Yongxin Zhao

Comparative Study on Separation and Purification of Isoflavones from the Seeds and Sprouts of Chickpea by High-Speed Countercurrent Chromatography

Abstract Chickpea is known as a plant that is rich in protein, carbohydrates, and nutrition, and its seeds and sprouts have been processed into various health foods. In the present study, four isoflavones were purified from the seeds and sprouts of chickpea by high-speed countercurrent chromatography (HSCCC) using two biphasic solvent systems composed of n-hexane–ethyl acetate–methanol–water (5:5:5:5, v/v) and ethyl acetate–water (1:1, v/v). The results indicated that 14.2 mg of formononetin, 15.7 mg of biochanin A, 9.1 mg of ononin, 11.3 mg of biochanin A-7-O-β-D-glucoside were obtained from 150 mg of sprout extracts with the purity of 92.26%, 95.86%, 95.32%, and 96.56%, respectively. Compared with the sprouts, separation of seed extracts yielded less amounts of biochanin A-7-O-β-D-glucoside and biochanin A with lower purity. The results indicate that four main isoflavones in chickpea, i.e., isoflavones, formononetin, biochanin A, ononin, and biochanin A-7-O-β-D-glucoside, are substantially increased by biosynthesis during the seed germination.

SEPARATION OF THE MINOR FLAVONOLS FROM FLOS GOSSYPII BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

An effective high-speed countercurrent chromatography (HSCCC) method was established for further separation and purification of four minor flavonols in addition to five major flavonols which were reported by our previous study from extracts of Flos gossypii. HSCCC was performed with three two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (7.5:15:6:7, v/v), (2.5:15:2:7, v/v), and (0:1:0:1, v/v). The separation was repeated three times, and 3.8 mg of 8-methoxyl-kaempferol-7-O-β-D-rhamnoside (HPLC purity 98.27%), 6.7 mg of astragalin (HPLC purity 94.18%), 3.3 mg of 4′-methoxyl-quercetin-7-O-β-D-glucoside (HPLC purity 94.30%), and 8.2 mg of hyperoside (HPLC purity 93.48%) were separated from 150 mg of the crude sample. The chemical structures of the flavonols were confirmed by MS, 1H NMR, and 13C NMR. Meanwhile, the results indicated that the target compound with smaller K value (<0.5) can be separated by increasing column length of HSCCC. And, four separation rules of flavonols were established according to the present study and references were summarized, which can be used as a useful guide for separation of flavonols by HSCCC.

Anti-Inflammatory Effect of Rosa rugosa Flower Extract in Lipopolysaccharide-Stimulated RAW264.7 Macrophages.

Rosa rugosa Thunb, a deciduous shrub of the genus Rosa, has been widely used to treat stomach aches, diarrhoea, pain, and chronic inflammatory disease in eastern Asia. In recent years, our research team has extensively studied the Rosa rugosa flower extract, and specifically undertook pharmacological experiments which have optimized the extraction process. Our methods have yielded a standard extract enriched in phenolic compounds, named PRE. Herein, we expand our efforts and evaluated the anti-inflammatory activity of PRE on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages. PRE significantly inhibited production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-a, interleukin (IL)-6, and interleukin 1β (IL-1β), as well as expression of their synthesizing enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase2 (COX-2). Furthermore, PRE inhibited activity of mitogen-activated protein kinases (MAPK) as well as nuclear factor-kappa B (NF-κB) signaling pathway. Our findings are the first to explain the anti-inflammatory mechanism by PRE in LPS-stimulated macrophages. Given these results, we propose that PRE has therapeutic potential in the prevention of inflammatory disorders.

Green synthesis and evaluation of isoquercitrin imprinted polymers for class-selective separation and purification of flavonol glycosides

A method for solid-phase extraction (SPE) of isoquercitrin (ISO) from natural plant extracts was proposed based on molecularly imprinted polymers (MIPs). The efforts in the present work aim at the emphasis on the topic of “green” chemistry, i.e., the use of green solvent and ionic liquid with a high percentage (63.2–69.3%) in the total volume of porogenic solvent. For the preparation of ISO-MIP monoliths, 4-vinylpyridine was used as the functional monomer, and ethylene glycol dimethacrylate was used as the cross-linking monomer, using a mixture of 1-butyl-3-methylimidazoliumtetrafluoroborate (ionic liquid)-N′N-dimethylformamide (DMF)-dimethyl sulfoxide as a porogen. It was found that the type of functional monomer, the ratio of template to functional monomer, the crosslinking degree, and the level of DMF, and the composition of the mobile phase greatly affected the retention of the template and performance of molecular recognition. The optimal MIPs were used as solid-phase extraction (SPE) sorbents for purification of ISO, hyperoside, and astragalin and a SPE protocol was optimised for the type of loading solvent, amount of MIPs, and washing and elution solvents. It was found that the most suitable solvents for loading, washing and elution steps were methanol–water (70:30, v/v), methanol–water (20:80, v/v) and acetonitrile–water (30:70, v/v), respectively. The highest recovery rate of ISO, hyperoside, and astragalin was 87.78%, 93.26% and 83.25%, respectively, from the crude extract of cotton flowers.

Preparation of phenylboronate affinity rigid monolith with macromolecular porogen

Boronate-affinity monolithic column was first prepared via polystyrene (PS) as porogen in this work. The monolithic polymer was synthetized using 4-vinylphenylboronic acid (4-VPBA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as crosslinker monomer, and a mixture of PS solution in tetrahydrofuran, the linear macromolecular porogen, and toluene as porogen. Isoquercitrin (ISO) and hyperoside (HYP), isomer diol flavonoid glycosides, can be baseline separated on the poly(VPBA-co-EDMA) monolith. The effect of polymerization variables on the selectivity factor, e.g., the ratio of monomer to crosslinker (M/C), the amount of PS and the molecular weight of macromolecular porogen was investigated. The surface properties of the monolithic polymer were characterized by scanning electron microscopy and nitrogen adsorption. The best polymerization condition was the M/C ratio of 7:3, and the PS concentration of 40 mg/ml. The poly(VPBA-co-EDMA) polymer was also applied to extract cis-diol flavonoid glycosides from the crude extraction of cotton flower. After treated by poly(VPBA-co-EDMA) for solid phase extraction, high purity ISO and HYP (>99.96%) can be obtained with recovery of 83.7% and 78.6%, respectively.

Anti-inflammatory effect of pomegranate flower in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages

Abstract Context: Punica granatum L (Punicaceae) flower is an important diabetes treatment in oriental herbal medicine. Objective: This study investigates the inflammation effects of pomegranate flower (PFE) ethanol extract in LPS-induced RAW264.7 cells. Materials and methods: PFE (10, 25, 50, 100 μg/mL) was applied to 1 μg/mL LPS-induced RAW 264.7 macrophages in vitro. Levels of nitric oxide (NO), prostaglandin E2 (PGE2) and pro-inflammatory cytokines interleukin (IL)-1β (IL-1β), interleukin (IL)-6 (IL-6) and tumor necrosis factor (TNF-α) in the supernatant fraction were determined using enzyme-linked immunosorbent assay (ELISA). Expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), phosphorylation of mitogen-activated protein kinase (MAPK) subgroups extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and P38, as well as nuclear factor-κB (NF-κB) activation in extracts were detected via Western blot. Results: 10–100 μg/mL PFE decreased the production of NO (IC50 value = 31.8 μg/mL), PGE2 (IC50 value = 54.5 μg/mL), IL-6 (IC50 value = 48.7 μg/mL), IL-1β (IC50 value = 71.3 μg/mL) and TNF-α (IC50 value = 62.5 μg/mL) in LPS-stimulated RAW 264.7 cells significantly. A mechanism-based study showed that phosphorylation of ERK1/2, p38, JNK and translocation of the NF-B p65 subunit into nuclei were inhibited by the PFE treatment. Discussion and conclusion: These results show that PFE produced potential anti-inflammatory effect through modulating the synthesis of several mediators and cytokines involved in the inflammatory process.

Cost‐effective imprinting combining macromolecular crowding and a dummy template for the fast purification of punicalagin from pomegranate husk extract

The combination of molecular crowding and virtual imprinting was employed to develop a cost-effective method to prepare molecularly imprinted polymers. By using linear polymer polystyrene as a macromolecular crowding agent, an imprinted polymer recognizable to punicalagin had been successfully synthesized with punicalin as the dummy template. The resulting punicalin-imprinted polymer presented a remarkable selectivity to punicalagin with an imprinting factor of 3.17 even at extremely low consumption of the template (template/monomer ratio of 1:782). In contrast, the imprinted polymer synthesized without crowding agent, did not show any imprinting effect at so low template amount. The imprinted polymers made by combination of molecular crowding and virtual imprinting can be utilized for the fast separation of punicalagin from pomegranate husk extract after optimizing the protocol of solid-phase extraction with the recovery of 85.3 ± 1.2%.

A strategy of improving the imprinting effect of molecularly imprinted polymer: Effect of heterogeneous macromolecule crowding

The topic in the present work is to prepare molecularly imprinted polymer (MIP) in heterogeneous crowding surrounding for improving imprinting effect. For the first time, heterogeneous crowding surrounding was made up of a tetrahydrofuran (THF) solution of low molecular weight polyethene glycol (PEG) and high molecular weight polystyrene (PS). The MIP prepared in heterogeneous crowding surrounding against procyanidin B2 can display apparent imprinting effect (imprinting factor of 5.13) in the situation of difficult imprinting at single crowding surrounding or non-crowding surrounding. The necessity of simultaneous use of low and high molecular weight crowding agents for enhanced imprinting effect was further proved. NMR peak shifts of active hydrogen of procyanidin B2 strongly suggested that the interaction between functional monomer and template can be enhanced due to the effect of heterogeneous crowding in the self-assembly process. The influence of molecular weight and amount of PEG, molecular weight of PS, as well as the ratio of procyanidin B2 to functional monomer on the imprinting factor was investigated. The MIP was then used to extract purify procyanidin B2 from extract of grape seed. The recovery of procyanidin B2 obtained was 87% with relative standard deviation of less than 5%.

Preparation of ionic liquid-mediated imprinted monolith for selective capture and purification of corilagin

A method for solid-phase extraction (SPE) of corilagin from natural plant extracts based on molecularly imprinted polymers (MIPs) was developed. For the preparation of corilagin-MIP monoliths, 4-vinylpyridine was used as functional monomer, and ethylene glycol dimethacrylate was used as cross-linking monomer, using a mixture of 1-butyl-3-methylimidazoliumtetrafluoroborate (ionic liquid)-N,N-dimethylformamide-dimethyl sulfoxide as a porogen. A morphological characteristic of the corilagin imprinted monolith was further studied by scanning electron microscopy and nitrogen sorption method. The greatest imprinting factor of COR was up to 9. The MIPs were used as solid-phase extraction (SPE) sorbents for purification of COR and the mean recoveries of corilagin was 78.0% with COR purity of 98.0% from the crude extract of phyllanthus urinaria L. The resulting COR-imprinted polymer also displayed the good performance of fragment imprinting polymer for gallic acid with the mean recoveries of 94.0% and purity of 99.7%.