N-(顺式-4-异丙基环己基-1-甲酰基)-D-苯丙氨酸和N-(反式-4-异丙基环己基-1-甲酰基)-L-苯丙氨酸的合成

目的合成N-(顺式-4-异丙基环己基-1-甲酰基)-D-苯丙氨酸和N-(反式-4-异丙基环己基-1-甲酰基)-L-苯丙氨酸.方法以(4-异丙基)环己基甲酸为原料,在二环己基碳二亚胺(DCC)作用下,与N-羟基琥珀酰亚胺反应得到(4-异丙基)环己基甲酸琥珀酰亚胺酯(3),柱色谱分离化合物3得到顺式和反式异构体.顺式体与D-苯丙氨酸甲酯发生酰化反应,碱水解后即得到N-(顺式-4-异丙基环己基-1-甲酰)-D-苯丙氨酸;而反式体与L-苯丙氨酸甲酯发生酰化反应,水解后得到N-(反式-4-异丙基环己基-1-甲酰)-L-苯丙氨酸.结果与讨论成功合成了目标化合物,反应总收率分别为39%和31%.     

缺氧诱导因子-1与宫颈癌关系的研究进展

缺氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)就是一个在缺氧状态下重要的转录调节因子.HIF-1在人体各种肿瘤中的过度表达,提示其与肿瘤进展和转移密切相关[1].近年来研究显示HIF-1与宫颈癌的关系密切,因此,了解HIF-1在宫颈癌中的研究进展具有重要意义.1缺氧诱导因子-1的生物学特性1.1结构缺氧诱导因子l(hypoxia-inducible factor1,HIF-1)于1992年在低氧诱导促红细胞生成素(erythropoietin,EPO)基因的转录激活中发现,能特异性地结合于促红细胞生成素编码区下游的缺氧反应元件(hypoxia response element,HRE)的特异性DNA序列.HIF-1为异二聚体蛋白聚合物,由120KD的α亚基和91-94KD3亚基组成.HIF-1β在任何状态下都表达,而HIF-1α只在氧浓度低于6％时才表达,正常氧浓度下IF-1的半衰期＜1-2min[2],所以HIF-1α是HIF-1的功能亚单位.1.2机制HIF-1α的401 -603氨基酸残基位有一个氧依赖性降解区,对HIF-1α的稳定性起重要作用.     

菊欧氏杆菌β-1,4-内切葡聚糖酶celY基因在大肠杆菌中的克隆、表达及活性分析

以菊欧氏杆菌(Erwinia chrysanthemi)基因组DNA为模板,通过PCR方法扩增了该菌的β-1,4-内切葡聚糖酶celY基因及其调控元件并克隆至pUC19载体。测序结果经分析,该基因编码的蛋白序列与菊欧氏杆菌Ech586的β-1,4-内切葡聚糖酶同源性达到98%。将celY的开放阅读框基因插入到表达载体pET-28a中,转化大肠杆菌E.coli BL21(DE3),采用LB-CMC平板刚果红染色筛选含celY基因的转化子。12%SDS-PAGE显示,重组菌经IPTG诱导后表达了目的蛋白,其相对分子质量与预期结果相符。CMCase活力测定显示重组pET-28a-celY阳性菌分泌到培养基中的CelY酶活力为84.4 U/L,周质中的酶活力为12.7 U/L。β-1,4-内切葡聚糖酶CelY工程菌的获得,为研究菊欧氏杆菌纤维素酶基因在菊欧氏杆菌同源转化中的表达及工程菌的酶学特性提供了重要的资料。     

人血浆中全反式维甲酸、13-顺式维甲酸和9-顺式维甲酸浓度的监测

目的建立高效液相色谱法同时测定人血浆中全反式维甲酸、13-顺式维甲酸和9-顺式维甲酸浓度。方法色谱柱为KromasilC18柱(4.6mm×250mm,5μm);柱温为室温。流动相A:甲醇;流动相B:0.01mol/L醋酸钠缓冲液(pH=5.7),梯度洗脱,流速1ml/min;检测波长340nm。结果全反式维甲酸、13-顺式维甲酸和9-顺式维甲酸血药浓度在1～200ng/ml范围内,浓度与峰面积比有良好的线性关系,最低检测浓度为0.5ng/ml。全反式维甲酸方法回收率为97.22%～108.80%,日内RSD≤8.24%,日间RSD≤11.34%;13-顺式维甲酸方法回收率为98.62%～104.80%,日内RSD≤8.02%,日间RSD≤11.70%;9-顺式维甲酸方法回收率为97.74%～102.24%,日内RSD≤7.72%,日间RSD≤9.17%。结论本方法简单、快速、灵敏、重现性好,适用于全反式维甲酸、13-顺式维甲酸和9-顺式维甲酸临床血药浓度监测及人体药代动力学研究。     

高效液相色谱法分析鉴定虾青素全反式、9-顺式、13-顺式几何异构体

目的 建立虾青素全反式、9-顺式和13-顺式异构体的高效液相色谱（HPLC）分析鉴定方法,优化一种碘诱导全反式虾青素异构化的方法。方法 采用高效液相色谱法,色谱柱为Shim-pack GIST C-18(1504.6 mm,5 μm),流动相为甲醇-乙腈-二氯甲烷-水（85:5.5:5:4.5）,检测波长为480 nm,流速为0.5 mL/min,进样量10 μL。为探究虾青素异构化条件,改良了一种碘诱导全反式虾青素异构化的方法,在曝光20 min后,分别对含碘量为5%~50%的10个虾青素样品溶液进行HPLC分析。结果 虾青素全反式、9-顺式和13-顺式异构体在该色谱条件下获得良好分离,经实验所得虾青素顺反异构体出峰顺序为全反式、9-顺式、13-顺式。异构化实验结果表明含碘量为15%的虾青素溶液经曝光后全反式、9-顺式、13-顺式异构体含量分别为对照品的68.5%、2436.7%和632.4%,为本实验的最佳比例。结论 本法准确、可靠,并较为便捷,在虾青素及其顺反异构体定量分析方面提供一定数据支撑,为虾青素制品在食品和医药方向的发展提供了理论依据。     

丹参4-羟基-3-甲基-2-丁烯基焦磷酸还原酶基因的全长克隆与诱导表达分析

本研究根据转录组数据提供的基因片段设计特异引物,利用cDNA快速末端扩增方法克隆丹参4-羟基-3-甲基-2-丁烯基焦磷酸还原酶全长cDNA(SmHDR),GenBank注册号JX233817,并进行蛋白结构预测、序列多重比对和构建进化树等生物信息学分析;然后采用实时荧光定量PCR(RT-PCR)检测该基因在Ag+诱导后的表达情况,使用UPLC检测对应样品的丹参酮类化合物含量。获得的SmHDR全长基因由1 647个核苷酸组成,编码463个氨基酸,蛋白分子质量为51.88 kD,等电点pI 5.72,二级结构中α-螺旋结构占35.64%、β-折叠占20.30%、无规则卷曲占44.06%。序列比对和系统进化分析表明,SmHDR与其他植物HDR家族具有较高的同源性,并与库洛胡黄连HDR两者的亲缘关系较近。RT-PCR结果显示,SmHDR受Ag+诱导后表达水平在12 h时急剧上升后显著下降,丹参酮类成分含量受Ag+诱导后先缓慢上升,在120 h时有明显提高,两者的增加趋势呈正相关。推测SmHDR基因可能参与丹参酮类成分的生物合成,这为进一步研究丹参酮类成分生物合成及其次生代谢调控机制奠定了基础。     

17β-甾醇酯肟-3的合成及其异构体的分离鉴定

为了寻找长效避孕药,我们采用了17β-甾醇酯与等重量的羟胺盐酸盐在吡啶存在下反应,合成了5个炔诺酮和18-甲基炔诺酮的17β-甾醇酯肟-3(Ⅱa-e)。所得的酮肟是顺式和反式的混合物,用HPLC测定了两种异构体的比例。其中四个光活酮肟(Ⅱ_(a,b,c,d))用旋转薄使仪分离得到了各自的顺式和反式异构体并研究了它们之间的不同处。     

β-内酰胺酶抑制短肽SIPIS04-01的表达、纯化与抑制作用测定

通过酵母双杂交系统从一个随机DNA片段文库中筛选到一个编码能与β-内酰胺酶结合的短肽SIPIS04—01的DNA序列，将它克隆到pGEX-4T-1的多克隆位点中，得到重组质粒pYG205。当用适量的IPTG诱导后，携带pYG205的E．coli DH5α能表达短肽SIPIS04-01-GST融合蛋白。利用谷胱甘肽Sepharose 4B亲和层析介质分离纯化短肽SIPIS04-01-GST融合蛋白，经凝血酶切割并分离纯化后，体外试验表明短肽SIPIS04-01具有抑制β-内酰胺酶的作用。     

人体β-防御素-3突变基因的构建及在大肠埃希菌中的融合表达

目的从人正常皮肤组织中提取β-防御索-3基因进行定点突变后在E．coli中融合表达。方法从人正常皮肤组织中提取总RNA，经RT-PCR扩增得到编码β-防御素-3成熟肽的cDNA序列，测序正确后，寻找合适的突变位点，设计含突变位点的引物，用PCR重叠延伸法对β-防御素-3成熟肽cDNA序列进行定点突变，将突变产物克隆至pUC18进行序列测定及在E．coli中融合表达。结果测序结果表明，经RT—PCR所得的β-防御素-3成熟肽序列与GeneBank中报道的编码人β-防御索-3成熟肽的cDNA序列完全-致。经过突变β-防御素-3成熟肽第29位谷氨酰胺密码子由CAG突变为精氨酸密码子CGA，其余核苷酸序列均未发生变化。将突变β-防御素-3基因在E．coli中诱导表达3～5h后可见突变β-防御素-3蛋白表达。结论成功构建并表达了β-防御素-3突变体基因，为进-步对突变体进行真核表达、生物活性研究奠定了基础。     

抗血吸虫病药物呋喃丙胺顺式异构体的研究

呋喃丙胺顺式异构体的合成,系按文献方法制备顺式β-(5-硝基-2-呋喃)-丙烯酸,然后于低温下与异丙胺经混合酸酐法,氨化为顺式与反式两种酰胺的混合物,再经分离获得顺式异构体。实验动物药理结果证明其生物活性与反式异构体相似。     

The cadin-2-en-1β-ol-1β-D-glucuronopyranoside suppresses TPA-mediated matrix metalloproteinase-9 expression through the ERK signaling pathway in MCF-7 human breast adenocarcinoma cells

A sesquiterpene glycoside, cadin-2-en-1β-ol-1β-D-glucuronopyranoside (known as CR4-1), was isolated from Catharanthus roseus (Apocynaceae) hairy root cultures. C. roseus is widely used as an ornamental and medicinal plant and is cultivated mainly for its alkaloids. C. roseus has been reported to have pharmacologic properties such as anti-cancer, enzymatic anti-oxidant, and anti-diabetic effects. In this study, we demonstrated that CR4-1 significantly inhibited the in vitro invasion of MCF-7 human breast adenocarcinoma cells induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA). Matrix metalloproteinases (MMPs) are known to be involved in cancer invasion and metastasis. Zymographic analysis showed that CR4-1 suppressed TPA-induced MMP-9 activity in a dose-dependent manner. We further demonstrated that CR4-1 suppressed the phosphorylation of extracellular signal-regulated protein kinase, but not p38 kinase or c-Jun N-terminal kinase (JNK). Moreover, CR4-1 attenuated TPA-induced degradation of κBα inhibitor (IκB-α). These results suggest that CR4-1 reduces the invasiveness of human cancer cells by suppressing MMP-9 expression through inhibition of the NF-κB signaling pathways.     

反相高效液相色谱法测定不同方法提取长春花中脱水长春碱的含量

目的:建立以反相高效液相色谱法测定不同方法提取长春花鲜叶中脱水长春碱含量的方法。方法:分别采用冷浸法、匀浆-空化法、超声法、热回流法对长春花鲜叶进行提取并比较含量。结果:长春花鲜叶以甲醇冷浸3h时脱水长春碱提取率最高。结论:本方法灵敏、准确,可为长春花鲜叶中脱水长春碱的定量分析提供科学、有效的检测手段。     

Alkaloid Production in Catharanthus roseus cell cultures VIII

A cell line of Catharanthus roseus (L.) G. Don coded PRL # 200, was characterized with respect to its biosynthetic capabilities for indolealkaloids, in particular catharanthine, in suspension cultures. Other alkaloids isolated are vallesiachotamine isomers, ajmalicine, h?rhammericine, h?rhammerinine, vindolinine, 19-epivindolinine and strictosidine lactam.     

Evaluation of hypolipidemic activity of leaf juice of Catharanthus roseus (Linn.) G. Donn. in guinea pigs

Our aim of the study was to evaluate the hypolipidemic activity of leaf juice of Catharanthus roseus (Linn.) G. Donn. in guinea pigs. Adult guinea pigs of either sex were divided into seven groups: group 1 - normal diet; group 2 - high fat diet; group 3 and 4 - normal diet plus leaf juice of Catharanthus roseus (Linn.) G. Donn. in the dose of 0.5 and 1 mL/kg, respectively; group 5 and 6- high fat diet with leaf juice of Catharanthus roseus (Linn.) G. Donn. in the dose of 0.5 and 1 mL/kg, respectively; group 7 - high fat diet plus atorvastatin (3 mg/kg). Above diet treatment was given for six weeks and drug was given during last three weeks. Serum lipid profile (total cholesterol, triglycerides, LDL-c, VLDL-c, HDL-c) was performed in each group of animals before and at the end of six weeks. Histological study of aorta, liver and kidney was done in group 1, 2, 6 and 7 and blood cell count was done in animals that were treated juice of C. roseus (Linn.) G. Donn. before and after juice administration. Simultaneous administration of leaf juice of C. roseus (Linn.) G. Donn. in the dose of 0.5 mL/kg prevents the rise of serum lipid parameters and decreases the fatty changes in the tissue induced by high fat diet, whereas in the dose of 1 mL/kg not only counteracts the elevation, but also significantly (p < 0.05) reduces the serum level LDL-c and the ratio of total cholesterol and HDL-c. Leaf juice of C. roseus (Linn.) G. Donn. possesses significant lipid lowering and anti atherosclerotic activity.     

Severe bone marrow depression induced by an anticancer herb Cantharanthus roseus

We report a 67-yr-old woman with hepatitis C-related liver cirrhosis and hepatoma who had developed severe bone marrow suppression after taking Cantharanthus roseus as an alternative anticancer treatment. The patient developed severe pancytopenia with initial presentations of vomiting, diarrhea, oral ulcer, and fever about 1 week after taking 5-days' course of Cantharanthus roseus. Bone marrow biopsy showed autolysis, which indicated massive necrosis of the hematopoietic cells. There was no malignant cell infiltration. The patient also had severe gastrointestinal disturbances, bacteremia, urinary tract infection, and impaired renal and liver function. Supportive care with broad-spectrum antibiotics, granulocyte colony-stimulating factor, repeated blood transfusions, and albumin supplement was given. She recovered and was discharged after 48 days hospitalization. Coadministration of Cantharanthus roseus and cisapride was noted, and these two drugs are both substrates of cytochrome P450 3A4 enzymes (CYP 3A4). Because the vinca alkaloids are extensively metabolized by the liver cytochrome P450 enzymes, poor hepatic function and drug-herb interaction might predispose the patient to develop the bone marrow toxicity. This case report demonstrated possible effect of oral dose of vinca alkaloids and also hinted that all the substrates and inhibitors of CYP 3A4 have propensity to interfere with metabolism of vinca alkaloids.     

长春花组织培养研究进展

长春花含有100多种吲哚类生物碱,具有抗肿瘤、降血压等多种生物活性,有较高的药用价值,但是含量偏低。长春花组织培养可从提高繁殖系数、调控次生代谢产物的累积等来提高长春花生物碱类成分含量。本文就外植体和培养基的选择、药用成分累积的影响因素、悬浮细胞培养应用、影响毛状根因素和基因工程技术应用等方面,综述了长春花组织培养的主要研究进展,为进一步开发利用长春花药物资源提供参考。     

长春花吲哚萜类生物碱代谢途径研究进展

长春花(Catharanthus roseus(L.)G.Don.)含有130余种生物碱,既是抗癌药物长春碱和长春新碱的唯一植物来源,也是研究吲哚萜类生物碱代谢途径的模式植物。生成长春花生物碱的前体环烯醚萜主要来源于2-C-甲基-D-赤藓醇4-磷酸途径(MEP),甲羟戊酸途径(MVA)可能在此过程中起辅助作用。吲哚生物碱代谢途径受茉莉酸甲酯正调控。文多灵途径过去被认为在长春花悬浮细胞中没有文多灵产生,而目前的研究发现文多灵在长春花C20hi细胞中可以微量合成。文章对近年来长春花吲哚生物碱代谢途径及部分关键酶研究进展进行了综述。     

DEC1 binding to the proximal promoter of CYP3A4 ascribes to the downregulation of CYP3A4 expression by IL-6 in primary human hepatocytes

In this study, we provided molecular evidences that interleukin-6 (IL-6) contributed to the decreased capacity of oxidative biotransformation in human liver by suppressing the expression of cytochrome P450 3A4 (CYP3A4). After human hepatocytes were treated with IL-6, differentially expressed in chondrocytes 1 (DEC1) expression rapidly increased, and subsequently, the CYP3A4 expression decreased continuously. Furthermore, the repression of CYP3A4 by IL-6 occurred after the increase of DEC1 in primary human hepatocytes. In HepG2 cells, knockdown of DEC1 increased the CYP3A4 expression and its enzymatic activity. In addition, it partially abolished the decreased CYP3A4 expression as well as its enzymatic activity induced by IL-6. Consistent with this, overexpression of DEC1 markedly reduced the CYP3A4 promoter activity and the CYP3A4 expression as well as its enzymatic activity. Using sequential truncation and site directed mutagenesis of CYP3A4 proximal promoter with DEC1 construct, we showed that DEC1 specifically bound to CCCTGC sequence in the proximal promoter of CYP3A4, which was validated by EMSA and ChIP assay. These findings suggest that the repression of CYP3A4 by IL-6 is achieved through increasing the DEC1 expression in human hepatocytes, the increased DEC1 binds to the CCCTGC sequence in the promoter of CYP3A4 to form CCCTGC-DEC1 complex, and the complex downregulates the CYP3A4 expression and its enzymatic activity.     

Biotransformation of triptolide and triptonide by cell suspension cultures of Catharanthus roseus

Catharanthus roseus cell suspension cultures were used to bioconvert both triptolide (1) and triptonide (2). The same reaction path was followed in both biotransformations. Two biotransformed products were obtained and their structures identified as triptriolide (3) and 12beta,13alpha-dihydroxytriptonide (4), respectively, from 1 and 2. Product 4 is a new compound.