Identification of Transient Receptor Potential Vanilloid 3 Antagonists from Achillea alpina L. and Separation by Liquid-Liquid-Refining Extraction and High-Speed Counter-Current Chromatography

Abstract

Bioassay-guided fractionation of the ethanol extract of whole herbs of Achillea alpina led to the isolation of isochlorogenic acids A and B as transient receptor potential vanilloid 3 (TRPV3) channel antagonists by using a calcium fluorescent assay. The structures were identified by spectroscopic analysis and the inhibitory activities of isochlorogenic acids A and B were confirmed by whole-cell patch clamp recordings of human embryonic kidney 293 (HEK293) cells expressing human TRPV3. Molecular docking results revealed that these two compounds reside in the same active pocket of human TRPV3 channel protein with lower binding energy than the agonist 2-aminoethoxydiphenyl borate (2-APB). High-speed counter-current chromatography (HSCCC) coupled with a liquid-liquid extraction approach was successfully established for the separation of isochlorogenic acids A and B from the whole herbs of A. alpina. Ethyl acetate and n-hexane-ethyl acetate-water (3:3:4 and 1:5:4, v/v/v) were selected as liquid-liquid extraction solvent systems to remove high- and low-polarity impurities in the mixture. Sixty g of ethanol extract was refined by solvent partition to yield 1.7 g of the enriched fraction, of which 480 mg in turn obtained 52.5 mg of isochlorogenic acid B (purity 98.3%) and 37.6 mg isochlorogenic acid A (purity 96.2%) after HSCCC with n-hexane-ethyl acetate-water containing 1% acetic acid (1:4:8, v/v/v).

Rapid enrichment and separation of two novel minor phenols from Malus hupehensis utilizing liquid–liquid extraction with three‐phase solvent system and high‐speed counter‐current chromatography based on the polarity parameter

“…As well known, the preparation of minor compounds is the challenge for various conventional solid sorbent-based chromatography techniques. HSCCC has been extensively applied for the separation of various natural compounds, and provided a nice candidate [8][9][10][11][12]. The selection of solvent system with "sweet point" (K = 1.0) is the foremost operation before the HSCCC separation.…”

“…In accordance with the concept "like dissolves like," some impurities are distributed in hdrophobic UP and hydrophilic LP. The remarkable enrichment of moderate polar molecules in moderately polar MP partly prevents serious loss due to repeated extraction in two-phase solvent systems [8,12,35]. Finding a suitable TPSS through repeated HPLC analysis is extremely difficult.…”

“…In the previous study, LLE showed potential in the isolation of target compounds from crude examples through efficient and simple means [11][12][13]. The traditional LLE mainly depends on two-phase solvents, and the resulting low selectivity in extraction causes a large loss of minor compounds [14][15][16][17].…”

“…Three-phase solvent system (TPSS), an emerging alternative, is divided into three mutually immiscible phases with different polarity, namely, the hydrophobic upper phase (UP), moderately polar middle phase (MP), and hydrophilic lower phase (LP). The rapid enrichment of four minor bufadienolides from toad meat and bafilomycin A1 from Streptomyces lohii fermentation indicates the feasibility of extracting target compounds with an optimized TPSS [9,12]. The essence of this method is to enrich target compounds in one phase on the basis of polarity (p).…”

Analytical, Preparative, and Industrial-Scale Separation of Substances by Methods of Countercurrent Liquid-Liquid Chromatography

“…The CCC methods are the most widely used in the field of pharmaceutical and natural product analytical and preparative-scale separations (alkaloids, peptides, drugs, chiral compounds, etc.) [ 17 , 27 , 28 , 29 , 30 , 38 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 ,…”