Separation and purification of quinolyridine alkaloids from seeds of Thermopsis lanceolata R. Br. by conventional and pH-zone-refining counter-current chromatography.

In this work, the preparative separation of quinolyridine alkaloids from seeds of T. lanceolata by conventional and pH-zone-refining counter-current chromatography. Traditional counter-current chromatography separation was performed by a flow-rate changing strategy with a solvent system of ethyl acetate-n-butanol-water (1:9:10, v/v) and 200 mg sample loading. Meanwhile, the pH-zone-refining mode was adopted for separating 2.0 g crude alkaloid extracts with the chloroform-methanol-water (4:3:3, v/v) solvent system using the stationary and mobile phases of 40 mM hydrochloric acid and 10 mM triethylamine. Finally, six compounds, including N-formylcytisine (two conformers) (1), N-acetycytisine (two conformers) (2), (-)-cytisine (3), 13-ß-hydroxylthermopsine (4), N-methylcytisine (5), and thermopsine (6) were successfully obtained in the two counter-current chromatography modes with the purities over 96.5%. Moreover, we adopted nuclear magnetic resonance and mass spectrometry for structural characterization. Based on the obtained findings, the pH-zone-refining mode was the efficient method to separate quinolyridine alkaloids relative to the traditional mode.

Extraction and purification of cis/trans asarone from Acorus tatarinowii Schott: Accelerated solvent extraction and silver ion coordination high-speed counter-current chromatography.

Acorus tatarinowii Schott is a traditional Chinese medicine used to treat memory and cognitive dysfunction. Because of their efficacy and lower toxic effects, research on α- and ß-asarone, the phytoconstituents, has attracted attention owing to their remarkable pharmacological activities. Silver ion coordination complexation high-speed counter-current chromatography was used to separate these isomers from A. tatarinowii extract, coupled with accelerated solvent extraction. Accelerated solvent extraction parameters were investigated with single-factor and orthogonal testing. A two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (2:1:2:1, v/v) with 0.50 mol/L silver ions was selected for separation. From 2.0 g crude extract, 1.4 g of ß-asarone and 0.09 g of α-asarone were obtained with purities over 98% by sequential sample loading in 20 h. The isolated compounds were identified by electrospray ionization mass spectrometry, 1H and 13C NMR. Silver ions significantly increased the separation factor and retention of the stationary phase. The chromatographic behavior indicated that cis-configuration was more strongly complexed with the silver ion. This was further demonstrated with the help of computational analysis. In conclusion, the established method could be employed to separate other cis-trans or E/Z isomers that form coordination complexes.

Flavonoid epimers from custard apple leaves, a rapid screening and separation by HSCCC and their antioxidant and hypoglycaemic activities evaluation.

Leaves of custard apple are widely used in many places as a popular dietary supplement for the treatment of diabetes. Flavonoids are known to have anti-diabetic activity. In this study, the main flavonoid epimers were separated. The crude extract was first screened by HPLC-DAD before and after incubation with DPPH method to evaluate the antioxidants. An efficient extraction method was employed to remove non-flavonoid components. Subsequently, five main flavonoids with two pairs of epimers including quercetin-3-O-robinobioside, rutin, quercetin-3-O-ß-D-glucoside, kaempferol-3-O-robinobioside, and kaempferol-3-O-rutinoside were successfully separated by high-speed counter-current chromatography with ethyl acetate/n-butanol/water (4:1:5, v/v) coupled with online-storage inner-recycling mode. The structures of the separated compounds were identified by spectral techniques. The purity of the separated flavonoid glycosides was over 98%, as determined by HPLC. The separated pure constituents were found to possess the antioxidant capacities following DPPH radical scavenging protocol. The compounds (1-3) exhibited better antioxidant activity. Furthermore, the glucose uptake of crude flavonoid extract had better results than the crude ethanol extract. The present study demonstrates that the efficacy of custard apple leaves in lowering glucose level, and antioxidant capacities of separated pure compounds probably appear to be predominantly responsible for hypoglycaemic properties on HepG2 cells.

A simple and efficient linear gradient coupled with inner-recycling high-speed counter-current chromatography mode for the preparative separation of flavonoid glycosides from leaves of custard apple.

In general, the simultaneous separation and isolation of compounds with a broad polarity range from natural products is a challenge by ordinary high-speed counter-current chromatography (HSCCC). Indeed, the complex solvent system screening methods limit the broader application of HSCCC. We describe herein a rapid and efficient linear gradient CCC (LGCCC) method that enables the separation of flavonoid glycosides with a broad range of KD values from custard apple leaves. Inner-recycling CCC (IRCCC) mode has been further applied for the separation of compounds with similar KD values. Similarly to binary gradient HPLC, the LGCCC mode is achieved by adjustment of the proportion between ethyl acetate (pump A) and n-butanol (pump B) in an ethyl acetate/n-butanol/water solvent system. Various separation factors have been investigated, including separation mode, rotation speed, flow rate, and sample loading. The IRCCC mode has been used for the secondary separation of two epimers with a simple ethyl acetate/water (1:1, v/v) solvent system. Finally, five main flavonoid glycosides have been successfully separated, namely quercetin-3-O-robinobioside (1, 4.8 mg) and rutin (2, 12.1 mg), quercetin-3-O-ß-d-glucoside (3, 4.2 mg), kaempferol-3-O-robinobioside (4, 9.6 mg), and kaempferol-3-O-rutinoside (5, 24.6 mg). The purities of the separated flavonoid glycosides were over 98%, as determined by HPLC. Our study indicates that a suitable combination of LGCCC and IRCCC modes is an effective strategy for separating flavonoid glycosides from custard apple leaves. The mathematical expression of the LGCCC was deduced to illuminate the separation mechanism. It may also be applied to obtain component fractions for the further screening of active compounds from complex natural products.

Novel ultrasonic-assisted vacuum drying technique for dehydrating garlic slices and predicting the quality properties by low field nuclear magnetic resonance.

A novel ultrasonic-assisted vacuum drying technique for dehydrating garlic slices to give high quality products was developed. Garlic slices were dried at 60 °C using four methods: ultrasonic-assisted vacuum drying (USVD), vacuum drying (VD), ultrasonic-assisted drying (USD), and convective drying (CD, the control with no vacuum or ultrasonic applied). Drying kinetics, water-content changes, and properties of the garlic slices were assessed. Univariate linear and partial-least-squares regression models were used to predict the properties from low-field nuclear magnetic resonance parameters. USVD gave the shortest drying time (180 min less than CD) and provided a better garlic color and texture, and allicin retention rate than the other methods. Higher correlations between low-field nuclear magnetic resonance parameters and quality properties were found by partial-least-squares regression (PLSR) than by univariate analysis, with the analysis results being credible. Overall, ultrasonic-assisted vacuum drying produced high-quality products with its properties predicted well by low-field nuclear magnetic resonance.

Rapid screening and purification of potential alkaloid neuraminidase inhibitors from Toddalia asiatica (Linn.) Lam. roots via ultrafiltration liquid chromatography combined with stepwise flow rate counter-current chromatography.

Toddalia asiatica (Linn.) Lam. is a medical plant traditionally used to treat coughs, fevers, and various diseases. Alkaloids are the main active ingredients in Toddalia asiatica (Linn.) Lam., but traditional methods for screening and separation are complex and labor-intensive. In this work, an efficient strategy was developed to rapidly screen, identify, and separate neuraminidase inhibitors from Toddalia asiatica (Linn.) Lam. Ultrafiltration, high performance liquid chromatography, and time-of-flight mass spectrometry were employed for rapid screening and identification of neuraminidase inhibitors. A two-phase solvent system comprising n-hexane/ethyl acetate/methanol/water (5:5:3:7, v/v) was then selected for separation by high-speed counter-current chromatography. A sample loading of 200 mg and a stepwise flow rate were achieved by increasing the flow rate from 2 to 4 mL/min after 4 h. Three main fluoroquinoline alkaloids (haplopine, skimmianine, and 5-methoxydictamnine) along with two coumarins were obtained via one-step separation and their structures were determined by mass spectrometry and nuclear magnetic resonance. In vitro assays revealed skimmianine with half-maximal inhibitory concentration of 16.2 ± 0.7 µmol/L was selected as the potential highest neuraminidase inhibitor. The results suggest that ultrafiltration high performance liquid chromatography-mass spectrometry combined with high-speed counter-current chromatography is efficient for the screening and isolation of neuraminidase inhibitors from complex natural products.

Chemical Constituents from Scindapsus officinalis (Roxb.) Schott. and Their Anti⁻Inflammatory Activities.

One new monoterpene glycoside (1), one new phenyl glycoside (2), one new caffeoyl derivative (3), were isolated from Scindapsus officinalis (Roxb.) Schott., along with four known compounds (4⁻7). Structures of the isolated compounds were elucidated by extensive analysis of spectroscopic data, especially 2D NMR data and comparison with literatures. All isolates were evaluated for anti-inflammatory activity against nitric oxide (NO) production in vitro. Compounds 3 and 7 exhibited moderate inhibitory effects on NO production with IC50 values of 12.2 ± 0.8 and 18.9 ± 0.3 µM, respectively.

Application of coordination agent in high-speed counter-current chromatography for the preparative separation and isolation ginkgolic acids from the sarcotesta of Ginkgo biloba L.

An efficient coordination high-speed counter-current chromatography method for the preparative separation of ginkgolic acids from the sarcotesta of Ginkgo biloba L was developed. The type, concentration, and mechanism of the coordination agent were investigated. Following the use of four types of metal salts including silver nitrate, copper chloride, ferric chloride, and aluminium nitrate, n-heptane/ethyl acetate/methanol/acetic acid 5:4:1:1, v/v with 0.20 mol/L silver nitrate as the coordination agent was chosen as the optimum two-phase solvent system. Five main ginkgolic acids including C13:0, C15:0, C15:1, C17:1, and C17:2 were successfully separated with purities greater than 98%. The sample loading was 500 mg, the flow-rate was 2.0 mL/min, rotation speed was 800 rpm and temperature was 20°C. The structures of the separated ginkgolic acids were identified by comparison with standard samples and electrospray ionization mass spectrometry. The introduction of coordination chemistry in high-speed counter-current chromatography is novel and effective for the preparative separation and isolation of ginkgolic acids from the sarcotesta of Ginkgo biloba L and could also be applied to separate compounds which form coordination bonds in other complex natural products.

Alkaloids from Scindapsus officinalis (Roxb.) Schott. and their biological activities.

Two new alkaloid glycosides (1-2), two new amide alkaloids (3 and 6), and two new amine alkaloids (4 and 5), were isolated from Scindapsus officinalis (Roxb.) Schott., along with four known alkaloids (7-10). Structural elucidation of the isolated compounds was established by spectroscopic analysis, especially 2D NMR techniques and comparison with literatures. The absolute configurations of compounds 1 and 2 were determined by the electronic circular dichroism calculations. Compound 1 represent the first example of alkaloid glycoside found to possess an unprecedented 2,5-methanocyclopenta[b]azepine core. All isolates were evaluated for cytotoxic activity against MCF-7 human breast cancer cell and anti-inflammatory activity against nitric oxide (NO) production in vitro. Compounds 4, 6 and 8 demonstrated moderate cytotoxic effects against MCF-7 cell (IC50 of 11.6, 25.3, and 20.5 µM, respectively), and 1, 7 and 9 exhibited moderate inhibitory effects on NO production (IC50 of 19.8, 30.3, and 11.8 µM, respectively).

Five new chromone glycosides from Scindapsus officinalis (Roxb.) Schott.

Five new chromone glycosides, officinalisides A (1), B (2), C (3), D (4) and E (5) were isolated from Scindapsus officinalis (Roxb.) Schott., along with six known chromone derivatives, 7-O-α-l-rhamnosyl-nereugenin (6), undulatoside A (7), drynachromoside A (8), drynachromoside B (9), 5,7-dihydroxy-2-methyl chromone(10), 5,7-dihydroxy-2-hydroxymethyl chromone (11). Structural elucidation of the isolated compounds was established by spectroscopic analysis, especially 2D NMR techniques and comparison with literatures. The isolates were evaluated for anti-inflammatory activities in a LPS-stimulated RAW 264.7 model using inhibition of nitric oxide (NO) production as an indicator. Compounds 2, 4 and 10 demonstrated potential anti-inflammatory activity with IC50 values of 16.1, 19.1, and 13.4µM, respectively, compared to the positive control dexamethasone.

Extraction and purification of five terpenoids from olibanum by ultrahigh pressure technique and high-speed countercurrent chromatography.

Five terpenoids, including two new ones, 3,7-dioxo-tirucalla-8,24-dien-21-oic acid (2) and 3α-acetoxyl-7-oxo-tirucalla-8,24-dien-21-oic acid (3), and three known ones, boscartol A (1), 11-keto-ß-boswellic acid (4), and acetyl-11-keto-boswellic acid (5), have been extracted by the ultrapressure extraction and purified by pH-zone-refining countercurrent chromatography and high-speed countercurrent chromatography from olibanum. For ultrapressure extraction, the optimal condition including 200 MPa of extraction pressure, ethyl acetate of extraction solvent, 1:20 (g/mL) of solid/liquid ratio, and 2 min of extraction time were obtained. For the separation, from 1.5 g of the terpenoid extract, 220.1 mg of 4, 255.5 mg of 5, and 212.3 mg of the mixture of 1, 2, and 3 were obtained by pH-zone-refining countercurrent chromatography under the solvent system of chloroform/ethyl acetate/methanol/water (3:1:3:2, v/v/v/v) with aqueous ammonia and trifluoroacetic acid as retention and eluter agents. The enriched mixture (210 mg) was further separated by conventional high-speed countercurrent chromatography with petroleum ether/ethyl acetate/methanol/water (1:0.8:1.1:0.6, v/v/v/v), yielding 30.1 mg of 1, 35.5 mg of 2, 12.3 mg of 3. The structures of these five terpenoids were elucidated by extensive spectroscopic methods.

Molecular evaluation of soil organic matter characteristics in three agricultural soils by improved off-line thermochemolysis: the effect of hydrofluoric acid demineralisation treatment.

The molecular composition of soil organic matter (SOM) in three agricultural fields under different managements, was evaluated by off-line thermochemolysis followed by gas chromatography mass spectrometry analysis (THM-GC-MS). While this technique enabled the characterization of SOM components in coarse textured soil, its efficiency in heavy textured soils was seriously affected by the interference of clay minerals, which catalyzed the formation of secondary artifacts in pyrolysates. Soil demineralization with hydrofluoric acid (HF) solutions effectively improved the reliable characterization of organic compounds in clayey soils by thermochemolysis, while did not alter significantly the results of coarse textured soil. A wide range of lignin monomers and lipids molecules, of plant and microbial origin, were identified in the pyrograms of HF treated soils, thereby revealing interesting molecular differences between SOM management practices. Our results indicated that clay removal provided by HF pretreatment enhanced the capacity of thermochemolysis to be a valuable and accurate technique to study the SOM dynamics also in heavy-textured and OC-depleted cultivated soils.

[Structural characteristics of humic acids from a long-term petroleum contaminated soil].

Petroleum contamination in soil decreases with the increase in the distance of soil to the drilling well. Accordingly, an abandoned petroleum well which had been exploited for about twenty years in Songyuan city of Jilin Province, China, was selected to investigate the structural characteristics of soil humic acids (HAs) under different petroleum contamination levels. Surface (0-20 cm) soil samples were collected at 0.5, 1.5, 3.5, 5.5 and 7.5 m deep from well head, and the petroleum contents were respectively 153.3, 148.4, 129.2, 50.5 and 5.62 g x kg(-1). HAs were extracted with 0.1 mol x L(-) NaOH and 0.1 mol x L(-1) Na4 P2O7 and were characterized with elemental analysis, Fourier transformed infrared (FTIR) spectroscopy and solid-state 13C cross polarization magic angle spinning nuclear magnetic resonance (13C CPMAS NMR) spectroscopy. Results showed that the atomic C/H, O/C and (N+O) /C ratios of HAs increased from 0.74, 0.41 and 0.45 for 7.5 m to 0.80, 0.83 and 0.88 for 0.5 m, respectively. The relative intensity of the peaks assigned to aliphatic carbon (2 921, 2 851 and 1 454 cm(-1)) in the FTIR spectra gradually decreased with increasing contamination levels, while that of the peak assigned to aromatic C(1 600 cm(-1)) increased, and the calculated absorption intensity ratio of 2 921 to 1 600 cm(-1) (2 921/1 600) declined from 0.22 for 7.5 m to 0.11 for 0.5 m. The solid-state 13C NMR data suggested that the relative content of alkyl C(0-50 ppm ) decreased from 49.9% for 7.5m to 30.9% for 0.5 m, while that of O-alkyl C(50-110 ppm), aromatic C(110-160 ppm) and carboxyl C(160-190 ppm) increased respectively from 20.1%, 13.1% and 14.3% to 28.0%, 18.8% and 19.3%. These results showed substantial chemical, structural, and molecular differences among these HAs. The aliphaticity and hydrophobicity of HAs decreased while aromaticity and polarity increased with the increase in petroleum content. Namely, HAs tended to become aged in molecular structure. Therefore, it is imperative to renew and activate the aged HAs by adopting appropriate measures for the remediation of petroleum contaminated soil.