gel undergoes a condensation reaction with silane coupling agent to introduce amino groups, which crosslink with glutaraldehyde molecules. One end of the free aldehyde functional group can be obtained on the surface of silica gel; The cell membrane contains a large number of amino groups, which are prone to undergo aldoamine condensation reactions at room temperature, thereby achieving the covalent connection between silica gel and the cell membrane. In chemically modified cell membrane bioaffinity chromatography, the carrier silica gel is covalently bonded to the cell membrane, which is relatively stable, and the cell membrane is not easily detached, thus extending the lifespan of the chromatography column.

Rhubarb is an authentic medicinal herb from Gansu Province and one of the commonly used traditional Chinese medicines in clinical practice; It has pharmacological effects such as antibacterial, anti-tumor, anti-inflammatory, antiviral, and lipid-lowering. Many scholars and research groups have confirmed the lipid-lowering effect of rhubarb in previous studies, and our research group has screened 30% ethanol extract as the effective part for lipid-lowering in hyperlipidemic rats, but its specific pharmacological substance basis is still unclear; The research group also prepared a physical adsorption L02 liver cell membrane biological affinity chromatography material, but its cell membrane detachment is severe and its usage is limited. In view of this, in this experiment, APTES modified silica gel was used as a carrier to prepare L02 liver cell membrane biological affinity chromatography material. The target on the cell membrane surface was used to specifically adsorb the chemical components in the 30% ethanol extract of rhubarb. HPLC analysis was performed on the changes in chromatographic peaks of each chemical component before and after adsorption. Based on the peak area change rate of the chromatographic peak, the lipid-lowering active components of rhubarb were quickly screened; And investigate the reusability of the material, providing new ideas for high-throughput screening of the substance basis of rhubarb’s lipid-lowering effect.

Silicone gel undergoes cross-linking and condensation reactions with APTES and glutaraldehyde, and is covalently linked to the cell membrane to prepare L02 cell membrane bio affinity material modified with APTES as a carrier. Scanning electron microscopy and infrared spectroscopy confirm the successful preparation. APTES modified L02 cell membrane bio affinity material was used to adsorb 30% ethanol extract of rhubarb, and the results showed that 11 components could be specifically bound; Including 4 anthraquinone glycosides, 5 anthraquinone glycosides, and 2 unknown components, among which aloe emodin 😯 – β – D-glucoside, rhein 😯 – β – D-glucoside, emodin 😯 – β – D-glucoside, and unknown component 1 are the main specific components that can be used as potential active ingredients for lipid-lowering activity verification. However, due to the numerous targets on the surface of cell membranes, this material is only suitable for preliminary screening of lipid-lowering active ingredients; And in the later stage, LC-MS/MS needs to be used for preliminary structural identification of two unknown components. Compared with the physical adsorption of L02 cell membrane bio affinity materials, this material introduces chemical covalent bonds to some extent, avoiding the detachment of the cell membrane and prolonging the usage times. It can be used for rapid screening of active ingredients in rhubarb for lowering blood lipids, and can also provide reference for rapid screening of other active ingredients for lowering blood lipids.