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Introduction: The diversity and complexity of components are important reasons for the unstable efficacy and safety of Chinese materia medica (CMM) in quality control. The good and stable quality control may be related to the quality markers with structural composition of multi-components., Objective: In the present study, we take Eclipta prostrata L. as a representative example. The 11 samples were collected from the different areas in China, and the discrepancy in bioactivity and chemical composition of these samples were compared. DOX (doxorubicin hydrochloride)-induced ICR mice were established for in vivo nephrotic syndrome experiments. The biochemical indicators including 24-h urine protein, triglyceride (TG), etc. were measured and the pathological change of kidney tissue was observed. MPC-5 cells damage model was induced to compare the difference of these samples in bio-activity. High-performance liquid chromatography (HPLC) method for 11 EEP (extract of Eclipta prostrata L.) samples were performed to analyse the content of the quality markers., Results: In vivo experiments, EEP could mitigate the content or activity of urine protein, TG, etc. compared with the positive group (TG content was 2.53 ± 0.39 mmol/L, urinary protein quantification on 35th day was 16.79 ± 2.32 mg). In vitro experiments, CCE (coumestans component of Ecliptae) and EEP had equivalent effects on biochemical indicators such as cell viability, etc. According to the HPLC analysis, the content of wedelolactone was 45.88% and demethylwedelolactone was 23.74% in CCE., Conclusion: The CCE with a ratio of 2:1 can be considered as a quality marker of Eclipta prostrata L.. This research may provide a perspective for quality control of CMM., (© 2020 John Wiley & Sons, Ltd.)