Comparison of contents variation of six isoflavones in black beans, semifinished and finished Sojae Semen Praeparatum / 药学实践杂志

Objective To compare the contents variation of six flavonoids includingdaidzin，glycitin, genistin, daidzein, glycitein and genisteinin black beans, semifinished and finished Sojae Semen Praeparatum．Methods The contents of flavonoids were determined by HPLC，the condition were Diamonsil C18 column (4.6×250 mm, 5 μm) , column temperature 30 ℃, detection wavelength 260 nm, mobile phase 0.2% acetic acid water (A) - methanol (B), gradient elution, flow rate 1.0 ml/min．Results The linearity of this method to determine 6 isoflavones was good (r≥0.9993) within the determination range, and the recovery rate met the requirements. The RSD of precision, repeatability and stability experiment was less than 4%, 3%and 3%. The results of HPLC showed that the contents of six flavonoidsin Sojae Semen Praeparatum increased significantly compared with black beans. And, the contents of six flavonoids in finished Sojae Semen Praeparatum were slightly more than those in semifinished Sojae Semen Praeparatum. Conclusion The HPLC method established in this study could accurately determine the content of 6 isoflavones in Sojae Semen Praeparatum. The content of six isoflavones in black beans could be increased by the fermentation, and the combined isoflavones were transformed into free isoflavones during the fermentation process.

Characteristic fingerprint and multi-components quantitative determination for Fuke Qianjin Capsules by HPLC / 中国中药杂志

To establish an HPLC characteristic fingerprint method of Fuke Qianjin Capsules,and determine the contents of its main components. The analysis was carried out on a Kromasil 100-5-C18 analytical column(4. 6 mm ×250 mm,5 μm) with gradient elution by acetonitrile(A)-0. 1% phosphoric acid aqueous solution(B),a flow rate at 1. 0 m L·min-1 and the detection wavelength of 254 nm.The column temperature was 30 ℃,and the injection volume was 10 μL. The determination method of genistin,jatrorrhizine,andrographolide and 14-deoxy-11,12-didehydroandrographolide index components were studied methodologically. The common mode of the characteristic fingerprint of Fuke Qianjin Capsules was set up with 8 common peaks,which were identified as genistin,jatrorrhizine,palmatine,berberine,andrographolide,14-deoxy-11,12-didehydroandrographolide,Z-ligustilide,and Z-3-butylidenephthalide,respectively,in comparison with the references. The similarities of 20 batches of Fuke Qianjin Capsules samples were above 0. 95. All of the above-mentioned 4 analytes could be well separated under the optimized chromatographic conditions. RSD of precision and repeatability experiment were both less than 1. 5%,and the sample solution was stable during 72 h. All of the compounds had a good linearity and linear range. The contents of genistin,jatrorrhizine,andrographolide,and 14-deoxy-11,12-didehydroandrographolide in 20 batches of Fuke Qianjin Capsules samples were 28. 66-56. 04,94. 77-197. 92,1 705. 33-4 148. 93 and 462. 16-1 225. 96 μg in each capsule,respectively. The developed HPLC characteristic fingerprint and quantitative analysis methods were reliable,accurate and sensitive,and could be used effectively evaluate the quality of Fuke Qianjin Capsules samples.

Determination of genistin and genistein contents in flemingia philippinensis by HPLC / 国际中医中药杂志

Objective To determine the contents of genistin and genistein in flemingia philippinensis by HPLC. Methods The contents of genistin and genistein in flemingia philippinensis were quantitatively determined by HPLC method. Results The linear relationship between genistin and genistein was good (r=0.9999, RSD=1.77％, 1.16％). There were differences in the contents of genistin and genistein in flemingia philippinensis of different regions, and the genistin content in Yangshuo city of Guangxi was the highest (0.575 mg/g), while the genistein content in Fuzhou city of Guangxi was the highest (0.264 mg/g). Conclusions The HPLC method has good reproducibility and stability in measuring the contents of genistin and genistein in flemingia philippinensis. The contents of genistin and genistein are different in flemingia philippinensis from different areas in Guangxi.

Efeitos pós-ingestivos de flavonóides no epitélio do intestino médio de larvas de Anticarsia gemmatalis (Hübner 1818) (Lepidoptera: Erebidae) / Post-ingestive effects of flavonoids in the midgut epithelium of Anticarsia gemmatalis (Hübner 1818) (Lepidoptera: Erebidae) larvae

An alternative control to soybean caterpillar has been the use of insect resistant plants that present phenolic flavonoids. The midgut is the main access pathway of food and chemical substances ingested. The present study examined morphological alterations in the midgut epithelium of the soybean caterpillar, after the ingestion of soybean genotypes containing the flavonoids rutin and genistin. The caterpillars and genotypes (BRS 257 - control, BR 16, Dowling, PI 229358, IAC 100 and PI 227687) were obtained from the insect rearing facility at the Embrapa Soja. The midguts were collected, fixed in Karnovsky, processed and analyzed under a light microscope. All treatments caused alterations in the midgut epithelial cells. These alterations in the columnar cells were more assiduous than the on other cell types, showing an increase of cytoplasmic protrusions and vacuoles. The goblet cells showed few changes for all genotypes tested, while the regenerative cells presented alterations mainly for the Dowling and PI 227687 treatments. The peritrophic membrane was absent for genotypes IAC100 and PI 227687. We conclude that the Dowling and PI 227687 genotypes were effective and promoted great morphological alterations in the midgut of the soybean caterpillars, being able to be very effective for the control of this plague.

Um controle alternativo para a lagarta da soja tem sido o uso de plantas resistentes a insetos que contém flavonoides fenólicos. O intestino médio é a principal via de acesso do alimento e substâncias químicas ingeridas. O presente estudo examinou as alterações morfológicas no epitélio do intestino médio da lagarta da soja, após a ingestão de genótipos de soja contendo os flavonoides rutina e genistina. As lagartas e os genótipos (BRS 257 - controle, BR 16, Dowling, PI 229358, IAC 100 e PI 227687) foram obtidos do laboratório de criação de insetos da Embrapa Soja. Os intestinos médios foram coletados, fixados em Karnovsky, processados e analisados ao microscópio de luz. Todos os tratamentos causaram alterações nas células epiteliais do intestino médio. As alterações foram mais assíduas nas células colunares do que nos demais tipos celulares. Essas apresentaram grande quantidade de protrusões citoplasmáticas e de vacúolos. As células caliciformes apresentaram poucas alterações para todos os genótipos testados, enquanto as células regenerativas apresentaram alterações principalmente nos tratamentos Dowling e PI 227687. A membrana peritrófica estava ausente para os genótipos IAC100 e PI 227687. Conclui-se que os genótipos Dowling e PI 227687 foram bastante efetivos e promoveram grandes alterações morfológicas no intestino médio das lagartas da soja, podendo ser bastante eficaz para o controle desta praga.

Pharmacological activities of genistin: Research advances / 国际药学研究杂志

Genistin, a kind of soy isoflavones with estrogen-like effect, is one of the effective components of soybean isoflavone. Recently, many researches have shown that genistin possesses a variety of pharmacological activities, however, the summary of its pharmacological activity is not enough. This review mainly focuses on the progress in the estrogen-like effects, anti-tumor activity, improvement of the myocardial ischemia, anti-inflammation activity, the regulation of metabolic syndrome related diseases and other pharmacological activities of genistin, so as to provide reference for the future studies and applications of genistin and other soy isoflavones.

Study on the Quality Standard of Flemingia philippinensis / 中国药房

OBJECTIVE:To establish the quality standard of Flemingia philippinensis. METHODS:The properties and micro-scopic characteristics were observed;TLC was adopted for qualitative identification of genistin and genistein;the moisture,total ash and extract were detected;HPLC was adopted for contents determination of genistin and genistein:the column was Thermo BE-TASIL C18 with mobile phase of Acetonitrile-0.5% glacial acetic acid(gradient elution)at a flow rate of 1.0 ml/min,the detection wavelength was 261 nm,column temperature was 25℃,and the injection volume was 10μl. RESULTS:The properties and micro-scopic characteristics of F. philippinensis and F. macrophylla showed strong specificity. TLC spots of genistin and genistein we- re clear and well separated,with no interference in negative control. The moisture was 3.69%-8.37%,total ash was 1.72%-6.74%,and extract was 5.89%-19.65%. The linear range was 0.012 9-2.588 μg for genistin(r=0.999 9)and 0.004 6-0.923 2 μg for genistein (r=0.999 9);RSDs of precision,stability and reproducibility tests were lower than 3.0%;recoveries were 99.63%-101.87%(RSD=0.82%,n=6)and 97.19%-100.34%(RSD=1.23%,n=6). CONCLUSIONS:The method is simple,accurate and specif-ic,and can be used for the quality control of F. philippinensis.

Analysis of the metabolites of genistin and enzyme kinetics of genistin in rat liver microsome / 中国药学杂志

OBJECTIVE: To characterize the metabolism of genistin and study its enzymatic kinetics in rat liver microsome by HPLC-MS. METHODS: Genistin was incubated with rat liver microsomal incubation system. HPLC-MS method was used to characterize the metabolites. A metabolite generation method was established for quantitative analysis of genistein with sulfamethoxazole as internal standard. The enzyme kinetics parameters Vmax and Km was calculated by the GraphPad Prism 5.0 software. RESULTS: The metabolites in vitro incubation system was identified as genistein. The optimal time in rat liver microsomes incubation time of 40 min, the optimal protein concentration of 1 mg · mL-1, a substrate concentration of 50 μmol · L-1. The enzyme kinetics parameters of genistein were as follows; Vmax=(0.1042 ± 0.0033) μmol · min-1 · mg(pro)-1, Km=(28.96 ± 2.80) μmol · L-1. CONCLUSION: The results indicate that genistin can be metabolited as the form of hydroxylation in rat liver microsome. Metabolite generation method is a reliable and simple method for determination of kinetic parameters of hepatic microsomal enzymes, and enzyme kinetic parameters obtained of genistin provide important parameters for further study.

Effects of different processing methods on content of flavonoids from Sophorae Fructus / 中草药

Objective: To compare the content of flavonoids from Sophorae Fructus by different processing methods. Methods: HPLC method with Zorbax Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm) was used in the experiment; Methanol-0.4% acetic acid was used as mobile phase, with gradient elution; Column temperature was set as 30℃; The flow rate was 1.0 mL/min and detection wavelength was 256 nm. Results: Genistin: crude (0.86%) > stir-frying with honey (0.67%) > carbonizing by stir-frying (0.48%); Rutin: crude (3.0%) > stir-frying with honey (2.2%) > carbonizing by stir-frying (0.88%); Sophoricoside: crude (8.08%) > stir-frying with honey (5.73%) > carbonizing by stir-frying (3.58%); Quercetin: crude (0.04%) < stir-frying with honey (0.05%) < carbonizing by stir-frying (0.12%); Genistein: crude (0.06%) < stir-frying with honey (0.08%) < carbonizing by stir-frying (0.21%); Kaempferide: crude (0.01%) < stir-frying with honey (0.02%) < carbonizing by stir-frying (0.54%). Conclusion: Among the flavonoids from Sophorae Fructus after processing, the content of flavonoid glycosides is reduced and the content of flavonoid aglycone is increased simultaneously, which may be related to the different functions of crude Sophorae Fructus, Sophorae Fructus stir-fried with honey, and Sophorae Fructus carbonized by stir-frying pieces.

Preparation of genistein from genistin-HP-β-CD by enzymatic hydrolysis method / 中国药学杂志

OBJECTIVE: To prepare genistein from genistin-HP-β-CD inclusion complex by enzymic hydrolysis method. METHODS: The method of saturated water solution was applied to prepare genistin-HP-β-CD, then, genistin inclusion complex was transformed into genistein by snailase. Taking the bioconversion rate as the index, different factors were studied to obtain the feasible reaction conditions. An HPLC method was established to compare the bioconversion rates between genistin and genistin-HP-β-CD under the optimum conditions. Hydrolysis product was identified by H-NMR and C-NMR. RESULTS: The optimum reaction conditions were determined just as follows; the reaction medium was acetic acid-sodium acetate buffer solution of pH 5.0, the ratio of snailase; genistin-HP-β-CD was 4:5, and the reaction time was 12 h at 50°C. Under the above mentioned condition, the conversion rate of genistin-HP-β-CD was (90.17±2.40)%, 0.95 times higher than that of the genistin not entrapped with HP-β-CD. CONCLUSION: Preparing genistein by enzymatic hydrolysis method after entrapping genistin with HP-β-CD can significantly shorten the reaction time, and is suitable for industrialization.

Effect of genistein on lipid accumulation in H4Ⅱ E cell induced by oleic acid / 国际中医中药杂志

Objective To observe the effects of genistein(GEN)on oleic acid(OA)induced lipid accumulationin in H4 ⅡE cells and to discuss the possible mechanism of GEN in the pointof AMPK.Methods H4ⅡE cells were cultured in vitro.The control group(NOR),OA treatment group(MOD group)and GEN treatment group were established according to the experimental requirements.The effects of GEN on the proliferation of H4 Ⅱ E cells were measured with MTT assay.The intraeellular TG mass was quantified spectrophotometrically using TG test kit.Cell protein was determined by DCTM Protein Assay kit.The intracellular TG concentration which was used to evaluate lipid accumulation was corrected using protein content as an internal standard.Western blotting was applied to determine the expression of AMPK and P-AMPK (Thr172).Accordingly the phosphorylation levels of AMPK was by means of P-AMPK(Thr172)/AMPK.Results OA treatment can induce lipid accumulation in H4 Ⅱ E cells while GEN treatment can decrease the intracellular TG concentration through up-regulate the phosphorylation levels of AMPK,though the effect was blocked by Compound C that is the inhibitor of AMPK.Conclusion GEN has anti-accumulation of lipid effect in H4ⅡE cell.The mechanism of GEN protective effect is partially due to AMPK.

Development of an analytical method to quantify total isoflavones in phytotherapic capsules using high-performance liquid chromatography

Isoflavones can be found in grains and leaves of soybean. Currently, these are sold in pharmacies as phytotherapic capsules. Isoflavones have been recommended by doctors, especially for women, due to their ability to relieve menopause symptoms, among other benefits. However, no method exists for the official control of isoflavone content in capsules sold in the Brazilian market. This study aims to develop an appropriate analytical method to determine the total isoflavone content (daidzin, glycitin, and genistin, and their respective aglycone forms) in phytotherapic capsules purchased in pharmacies in Curitiba, Parana State, Brazil, using the technique of high-performance liquid chromatography with UV detection (UV-HPLC). The HPLC system consisted of a quaternary pump, an autosampler, and Waters reversed-phase C18 column (5 μm × 300 mm. Analyses were carried out at 40 °C, using a flow rate of 1.0 mL/min (acetonitrile and acetic acid 0.1%), and detection was performed at 254 nm. The method was validated as required by ANVISA and showed to be reliable for the following parameters: linearity (r² >0.99), selectivity (correlation between 0.99 and 1.00), precision (relative standard derivation <1.59%), accuracy (from 80% to 111.63% intraday and from 80% to 117.88% interday recovery), and robustness.

In vivo metabolism of Talosin A, new isoflavonol glycoside from Kitasatospora kifunensis, in rats

The isoflavonol glycoside Talosin A, genistein (GT)-7-alpha-L-6-deoxy talopyranose (GT-Tal), was first isolated from the culture broth of Kitasatospora kifunensis MJM341. The aim of the present study was to evaluate the oral absorption and metabolism of the newly isolated isoflavonol glycoside, GT-Tal compared to genistin (GT-7-O-beta-D-glucopyranoside; GT-Glu). Free GT-Glu and GT-Tal could not be detected prior to enzymatic hydrolysis of the corresponding conjugates in rat plasma. Following oral administration of GT-Tal (15 min), GT-Tal was rapidly absorbed through the gastrointestinal tract and metabolized into GT-Tal conjugates with a mean Cmax of 2.74 microg/mL. GT-Tal was further metabolized to its aglycone, free GT and conjugated GT. After oral administration, GT-Glu was absorbed after being convereted to its aglycone and then further metabolized into its conjugate metabolites (free GT with a mean Cmax of 0.24 mg/mL at 1.25 h; conjugated GT with a mean Cmax of 1.31 mg/mL at 2.00 h). Significant differences in absorption and metabolism of GT-Tal and GT-Glu were observed. GT-Tal was metabolized into its corresponding conjugates or underwent deglycosylation to form GT, whereas GT-Glu was metabolized into its aglycone, GT.

RP-HPLC determination of daidzein, genistein, daidzin and genistin in soybean extract / 中草药

Object A method for the determination of daidzein, genistein, daidzin and genistin in soybean extract by RP HPLC was established Methods Zorbax SB C 18 column (4 6?15 mm, 5 ?m) was used Daidzein and genistein were separated with mobile phase Ⅰ∶methanol acetonitrile 0 1% phosphoric acid solution (30∶8∶62) Daidzin and genistin were separated with mobile phase Ⅱ∶methanol acetonitrie 0 1% phosphoric acid solution (16∶24∶60) The UV detection wavelength was 254 nm Results The average recoveries of daidzein, genistein, daidzin and genistin were 98 7%, 99 5%, 98 4%, 98 6% and the precision (RSD) were 1 4%, 0 9%, 0 9%, 1 7% (n=4) respectively Conclusion: The method was accurate, rapid and with good reproducibility

Separation of soybean isoflavone glucosides from Semen Sojae Praeparatum by preparative reversed-phase high performance liquid chromatography / 第二军医大学学报

Objective:To search for a rapid and efficient method for the isolation of 3 kinds of isoflavone glucosides from the ethanol extract of Semen Sojae Praeparatum.Methods: The crude isoflavones were extracted by 75% aqueous ethanol after removing the oil by Soxhlet extraction.Then 1300 macroporous resin,water and different concentrations of aqueous ethanol(10%,20%,30%,40%,50%,70%,and 95%) were used to separate and elute the crude isoflavones.The fraction eluted by 40% aqueous ethanol was subjected to preparative HPLC analysis.The chromatographic conditions: A YWG C18(10.0 mm?200 mm,i.d.10 ?m) column was used;the mobile phase consisted of acetonitrile-water-acetic acid at volume ratio of 25751(V/V/V) and a flow rate of 3.0 ml/min;the injection volume was 750 ?l;and the detection wave length was set at 260 nm.Results: Daidzin,glycitin and genistin were rapidly separated with the purities over 99% as determined by external standard HPLC.Conclusion: This technique is simple and suitable for the isolation of daidzin,glycitin and genistin from Semen Sojae Praeparatum.