Separation of α‐glucosidase inhibitors from Potentilla kleiniana Wight et Arn using solvent and flow‐rate gradient high‐speed counter‐current chromatography target‐guided by ultrafiltration HPLC‐MS screening

Abstract

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.

Application of choline chloride deep eutectic solvents and high‐speed counter‐current chromatography to the extraction and purification of flavonoids from the thorns of Gleditsia sinensis Lam

“…Generally, a suitable composition for the twophase solvent system is crucial for a perfect HSCCC separation, which relied on satisfactory partition coefficient (K D ) values ranging from 0.5 to 2.0 for the target components. 25,26 Previous studies have identified the flavonoids from the thorns of G. sinensis mainly as flavonoid aglycones as well as a few flavonoid glycosides. Thus, several solvent systems of n-hexane-ethyl acetatemethanol-water, n-hexane-ethyl acetate-n-butanol-water, chloroform-methanol-water, dichloromethane-methanol-water, and dichloromethane-methanol-n-butanol-water with different composition ratios, were applied for testing the K D values of the five main constituents (1)(2)(3)(4)(5) in the DES extracts (Table 2).…”

“…It was noted that extraction with DES-1 achieved a higher yield of flavonoids with peaks 1-4 being 51.6%, 69.8%, 59.1%, and 28.6% higher than the control sample, respectively, while peak 5 being 26.7% less than the control sample. Overall, DES-1 was identified as the most efficient alcohol-based DES for the flavonoids.F I G U R E 1 HPLC chromatogram of flavonoids from the thorns of Gleditsia sinensis at 300nm, extracted by means of different eutectic solvents DES1DES5, and a control extract obtained with methanolwater (75:25)…”

On‐line coupling pressurised liquid extraction with two‐dimensional counter current chromatography for isolation of natural acetylcholinesterase inhibitors from Astragalus membranaceus

“…16,17 Compared with immobilised enzyme screening assay, complex synthesis procedures can be avoided. 18,19 PC12 rat pheochromocytoma cells have been widely used as cellular models of neurodegenerative diseases. 20 Aβ is widely considered to play a central role in the pathogenesis of AD.…”

Rapid screening and purification of potential inhibitors from Medicago sativa by ultrafiltration‐liquid chromatography combined with stepwise flow rate counter‐current chromatography

“…Conversely, in vitro evaluation methods are fast and require only a small amount of samples, making them suitable for analysing a wide variety of active components found in foods and herbs. To overcome these limitations and enhance drug discovery throughput, molecular methods based on high‐affinity and high‐selectivity protein–ligand interactions have been successfully applied 20 . In particular, combined ultrafiltration‐liquid chromatography‐mass spectrometry (UF‐LC–MS) is a powerful method for screening a wide range of biologically active compounds from botanical extracts because the UF step facilitates the separation of ligand–receptor complexes from unbound compounds, while the subsequent LC–MS step can identify the ligands 21 …”