An Efficient Strategy Based on Liquid–Liquid Extraction with Three-Phase Solvent System and High Speed Counter-Current Chromatography for Rapid Enrichment and Separation of Epimers of Minor Bufadienolide from Toad Meat

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).

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Efficient Preparation of Bafilomycin A1 from Marine Streptomyces lohii Fermentation Using Three-Phase Extraction and High-Speed Counter-Current Chromatography

Marine Drugs ◽

10.3390/md18060332 ◽

2020 ◽

Vol 18 (6) ◽

pp. 332

Author(s):

Ye Yuan ◽

Xiaoping He ◽

Tingting Wang ◽

Xingwang Zhang ◽

Zhong Li ◽

...

Keyword(s):

Crude Extract ◽

High Speed ◽

Solvent System ◽

Bafilomycin A1 ◽

Middle Phase ◽

Two Phase ◽

Rapid Separation ◽

Lower Phase ◽

Three Phase ◽

Counter Current

An efficient strategy was developed for the rapid separation and enrichment of bafilomycin A1 (baf A1) from a crude extract of the marine microorganism Streptomyces lohii fermentation. This strategy comprises liquid−liquid extraction (LLE) with a three-phase solvent system (n-hexane–ethyl acetate–acetonitrile–water = 7:3:5:5, v/v/v/v) followed by separation using high-speed counter-current chromatography (HSCCC). The results showed that a 480.2-mg fraction of baf A1-enriched extract in the middle phase of the three-phase solvent system was prepared from 4.9 g of crude extract after two consecutive one-step operations. Over 99% of soybean oil, the main hydrophobic waste in the crude extract, and the majority of hydrophilic impurities were distributed in the upper and lower phase, respectively. HSCCC was used with a two-phase solvent system composed of n-hexane–acetonitrile–water (15:8:12, v/v/v) to isolate and purify baf A1 from the middle phase fraction, which yielded 77.4 mg of baf A1 with > 95% purity within 90 min. The overall recovery of baf A1 in the process was determined to be 95.7%. The use of a three-phase solvent system represents a novel strategy for the simultaneous removal of hydrophobic oil and hydrophilic impurities from a microbial fermentation extract.

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Preparative Separation of Alkaloids from Stem of Euchresta tubulosa Dunn. by High-Speed Counter-Current Chromatography Using Stepwise Elution

Molecules ◽

10.3390/molecules24244602 ◽

2019 ◽

Vol 24 (24) ◽

pp. 4602 ◽

Cited By ~ 3

Author(s):

Weixin Li ◽

Huan Wang ◽

Aiwen Dong

Keyword(s):

High Speed ◽

Methylene Chloride ◽

Liquid Extraction ◽

Preparative Separation ◽

Target Compound ◽

Two Phase ◽

Chinese Herbal ◽

Liquid Liquid Extraction ◽

Counter Current ◽

First Time

Euchresta tubulosa Dunn. is a Chinese herbal medicine with biological activity, but there are few studies on its components at present. Alkaloids in the stem of Euchresta tubulosa Dunn. were isolated and purified by high-speed counter-current chromatography (HSCCC) using stepwise elution. First of all, liquid-liquid extraction (methylene chloride-methanol-water, 5:1:4, v/v) was used for the preliminary enrichment. According to the partition coefficient (K) of a target compound in a series of different two-phase solvents, the final result was that carbon tetrachloride-methylene chloride-methanol-water (2:3:3:2, v/v) (1) and methylene chloride-methanol-water (5:3:2, v/v) (2) were suitable for the HSCCC using stepwise elution. As a result, the purity was all higher than 93% and matrine (1), oxymatrine (2), N-formyl cytisine (3), and N-acetyl cytisine (4) can be eluted at one time by this mode. Cytisine-type alkaloids were isolated for the first time in this plant. Finally, the applicability of the mode was verified.

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The separation of flavonoids from Pongamia pinnata using combination columns in high-speed counter-current chromatography with a three-phase solvent system

Journal of Chromatography A ◽

10.1016/j.chroma.2013.09.010 ◽

2013 ◽

Vol 1315 ◽

pp. 80-85 ◽

Cited By ~ 17

Author(s):

Hao Yin ◽

Si Zhang ◽

Lijuan Long ◽

Hang Yin ◽

Xinpeng Tian ◽

...

Keyword(s):

High Speed ◽

Solvent System ◽

Pongamia Pinnata ◽

Three Phase ◽

Counter Current

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Liquid–liquid/solid three-phase high-speed counter-current chromatography based on O-carboxymethyl chitosan-functionalized multi-walled carbon nanotubes as solvent additive

RSC Advances ◽

10.1039/c4ra03222e ◽

2014 ◽

Vol 4 (50) ◽

pp. 26231-26239 ◽

Cited By ~ 3

Author(s):

Yanjuan Liu ◽

Yongfeng Liu ◽

Gaohong Wang ◽

Duolong Di

Keyword(s):

High Speed ◽

Solvent System ◽

Carboxymethyl Chitosan ◽

System P ◽

Three Phase ◽

Multi Walled Carbon Nanotubes ◽

Carbon Nano Tubes ◽

Solvent Additive ◽

Walled Carbon Nanotubes ◽

Counter Current

A new liquid–liquid/solid three phase HSCCC system was established based on O-carboxymethyl chitosan-functionalized multi-walled carbon nano tubes as additive of biphasic solvent system.

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