Magnetic molecularly imprinted polymers(MMIPs) were prepared with ZL006 as template, acrylamide(AA) as the functional monomer, and acetonitrile as pore-forming agent; then Fourier transform infrared spectroscopy(FT-IR) and scanning electron microscopy(SEM) were used to characterize their forms and structures. Simultaneously, the MMIPs prepared previously were used as sorbents for dispersive magnetic solid phase extraction(DSPE) to capture and identify potential nNOS-PSD-95 uncouplers from extracts of Trifolium pratense and the the activities of the screened compounds were evaluated by the neuroprotective effect and co-immunoprecipitation test. The experiment revealed that the successfully synthesized MMIPs showed good dispersiveness, suitable particle size and good adsorption properties. Formononetin, prunetin and biochanin A were separated and enriched from Trifolium pratense by using the MMIPs as artificial antibodies and finally biochanin A was found to have higher cytoprotective action and uncoupling action according to the neuroprotective effect and co-immunoprecipitation test.
Adsorption ; Genistein ; chemistry ; Molecular Imprinting ; Phytochemicals ; chemistry ; Polymers ; chemistry ; Solid Phase Extraction ; Spectroscopy, Fourier Transform Infrared ; Trifolium ; chemistry

OBJECTIVETo study the methods of determining the amount of Isoflavones in red clover extractive.

METHODRP-HPLC is employed to determine the Isoflavones, with a C18 RP Column, a moble phase of MeOH-CH3CN-0.1%H3PO4 and a detection wavelength of 260 nm.

RESULTAfter being water-solved, the four groups of flavoes elements satisfactorily separated, the amount of feeding in a range of 0.024-0.336 microg which has a good linear relationship with the peaks, and the total isoflavones determining results repetition, RSD=10.1%.

CONCLUSIONA simple, reliable and effective quality-control method is given.

Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Hydrolysis ; Isoflavones ; analysis ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control ; Trifolium ; chemistry

OBJECTIVETo establish a high performance liquid chromatography method with spectrofluorescence detector for the quantitative analysis of benzopyrene in red clover extract.

METHODAnalysis was performed on Hypersil C18 column (4. 6 mm x 200 mm, 5 microm) with acetonitrile and water (70: 30) as mobile phase; The excitation wavelength and emission wavelengh were 368 nm and 405 nm, respectively; Flow rate was 1.0 mL x min(-1).

RESULTThe linear range was 0.93-37.2 ng x mL(-1), and r = 0.9992 (n = 5). The average recovery of benzopyrene was 85.2% (n = 9).

CONCLUSIONThe method was convenient and accurate with good recovery.

Benzo(a)pyrene ; analysis ; isolation & purification ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Fluorescence ; Plants, Medicinal ; chemistry ; Reproducibility of Results ; Trifolium ; chemistry

This study was designed to investigate the potential estrogenic effects of perinatal dietary phytoestrogens on the rat uterus. Pregnant rats were divided to three groups provided the following diets: (1) rat chow, (2) rat chow with 7.5% Trifolium (T.) pratense, or (3) rat chow supplemented with 17beta-estradiol (0.5 mg/kg). The dams in each group were kept on the same diet during pregnancy and lactation. Female offspring were euthanized on day 21 at which time body and organ weights were recorded and tissue samples were taken for histology. Immunohistochemistry was performed to detect estrogen receptor alpha (ERalpha) and progesterone receptor (PR) levels. Our results revealed estrogen-like biological effects of perinatal T. pratense exposure. Relative uterus and ovary weights in the experimental groups were increased compared to control. The number of uterine glands and luminal epithelium heights were also increased. However, there were no statistically significant changes detected in the immunostaining intensity of ERalpha and PR between the groups.
Animals ; Animals, Suckling ; Body Weight/drug effects ; Estrogen Receptor alpha/*metabolism ; Female ; Immunohistochemistry ; Isoflavones/*pharmacology ; Lactation ; Maternal Exposure ; Organ Size/drug effects ; Phytoestrogens/*pharmacology ; Plant Components, Aerial/chemistry ; Pregnancy ; Rats ; Rats, Wistar ; Receptors, Progesterone/*metabolism ; Trifolium/*chemistry ; Uterus/*drug effects

OBJECTIVETo evaluate the effects of red clover isoflavones on the proliferation and apoptosis of human benign prostatic hyperplasia (BPH) stromal cells.

METHODSWe treated human prostate stromal cells with red clover isoflavones at the concentration of 12.5, 25, 50 and 100 microg/ml, and established a PBS blank control, a dimethyl sulphoxide (DMSO) negative control and four finasteride positive control groups (at the concentration of 12.5, 25.0, 50.0 and 100.0 microg/ml). We determined the effects of different concentrations of red clover isoflavones on the proliferation of the cells by MTT assay and on their apoptosis by Annexin V/PI double staining flow cytometry.

RESULTSRed clover isoflavones inhibited the proliferation of the BPH stromal cells by 18.86% at 25.0 microg/ml, compared with 5.17% in the blank control group (P < 0.05), and more obviously at a higher concentration. At 50.0 microg/ml, red clover isoflavones exhibited a weaker inhibitory effect than finasteride (28% vs 69.88% , P < 0.05). Annexin V/PI double staining flow cytometry showed that red clover isoflavones at 25.0 microg/ml induced the apoptosis of the prostate stromal cells by (18.54 +/- 2.5)%, with significant differences from the negative control and blank control (P < 0.01).

CONCLUSIONRed clover isoflavones can inhibit the proliferation and promote the apoptosis of human BPH stromal cells.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Humans ; Isoflavones ; pharmacology ; therapeutic use ; Male ; Plant Extracts ; pharmacology ; therapeutic use ; Prostate ; cytology ; drug effects ; Prostatic Hyperplasia ; drug therapy ; pathology ; Stromal Cells ; drug effects ; Trifolium ; chemistry

Effects of six kinds of Chinese herb extracts, including Folium Crataegi extract, Herba Epimedii extract, Folium Acanthopanacis Senticosi extract, Trifolium pratense L. extract, Folium Ginkgo extract and Radix Puerariae extract, on the activities of CYP450 isozymes (CYP1A2, CYP2C, CYP2E1, CYP2D, CYP3A) in rat hepatic microsomals were studied by using a UPLC-MS/MS (MRM) and cocktail probe substrates method. The results showed that effects of six kinds of Chinese herb extracts on each CYP450 isozyme activity were inhibitory. The IC50 of Folium Crataegi extract for the inhibition of rat microsomal CYP2D activity was only for 4.04 microg x mL(-1), which showed the highest inhibition; Trifolium pratense L. extract had strong inhibitory action to CYP2D, the IC50 value was 5.73 microg x mL(-1); Folium Crataegi extract also had strong inhibitory action on CYP2E1, the IC50 value was 10.91 microg x mL(-1). Furthermore, the IC50 of Folium Ginkgo extract for the inhibition of rat microsomal CYP3A, 2D, 2E1 activities were 45.12, 35.45 and 22.41 microg x mL(-1), respectively, and the IC50 of Folium Acanthopanacis Senticosi extract on the inhibition of rat microsomal CYP2E1 activity was 32.89 microg x mL(-1). In addition, mechanism of inhibition experimental results showed that the inhibiting abilities of Folium Crataegi extract and Radix Puerariae extract on each CYP450 isozyme increased with the increasing of the preincubation time, therefore, the inhibitory effects were a mechanism-based inhibition.
Animals ; Chromatography, High Pressure Liquid ; Crataegus ; chemistry ; Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 CYP2E1 ; metabolism ; Cytochrome P-450 CYP3A ; metabolism ; Cytochrome P-450 Enzyme System ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Eleutherococcus ; chemistry ; Epimedium ; chemistry ; Ginkgo biloba ; chemistry ; Inhibitory Concentration 50 ; Male ; Microsomes, Liver ; enzymology ; Plants, Medicinal ; chemistry ; Pueraria ; chemistry ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry ; Trifolium ; chemistry

The study investigated the effects of red clover extract (RCE) on mouse T macrophages and lymphocytes in vitro. The cell toxic effect of RCE was estimated by MTT assay. Multiple-fluorescence staining plus flow cytometry were used to detect the effect of RCE on CD69/CD25/CD71 expression of mouse T lymphocytes stimulated by Con A; CFDA-SE staining plus flow cytometry were used to analyze the effect of RCE on proliferation of T lymphocytes activated by Con A; The effect of RCE on nitric oxide (NO) secretion of mouse macrophages stimulated by lipopolysaccharide (LPS) for 24 h was assayed by Griess reagent system. We found that RCE had potent anti-inflammatory effects on mice. RCE had little cell toxic effect on mouse lymphocytes and macrophages. RCE strongly inhibited the excessive production of inflammatory mediators (NO, CD69, CD25, CD71), in a dose-dependent manner, like cyclosporine A injection. RCE could inhibit proliferation of CD3+ T lymphocytes. These data suggested that RCE might exhibit anti-inflammatory effect by inhibiting the activation and proliferation of mouse lymphocytes and the NO secretion of mouse macrophages.
Animals ; Anti-Inflammatory Agents ; isolation & purification ; pharmacology ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; CD3 Complex ; analysis ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Lectins, C-Type ; Lymphocyte Activation ; drug effects ; Macrophages ; cytology ; secretion ; Male ; Mice ; Mice, Inbred BALB C ; Nitric Oxide ; secretion ; Plants, Medicinal ; chemistry ; Receptors, Transferrin ; metabolism ; T-Lymphocytes ; cytology ; drug effects ; metabolism ; Trifolium ; chemistry