Traditional Chinese medicine is rich in chemical components with diverse structures, exhibiting a wide range of activities, high efficiency, and low toxicity. Screening active ingredients from traditional Chinese medicine and natural drugs is an important direction in Chinese medicine research. With the continuous development of biotechnology and chemical separation techniques, many methods and technologies have been applied to the screening of active ingredients in traditional Chinese medicine, such as methods based on the separation and identification of active ingredients, serum pharmacochemistry and serum pharmacology, metabolomics, molecular docking technology, and biochromatography-based screening methods. These new methods for screening active ingredients provide new candidate compounds for new drug development and open up new avenues.

1. Screening Based on Traditional Component Separation and Activity Tracking

The traditional approach to component separation research involves first using phytochemical methods to extract, separate, and identify the chemical components in traditional Chinese medicine. Then, various biological methods are employed along with pharmacological models (such as whole animals, isolated organs, cells, and molecular models) to screen each chemical component for biological activity. Finally, the active ingredients are identified, clarifying the material basis for the efficacy of traditional Chinese medicine. This methodology has been systematically applied to the separation and corresponding biological activity research of chemical components in most single-herb traditional Chinese medicines, achieving remarkable results and successfully discovering new anti-malarial drugs like artemisinin, treating cardiovascular and cerebrovascular diseases with ligustrazine, and treating Alzheimer's disease with huperzine A.

However, this research method is based on the most fundamental level of studying the effective substances of traditional Chinese medicine. It is not only highly blind but also ignores the systematic nature, overall effect, and mutual influence of the biological environment of traditional Chinese medicine. Therefore, the following two issues often arise in the application of this method: first, the loss of biologically active or effective ingredients during the process of separation and purification; second, the abnormal phenomenon of lower activity as the chemical components of traditional Chinese medicine become more purified. For the study of the material basis of efficacy of single traditional Chinese medicines, this research model still plays an important role because it simplifies the research object and is highly operable. Due to the complex targets and wide range of functions and indications of traditional Chinese medicine, screening with a single or limited number of activity indicators cannot fully reflect the material basis of its efficacy, especially in the study of the material basis of traditional Chinese medicine compound formulations. For compound formulations with many herbs, using this research method cannot elucidate the compatibility characteristics of traditional Chinese medicine compounds or reflect the advantages of compound formulations in treating diseases. To address the issues of this research model, some scholars have expanded the level of separation and analysis, considering the complex components of single herbs or traditional Chinese medicine formulas as combinatorial chemical libraries or chemical substance groups. They analyze and standardize different substance groups using modern instrumental analysis methods, then screen for active ingredients in each substance group to identify the active components.

2.Screening Based on Serum Pharmacochemistry and Serum Pharmacology

Based on the hypothesis that blood-entering components may be the active ingredients of traditional Chinese medicine, serum pharmacochemistry research employs the method of collecting serum samples after oral administration of traditional Chinese medicine and separating and identifying the migrating components from the serum. Some scholars have conducted preliminary exploration on the serum of rats after intragastric administration of Shenghua Tang (a traditional Chinese medicine formula) and discovered nine migrating components in the blood, six of which are prototype components and three are metabolites. These migrating components in the blood may be the basis for the action of Shenghua Tang in the human body, and further research into them will help elucidate the effective ingredients and mechanism of action of Shenghua Tang. Using HPLC-DAD/MS methods, the blood-entering components of Danggui Buxue Tang (a traditional Chinese medicine formula) were studied, and 46 blood-entering components were found in the serum of rats after oral administration. Among them, 10 prototype components were identified, and 21 potential metabolic components were distinguished. After administering Shuangdan Granules to rats, the migrating components in the rat serum were analyzed, and 16 prototype components and 5 metabolic components from Shuangdan Granules were identified, further elucidating the material basis for Shuangdan Granules' pharmacological effects. To a certain extent, the research methods of serum pharmacochemistry and serum pharmacology reflect the change process of traditional Chinese medicine chemical components in the body and the interaction of each component with the organism. However, there are still some limitations: ① There are certain technical bottlenecks in the enrichment, separation, and measurement of components with low content and low blood concentration; ② Not all blood-entering components are responsible for the pharmacological effects of drugs, and further research is needed on the identified blood-entering components to clarify the material basis and mechanism of pharmacological effects; ③ Due to the use of animals in experiments, there are many influencing factors, such as time, blood collection volume, and individual differences; ④ Metabolites in the body undergo corresponding metabolic changes over time.

3.Screening Based on Molecular Docking Technology

Molecular docking technology is an important technique in the field of computer-aided drug design. It can not only study the interaction between active ingredients in drugs and targets, but also be used to discover and optimize lead compounds. Molecular docking technology, also known as molecular docking virtual screening technology, utilizes one or more protein targets to screen traditional Chinese medicine chemical components or natural product databases, aiming to find compounds that specifically bind to the target protein and ultimately select lead compounds with certain activity. Currently, molecular docking has been widely applied in the screening of active ingredients in traditional Chinese medicine. Traditional Chinese Medicine Chemical Database (TCMD) and China Natural Product Database (CNPD) have been successfully developed and applied to the design of lead compounds, becoming reliable tools for virtual screening. Some scholars have established a pharmacophore model of matrix metalloproteinase-9 (MMP-9) inhibitors based on experimental structures of multiple receptor-ligand complexes. Using this pharmacophore model and molecular docking for virtual screening, four novel MMP-9 inhibitors were discovered from natural product databases. This technology is also currently being applied to virtual screening of active ingredients in compound prescriptions. Some scholars have constructed a structural ligand molecular library of 21 known major chemical components and metabolites in Liuwei Dihuang Pill (a traditional Chinese medicine formula) and analyzed the effective substances that inhibit α-glucosidase in Liuwei Dihuang Pill using virtual screening techniques. Two tetracyclic triterpenoid compounds with low binding energy were found, successfully predicting the active ingredients of Liuwei Dihuang Pill in the treatment of type 2 diabetes. However, current molecular docking predictions for highly flexible ligands are not very reliable. Additionally, as the experiments are conducted using computers, the binding sites of proteins cannot be accurately determined.

4.Screening and Analysis of Active Compounds using Biochromatography

Biochromatography is a chromatographic technique that immobilizes biological macromolecules such as target proteins, receptors, and enzymes onto a specific carrier as the stationary phase of the chromatography. By adding corresponding traditional Chinese medicine extracts to the mobile phase, this technique enables the elution and separation of natural drug chemical components that specifically bind to the target biological macromolecules. According to literature reports, α-acid glycoprotein and human serum albumin (HSA) are commonly used as the stationary phases in biochromatography.

Cell membrane chromatography involves immobilizing cell membranes onto a material carrier while retaining their biological characteristics as the stationary phase. This fixed phase is used to study the interaction between drugs and membrane receptors, specifically screening for effective chemical components in traditional Chinese medicine, and analyzing them using membrane chromatography techniques. This technology is widely used in the study of drug-cell membrane receptor affinity and interaction, thereby screening for the pharmacodynamic basis of traditional Chinese medicine. For example, using cardiac muscle cell membrane chromatography, the retention characteristics of four chemical components in the traditional Chinese medicine Chuanxiong have been screened and analyzed, and the types and characteristics of related receptor interactions have been studied.

Cell extraction is a target cell extraction method based on whether the chemical components of traditional Chinese medicine have specific affinity with cells, using active cells as the separation carrier. When combined with high-performance liquid chromatography techniques, the chemical components before and after binding can be separated, analyzed, and compared by observing changes in the peak areas of biological fingerprint spectra. By comparing the components bound to active cells with those in the cell lysate, and using corresponding analytical methods for identification, potential pharmacodynamic components that interact with active cells can be screened from complex traditional Chinese medicine components.

5.Screening Based on Metabolomics Methods

Metabolomics, following genomics and proteomics, is one of the indispensable and fundamental disciplines in systems biology research. According to research reports, metabolomics strategies have been used to analyze the metabolite profiles of rats, the chemical fingerprint of Epimedium brevicornum, and the components and metabolite profiles after their entry into the body. By further comparing and analyzing the metabolic profiles of serum and urine samples from normal, model, and Epimedium brevicornum ethanol extract-treated rat groups, it was discovered that icariin and epimedin C may be the main pharmacodynamic components of Epimedium brevicornum.

A study was conducted to investigate the metabolic distribution in the liver of rats before and after administering Chuanxiong (Ligusticum wallichii). Metabolites such as pinosylvin and ethyl ferulate were identified using HPLC-MS, UV-Vis, and IR techniques. The anti-tumor activities of these two metabolites were evaluated using the MTT method, and the results showed that pinosylvin exhibited strong anti-tumor activity, while ethyl ferulate did not show significant activity.

As China's strategy for modernizing traditional Chinese medicine (TCM) progresses, a group of TCMs with clinically proven efficacy and high safety are gradually gaining international recognition. However, the research on the pharmacodynamic substances of TCM is a major bottleneck constraining the development of TCM modernization and is also a hot and difficult topic in the field of TCM research. Given the characteristics of TCM, which includes multiple components, multiple targets, and synergistic effects, the rapid and efficient development of screening and identification technologies for multiple active ingredients is of significant importance in advancing the modernization of TCM.

References

[1] Bi Suxia, Wang Ying, Li Dapeng, et al. Research progress on screening methods for active ingredients of traditional Chinese medicine [J]. Strait Pharmaceutical Journal, 2018, 30(11): 43-46.

[2] Xu Qing, Li Zhi, Wan Meixu, et al. Research progress in new screening technologies for active ingredients of traditional Chinese medicine [J]. Drug Evaluation Research, 2021, 44(07): 1541-1547.

[3] Fu Renjie, Qi Jin, Yu Boyang. Research progress in screening techniques for pharmacodynamically active ingredients of traditional Chinese medicine [J]. Guangzhou Chemical Industry and Technology, 2020, 48(17): 1-3+23.

About the Author

Xiaomichong, a pharmaceutical quality researcher, has been committed to pharmaceutical quality research and drug analysis method validation for a long time. Currently employed by a large domestic pharmaceutical research and development company, she is engaged in drug inspection and analysis as well as method validation.