葛根药材HPLC指纹图谱与6种成分含量测定研究

目的 建立葛根Pueraria lobate药材HPLC指纹图谱,并测定不同产地葛根中6种成分(3’-羟基葛根素、葛根素、3’-甲氧基葛根素、葛根素芹菜糖苷、大豆苷、大豆苷元)的含量,为葛根优质药材产地与质量控制提供参考。方法 采用HPLC法,葛根药材30%乙醇提取液的分析采用Hypersil BDS C₁₈色谱柱(250 mm×4.6 mm,5μm);0.1%甲酸水-乙腈为流动相,梯度洗脱;体积流量1.0 mL/min;检测波长250 nm;柱温25℃。建立32批葛根药材指纹图谱,并测定不同产地葛根药材6种成分的含量。结果 建立了葛根药材HPLC指纹图谱,共提取出10个共有峰,指认6个共有峰;建立了含量测定方法,并对不同产地来源的32批葛根药材中6种成分含量测定,不同产地葛根质量对比综合评分结果显示河南最高,其次为陕西。结论 建立了葛根HPLC指纹图谱方法,通过对比,各样品与对照图谱相似度均大于0.9,为葛根道地药材质量标准提供整体质量控制依据。结合产地调研信息,可将葛根优质原料药材产地优选为陕西、河南秦岭山脉。     

高效液相色谱法测定不同产地葛根中葛根素、大豆苷及大豆苷元的含量

目的 :建立同时测定葛根中葛根素、大豆苷及大豆苷元的含量测定方法。方法 :选用SYMMETRY C₁₈色谱柱 (3.9mm× 15 0mm ,5 μm) ,甲醇 1%醋酸水溶液为流动相梯度洗脱 ,紫外检测波长为 2 5 0nm ,HPLC法。结果 :平均回收率分别为 10 1.7% ,10 0 .7% ,10 1.7% (n=3) ,RSD分别为 0 .43 % ,0 .82 % ,1.5 0 % (n=3)。结论 :本法简便 ,准确 ,快速 ;适用于含葛根素、大豆苷及大豆苷元的葛根药材及制剂的质量控制。     

Inclusion complexes of isoflavones with two commercially available dendrimers: Solubility, stability, structures, release behaviors, cytotoxicity, and anti-oxidant activities

We prepared and characterized the inclusion complexes of daidzein with poly(amidoamine) (PAMAM) and poly(propylene imine) (PPI) dendrimers. Aqueous solubility of daidzein was significantly enhanced by both PAMAM and PPI (186- and 650-fold at 0.36mM, respectively). Daidzein in G3 PAMAM solution is more stable than that in G4 PPI. NMR studies reveal the encapsulation of daidzein within the interior cavities of PPI through hydrophobic interactions. Daidzein exhibits a slower release behavior from PPI than that from PAMAM. PPI/daidzein complex is much more toxic than PAMAM/daidzein complex on several cell lines. PAMAM/daidzein complexes showed similar protective effect on oxidative stress-induced cytotoxicity as compared to free daidzein. These results suggest that the inclusion of daidzein with dendrimer can effectively improve the solubility, prolong the delivery, and maintain the anti-oxidant activity of daidzein. This research provides new insights into dendrimer-based drug delivery systems and will be helpful for the design of novel dendrimer/drug formulations.     

HPLC法测定大鼠血浆中大豆苷元的浓度

目的:建立测定大鼠血浆中大豆苷元浓度的高效液相色谱法。方法:用乙酸乙酯提取血浆样品中大豆苷元,采用高效液相色谱法进行测定。色谱柱:Hypersil ODS C₁₈柱（250mm×4.6mm,5μm）,流动相:甲醇-0.05%磷酸溶液（60:40）,检测波长:260nm,柱温:30℃,流速:1ml·min^-1,进样量:20μl。结果:大豆苷元的线性范围为0.01～1.00μg·ml^-1（r=0.9998）;日内和日间精密度RSD均<4.1%;相对回收率97.11%～101.82%,绝对回收率>90.0%。结论:本法专属性强,灵敏度高,重复性好,适用于大豆苷元的药物动力学研究。     

Genotoxicity assessment of S-equol in bacterial mutation,chromosomal aberration,and rodent bone marrow micronucleus tests

Intervention studies in man have shown that dietary soy isoflavones may provide certain health benefits. One of the possible reasons for this benefit is that the daidzein contained in soy is converted to S-equol. As part of a drug development program for S-equol, three genotoxicity studies were conducted. The studies comprised bacterial mutation, chromosomal aberration, and in vivo bone marrow micronucleus tests conducted according to Good Laboratory Practices (GLP). No evidence of genotoxic activity was observed in the in vitro tests at concentrations up to those associated with cell toxicity. In addition, no evidence of cytotoxicity or genotoxicity was seen in the rat bone marrow micronucleus test in animals dosed at levels up to the standard limit of 2000 mg/kg. It is concluded that S-equol is not active in the standard battery of genotoxicity assays recommended by the International Conference on Harmonisation (ICH) for registration of new pharmaceuticals. The current results support the further development of S-equol.     

Assessment of the estrogenicity of isoflavonoids,using MCF-7-ERE-Luc cells

In the current study, our research focused on the estrogenic activity of isoflavonoids, mainly genistein, biochanin A and daidzein. Genistein enhanced the reporter gene expression of MCF-7-ERE-Luc cells, at a concentration as low as 10 nM, with a concentration of 100 nM the achieved gene expression effects were similar to those of 10 pM 17beta-estradiol, Based on the estrogenic activities of biochanin A and daidzein, hydroxyl groups at the 4' and 5 positions are needed for the maximal effect of the genistein. The estrogenic effects of these isoflavonoids were inhibited by the concomitant treatment with tamoxifen. The data showed that the estrogenic effects of isoflavonoids were mediated through estrogen receptors. When the isoflavonoids were tested as mixtures, the estrogenic effects were lower than the arithmetic sum of those induced by each individual isoflavonoid. The estrogenic potency of each isoflavonoid was presented at EC50 levels with a 17beta-estradiol equivalent concentration (EEQ) based on the dose response of each chemical. The EC50s and EEQs of genistein, biochanin A and daidzein were 4.15, 0.89 and 0.18 microM, and 15.0, 5.12 and 1.83 microM/M, respectively. Our data clearly demonstrated that the pERE-luciferase reporter gene assay was suited for the sensitive and quantitative measurement, and large scale screening, of the estrogenicity of chemicals in vitro.     

Genistein and daidzein, in combination, protect cellular integrity during 7,12-dimethylbenz[a]anthracene (DMBA) induced mammary carcinogenesis in Sprague-Dawley rats

The status of glycoconjugates (protein bound hexose, hexosamine, sialic acid and fucose) in plasma or serum serve as potential biomarkers for assessing tumor progression and therapeutic interventions. Aim of the present study was to investigate the protective effect of two major soy isoflavones, genistein and daidzein, in combination on the status of glycoconjugates in plasma, erythrocyte membrane and mammary tissues during 7,12-dimethylbenz[a]anthracene (DMBA) induced mammary carcinogenesis in female Sprague-Dawley rats. A single subcutaneous injection of DMBA (25 mg rat(-1)) in the mammary gland developed mammary carcinoma in female Sprague-Dawley rats. Elevated levels of plasma and mammary tissue glycoconjugates accompanied by reduction in erythrocyte membrane glycoconjugates were observed in rats bearing mammary tumors. Oral administration of genistein + daidzein (20 mg + 20 mg kg(-1) bw/day) to DMBA treated rats significantly (p< 0.05) brought back the status of glycoconjugates to near normal range. The present study thus demonstrated that genistein and daidzein in combination protected the structural integrity of the cell surface and membranes during DMBA-induced mammary carcinogenesis.     

葛根中总黄酮的导数脉冲极谱测定

葛根为豆科植物,是中医常用的祛风解表药,多年来临床试验证明葛根制剂对伴有颈项强痛、头痛、头晕的高血压病、心绞痛、早期耳蜗性聋、偏头痛等病症均有较好的疗效。通过化学、药理和临床试验证明,葛根的有效成分是葛根素(puerarin)、大豆甙(daidzin)和大豆甙元(daidzein)等异黄酮类化合物,其结构如下: