OBJECTIVETo establish a highly sensitive screening method for phytoestrogen active constituents and to primarily screen the phytoestrogenic active constituents from the chickpea extractions by the method.

METHODHuman ERalpha cDNA was cloned using MCF-7 total RNA as the template by RT-PCR and then was constructed into a pcDNA3 and named as pERalpha. The cell line MCF-7 was co-transfected with pERalpha and the reporter plasmid pERE-Luc which carrying the estrogen response element (ERE) plus the luciferase reporter gene. The luciferase activity was then assayed. The model was optimized by changing the ratio of two plasmids. The feasibility of the optimized model was further proved by the several known phytoestrogen compounds including fermononetin, biochanin A and genistein, et al. As an application of the model, the phytoestrogen activity of the extracts of the chickpea was assayed.

RESULTThe recombinant plasmid (pERalpha) can enhance luciferase activities of pERE-Luc transfected MCF-7 cells. The highest transfection efficiency and luciferase activity were found at the ratio of 10:1 (pERE-Luc: pERalpha), the luciferase activity was improved five times as high as the unique pERE-Luc transfection. The co-transfection screening model also indicated that fermononetin, biochanin A and genistein could induce ERE-driven luciferase activity and ICI 182,780 suppressed the induced transcription. As the application of the model, the results showed that the ethanol (70%) total extraction, the ethyl acetate extraction and the ligarine extraction of the chickpea can induce ERE-driven luciferase activity. Concurrent treatment with ICI 182,780 abolished the induced luciferase activity.

CONCLUSIONA phytoestrogen active constituent screening mode have been established based on co-transfection method. It is sensitive to assay the phytoestrogen active constituents and can be applied to screen the active component of phytoestrogens.

Cell Line, Tumor ; Cicer ; chemistry ; metabolism ; Drug Evaluation, Preclinical ; methods ; Estrogen Receptor alpha ; genetics ; metabolism ; Genes, Reporter ; drug effects ; Genetic Vectors ; metabolism ; Genistein ; chemistry ; pharmacology ; Humans ; Luciferases ; drug effects ; metabolism ; Phytoestrogens ; analysis ; pharmacology ; Plant Extracts ; chemistry ; metabolism ; pharmacology ; Plasmids ; drug effects ; metabolism ; Transfection ; methods

Objective:To study the material basis of the drug effect of the raw material of lamb′s tripe and vitamin B12 capsule.Methods:Rennet and pepsin were extracted with 0.9% sodium chloride and were purified by saturated ammonium sulfate precipitation, diethylaminoethyl-cellulose 52 and high pressure chromatography (HPLC) chromatography. Relative molecular weight was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and liquid chromatography-triple quadrupole mass spectrometer. The amino acid composition and antioxidant activity of raw materials were analyzed with HPLC. The raw materials were completely extracted with water, phosphate buffer and sodium bicarbonate in turn, and in vitro 1, 1-diphenyl-2-picrylhydrazyl radil 2, 2-diphenyl-1-(2, 4, 6-trinitrophenyl)hydrazyl (DPPH) and 2, 2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) radical activity were measured. Glycoprotein from raw material was extracted with hot water and determined its growth-promoting activity gaginst Bifidobacterium adolescentis , Lactobacillus delbrueckii subsp. Bulgaricus and Enterococcus faecalis was measured. The composition of amino acid and monosaccharide were analyzed by HPLC and gas chromatography-mass spectrometry, respectively. Results:The enzyme activity of two purified rennet F6-2 and F7-2 which had different ionic strengths and pepsin F7-1 were 27 557.10, 17 532.60 and 17 728.15 U/g, respectively. The results of SDS-PAGE indicated that the molecular weights of three enzymes were similar, ranging from 35 000 to 40 000. The raw material contained 16 kinds of amino acids, of which hydrophobic amino acids accounted for 33.03% of the total amino acid content. When the sample concentration was 5 mg/mL, ABTS free radical scavenging activity of the three extracts was (37.80±0.45)%, (23.20±0.78)% and (62.80±0.74)%; DPPH free radical scavenging activity was (57.87±0.55)%, (5.03±0.25)% and (26.67±3.10)%, respectively. Glycoprotein extracts had promoted the growth of Bifidobacterium adolescentis, Lactobacillus delbrueckii subsp. Bulgaricus and Enterococcus faecalis, and there was statistically significant difference in the promotion of Lactobacillus delbrueckii subsp. Bulgaricus and Enterococcus faecalis ( P<0.05). The protein chain of glycoprotein was composed of 15 amino acids and the polysaccharide chain was composed of two monosaccharides, glucose and lactose. Conclusions:Two rennet and one pepsin are isolated and analyzed from raw material of lamb abomasum by various chromatographic methods. The raw material is rich in antioxidant active ingredients. The glycoprotein components of lamb abomasum has the activity of promoting the growth of probiotics.

Twelve alkaloids were isolated from the bulbs of Fritillaria yuminensis by column chromatography over silica gel, ODS, and Sephadex LH-20, as well as RP-HPLC. Their structures were identified mainly by NMR and MS analyses as yubeinine(1), imperialine(2), delavinone(3), tortifoline(4), hupehenizioiside(5), imperialine-β-D-glucoside(6), kuroyurinidine(7), pengbeisine A(8), walujewine A(9), peimisine-3-O-β-D-glucopyranoside(10), solanidine-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside(11), and solanidine-3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-glucopyranoside(12). Compounds 4-12 were obtained from F. yuminensis for the first time.
Alkaloids ; analysis ; Chromatography, High Pressure Liquid ; Fritillaria ; chemistry ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Phytochemicals ; analysis ; Plant Roots ; chemistry

OBJECTIVE：To optimize the extractio n technology of Fritillaria pallidiflora polysaccharides（FPSP），and to study its structure. METHODS ：Using the yield of FPSP as response value ，Box-Behnken design-response surface methodology was adopted to optimize solid-liquid ratio ，extraction temperature and extraction time of FPSP extraction technology. Structural properties of FPSP was characterized by UV spectrum ，FTIR，GC-MS，Congo red staining ，SEM，XRD and thermogravimetric analysis. RESULTS：The optimal technology parameters of FPSP were solid-liquid ratio of 1∶28（g/mL），extraction temperature of 94 ℃， extraction time of 2.5 h；the yield of FPSP was 16.25％（n＝3），the error of which to theoretical yield （16.58％）was 0.33％. FPSP contained xylose ，glucose and galactose with a molar ratio of 1∶58.02∶0.73，and trace amount of mannose ；there was a weak absorption peak near the wavelength of 280 nm；belonged to α-configuration pyranopolysaccharide. FPSP was in triple-helical structure. The surface of FPSP was a network structure formed by irregular particles. FPSP had both crystalline and amorphous structures. FPSP had good thermostability. CONCLUSIONS ：The optimized extraction technology of FPSP is reasonable ，and has high extraction yield. The research might provide reference for the further development and utilization of F. pallidiflora .