Separation of polysaccharides from Lycium barbarum L. by high-speed countercurrent chromatography with aqueous two-phase system.

The traditional method for isolation and purification of polysaccharides is time-consuming. It often involves toxic solvents that destroy the function and structure of the polysaccharides, thus limiting in-depth research on the essential active ingredient of Lycium barbarum L. Therefore, in this study, high-speed countercurrent chromatography (HSCCC) and aqueous two-phase system (ATPS) were combined for the separation of crude polysaccharides of Lycium barbarum L. (LBPs). Under the optimized HSCCC conditions of PEG1000-K2HPO4-KH2PO4-H2O (12:10:10:68, w/w), 1.0 g of LBPs-ILs was successfully divided into three fractions (126.0 mg of LBPs-ILs-1, 109.9 mg of LBPs-ILs-2, and 65.4 mg of LBPs-ILs-3). Moreover, ATPS was confirmed as an efficient alternative method of pigment removal for LBPs purification, with significantly better decolorization (97.1 %) than the traditional H2O2 method (88.5 %). Then, the different partitioning behavior of LBPs-ILs in the two-phase system of HSCCC was preliminarily explored, which may be related to the difference in monosaccharide composition of polysaccharides. LBPs-ILs-1 exhibited better hypoglycemic activities than LBPs-ILs-2 and LBPs-ILs-3 in vitro. Therefore, HSCCC, combined with aqueous two-phase system, was an efficient separation and purification method with great potential for separating and purifying active polysaccharides in biological samples.

Identification of anti-photoaging components of Olea europaea leaves based on spectrum-effect relationship.

In this study, to identify bioactive components of Olea europaea leaves extract (OLE), chemometrics analyses including bivariate correlation analysis and partial least squares regression were used to establish the relationships between the chromatograms and anti-photoaging effect of OLE samples. Firstly, the fingerprint of olive leaves extract was determined by high-performance liquid chromatography (HPLC). Photoaging models of HaCaT cells were established by UVB irradiation. The photoaging resistance of OLE was evaluated by cell viability using the MTT assay. Chemometrics analyses showed that compounds 14, 19, 20, 24, 26, and 28 might be the major anti-photoaging components of OLE. Furthermore, after separation by HSCCC and NMR identification, compound 19 is luteoloside and compound 24 is oleuropein. Oleuropein and luteoloside were docked with collagenase (MMP-1), stromelysin (MMP-3), and gelatinase (MMP-9), respectively. The results showed that oleuropein and luteoloside inhibited their activity by directly interacting with MMP-1, MMP-3, and MMP-9, thereby exhibiting anti-photoaging activity. The current bioassay and spectrum-effect relationships are proper for associating sample quality with the active ingredient, and our finding would provide foundation and further understanding of the quality evaluation and quality control of Olea europaea.

Preparation of highly purified oleuropein by combinative technology off line of HSCCC-PHPLC based on dual wavelength.

Oleuropein is the main active substance in foods or functional foods produced from olive (Olea europaea L.) leaves. In the present study, the combinative technology off line of HSCCC-PHPLC based on dual wavelength was used to separate highly purified oleuropein from oleuropein extract. Response surface methodology was used to optimize the conditions of HSCCC. Furthermore, a large amount of higher purified oleuropein was obtained through HSCCC at the wavelength of 254 nm, and oleuropein with the purity greater than 98.5% was obtained by PHPLC at the wavelength of 300 nm. Finally, the purity and structure identification of highly purified oleuropein were determined by various methods and its stability was investigated. As a result, oleuropein was stable in solution, and had good stability under the condition of dark storage at 4°C within a week or under the condition of dark storage at -20°C within one year. PRACTICAL APPLICATION: In this study, an efficient method for purification and refining of oleuropein by combinative technology off line of HSCCC-PHPLC based on dual wavelength was established. Oleuropein with the purity greater than 98.5% was macro-obtained via the technology. The highly purified oleuropein could be used to control the quality of olive products.

Recovery and recycling of solvent of counter-current chromatography: The sample of isolation of zeaxanthin in the Lycium barbarum L. fruits.

An efficient method of recovering and recycling solvent for counter-current chromatography was established by which zeaxanthin was separated from Lycium barbarum L. fruits. A column with activated carbon combined with high performance counter-current chromatography formed the recovering and recycling solvent system. Using the solvent system of n-hexane-ethyl acetate-ethanol-water (8:2:7:3, v/v) from the references, five injections were performed with an almost unchanged purity of zeaxanthin (80.9, 81.2, 81.5, 81.3, and 80.2% respectively) in counter-current chromatography separation. Meanwhile, the mobile phase reduced by half than conventional counter-current chromatography. By this present method, an effective improvement of counter-current chromatography solvent utilization was achieved.

Strategy for the separation of strongly polar antioxidant compounds from Lycium barbarum L. via high-speed counter-current chromatography.

In the separation of strongly polar antioxidant compounds from natural products using high-speed counter-current chromatography that is target-guided by 2,2-diphenyl-1-picrylhydrazyl high-performance liquid chromatography experimentation, low adsorption ability is encountered due to the strong polarity of the target compounds. In this study, a strategy of novel partition coefficient value calculation was proposed for overcoming this problem. The partition coefficient value was expressed as the ratio of the antioxidant activities of the upper phase and the lower phase. This strategy was used in high-speed counter-current chromatography with a hydrophilic organic/salt-containing aqueous two-phase system for bioassay-guided separation of strongly polar antioxidant compounds from Lycium barbarum L. The antioxidant activity was determined via the radical scavenging activity method using 2,2-diphenyl-1-picrylhydrazyl radicals. A hydrophilic organic/salt-containing aqueous two-phase system of 95% EtOH - sat. (NH4)2SO4 (1:1.8, v/v) was successfully used to separate Lycium barbarum L. extract. Five fractions were collected via high-speed counter-current chromatography separation. The antioxidant activity of the third fraction was the highest. Three compounds were separated via MCI gel column chromatography and Sephadex LH-20 column chromatography from the third fraction, and their antioxidant activities were determined. The antioxidant activities of the three compounds were higher than that of the third fraction. These results demonstrate that this strategy can be used to separate strongly polar antioxidant compounds from natural products.

Preparative Separation of Flavonoids from Goji Berries by Mixed-Mode Macroporous Adsorption Resins and Effect on Aß-Expressing and Anti-Aging Genes.

Flavonoids are the main constituents of Goji berries and have good biological and pharmacological activities. The mixed-mode macroporous adsorption resins (MARs) for purification of flavonoids from Goji berries through computer-assisted calculation of the molecular size of flavonoids and the precise matching of MAR physical and chemical properties was firstly developed in the present study. Ten varieties of MARs with suitable molecular dimensions and polarities were used for investigating the adsorption/desorption behaviors of the flavonoids. Both AUKJ-1 and BWKX-1 showed higher separation efficiency than other MARs and then were mixed in different ratios to constitute a mixed-mode macroporous adsorption resin to obtain the optimal adsorption phase. Under optimal conditions, total flavonoid content of purified flavonoid (p-FLA) extract increased from 0.97% to 36.88% after one purification. The p-FLA extract from Goji berries significantly improved the expression of six genes with anti-aging effects and played an important role in aging-related Alzheimer's disease by down-regulating Aß expression.

Continuous separation of maslinic and oleanolic acids from olive pulp by high-speed countercurrent chromatography with elution-extrusion mode.

In this work, a continuous high-speed countercurrent chromatography method has been developed on the basis of elution-extrusion mode and this method was successfully applied to the separation of maslinic and oleanolic acid from the extract of olive pulp. In the process of 'elution', the sample solution was continuously loaded into the column and the maslinic acid was steadily eluted out in this step while highly retained oleanlic acid always stayed in the column. In the process of 'extrusion', the oleanlic acid was pushed out of the column with the stationary phase. In this way, we achieved a large sample loading. A total of 120 mL sample solution (about 89.55% of the column volume) which contains 600 mg olive pulp extract was pumped in the apparatus by a constant-flow pump and the maslinic and oleanolic acids were largely separated within 120 min. Both of these two compounds presented high yields and high purities (271.6 mg for maslinic acid with 86.7% and 83.9 mg oleanolic acids with 83.4%).

Establishment and application of a method for screening the therapeutic drugs of ethanol-induced liver injury based on cellular metabonomics.

A drug-screening method to test the capacity of drugs to protect against ethanol-induced liver injury based on cellular metabonomics was established and applied in this study. It screens for the ability to protect against ethanol-induced liver injury by considering changes in the cellular metabolites of human normal liver L-02 cells subjected to ethanol treatment. This method considers cellular metabolites as the main analytical index, principal component analysis and orthogonal partial least squares discriminant analysis as the main multi- and megavariate data analysis methods, and vitamin C as the standard substance to determine the ability to protect against ethanol-induced liver injury. Ability to protect against ethanol-induced liver injury unit = [190 - 50× (14.318 - 10 × Y predictive value)1/2 ] × ability 1 µg/mL vitamin C. Olive leaf extract, Lycium barbarum L extract and fish roe peptide were screened using the established methods. Olive leaf OP phase had the strongest ability to protect against ethanol-induced liver injury, at 81.88. The value for L. barbarum L was 37.56. The fish roe peptide water phase was 63.07. All three have the ability to protect against ethanol-induced liver injury. The drug-screening method for ability to protect against ethanol-induced liver injury based on cell metabonomics is a fast, accurate and effective method for quantitative detection of ability to protect against ethanol-induced liver injury.

Development of an HPLC-DAD method for the determination of five alkaloids in Stephania yunnanensis Lo and in rat plasma after oral dose of Stephania yunnanensis Lo extracts.

For the rational utilization and the quantitative quality control of the Stephania yunnanensis Lo, an HPLC-DAD method was developed for the quantitative and simultaneous determination of five alkaloids in rat plasma (stepharine, sinomenine, palmatine, isocorydine and tetrahydropalmatine), which were the main active chemical constituents of this plant and belong to four kinds of isoquinoline-type alkaloids (protoberberine, morphine, aporphine and protaporphine alkaloids). The contents of five alkaloids ranged from 0.09 to 2.32% (w/w). The method validation was tested for the linearity (r2 > 0.9975), precision (intra-day RSD < 4.8% and inter-day RSD < 4.9%), extraction recovery (85.49 ± 2.29% to 99.21 ± 1.48%) and stability (98.5 ± 5.3% to 101.2 ± 3.4%). We developed an HPLC-DAD method to simultaneously measure these alkaloids in rat plasma after oral administration of the extract of this plant to rats. The results supported the hypothesis that isoquinoline alkaloids were the compounds responsible for the main pharmacological activities for anti-inflammatory and analgesic.

Antibacterial Activity of Hydroxytyrosol Acetate from Olive Leaves (Olea Europaea L.).

Vibrio spp. are pathogens of many bacterial diseases which have caused great economic losses in marine aquaculture. The strategy of alternative medical treatment that is utilised by herbalists has expanded in the past decade. The aim of our study is to discover the antibacterial molecules against Vibrio spp. Bacterial growth inhibition, membrane permeabilisation assessment and DNA interaction assays, as well as agarose gel electrophoresis, were employed to elucidate the antibacterial activity of hydroxytyrosol acetate. Results showed that hydroxytyrosol acetate had antibacterial activity against Vibrio spp. and it played the role via increasing bacterial membrane permeabilisation. The DNA interaction assay and agarose gel electrophoresis revealed that hydroxytyrosol acetate interacted with DNA. Hydroxytyrosol acetate enhanced the fluorescent intensity of DNA binding molecules and mediated supercoiled DNA relaxation. The present study provides more evidence that hydroxytyrosol acetate is a novel antibacterial candidate against Vibrio spp.

[Research on anticancer activity of isocorydine and its derivatives].

Isocorydine and its analogs were extracted from Dicranostigma leptopodum and Stephania yunnanensis through the method of natural products chemistry. Its derivatives were prepared by chemical structure modifications from isocorydine. MTT method was used to study the inhibitory effect of those compounds on the growth of HepG2, HeLa and MGC-803 cancer cell lines in vitro. The results showed that isocorydine and its analogs all have the growth inhibition for those cancer cell lines. This paper investigated the structure-activity relationship of isocorydine and its derivatives with anticancer activity in the aspect of stereochemical structure, functional groups positions of the compounds and the electron density of aromatic rings based on the single crystal diffraction structure and the molecular docking of EGFR and isocorydine.

Three-phase solvent systems for the comprehensive separation of a wide variety of compounds from Dicranostigma leptopodum by high-speed counter-current chromatography.

A three-phase solvent system was efficiently applied for high-speed counter-current chromatography to separate secondary metabolites with a wide range of hydrophobicity in Dicranostigma leptopodum. The three-phase solvent system of n-hexane/methyl tert-butyl ether/acetonitrile/0.5% triethylamine (2:2:3:2, v/v/v/v) was selected for high-speed counter-current chromatography separation. The separation was initiated by filling the column with a mixture of intermediate phase and lower phase as a stationary phase followed by elution with upper phase to separate the hydrophobic compounds. Then the mobile phase was switched to the intermediate phase to elute the moderately hydrophobic compounds, and finally the polar compounds still retained in the column were fractionated by eluting the column with the lower phase. In this research, 12 peaks were eluted out in one-step operation within 110 min, among them, eight compounds with acceptable purity were obtained and identified. The purities of ß-sitosterol, protopine, allocryptopine, isocorydione, isocorydine, coptisine, berberrubine, and berberine were 94.7, 96.5, 97.9, 86.6, 98.9, 97.6, 95.7, and 92.8%, respectively.

Simultaneous determination of the content of isoquinoline alkaloids in Dicranostigma leptopodum (Maxim) Fedde and the effective fractionation of the alkaloids by high-performance liquid chromatography with diode array detection.

A simple and efficient method was developed for the simultaneous determination of eight isoquinoline alkaloids in methanol extracts of Dicranostigma leptopodum (Maxim) Fedde and the effective fractionation of the alkaloids of D. leptopodum by high-performance liquid chromatography with diode array detection. The chromatographic conditions were optimized on a SinoChrom ODS-BP column to obtain a good separation of the four types of alkaloid analytes, including two aporphines (isocorydine, corydine), two protopines (protopine and allocryptopine), a morphine (sinoacutine), and three quaternary protoberberine alkaloids (berberrubine, 5-hydroxycoptisine, and berberine). The separation of these alkaloids was significantly affected by the composition of the mobile phase, and particularly by its pH value. Acetonitrile (A) and 0.2% phosphoric acid solution adjusted to pH 6.32 with triethylamine (B) were selected as the mobile phase with a gradient elution. With this method, a new quaternary protoberberine alkaloid was isolated and the two structural isomers (isocorydine and corydine) were baseline separated. The appropriate harvest period for D. leptopodum was also recommended based on our analysis. The method for the effective fraction of the alkaloids of D. leptopodum was optimized under this method with regard to the varying significant pharmacological activities of the alkaloids.

A new quaternary protoberberine alkaloid isolated from Dicranostigma leptopodum (Maxim) Fedde.

Phytochemical investigation of the whole plants of Dicranostigma leptopodum (Maxim) Fedde has led to the isolation of two quaternary protoberberine alkaloids 5-hydroxy-coptisine (1) and berberrubine (2). This type of alkaloids was isolated from the genus Dicranostigma for the first time and the new compound structure (1) was elucidated by various spectroscopic methods including 2D NMR techniques (gCOSY, HMQC and HMBC) and HR-ESI-MS.

[Extraction and purification of isocorydine from Dicranostigma leptopodum].

OBJECTIVE: To optimize the conditions of extraction and purification of isocorydine from Dicranostigma leptopodum. METHODS: Extraction conditions of isocorydine were selected on the basis of orthogonal experimental design, the static adsorption/desorption experiments were used to evaluate the optimum resin. RESULTS: The optimum extraction parameters were as follows: the ratio of raw materials to solvent 1:15 (g/mL), extraction solvent 1% vitriol, the extraction three times and 1 h each time. LX28 resin exhibited higher adsorption efficiency. CONCLUSION: Under the above optimum conditions, the extraction yield of isocorydine is 0.88%. The purity of isocorydine can reach 85.34% with a yield rate of 68.64%.

Activity-screening-guided isolation and purification for vasodilative effects compounds from Radix Astragali by high-speed counter-current chromatography using gradient elution.

The animal models were conducted to activity screening of different ethanol fraction from Radix Astragali. Eluted from the macroporous adsorptive resins, the potential active fraction that had vasodilative effects was isolated and purified by high-speed counter-current chromatography (HSCCC) using gradient elution. The solvent system was composed of chloroform-methanol-acetic acid-water (2 : 1 : 1 : 1, v/v/v/v). The flow rate was 1.0 mL min(-1) initially and changed to 2.0 mL min(-1) at 300 min. Four flavonoids named calycosin-7-O-ß- D -glycoside (a), formononetin-7-O-ß- D -glycoside (b), calycosin (c) and formononetin (d) with purity over 94% were obtained and then identified by (1)H NMR and (13)C NMR.

Online extraction and isolation of highly polar chemical constituents from Brassica napus L. pollen by high shear technique coupled with high-performance counter-current chromatography.

High shear technique coupled with high-performance counter-current chromatography was successfully used for the extraction and online isolation of seven highly polar chemical constituents from the Brassica napus L. The lower phase of ethyl acetate-n-butanol-water (1:4:5, v:v:v) was used as both the high shear technique solvent and high-performance counter-current chromatography mobile phase. Seven compounds of 14.2 mg of uridine, 4.6 mg of xanthosine, 7.8 mg of guanosine, 5.3 mg of adenosine, 19.5 mg of kaempferol-3,4'-di-O-ß-D-glucopyranoside, 17.7 mg of kaempferol-3-O-(2-O-ß-D-glucopyranosy1)-ß-D-glucopyranoside, and 25.7 mg of an unknown compound, with a high-performance liquid chromatography (HPLC) purity over 95.0%, were obtained in a one-step extraction-separation process within 130 min from 20.0 g of raw material of pollen of Brassica napus L. Moreover, the mode of elution-extrusion was employed for the separation of the last one compound. The isolated compounds were analyzed by HPLC, and the chemical structures of the compounds mentioned above were identified by UV and NMR. It is the first time to combine the high shear technique and high-performance counter-current chromatography for the online isolation of the nature products.

Adsorption characteristics of (-)-epigallocatechin gallate and caffeine in the extract of waste tea on macroporous adsorption resins functionalized with chloromethyl, amino, and phenylamino groups.

According to the Friedel-Crafts and amination reaction, a series of macroporous adsorption resins (MARs) with novel structures were synthesized and identified by the Brunauer-Emmett-Teller (BET) method and Fourier transform infrared (FTIR) spectra, and corresponding adsorption behaviors for (-)-epigallocatechin gallate (EGCG) and caffeine (CAF) extracted from waste tea were systemically investigated. Based on evaluation of adsorption kinetics, the kinetic data were well fitted by pseudo-second-order kinetics. Langmuir, Freundlich, Temkin-Pyzhev, and Dubinin-Radushkevich isotherms were selected to illustrate the adsorption process of EGCG and CAF on the MARs. Thermodynamic parameters were adopted to explain in-depth information of inherent energetic changes associated with the adsorption process. The effect of temperature on EGCG and CAF adsorption by D101-3 was further expounded. Van der Waals force, hydrogen bonding, and electrostatic interaction were the main driving forces for the adsorption of EGCG and CAF on the MARs. This study might provide a scientific reference point to aid the industrial large-scale separation and enrichment of EGCG from the extracts of waste tea using modified MARs.

Preparative separation and purification of Rebaudioside A from Stevia rebaudiana Bertoni crude extracts by mixed bed of macroporous adsorption resins.

The separation and purification of Rebaudioside A (RA) from Stevia rebaudiana Bertoni crude extracts (Steviosides) by macroporous adsorption resin (MAR) mixed bed were systematically investigated. MAR mixed bed of HPD750-LSA40-LSA30-DS401 was selected due to its better separation degree. Based on the kinetics/thermodynamics experiment of the mixed bed, it was found that the experimental data fitted better to the pseudo-second-order model, and intra-particle diffusion was rate-limiting step. The adsorption isotherm was consistent with IV equilibrium adsorption isotherm classified by Brunauer. Furthermore, the influencing factors for the separation of RA based on HPLC were also investigated. Under the optimal conditions, the separation degree for RA (DAS) increased from 0.771 to 1.54. Moreover, the experimental results showed that the purity of the obtained product increased from 60% to 97%.

Efficient method for screening and identification of radical scavengers in the leaves of Olea europaea L.

In this article, an efficient method was developed to screen, isolate and identify the major radical scavengers in the leaves of Olea europaea L. by DPPH-HPLC-DAD, HSCCC and NMR. The method of DPPH-HPLC-DAD was used to screen the major radical scavengers. It was found that three major constituents (A, B, C) in the extract of the leaves of O. europaea L. possessed potential antioxidant activities. In order to identify the chemical structures of those compounds, the HSCCC method with a two-phase solvent system composed of petroleum ether-ethyl acetate-water at an optimized volume ratio of 6:600:700 (v/v/v) together with column chromatography was developed to isolate and purify the active compounds. Pure compounds A (225 mg), B (10 mg) and C (12 mg) with purities 92.6, 95.1 and 96.4%, respectively, were obtained from the crude sample (500 mg). Their structures were identified as oleuropein (A), luteolin-7-O-glucoside (B) and verbascoside (C) by (1) H-NMR and (13) C-NMR.