Discovery of anti-renal fibrosis quality markers from Piperis longi fructus: An integrated strategy of hollow fibre cell trapping method coupled with liquid chromatography-mass spectrometry and in vitro efficacy verification

Modern pharmacological research has shown that the extract of traditional Chinese medicinal herb of Piperis longi fructus has anti-renal fibrosis activity, which made it potentially useful as a therapeutic medicine for renal fibrosis. To enhance the quality control of Piperis longi fructus, a hollow fiber cell trapping (HFCT) was established to investigate its anti-renal fibrosis quality markers (Q-markers). Human kidney 2 cells or transforming growth factor-β (TGF-β)-induced renal fibrosis model cells were loaded into the lumen of the hollow fiber with scanning electron microscopy confirming the cells adherence and proliferation on the inner wall of the fiber. Then the fiber was immersed in the extract of Piperis longi fructus to screen the active components. Cell counting kit-8 assay and flow cytometry were used to detect the effects of Piper longum extract on cell proliferation and HFCT procedure on Apoptosis, respectively. The critical variables were investigated and optimized to ensure cell viability and reliable active screening. Trapped components were separated and identified using liquid chromatography-mass spectrometry, and cell trapping factor with an asterisk (CTF*) was proposed to assess the binding strength between the model cell and the active ingredients. Western blot analysis was used to study the active effects of high-CTF* components (piperlonguminine, piperlongumine and piperine) on renal fibrosis-related proteins including Collagen type I (COL-I) and α-smooth muscle actin (α-SMA). Finally, piperlonguminine and piperlongumine were determined as anti-renal fibrosis Q-markers of Piperis longi fructus. This study demonstrated that HFCT is a simple and cost-effective technique for rapid screening of quality markers in traditional Chinese medicines.