Large-Scale Preparative Isolation of Rosavin from Rhodiola rosea via Ion Liquids MAE and Subsequent Flash Adsorption Chromatography

Rosavin was a major active compound from Rhodiola rosea. It is known that it possesses stimulant, antidepressant, and anticancer properties. In the present study, a two-step flash column chromatography has been developed for the large-scale preparative separation and purification of rosavin from the extracts of Rhodiola rosea, which were obtained by microwave assisted extraction (MAE) using ion liquids (ILs) as extraction solvent under the optimized conditions (microwave power 1200 W, extraction time 6 min, solid-liquid ratio 1:20, microwave temperature 120°C) resulting from the analysis of the D-optimal design. In the first separation, the Rhodiola rosea extract was loaded into polyamide and HPD-200 macroporous resin flash columns in series for efficient enrichment of rosavin. In the second one, the rosavin-rich crude sample after the serial column chromatography was subjected to final purification by silica gel flash chromatography. After the two-step separation, 529.1 mg of rosavin was obtained at a purity of 98.2% and a recovery of 60.6%. The separation process can provide a new method for large-scale separation and purification of rosavin for its pharmaceutical and practical use.     

Extraction of Astaxanthin from Shrimp Waste using Response Surface Methodology and a New Hybrid Organic-Inorganic Monolith

A 17-run Box-Behnken design (BBD) with three factors was used to improve the conditions for extracting astaxanthin from shrimp waste. The astaxanthin level was determined by high performance liquid chromatography (HPLC) using a C₁₈ column and a UV detector at 476 nm. The mean extraction yield of astaxanthin at the improved conditions (extraction time 1.4 h, extraction temperature 72.4°C, and liquid-solid ratio 27.3) was 98.7 ± 2.6 µg g^?1. A solid-phase extraction (SPE)-HPLC method was developed with a new hybrid organic-inorganic hybrid monolith for further purification of astaxanthin from shrimp waste. The SPE recoveries were ranging from 81.3 ± 2.4% to 86.5 ± 3.3%, and the intra-day and inter-day relative standard deviations (RSDs) of the proposed method were less than 4.3 ± 0.5% and 4.8 ± 0.2%, respectively. BBD increased the extraction efficiency and shortened extraction times significantly. SPE-HPLC with hybrid monolith showed good selectivity and purified astaxanthin from shrimp waste.     

Separation and Purification of Two Isomeric Saponins from Albiziae Cortex by High-Speed Counter-Current Chromatography and Preparative High-Performance Liquid Chromatography

Following constituents’ enrichment steps with the AB-8 macroporous resin, silica gel, and ODS columns, high-speed counter-current chromatography (HSCCC) and preparative HPLC were successfully used for the isolation and purification of two complex isomeric saponins including a new one from albiziae cortex. The two-phase solvent system used for separation was composed of n -hexane/ n -butanol/water (1:10:5, v/v/v ). A total of 8.2 mg julibroside J _{5 a} and 11.6 mg julibroside J ₅ with purity of higher than 98%, respectively, as determined by HPLC-ELSD were obtained from the constituents enriched fraction (475.4 mg) of albiziae cortex. Their structures were identified by HR-MS, ¹ H NMR ¹³ C NMR, and 2D NMR. This is the first ever report on the separation of complex isomeric saponins from albiziae cortex by HSCCC.     

One Step Purification of Piperine Directly from Piper nigrum L. by High Performance Centrifugal Partition Chromatography

Abstract

In this study, a preparative high performance centrifugal partition chromatography (HPCPC) method for isolation and purification of the bioactive component piperine directly from the ethanol extract of Piper nigrum L. was successfully established by using n-hexane-ethyl acetate-methanol-water as the two-phase solvent system. The upper phase of n-hexane-ethyl acetate-methanol-water (6:5:6:5, v/v) was used as the stationary phase of CPC. Under the optimum conditions, 40 mg of piperine at 98.5% purity, as determined by HPLC, was yielded from 300 mg of the crude extract in a single CPC separation. The peak fraction of CPC was identified by ¹H NMR and ¹³C NMR.     

Preparative Separation of Glabridin from Glycyrrhiza glabra L. Extracts with Macroporous Resins

Abstract

In the present study, the performance and adsorption characteristics of five macroporous resins for the separation of glabridin from Glycyrrhiza glabra L. have been evaluated. The adsorption and desorption properties of glabridin on macroporous resins including HPD100, HPD300, HPD800, NKA and H103 were compared. HPD100 resin offered the best adsorption and desorption capacities based on the research results. Both Langmuir and Freundlich isotherms were used to describe the interactions between the solutes and resins at different initial concentrations. Dynamic adsorption and desorption experiments on HPD100 resin packed column were conducted to optimize the separation process of glabridin from licorice extracts. After the treatment with stepwise elution on HPD100 resin, the content of glabridin in the product increased from 0.21% to 32.2% which is 153-fold higher than it in G. glabra L. roots and the recovery yield was 79.7%. The results indicated the good ability of HPD100 resin for separation glabridin and the study may provide scientific references for the large-scale glabridin production from G. glabra L. or other plants extracts.     

Development of a pre‐purification process for homoharringtonine

A novel pre‐purification method was developed for producing homoharringtonine from Cephalotaxus koreana, giving high purity and yield. The simple, efficient procedure involved biomass extraction, liquid–liquid extraction, and synthetic adsorbent treatment, followed by low‐pressure chromatography. The use of active clay treatment and silica gel low‐pressure chromatography in the pre‐purification process allowed for the rapid, efficient separation of homoharringtonine from interfering compounds and, compared with alternative processes, increased the yield and purity of crude homoharringtonine for subsequent high‐performance liquid chromatography (HPLC) purification. Homoharringtonine of over 52% purity could be obtained simply with high yield from biomass using this pre‐purification method, while minimizing solvent use and the scale and complexity of HPLC operations for homoharringtonine purification. Copyright © 2005 Society of Chemical Industry     

Extraction of Glycyrrhizic Acid and Glabridin from Licorice

The extraction and separation conditions of glycyrrhizic acid and glabridin from licorice were investigated. By changing the different extraction solvents, procedures, times and temperature, the optimum extraction condition was established: the used of ethanol/water (30:70, v/v) as an extraction solvent, and 60 min dipping time under 50°C. The extracts of licorice were separated and determined by reversed-phase high performance liquid chromatography with a methanol/water (70:30, v/v, containing 1% acetic acid) as the mobile phase. Under the optimum extraction condition, 2.39 mg/g of glycyrrhizic acid and 0.92 mg/g of glabridin were extracted from Chinese licorice and the recoveries were 89.7% and 72.5% respectively.     

Using High-Performance Counter-Current Chromatography Combined with Preparative High Performance Liquid Chromatogramphy for the Separation of Bioactive Compounds from the Water Extract of Gentiana macrophylla Pall

In this paper, a combined high performance counter-current chromatography (HPCCC) and preparative high-performance liquid chromatography (HPLC) method was employed for rapid separation and enrichment of bioactive constituents from a water extract of Gentiana macrophylla Pall. With a two phase solvent system composed of ethyl acetate-n-butanol-water-acetic acid (2: 3: 5: 0.6, v/v), the water extract of G. macrophylla Pall was fractionated into six fractions with three targets isolated and four others highly concentrated, which were then further purified by preparative-HPLC. As a result, 37 mg deglucoserrulatoside, 22.4 mg loganic acid, 3.9 mg isoorientin, 22.4 mg swertiamarin, 52.3 mg gentiopicroside, 27.5 mg sweroside, and 7.9 mg macrophylloside D with the purity of 95.3%, 90.2%, 98%, 98%, 99.2%, 98.8%, and 98.4%, respectively, were isolated from the water extract of Gentiana macrophylla Pall. The structures were confirmed by UV spectra, MS, as well as NMR measurements.     

One Step Large-Scale Separation and Purification of Rutin from Boenninghausenia sessilicarpa by Countercurrent Chromatography

In this study, a preparative countercurrent chromatography (CCC) method for isolation and purification of the bioactive component rutin directly from the ethanol extract of Boenninghausenia sessilicarpa was successfully established by using n-butanol-ethyl acetate-water as the two-phase solvent system. The upper phase of n-butanol-ethyl acetate-water (4:1:5, v/v) was used as the stationary phase of CCC. Under the optimum conditions, 112 mg of rutin at 98.6% purity was obtained from 2.0 g of the crude extract in a single CCC separation. The peak fraction of CCC was identified by negative ESI, ¹H NMR, and ¹³C NMR.     

An Efficient Strategy Based on Macroporous Resins and Semi-Preparative High Performance Liquid Chromatography for Rapid Separation of Five Flavonoids Components From Flaveria Bidentis(L.) Kuntze

In this study, a simple, rapid, and efficient purification method of five major flavonoids from crude Flaveria bidentis extracts was established. Five flavonoids components were initially obtained by ethanol-water extraction of Flaveria bidentis (L.) Kuntze, followed by using D4020 resin-based column chromatography and semi-preparative high performance liquid chromatography. 9.3 mg hyperoside, 6.5 mg patuletin-3-O-glucoside, 1.7 mg isorhamnetin 3-sulphate, 8.4 mg astragalin, and 4.9 mg 6-methoxykaempferol-3-O-galactoside at high purity of over 94% were obtained from 580 mg Flaveria bidentis extracts. This method is more efficient than HSCCC comparing with the results of these two methods regarding analysts’ purity, recovery, and running time.     

Application of High-Speed Counter-Current Chromatography Preparative Separation of Flavone C-Glycosides From Lophatherum gracile

Preparative high-speed counter-current chromatography (HSCCC) was used to separate and purify bioactive constituents from the stems and leaves of Lophatherum gracile Brongn. Six flavone C-glycosides each at over 95% purity including two new compounds were obtained in one-step separation by HSCCC with an optimized two-phase solvent system composed of ethyl acetate-n-butanol-ethanol-water at volume ratio of 4:2:1.5:8.5 (v/v/v/v). The experiment yielded 19.9 mg of luteolin 6-C-β-D-galactopyranosiduronic acid (1→2)-β-D-glucopyranoside (1), 28.5 mg of luteolin 6-C-α-L-arabinopyranosyl-7-O-β-D-glucopyranoside (2), 31.5 mg of isoorientin (3), 44.8 mg of orientin (4), 25.3 mg of swertiajaponin (5) and 12.1 mg of apigenin 6-C-β-D-galactopyranosiduronic acid (1→2)-β-D-glucopyranoside (6) from 500 mg of crude extracts. The purity of these compounds was determined by high-performance liquid chromatography (HPLC). Their chemical structures were identified by electron spray ionization mass spectroscopy (ESI-MS), ¹H and ¹³C nuclear magnetic resonance spectroscopy (NMR).     

Isolation and Purification of Two Constitutes from Dendrobium fimbriatum Hook by High-Speed Counter-current Chromatography Using Stepwise Elution

Abstract

Preparative high-speed counter-current chromatography (HSCCC) was successfully used for the isolation and purification of 2-hydroxyethyl caffeate and denhydroshizukanolide from Dendrobium fimbriatum Hook using stepwise elution with a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at (1:1:1:1, v/v) and (3:1:3:1, v/v). Using a preparative unit of the HSCCC centrifuge, about a 100 mg amount of the sample was separated, yielding 13.3 mg of 2-hydroxyethyl caffeate and 18.0 mg of denhydroshizukanolide at a high purity of over 95%. The peak fraction of HSCCC was identified by ¹H NMR and ¹³C NMR.     

Preparative separation of nine flavonoids from Pericarpium Citri Reticulatae by preparative-HPLC and HSCCC

An economical and systematized chromatographic separation process to isolate and purify flavonoids from Pericarpium Citri Reticulatae was developed. Solvent refluxing extraction, macroporous resin column, preparative HPLC and HSCCC (high-speed counter-current chromatography) were used for extraction, fraction, isolation, and purification of nine flavonoids including vicenin-2(42 mg), hesperidin (108 mg), isosinensetin (15 mg), sinensetin (10 mg), tetramethyl-O-isoscutellarein (11 mg), nobiletin (121 mg), 3, 5, 6, 7, 8, 3′,4′-heptamethoxyflavone (34 mg), tangeretin (114 mg), and 5-demethylnobiletin (13 mg) from Pericarpium Citri Reticulatae, and their purities were all above 98%. All these constituents were identified by UV, MS, ¹H NMR, and ¹³C NMR. The procedure and techniques described here can provide a useful reference for the preparative-scale isolation of some flavonoids in a fast, cost-effective, and environmentally-friendly method.     

银杏内酯A和B的分离纯化研究

In this paper a simple preparative method for isolation and purification of ginkgolides A and B was developed,As starting material,a commercially available standardized ginkgo extract (EGb761,containing 24% flavonoid and 6% terpene trilactones) was used,After a pretreatment step,optimized by the uniform design method ,the concentrated intermediate extract with high content of GA and gb( 90%) was separated into the individual terpenes by preparative liquid chromatography eluted with petroleum ether-ethylacetate,Analysis of products was carried out by means of HPLC-ELSD(evaporative light -scattering detector),The results show that ginkgolides A and B are obtained in higher yield and better purity.     

Effective and Preparative Separation of Bioactive Flavonoids From Gynostemma Pentaphyllum Tea Using Elution-Extrusion Counter-Current Chromatography

Elution-extrusion counter-current chromatography (EECCC) was successfully applied for screen and separation of four flavonoids from Gynostemma pentaphyllum tea, a popular herbal tea extract in China. With the hexane/ethyl acetate/methanol/water 5/6/5/6 (v/v) system, 300 mg of G. pentaphyllum tea extract were fractionated on a 180 mL-capacity preparative hydrodynamic CCC column. Satisfactory separation efficiency was achieved, producing milligram-amounts of quercetin, isorhamnetin, and cirsiliol over 90% pure in one EECCC process. Due to the hydrophilic property, the major flavonoid glycoside, rutin, was co-eluted with the solvent front as a mixture. Therefore, another carefully selected biphasic liquid system composed of ethyl acetate/n-butanol/water (4/1/5, v/v) was employed, yielding 35 mg of rutin with 97.1% purity. Structures of all separated compounds were identified by ESI-MS, ¹H NMR, and ¹³C NMR.     

Synthesis of Ultra-high Weight Average Molecular Mass of Poly-L-lactide

High purity in high yield L-lactide was prepared using a new purification method, and poly-L-lactide (PLLA) with ultra-high weight average molecular mass and narrow polydispersity index was synthesized by ring-opening polymerization. The effects of the purification method on the purity and yield of L-lactide were investigated, and the influences of initiator concentration, polymerization temperature and polymerization time on the weight average molecular mass of PLLA were also studied. A synthetic purification method involving a water bath and two times recrystallization could improve the purity of L-lactide to 100%. The yield of L-lactide reached 40.6% and increased 12.1% compared with the recrystallization method. Poly-L-lactide with a weight average molecular mass of about 102.4 × 10⁴ and a polydispersity index of 1.16 was obtained when polymerization was conducted with molar ratio of monomer to initiator ([M]/[I]) of 12000 for 24 h at 140°C.     

Acid-Alkali Extraction of Triterpene Acids from Poria and Preparative Separation by High-Speed Counter-Current Chromatography

A method for the acid-alkali extraction and preparative separation of triterpene acids from poria was established. The triterpene acids were enriched and separated into two fractions after extraction at the optimized pH value. The two fractions were subjected to high-speed counter-current chromatography for the preparative separation of triterpene acids, separately. As a result, dehydropachymic acid, pachymic acid, 3-epi-dehydropachymic acid, poricoic acid B, dehydrotumulosic acid, and 3-epi-dehydrotumulosic acid were obtained with purities of 94.1%, 96.2%, 93.5%, 85.9%, 80.1%, and 93.1%, respectively. The structures were identified by ESI-MS, ¹H NMR, and ¹³C NMR.     

A simple method for extraction and purification of pedunculoside from the dried barks of Ilex rotunda and its inhibitory effect on pancreatic lipase in vitro

A simple and efficient method was developed for the preparation of pedunculoside from the dried barks of Ilex rotunda. Pedunculoside was extracted by heat reflux. The optimal conditions were determined by the response surface methodology (40% ethanol concentration, 14 mL/g solvent/material loading level and 90 min extraction time with the dried barks being extracted twice). After extraction and condensing the crude extract, pedunculoside was directly purified through crystallization in water with the addition of ethyl acetate. Pedunculoside (96.9% purity) was obtained with 48.2% recovery after the extraction and purification. Compared with the reported chromatographic methods, this strategy is simple, eco-friendly, and economical. The purified pedunculoside showed an inhibitory effect on pancreatic lipase in vitro (IC₅₀ = 80.8 μg/mL), suggesting its anti-obesity potential.     

Synthesis and purification of trisulphoindigo and reversed-phase high performance liquid chromatographic determination of trisulphoindigo in FD & C Blue No.2

The effect of temperature on product formation during the sulphonation of indigo with concentrated sulphuric acid and 30 % fuming sulphuric acid was studied with the aid of high performance liquid chromatographic (HPLC) analysis. Trisulphoindigo was prepared by the sulphonation of indigo with concentrated sulphuric acid at 160°C. The di- and tetra-sulphoindigo side products formed during the reaction were removed by preparative HPLC to yield a product of high chromatographic purity. The trisulphoindigo content of the food dye FD & C Blue No. 2 and the purity of commercially available trisulphoindigo were determined by HPLC analysis.     

Separation and Purification of Three High-Purity Isoflavonoids from Belamcanda chinensis (L.) DC. by Supercritical Fluid Extraction and High-Speed Counter-Current Chromatography

Supercritical fluid extraction (SFE) was used to extract three isoflavonoids including irigenin, irisfloretin and dichtomitin from Belamcanda chinensis (L.) DC. The parameters including pressure, temperature, sample particle size, and flow rate of CO₂ were optimized with an orthogonal test. Under the optimized conditions of 15 MPa, 55°C, a sample particle size of 20–40 mesh and CO₂ flow rate of 40 L h^?1. The process was then scaled up by 10 times using a preparative SFE system. The yield of the crude extract from SFE was 4.1%, which contained irigenin, irisfloretin, and dichtomitin 0.71%, 0.49%, and 0.05%, respectively. To compare the extraction methods, Soxhlet Extraction (SE) was performed. The results indicated that SFE was better than SE. Irigenin, irisfloretin, and dichtomitin in the SFE extract were then separated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (2:4:3:3, v/v). From 5.0 g of dry crude extract, 27.8 mg irigenin, 16.4 mg irisfloretin, and 2.1 mg dichtomitin were obtained at purities of 97.1%, 96.4%, and 98.0%, respectively, as determined by HPLC-PDA. These results well indicate that SFE and HSCCC are very powerful techniques for the extraction and purification of irigenin, irisfloretin, and dichtomitin from B. chinensis.