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Traditional bioassay‐guided investigation of bioactive compounds from natural products comprises critical steps, such as extraction, repeated column separation, and activity assay. Thus, the development of facile, rapid, and efficient technology is critically important. Here, a HepG2 cell‐based extraction method was first developed to rapidly screen potential antitumor compounds from the seeds ofCassia obtusifolia. Then, an online extraction and enrichment–high‐speed counter‐current chromatography (HSCCC) strategy was fabricated to facilely and efficiently isolate target antitumor compounds, which included direct extraction from solid C. obtusifolia, removal of polar interferences, enrichment of target compounds, and preparative isolation by HSCCC using flow rate stepwise increasing mode. After further purification by Sephadex LH‐20 column, five antitumor anthraquinones, aurantio‐obtusin, 1‐desmethylaurantio‐obtusin, chryso‐obtusin, obtusin, and questin, were obtained for structural characterization and bioassay verification. The results may not only provide new perspectives for facile and rapid investigation of bioactive compounds from complex natural products, but also offer a scientific basis for the potential applications of C. obtusifolia.

Prunella vulgaris L. is a well-known traditional Chinese medicine for blood glucose homeostasis and antioxidant potential. Ethyl acetate fraction of P. vulgaris L. demonstrated higher phenolic content (85.53 ± 6.74 mg gallic acid equivalents per gram dry weight), α-glucosidase inhibitory (IC 50 , 69.13 ± 2.86 μg/ml), and antioxidant (IC 50 , 8.68 ± 1.01 μg/ml) activities. However, the bioactive polyphenols responsible for the beneficial properties remain unclear.Here, bioreaction-HPLC-quadrupole-time-of-flight-MS/MS method was developed for rapid, accurate, and efficient screening and identification of polyphenols with α-glucosidase inhibitory and antioxidant activities from P. vulgaris L.Bioactive polyphenols can specifically bind with α-glucosidase or react with 1,1-diphenyl-2-picryl-hydrazyl radical, which was easily discriminated from nonactive compounds. Subsequently, 20 bioactive polyphenols (16 phenyl propionic acid derivatives and four flavonoids) were screened and identified. Furthermore, molecular docking analysis revealed that screened 20 polyphenols bind with the active sites of α-glucosidase through hydrogen bonding and π-π stacking. Density functional theory calculations demonstrated their electron transport ability and chemical reactivity. The in silico analysis confirmed the screened results. In summary, this study provided a valuable strategy for rapid discovering bioactive compounds from complex natural products and offered scientific evidence for further development and application of P. vulgaris L.

Efficient and targeted screening and isolation of bioactive compounds from complex natural products is still a challenging work. Herein, diagnostic ion filtering based high‐performance liquid chromatography–quadrupole time‐of‐flight−tandem mass spectrometry was firstly developed to screen six main iridoid glycosides from Hedyotis diffusa. Then, online extraction‒high‐speed counter current chromatography was proposed for targeted enrichment and preparative isolation using ethyl acetate/n‐butanol/water (4.5:0.5:5, v/v/v) as solvent system. After that, Sephadex LH‐20 column chromatography using methanol as solvent system was selected for further purification of six iridoid glycosides with purities over 98%. They were finally identified as monotropein, desacetylasperuloside acid, asperuloside, 6‐O‐(Z)‐p‐coumaroyl scandoside methyl ester, 6‐O‐(Z)‐feruloyl scandoside methyl ester, and 6‐O‐(E)‐p‐coumaroyl scandoside methyl ester. And their anti‐inflammatory activities were evaluated and confirmed by lipopolysaccharide activated RAW 264.7 macrophages. Obviously, the results provide a scientific basis for the potential applications of H. diffusa, and the developed methodology is efficient and reliable for targeted screening and isolation of bioactive compounds from natural products.

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