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In this work, pH-zone-refining countercurrent chromatography was investigated in the preparative separation of two bioactive components, oleanolic acid and ursolic acid, from three different natural products, Aralia chinensis, apple peels and Eriobotrya japonica Thunb. Oleanolic acid and ursolic acid are structurally isomeric pentacyclic triterpene acids that are widely distributed in many natural products. However, it was difficult to separate these components with high purity by conventional methods. A biphasic solvent system composed of n-hexane–dichloromethane–methanol–water (7 : 3 : 2 : 8, v/v) was selected, in which an optimized concentration of 10 mmol L−1 trifluoroacetic acid was added in the upper phase as the retainer and 10 mmol L−1 ammonia (with 25–28% NH3) was added in the aqueous phase as the eluter. Consequently, 38.56 mg of oleanolic acid with 99.01% purity was separated from 100 mg of the crude extract of Aralia chinensis, while 65.6 mg of a mixture of ursolic acid (90.98%) and oleanolic acid (6.51%) and 46.6 mg of a mixture of ursolic acid (74.35%) and oleanolic acid (23.61%) were separated from 100 mg of the crude extract of apple peels and 100 mg of the crude extract of Eriobotrya japonica Thunb., respectively, by pH-zone-refining countercurrent chromatography using the above selected biphasic solvent system. The results showed that pH-zone-refining countercurrent chromatography is an efficient method for the preparative separation of pentacyclic triterpene acids from natural products.

An efficient and target-oriented pH-peak-focusing countercurrent chromatographic method was established for large-scale separation of baicalin and wogonoside from the crude exact of traditional Chinese medicinal herb Scutellaria baicalensis Georgi. An optimized two-phase solvent system composed of n-butanol-ethyl acetate-methanol-water (1:4:0.5:5, v/v) was selected. Trifluoroacetic acid (10 mmol/L) was added to the upper organic phase, used as the stationary phase. One liter of the aqueous lower phase was used as the mobile phase for 0-350 min, and then 10 mmol/L ammonia was added to remaining 1 L of the aqueous lower phase and used as the mobile phase for 350-600 min. In total, 493.2 mg of baicalin with 98.6% purity and 88.6 mg of wogonoside with 98.9% purity were obtained from 1.0 g of crude exact of S. baicalensis by countercurrent chromatography in a single run. The acid dissociation constant (pKa) and oil-water partition coefficient values of two components were measured to better understand the mechanism of separation. Results showed that pH-peak-focusing countercurrent chromatography with a polar solvent system added with trifluoroacetic acid could be an efficient method for large-scale isolation of organic acids, which are difficult to separate with conventional countercurrent chromatography due to their poor solubility in non-polar solvents.

Retention behavior of two structural isomeric pentacyclic triterpenic acids, maslinic acid and corosolic acid, was investigated by reverse phase high performance liquid chromatography (HPLC) with hydroxypropyl-β-cyclodextrin (HP-β-CD) as mobile phase additive. Inclusion complexation of maslinic acid, corosolic acid with hydroxypropyl-β-cyclodextrin was evaluated under different concentration of hydroxypropyl-β-cyclodextrin. Apparent formation constant (Km) between methanol and hydroxypropyl-β-cyclodextrin was determined to be 13.82 L mol−1 under 25 °C using UV-spectrophotometry. Two retention models were employed individually for evaluation of inclusion complexation between the two pentacyclic triterpenic acids and hydroxypropyl-β-cyclodextrin. It was found that a higher apparent formation constant (Kf) for corosolic acid and hydroxypropyl-β-cyclodextrin was obtained, 19115 L mol−1, indicating that a greater affinity of hydroxypropyl-β-cyclodextrin with corosolic acid was produced compared with that of maslinic acid, 11775 L mol−1, in the selected mobile phase, and stoichiometric ratio for both of inclusion complex was found to be 1:1. Thermodynamic analysis showed that a negative standard enthalpy change (ΔH) and an entropy change (ΔS*) for analyte transfer were obtained, where ΔH of maslinic acid and corosolic acid was found to be −10.188 kJ mol−1 and −10.650 kJ mol−1, ΔS* of two compounds was −2.092 and −2.180, respectively, indicating that transfer of structural isomers from mobile phase to stationary phase was enthalpically driven. Meanwhile, positive values were obtained for standard enthalpy change and standard entropy change, 136 kJ mol−1 and 274 kJ mol−1 and 536 J mol−1 K−1and 1004 J mol−1 K−1, for inclusion complexation between maslinic acid, corosolic acid and hydroxypropyl-β-cyclodextrin, while negative values were obtained for Gibbs free energy during formation of inclusion complex, −160 kJ mol−1 and −299 kJ mol−1, indicating a spontaneous inclusion reaction happened.

In this work, a three-layer TiO2 composite film consisting of flower-like TiO2 (Flo-TiO2) as overlayer, TiO2 nanotube arrays as interlayer and TiO2 nanoparticle (P25) as underlayer was fabricated as the photoelectrode of dye-sensitized solar cells (DSSCs). Due to the introduction of Flo-TiO2, the three-layer composite film has strong light-scattering ability. Then, we have investigated and compared the photoelectric conversion properties of DSSCs based on three-layer structure (P25/TNT arrays/Flo-TiO2) photoelectrode and double-layer film (P25/TNT arrays) photoelectrode. It is found that DSSCs based on three-layer structure exhibit a high power conversion efficiency of 6.48% compared with the DSSCs composed of double-layer film (5.11%).

In this study, a novel three-layer photoanode with TiO2 nanoparticles (P25) as the underlayer, TiO2 nanotube (TNT) arrays as the interlayer and the hollow TiO2 boxes (TiO2-HB) as overlayer film have been fabricated for application in dye sensitized solar cells (DSSCs). The TiO2-HB was introduced as light-scattering layer due to the hollow structure in DSSCs, which increases the transmission distance of light in the TiO2 nanostructure photoanode and enhances light harvesting efficiency. It was obviously that the three-layer film photoanode cell achieved the higher conversion efficiency and attained the value of 6.03 %, which is 20.6 % higher than the double-layer film cell (η = 5.01 %)that of the P25 particles film as the underlayer and TNT arrays as the overlayer film under simulated AM 1.5 sunlight (constant irradiation of 100 mWcm−2).

Essential oils are an important source of natural antioxidants and multiple methods have been established for evaluation of their overall antioxidant activity, however, the antioxidant activities of their compounds are less investigated. In the present study, the hyphenation of 2,2&prime, diphenyl-1-picrylhydrazyl (DPPH)-gas chromatography (GC)-mass spectrometry (MS) offline and high-speed countercurrent chromatography (HSCCC) is established for efficient screening, identification, and isolation of antioxidants from essential oils and applied to the essential oil of Curcuma wenyujin Y.H. Chen et C. Ling. Five compounds are preliminarily screened as antioxidants using DPPH-GC according to the reduction of GC peak areas of each compound after reaction with DPPH and then identified as eucalyptol (7.66%), camphor (2.34%), &delta, elemene (1.15%), &beta, elemene (7.10%), and curzerene (15.77%) using GC-MS. Moreover, these five compounds are isolated by HSCCC using two solvent systems, n-hexane-acetonitrile-ethanol (5:3:2, v/v) and n-hexane-acetonitrile-acetone (4:3:1, v/v), and subjected to DPPH scavenging assay. Camphor, &delta, elemene, and &beta, elemene show weak DPPH scavenging activity, while curzerene and eucalyptol show moderate DPPH scavenging activity. Notably, a significant synergistic effect on DPPH scavenging is found between curzerene and eucalyptol. The result demonstrated that off-line DPPH-GC-MS coupling CCC is an efficient method for screening, identification, and separation of antioxidant compounds in essential oil

Triterpenoids are the major secondary metabolites and active substances in Ganoderma, considered as the “marker compounds” for the chemical evaluation or standardization of Ganoderma. A response surface methodology was used to optimize the ultrasonic-assisted extraction of triterpenoids. The extraction rate was 7.338 ± 0.150 mg/g under the optimum conditions: 87% ethanol, ratio of solid to liquid (w : v) 1 : 28, and ultrasound extraction time 36 min. Based on the high sensitivity and selectivity of HPLC-LTQ-Orbitrap-MSn, 24 components of triterpenoids were tentatively identified in the negative mode. Then, the global chemical profiling consisting of HPLC and TLC fingerprints generated by ChemPattern™ software was developed for evaluation of Ganoderma species. For fingerprint analysis, 11 peaks of triterpenoids were selected as the characteristic peaks to evaluate the similarities of different samples. The correlation coefficients of similarity were greater than 0.830. The cluster analysis showed a clear separation of three groups, and 11 peaks played key roles in differentiating these samples. The developed global chemical profiling method could be applied for rapid evaluation, quality control, and authenticity identification of Ganoderma and other herbal medicines.

Preparative enantioseparations of α-cyclopentylmandelic acid and α-methylmandelic acid by high-speed countercurrent chromatography (HSCCC) and high performance liquid chromatography (HPLC) were compared using hydroxypropy-β-cyclodextrin (HP-β-CD) and sulfobutyl ether-β-cyclodextrin (SBE-β-CD) as the chiral mobile phase additives. In preparative HPLC the enantioseparation was achieved on the ODS C18 reverse phase column with the mobile phase composed of a mixture of acetonitrile and 0.10 mol L−1 phosphate buffer at pH 2.68 containing 20 mmol L−1 HP-β-CD for α-cyclopentylmandelic acid and 20 mmol L−1 SBE-β-CD for α-methylmandelic acid. The maximum sample size for α-cyclopentylmandelic acid and α-methylmandelic acid was only about 10 mg and 5 mg, respectively. In preparative HSCCC the enantioseparations of these two racemates were performed with the two-phase solvent system composed of n-hexane-methyl tert.-butyl ether-0.1 molL−1 phosphate buffer solution at pH 2.67 containing 0.1 mol L−1 HP-β-CD for α-cyclopentylmandelic acid (8.5:1.5:10, v/v/v) and 0.1 mol L−1 SBE-β-CD for α-methylmandelic acid (3:7:10, v/v/v). Under the optimum separation conditions, total 250 mg of racemic α-cyclopentylmandelic acid could be completely enantioseparated by HSCCC with HP-β-CD as a chiral mobile phase additive in a single run, yielding 105-110 mg of enantiomers with 95-98% purity and 85-90% recovery. But, no complete enantioseparation of α-methylmandelic acid was achieved by preparative HSCCC with either of the chiral selectors due to their limited enantioselectivity. In this paper preparative enantioseparation by HSCCC and HPLC was compared from various aspects.

This work concentrates on the separation mechanism and application of chiral ligand exchange high-speed countercurrent chromatography (HSCCC) in enantioseparations, and comparison with traditional chiral ligand exchange high performance liquid chromatography (HPLC). The enantioseparation of ten aromatic α-hydroxyl acids were performed by these two chromatographic methods. Results showed that five of the racemates were successfully enantioseparated by HSCCC while only three of the racemates could be enantioseparated by HPLC using a suitable chiral ligand mobile phase additive. For HSCCC, the two-phase solvent system was composed of butanol-water (1:1, v/v), to which N-n-dodecyl-L-proline was added in the organic phase as chiral ligand and cupric acetate was added in the aqueous phase as a transition metal ion. Various operation parameters in HSCCC were optimized by enantioselective liquid-liquid extraction. Based on the results of the present studies the separation mechanism for HSCCC was proposed. For HPLC, the optimized mobile phase composed of aqueous solution containing 6 mmol L−1 L-phenylalanine and 3 mmol L−1 cupric sulfate and methanol was used for enantioseparation. Among three ligands tested on a conventional reverse stationary phase column, only one was found to be effective. In the present studies HSCCC presented unique advantages due to its high versatility of two-phase solvent systems and it could be used as an alternative method for enantioseparations.

The optimal extraction condition for extracting quaternary ammonium alkaloid dehydrocorydaline from Corydalis yanhusuo W. T. Wang was investigated using orthogonal experimental design. pH-zone-refining counter-current chromatography (CCC) with normal phase elution was successfully applied to preparative separation of alkaloids from the crude extract of Corydalis yanhusuo. The separation was performed with a biphasic solvent system composed of chloroform (CHCl(3))-methanol (MeOH)-water (2:1:1, v/v), in which the lower organic phase containing 10 mM of triethylamine was used as the mobile phase, while the upper aqueous phase containing 10 mM of hydrochloric acid was used as the stationary phase. The separation mechanism of quaternary ammonium alkaloids using pH-zone-refining CCC was discussed in comparison with standard high-speed CCC. In the present study, the separation of 1.200 g of crude sample yielded 129 mg of dehydrocorydaline and 12 mg of palmatine at a high purity of 94 and 92%, respectively. Recovery for dehydrocorydaline and palmatine was 85 and 86%, respectively.

This work concentrates on a chiral separation technology named biphasic recognition applied to resolution of α-cyclohexylmandelic acid enantiomers by high-speed counter-current chromatography (HSCCC). The biphasic chiral recognition HSCCC was performed by adding lipophilic (−)-2-ethylhexyl tartrate in the organic stationary phase and hydrophilic hydroxypropyl-β-cyclodextrin in the aqueous mobile phase, which preferentially recognized the (−)-enantiomer and (+)-enantiomer, respectively. The two-phase solvent system composed of n-hexane-methyl tert-butyl ether–water (9:1:10, v/v/v) with the above chiral selectors was selected according to the partition coefficient and separation factor of the target enantiomers. Important parameters involved in the chiral separation were investigated, namely the types of the chiral selectors (CS); the concentration of each chiral selector; pH of the mobile phase and the separation temperature. The mechanism involved in this biphasic recognition chiral separation by HSCCC was discussed. Langmuirian isotherm was employed to estimate the loading limits for a given value of chiral selectors. Under optimum separation conditions, 3.5–22.0 mg of α-cyclohexylmandelic acid racemate were separated using the analytical apparatus and 440 mg of racemate was separated using the preparative one. The purities of both of the fractions including (+)-enantiomer and (−)-enantiomer from the preparative CCC separation were over 99.5% determined by HPLC and enantiomeric excess reached 100% for the (±)-enantiomers. Recovery for the target compounds from the CCC fractions reached 85–88% yielding 186 mg of (+)-enantiomer and 190 mg of (−)-enantiomer. The overall experimental results show that the HSCCC separation of enantiomer based on biphasic recognition, in which only if the CSs involved will show affinity for opposite enantiomers of the analyte, is much more efficient than the traditional monophasic recognition chiral separation, since it utilizes the cooperation of both of lipophilic and hydrophilic chiral selectors.

Analytical enantioseparation of five β-substituted-2-phenylpropionic acids by high-performance liquid chromatography with hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral mobile phase additive was established in this paper, and chromatographic retention mechanism was studied. The effects of various factors such as the organic modifier, different ODS C18 columns and concentration of HP-β-CD were investigated. The chiral mobile phase was composed of methanol or acetonitrile and 0.5% triethylamine acetate buffer at pH 3.0 added with 25 mmol L(-1) of HP-β-CD, and baseline separations could be reached for all racemates. As for chromatographic retention mechanism, it was found that there was a negative correlation between the concentration of HP-β-CD in mobile phase and the retention factor under constant pH value and column temperature.

Acteoside,a chemical component of the traditional Chinese medicinal herb Rehmannia glutinosa,was separated from the plant by a combination of macroporous resin column chromatography and high-speed countercurrent chromatography.The static adsorption and desorption of acteoside in the crude extract were in-vestigated using four kinds of macroporous resin,among which the D101 macroporous resin presented the best adsorption and desorption rates toward the compound.The crude extract was then gradient-eluted with increa-sing volume percentages of ethanol,where it was found that the highest content of acteoside was obtained when using 10%(v/v) ethanol eluent,in which the purity of acteoside was increased from 4.9% to 32.6%.Then,the partially purified crude extract (165 mg) was further purified by high-speed countercurrent chroma-tography using a two-phase solvent system consisting of ethyl acetate-n-butanol-water (1:4:5,v/v/v),yielding 45 mg of acteoside with 96% purity.

A method was established for the enantioseparation of atrolactic acids using high performance liquid chromatography with sulfobutyl ether-beta-cyclodextrin (SBE-beta-CD) as the chiral mobile phase additive, and a C18 reversed-phase column. The influences of the different types of cyclodextrin derivatives, the concentration of chiral mobile phase additive, pH value of the mobile phase, the flow rate and column temperature on the peak resolution were investigated. The separation mechanism for atrolactic acids by high performance liquid chromatography using chiral mobile phase additive, as well as the formation of inclusion complex between SBE-beta-CD and atrolactic acid was studied. In addition, the formation constant was calculated. The enantioseparation was carried out on a reversed-phase column YMC-Pack ODS-A C18 (250 mm x 4.6 mm, 5 microm) with chiral mobile phase consisted of 0.1 mol/L phosphate buffer at pH 2.68 containing 25 mmol/L SBE-beta-CD-acetonitrile (90:10, v/v). The detection wavelength was 220 nm. The flow rate was 0.6 mL/min and the column temperature was 30 degrees C. The retention times of atrolactic acids were 26.65 and 28.28 min, and the peak resolution of 1.68 was achieved. The above established method proves to be a simple and economical way for analytical enantioseparation of atrolactic acid with high peak resolution.

Spiral tube assembly counter-current chromatography was successfully applied in enantioseparation of dl -tryptophan using bovine serum albumin as chiral selector. An improved biphasic aqueous–aqueous solvent system 12.0% (w/w) polyethyleneglycol 8000-9.0% (w/w) dibasic potassium phosphate-0.1% ammonia-78.9% water was used as the solvent system for counter-current chromatography, in which bovine serum albumin was predominantly distributed in the lower phase of the two-phase aqueous system. The aqueous–aqueous solvent system gave a very high enantioselectivity for d - and l -tryptophan at α = 2.605 along with distribution ratio D D = 1.200 and D L = 0.461. High peak resolution was obtained for enantioseparation of 2.0 mg of dl -tryptophan by spiral tube assembly counter-current chromatography under room temperature. It was found that 0.1% ammonia added in the aqueous–aqueous solvent system greatly improved the enantioseparations. An unusual extremely broad peak for l -tryptophan was observed during enantioseparations. In order to give an explanation, mass transfer rates of d - and l -enantiomers through the interface between the two phases were measured. It was found that l -tryptophan showed lower mass transfer rate than d -tryptophan. Further discussions were proposed for possible reasons for mass transfer rate difference between the enantiomers.

The enantioseparation of ten mandelic acid derivatives was performed by reverse phase high performance liquid chromatography with hydroxypropyl-β-cyclodextrin (HP-β-CD) or sulfobutyl ether-β-cyclodextrin (SBE-β-CD) as chiral mobile phase additives, in which inclusion complex formations between cyclodextrins and enantiomers were evaluated. The effects of various factors such as the composition of mobile phase, concentration of cyclodextrins and column temperature on retention and enantioselectivity were studied. The peak resolutions and retention time of the enantiomers were strongly affected by the pH, the organic modifier and the type of β-cyclodextrin in the mobile phase, while the concentration of buffer solution and temperature had a relatively low effect on resolutions. Enantioseparations were successfully achieved on a Shimpack CLC-ODS column (150 × 4.6 mm i.d., 5 μm). The mobile phase was a mixture of acetonitrile and 0.10 mol L −1 of phosphate buffer at pH 2.68 containing 20 mmol L −1 of HP-β-CD or SBE-β-CD. Semi-preparative enantioseparation of about 10 mg of α-cyclohexylmandelic acid and α-cyclopentylmandelic acid were established individually. Cyclodextrin-enantiomer complex stoichiometries as well as binding constants were investigated. Results showed that stoichiometries for all the inclusion complex of cyclodextrin-enantiomers were 1:1.

The bioactive iridoid component catalpol was successfully separated by high-speed countercurrent chromatography with high purity from the partially purified crude extract of Rehmannia glutinosa. A polar two-phase solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v) was selected by thin-layer chromatography and run on a preparative scale where the lower aqueous phase was used as the mobile phase with a head-to-tail elution mode. A 105 mg quantity of the partially purified sample containing 39.2% catalpol was loaded on a 270-mL capacity high-speed countercurrent separation column, yielding 35 mg of catalpol at 95.6% purity. The chemical structure of catalpol was determined by comparison with the high-performance liquid chromatography retention time of standard substance as well as the (1)H NMR spectrum.

Camptothecin-20(s)-O-(2-pyrazolyl-1)acetic ester (CPT6) is a novel semi-synthetic analog of camptothecin. In a previous report, CPT6 possessed higher cytotoxic activity in vitro towards human breast tumor MCF-7 cells than topotecan. In this study, the antitumor activity of CPT6 on the human breast tumor MCF-7 cell line was analyzed using the MTT method. The underlying mechanism of CPT6 action was investigated by analyzing the cell cycle distribution, apoptotic proportion, changes in mitochondrial membrane potential, and intracellular Ca2+ concentration using flow cytometry. Nuclear and mitochondrial morphologies were also observed by laser scanning confocal and transmission electron microscopy. DNA damage was observed in MCF-7 cells treated with CPT6. Low-dose CPT6 had a significant cytotoxic effect and could inhibit proliferation and induce apoptosis in MCF-7 cells, possibly through cell nucleus fragmentation and DNA damage. CPT6 thus appears to display potent antitumor activity against human breast tumor MCF-7 cells via the induction of apoptosis, and may be a useful alternative drug for breast cancer therapy.

In an effort to decrease the toxicity of camptothecin (CPT) and improve selectivity for hepatoma and colon cancer cells, bile acid groups were introduced into the CPT 20 or 10 positions, resulting in the preparation of sixteen novel CPT-bile acid analogues. The compounds in which a bile acid group was introduced at the 20-hydroxyl group of CPT showed better cytotoxic selectivity for human hepatoma and colon cancer cells than for human breast cancer cells. Fluorescence microscopy analysis demonstrated that one compound (E2) entered human hepatoma cells more effectively than it did human breast cancer cells. Compound G4 exhibited the best anti-tumour activity in vivo. These results suggested that introduction of a bile acid group at the 20-position of CPT could decrease toxicity in vivo and improve selectivity for hepatoma cells.

A new coumarin, edgeworic acid (1), was isolated from the flower buds of Edgeworthia chrysantha, together with the five known coumarins umbelliferone (2), 5,7-dimethoxycoumarin (3), daphnoretin (4), edgeworoside C (5), and edgeworoside A (6). Their structures were established on the basis of spectral data, particularly by the use of 1D NMR and several 2D shift-correlated NMR pulse sequences (1H-1H COSY, HSQC and HMBC), in combination with acetylation reactions.

An ethanol extract of air-dried flowers of Edgeworthia chrysantha Lindl. was partitioned between water and petroleum, ethyl acetate, and n-butanol. The n-butanol extraction was initially purified by silica gel column chromatography to give a partially purified sample. The bioactive compound rutin, along with nicotiflorin, were successfully separated from the partially purified sample by high-speed counter-current chromatography. The two compounds were isolated from the plant of Edgeworthia genus for the first time. The two-phase solvent system used was composed of ethyl acetate-n-butanol-water at an optimized ratio of 4:1:5 (v/v/v). High-speed counter-current chromatography yielded, from 108 mg of the partially purified extract, 53 mg rutin and 32 mg nicotiflorin with 92.5% and 92.2% recovery, with each at over 96.5% purity by high-performance liquid chromatography analysis. Their structures were identified by 1H NMR and 13C NMR.