A new strategy by converging ultrafiltration high‐performance liquid chromatography with ultraviolet and mass spectrometry and pH‐zone‐refining counter‐current chromatography was developed for the rapid screening and separation of potential acetylcholinesterase inhibitors from the crude alkaloidals extract of Zanthoxylum nitidum. An optimized two‐phase solvent system composed of chloroform/methanol/water (4:3:3, v/v) was used in this study. And, in the optimal solvent system, 45 mM hydrochloric acid was added to the aqueous stationary phase as the retainer, while 5 mM triethylamine was added to the organic mobile phase as the eluter. As a result, with the purity of over 95%, five alkaloids including jatrorrhizine (1, 340 mg), columbamine (2, 112 mg), skimmianine (3, 154 mg), palmatine (4, 226 mg), and epiberberine (5, 132 mg) were successfully purified in one step from 3.0 g crude alkaloidals extract. And their structures were identified by ultraviolet, mass spectrometry, 1H and 13C NMR spectroscopy. Notably, compounds 2, 4 and 5 were firstly reported in Z. nitidum. In addition, acetylcholinesterase inhibitory activities of compounds 1–5 were evaluated, and compounds 3, 4 and 5 exhibited stronger acetylcholinesterase inhibitory activity (IC50 values at 8.52 ± 0.64, 14.82 ± 1.21 and 3.12 ± 0.32 μg/mL, respectively) than berberine (IC50 value at 32.86 ± 2.14 μg/mL, positive control). The results indicated that the proposed method is an efficient technique to rapidly screen acetylcholinesterase inhibitors from complex samples, and could be served as a large‐scale preparative technique for separating ionizable active compounds.

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Introduction Potentilla kleiniana Wight et Arn is widely used as a herbal medicine to treat type 2 diabetes. However, detailed information about its active compounds is lacking. Objective To develop an efficient method for the rapid screening and separation of α‐glucosidase inhibitors from Potentilla kleiniana Wight et Arn. Methodology Potential α‐glucosidase inhibitors from Potentilla kleiniana Wight et Arn were rapidly screened out through ultrafiltration high‐performance liquid chromatography mass spectrometry (HPLC‐MS), and then followed by a target‐guided high‐speed counter‐current chromatography (HSCCC) separation using two‐phase solvent systems composed of n‐hexane/ethyl acetate/methanol/water (1:10:1:10, v/v/v/v and 1:10:5:6, v/v/v/v), and adopting increasing flow‐rate from 1.5 to 3.0 mL/min after 200 min. Their structures were identified by ultraviolet (UV), MS, proton nuclear magnetic resonance (1H‐NMR) and carbon‐13 (13C)‐NMR, and their α‐glucosidase inhibitory activities were assessed by in vitro assay. Results Five α‐glucosidase inhibitors including gallic acid (25.7 mg, 98.2%, 1), brevifolincarboxylic acid (9.86 mg, 95.3%, 2), ethyl evifolincarboxylate (13.26 mg, 97.6%, 3), 3,3′‐di‐O‐methylellagic acid‐4′‐O‐β‐d‐glucopyranoside (16.26 mg, 95.1%, 4), and 3,3′‐di‐O‐methylellagic acid (10.54 mg, 96.8%, 5) were successfully purified from 250 mg n‐butanol extract in a single run. Compounds 1, 2, 4 and 5 exhibited stronger α‐glucosidase inhibitory activities[half maximal inhibition concentration (IC50) values at 173.41 ± 6.35, 323.46 ± 8.08, 44.63 ± 2.50, and 20.73 ± 2.56 μM, respectively] than acarbose (IC50 value at 332.12 ± 5.52 μM, reference compound). Conclusions Notably, compounds 2–5 were reported in the Potentilla kleiniana Wight et Arn for the first time. The results indicated that the proposed method could be applied for the rapid screening and preparative separation of α‐glucosidase inhibitors from a complex matrix.

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The leaves of Malus hupehensis have a strong antioxidant activity and are commonly consumed as a healthy tea. However, detailed information about its antioxidants is incomplete. Herein, we developed an effective strategy based on combining off-line two-dimensional high-performance liquid chromatography with ultraviolet and tandem mass spectrometry detection with a 1,1'-diphenyl-2-picrylhydrazyl assay to rapidly screen and identify the antioxidants from the leaves of M. hupehensis. In the orthogonal two-dimensional liquid chromatography system, a Venusil HILIC column was used for the first dimension, while a Universil XB-C column was installed in the second dimension. As a result, 32 antioxidants, including ten dihydrochalcones, two flavanones, nine flavonols, four flavones, and seven phenolic acids were tentatively identified, out of which 23 compounds, as far as we know, were isolated and characterized from the leaves of M. hupehensis for the first time. To the best of our knowledge, this is the first systematic investigation of the antioxidants from the leaves of M. hupehensis. The results indicated that the proposed method is an efficient technique to rapidly investigate antioxidants, especially for coeluted and minor compounds in a complex system.

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