β-双氢青蒿素在不同溶剂中的核磁共振研究

目的研究β-双氢青蒿素在不同测试溶剂中的转化并对其核磁共振信号进行全归属。方法利用HSQC,HMBC等二维核磁共振技术,对β-双氢青蒿素进行测试,利用1H-NMR研究双氢青蒿素β-体与α-体的相互转化。结果首次利用二维核磁共振技术对β-双氢青蒿素的核磁共振信号进行了全归属,β-双氢青蒿素在DMSO中只以β-体一种构型存在。结论β-双氢青蒿素在DMSO中不存在异构体的转化,其核磁信号可以以DMSO-d6为溶剂而得到准确归属。     

科研文摘

用碘二氟醋酸酯(1)、铜(2)和各种有机卤化物(3)在DMSO 中搅拌反应(A 法),或1、2和 DMSO 先在室温反应完全后再加入3(B 法)均可合成标题物(4)。列举14例,炔基卤化物的产率较低为21%,其余为37～88%。链烯卤化物,除顺溴苯乙烯外,均立体专一地得到构型保持不变的产物。     

产紫青霉G59的庆大霉素抗性突变株新产抗肿瘤活性产物研究

目的阐明真菌无活性野生株产紫青霉G59的庆大霉素抗性突变株2-5-3-1新产抗肿瘤活性产物。方法在活性跟踪和薄层检测指导下,通过与原始菌样品直接对照,利用液液萃取、柱层析、制备薄层层析、重结晶等技术,分离纯化活性产物。根据理化常数和波谱数据鉴定化合物结构。用MTT法测试样品对K562细胞的抑制活性。结果从突变株2-5-3-1发酵物中分离鉴定了麦角甾醇（1）、fructigenine A（2）、rugulosuvine A（3）、大黄素（4）和ω-羟基大黄素（5）。化合物1~5在100μg/ml浓度下对K562细胞的抑制率分别为52.8%、60.8%、41.2%、84.6%和37.1%,其中1、2和4的IC50分别为93.1、58.4和30.0μg/ml。结论化合物1~5均为产紫青霉G59不生产而突变株新产抗肿瘤活性产物。用DMSO介导的庆大霉素抗性筛选方法可以将无活性真菌野生株转化成活性突变株并供新产活性产物研究。     

二氢去氢双松柏醇的微生物转化研究

利用微生物转化的方法,以Bacillus subtilisYD 016为转化菌株,对二氢去氢双松柏醇的转化进行研究。该菌株可以将二氢去氢双松柏醇完全转化,主要得到3种转化产物（记为HMZG-1、HMZG-2、HZMG-3）。通过硅胶柱层析和制备型HPLC方法对转化产物进行分离纯化,经ESI-MS、¹H-NMR和¹³C-NMR分析,转化产物分别鉴定为二氢去氢双松柏醇-4-O-β-D-吡喃葡萄糖苷（HMZG-1）、二氢去氢双松柏醇-9’-O-β-D-吡喃葡萄糖苷（HMZG-2）、二氢去氢双松柏醇-9-O-β-D-吡喃葡萄糖苷（HMZG-3）。通过高效液相色谱法对生物转化情况进行检测,建立转化产物和底物的标准曲线。根据HPLC结果计算可知,3种转化产物的转化率分别为:17.34%,14.29%,66.41%。该研究丰富了Bacillus subtilis YD 016的底物作用范围,为二氢去氢双松柏醇葡萄糖苷的合成提供了一条新途径。     

维生素D制剂的药理机制与临床应用

维生素D是一类具有抗佝偻病作用的类固醇类化合物。随着研究的深入，人们提出了维生素D内分泌系统(vitamin D endocrine system)的概念。在这个系统中，肾脏是生成活性维生素D-1α，25-(OH)2D3的内分泌组织。肝脏将维生素D原转化为25-(OH)D3，再在肾脏转化为1α，25-(OH)2D3和24R，25-(OH)2D3以及34种其他维生素D代谢产物。     

基于脱酰基酶转化阿尼芬净前体echinocandin B条件的研究

脱酰基酶可以将echinocandin B(ECB)脱去酰基侧链得到ECB母核,用于化学合成抗真菌药阿尼芬净。本研究考察了以二甲基亚砜(DMSO)助溶剂的转化体系脱酰基酶对ECB转化的各因素,建立了转化工艺(磷酸缓冲浓度：0.02mol/L,pH7.0,KCl浓度：0.68mol/L,DMSO浓度：15%,ECB浓度：450mg/L,加酶量：10%,转化温度：40℃)。为了提高转化效率,探索了新的转化体系即以β-环糊精助溶剂的转化体系,其优势为：当ECB与β-环糊精的浓度比为1:6时,ECB可在体系中完全溶解,底物浓度可以达到2g/L。     

取代2—羟甲基苯甲酰胺和1—亚氨基—1,3—二氢异苯并呋喃的合成

取代苯酞在三乙胺/三氯化铝存在下氨解生成取代2-羟甲基苯甲酰胺(3)和1-亚氨基-1,3-二氢异苯并呋喃(4),苯酞3-位以环状/非环状基团取代可能影响反应过程和产物的生成,当回流反应时只生成产物4,不生成产物3,且4的收率很低,1-苄亚氨基-3-,二取代-1,3-二氢异苯并呋喃在空气中能缓慢氧化为相应氢过氧化物,氢过氧化物结构用光谱法,化学分析法和单昌X-射线衍射确证,并推测了生成氢过氧化物的机理,化合物7米的1H-NMR表示6,34处有一单峰,归属为PhCHOOH氢质子,HMBC表明92\81处有吸收峰,归属为NCH(OOH)Ph碳质子,IR表明845cm^-1有吸收峰,归属为-O-O-吸收.     

海葵来源真菌Cochliobolus lunatus（TA26-46）对土霉素生物转化的研究

目的 研究海葵来源真菌Cochliobolus lunatus (TA26-46)对土霉素的生物转化作用。方法 通过在察氏固体培养基平板中添加土霉素,利用海洋真菌体内特殊的转化酶系统对土霉素进行生物转化;利用HPLC-DAD进行转化产物的追踪,并运用硅胶柱层析和半制备型HPLC等分离纯化转化产物,利用核磁、质谱等现代波谱分析方法对转化产物进行结构鉴定,并测试转化产物的抗菌活性。 结果 海洋真菌C. lunatus (TA26-46)对土霉素(1)产生了生物转化作用,从发酵物中分离鉴定得到2个土霉素的降解产物hemi-cyclines A 和B (2和3)。抗菌活性测试结果表明,降解产物未显示抗菌活性。结论 通过生物学方法,利用海洋真菌C. lunatus (TA26-46)成功对四环类抗生素土霉素进行了生物转化,获得了无抗菌活性的降解产物,为解决环境中的抗生素污染问题提供了借鉴。     

采用微生物转化法合成5—羟基普萘洛尔

采用短刺小克银汉霉菌（Cunninghamella blakesleana AS3.153）对普萘洛尔进行了代谢转化研究。液相色谱-多级质谱和核磁分析结果证明，转化液中主要含有5-羟基普萘洛尔转化产物；考察了单羟化物形成的影响因素，发现在pH6.5、底物浓度0.25%和转化反应持续48h等条件下，5-羟基普萘洛尔的产率可达76%，为活性药物代谢产物的合成开辟了一条新途径。     

2-APB通过下调TRPM7在小鼠皮肤创伤愈合中的作用

目的:探究2-氨基乙基二苯基硼酸酯(2-APB)对小鼠皮肤创伤愈合的促进作用及其可能的机制。方法:将实验小鼠分为5组：Control组、二甲基亚砜(DMSO)组、低(50 mg/L)、中(100 mg/L)和高(200 mg/L)浓度2-APB组,在每只小鼠皮肤背部脊正中线两侧1 cm左右用圆形打孔器各打一个直径10 mm、深至皮下的皮肤创口,其中Control组仅用纱布包扎,不敷用各种药剂;DMSO组每天敷1 g DMSO/凡士林膏剂;2-APB组每天敷1 g相对应浓度的2-APB/凡士林膏剂,隔天拍照观察愈合情况,并于第21天取材,使用HE染色观察创面病理形态以及Western blot检测TRPM7、转化生长因子-β(TGF-β)、I型胶原(Collgen-I, Col-I)以及IL-1β表达情况。结果:与Control组和DMSO组相比,不同浓度的2-APB均可显著促进小鼠皮肤伤口愈合(P<0.01),而DMSO组与Control组之间创伤愈合率无明显差异。HE染色结果显示,与Control组和DMSO组相比,2-APB可增加创面胶原含量以及真皮层的厚度(P<0.0...     

In vitro metabolism of the new cardiotonic agent denopamine (TA-064) by rat and rabbit liver preparations. Oxidation, methylation, and glucuronidation

The metabolic pathways of the cardiotonic agent denopamine, (-)-(R)-1-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol, were studied in vitro with rat and rabbit liver preparations. 4'-O-Demethylated (M-1), 3'-O-demethylated (iso-M-1), and 3-hydroxylated (M-4) metabolites of denopamine were formed by incubation of denopamine with the rat liver microsomal fraction containing the NADPH-generating system. The ratio of M-1 to iso-M-1 formed in this system was 33:1. 3-Methoxydenopamine (M-2) and 3-hydroxy-4-O-methyldenopamine (iso-M-2) were formed via the catechol intermediate M-4, when denopamine was incubated with the rat liver 9000g supernatant fraction in the presence of the NADPH-generating system and S-adenosyl-L-methionine. The ratio of M-2 to iso-M-2 in this system was 7:1. Conversion of iso-M-2 to M-2, i.e. 4-O-demethylation followed by 3-O-methylation, but not vice versa, took place in this system. M-2 was demethylated at 4' to form M-3 by the above microsomal system. M-1 was not ring-hydroxylated by this system, excluding the metabolic route to M-3 via M-1. Denopamine, M-1, M-2, and M-3 were glucuronidated in vitro by the rabbit liver microsomal fraction. The glucuronides of denopamine and M-2 were conjugated at the 4-phenolic hydroxy group, and the glucuronides of M-1 and M-3, which possess two phenolic hydroxy groups, were preferentially conjugated at the 4'-hydroxy group. The order of the rates of in vitro glucuronidation was M-3 greater than M-1 greater than M-2 greater than denopamine.     

Reproducibility over time of the urinary diclofenac/4'-OH diclofenac ratio among different CYP2C9 genotypes.

Diclofenac has been used for the evaluation of CYP2C9 activity in vitro as well as in vivo with varying results. The present study was aimed at evaluating the reproducibility of the urinary diclofenac/4'-OH diclofenac ratio among different CYP2C9 genotypes in healthy volunteers. The study of CYP2C9 genotypes in the family of a CYP2C9*3/*3 subject is also reported. The urinary diclofenac/4'-OH diclofenac ratio was determined on two occasions within a period of 9-12 months, and was found to be correlated (r = 0.83, p < 0.05). The mean (+/- SD) of diclofenac/4'-OH diclofenac ratio was 1.5 times higher among subjects carrying CYP2C9*3 allele (CYP2C9*1/*3 and CYP2C9*2/*3 genotypes) (0.91 +/- 0.28), compared to CYP2C9*1/*1 subjects (0.60 +/- 0.11). The results show that the urinary diclofenac/4'-OH diclofenac ratio might be used to study CYP2C9 in humans. The data agree with previous studies showing that the CYP2C9*3 allelic variant seems to cause a decreased CYP2C9 hydroxylation capacity.     

Therapeutic possibilities of thymopoietin fragments (TP3 and TP4) based on experimental animal models

The effects of thymic hormones are focused on the induction of T-cell subpopulations and restoration of the reactivity of an impaired immune system. TP3 and TP4 (corresponding to thymopoietin 32-34 and 32-35) exert a thymic hormone substitution effect. These peptides elicit dissimilar quantitative and qualitative effects. The aim of the present experiments was to investigate: (a) the effect of thymopoietin fragments in mice with unbalanced immune systems caused by experimental manipulation; and (b) the ratio of target cells after treatment. The distribution of Thy1, Lyt1, Lyt2 positivity was determined in a direct complement mediated cytotoxicity test. Autoantibody production was measured by Coombs' test. A count of Lewis Lung Tumour (LLT) metastases was made after two weeks of inoculation. Groups of mice were thymectomized and/or injected with cyclophosphamide (CY) (240 mg/kg) 96 h before tumour cell inoculation. The number of LLT metastases was decreased by treatment with peptides (TP3 = 72, TP4 = 97, TP5[thymopoietin 32-36] = 83.1 in %) and immunosuppression produced by CY was partly restored. After thymectomy, however, only TP3 treatment caused a decreasing effect (97.4%) on CY immunotoxicity independently of thymectomy. Inhibition of autoantibody production was detected with TP3 (5-6 weeks earlier than in mice treated with TP5). The ratio of Thy1+ and Lyt2+ cells was increased by treatment with TP3 and TP4, but the ratio of Lyt1+ cells was decreased by application of TP5. After TP3 treatment of nude mice the Lyt1+/Lyt2+ ratio increased both in bone marrow and spleen. No effect of TP4 was observed on Lyt 1+ cells, but the number of Lyt2+ increased in bone marrow.(ABSTRACT TRUNCATED AT 250 WORDS)     

碱和水份对7-氯-6-氟-4-羟基喹啉-3-羧酸乙酯的N-或O-乙基化产物比例的影响

7-氯-6-氟-4-羟基喹啉-3-羧酸乙酯(3)的乙基化使用无水 K_2CO_3或无水 Na_2CO_3作脱酸剂,用HPLC 法测定 N-及 O-乙基化物(2与1)的相对含量。考察了溶剂中水份对2和1生成比例的影响。     

如何优化番木鳖碱脂质体制备工艺

目的优化制备番木鳖碱脂质体的处方。方法采用乙醇注入式硫酸铵梯度法制备番木鳖碱脂质体;并以番木鳖碱脂质体的包封率为主要评价指标,采用正交设计法优化番木鳖碱脂质体的配方。结果获得了番木鳖碱脂质体的工艺处方：卵磷脂与胆固醇的重量比为6：1．番木鳖碱和卵磷脂的重量比为1：30,当硫酸铵浓度达到0．15mol·L^-1,卵磷脂的重量与硫酸铵溶液的体积比为12：1。按该处方工艺制备3批番木鳖碱脂质体。包封率平均值为92．17％。结论按照优化处方,采用乙醇注入式硫酸铵梯度法可制得包封率稳定,粒径较小且分布较窄的番木鳖碱脂质体。     

Involvement of cytochromes P-450 2E1 and 3A4 in the 5-hydroxylation of salicylate in humans.

Hydroxylation of salicylate into 2,3 and 2,5-dihydroxybenzoic acids (2,3-DHBA and 2,5-DHBA) by human liver microsomal preparations was investigated. Kinetic studies demonstrated that salicylate was 5-hydroxylated with two apparent Km: one high-affinity Km of 606 microM and one low-affinity Km greater than 2 mM. Liver microsomes prepared from 15 human samples catalyzed the formation of 2,5-DHBA at metabolic rate of 21.7 +/- 8.5 pmol/mg/min. The formation of 2, 3-DHBA was not P-450 dependent. Formation of 2,5-DHBA was inhibited by 36 +/- 14% following preincubation of microsomes with diethyldithiocarbamate, a mechanism-based selective inhibitor of P-450 2E1. Furthermore, the efficiency of inhibition was significantly correlated with four catalytic activities specific to P-450 2E1, whereas the residual activity was correlated with three P-450 3A4 catalytic activities. Troleandomycin, a mechanism-based inhibitor selective to P-450 3A4, inhibited by 30 +/- 12% the 5-hydroxylation of salicylate, and this inhibition was significantly correlated with nifedipine oxidation, specific to P-450 3A4. The capability of seven recombinant human P-450s to hydroxylate salicylate demonstrated that P-450 2E1 and 3A4 contributed to 2, 5-DHBA formation in approximately equal proportions. The Km values of recombinant P-450 2E1 and 3A4, 280 and 513 microM, respectively, are in the same range as the high-affinity Km measured with human liver microsomes. The plasmatic metabolic ratio 2,5-DHBA/salicylate, measured 2 h after ingestion of 1 g acetylsalicylate, was increased 3-fold in 12 alcoholic patients at the beginning of their withdrawal period versus 15 control subjects. These results confirm that P-450 2E1, inducible by ethanol, is involved in the 5-hydroxylation of salicylate in humans. Furthermore, this ratio was still increased by 2-fold 1 week after ethanol withdrawal. This finding suggests that P-450 3A4, known to be also inducible by alcoholic beverages, plays an important role in this increase, because P-450 2E1 returned to normal levels in less than 3 days after ethanol withdrawal. Finally, in vivo and in vitro data demonstrated that P-450 2E1 and P-450 3A4, both inducible by alcohols, catalyzed the 5-hydroxylation of salicylate.     

High performance gene delivery polymeric vector: nano-structured cationic star polymers (star vectors)

Nano-structured hyperbranched cationic star polymers, called star vectors, were molecularly designed for a novel gene delivery non-viral vector. The linear and 3, 4 or 6 branched water-soluble cationic polymers, which had same molecular weight of ca. 18,000, were synthesized by iniferter (initiator-transfer agent-terminator)-based photo-living-radical polymerization of 3-(N,N-dimethylamino)propyl acrylamide, initiated from respective multi-dithiocarbamate-derivatized benzenes as an iniferter. All polymers produced polyion complexes 'polyplexes' by mixing with pDNA (pGL3-control plasmid), in which the particle size was ca. 250 nm in diameter [the charge ratio < 2/1 (vevtor/pDNA)] and ca. 150 nm (the charge ratio > 2.5/1), and the zeta-potential was ca. +10 mV (the charge ratio > 1/1). When COS-1 cells were incubated with the polyplexes 12 h after preparation under the charge ratio of 5/1, higher gene expression was obtained as an increase in branching, with a little cytotoxicity. The relative gene expression to the linear polymer was about 2, 5, and 10 times in 3-, 4-, and 6-branched polymers, respectively. The precise change in branching of polymers enabled the control of the gene transfer activity.     

N-hydroxy-N-arylacetamides. V. Differences in the mechanism of haemoglobin oxidation in vitro by N-hydroxy-4-chloroacetanilide and N-hydroxy-4-chloroaniline

1. Autoxidation of N-hydroxy-4-chloroaniline(I) in buffer pH 7.4 was rapid and yielded 4,4'-azoxybischlorobenzene, 4-chloronitrosobenzene, 4-chloronitrobenzene, and 4-chlorophenyl nitroxide. In contrast, autoxidation of N-hydroxy-4-chloroacetanilide(II) was very slow, since in ether and water 78 and 92%, respectively, had decomposed in six months. 2. Haemoglobin(HbO2)-catalysed autoxidation of (I) occurred at a molar ratio of haemoglobin-Fe2+ to (I) of less than 0.25 and was accompanied by ferrihaemoglobin(HbFe3+)-formation and oxygen consumption. Coupled oxidation of HbO2 with (I) occurred at a molar ratio of greater than 0.2 and was accompanied by liberation of oxygen and the formation of HbFe3+, haemoglobin-4-chloronitrosobenzene complex, HbO2, desoxyhaemoglobin, 4-chloronitrosobenzene, 4-chloronitrobenzene, 4-chloroaniline, 4,4'-azoxybischlorobenzene, and 4-chlorophenyl nitroxide. At an equimolar ratio of 10(-3) M haemoglobin-Fe2+ to (I), 96% HbO2 was converted into HbFe3+ (50%) and haemoglobin-4-chloronitrosobenzene complex in the initial fast phase of the reaction, but only 34% of the bound oxygen was liberated, the rest was sequentially reduced to water. (I) completely disappeared, and 4-chloronitrosobenzene was the major metabolite, mainly bound to haemoglobin. 3. Chemical oxidation of (II) by PbO2 in benzene produced acetyl 4-chlorophenyl nitroxide, whose spontaneous decomposition gave 38% 4-chloronitrosobenzene, 33% N-acetoxy-4-chloroacetanilide, 10% 4-chloroacetanilide, and 8% 4-chloronitrobenzene. Its spontaneous decomposition in water also followed second order kinetics, K = 350 l mol-1 sec-1 and yielded N-(2-acetylamino-5-chlorophenyl)-p-benzo-quinoneimine-N-oxide in addition. 4. In the coupled oxidation of 10(-3) M haemoglobin-Fe2+ with 10(-3) M (II), 75% HbFe3+ was formed after 1 h, but only one third of the equivalent of oxygen was released, and two thirds were reduced to water. Concentration of (II) decreased by 5% only, indicating that one mol of (II) had catalysed the oxidation of 15 equivalents of haemoglobin-Fe2+. The identity of the product pattern formed with HbO2 with that produced by chemical one-electron oxidation indicated that oxygen bound to haemoglobin also functions as an acceptor for electrons from (II) as from (I), but the different redox potentials can explain why the secondary aromatic nitroxide was catalytically active and the primary nitroxide was not.     

人参皂苷Rg1可能通过CDK4-pRB-E2F1通路减少神经元凋亡

目的　探讨人参皂苷Rg1是否通过CDK4 pRB E2F1通路对抗Aβ1～ 4 0 诱导的神经元凋亡。方法　用TUNEL染色、DNA琼脂糖凝胶电泳方法观察神经元凋亡形态学和生化改变 ;免疫印迹方法检测细胞周期相关的周期依赖性激酶4(Cyclindependentkinase 4,CDK4)、磷酸化的视网膜神经胶质瘤蛋白 (Retinoblastomaprotein ,pRB)水平 ;RT PCR技术检测E2F1mRNA表达水平 ;荧光分光光度计法检测凋亡效应分子Caspase 3活力的变化。结果　人参皂苷Rg1预处理可降低Aβ1～ 4 0 诱导的大鼠皮层神经元的凋亡率 ;凋亡细胞特有的DNA梯形条带消失 ;CDK4、磷酸化pRB水平降低 ;E2F1基因表达下调 ;Caspase 3活力下降。结论　人参皂苷Rg1可通过抑制CDK4 pRB E2F1信号传导通路及Caspase 3的激活 ,减少Aβ1～ 4 0 诱导的大鼠皮层神经元凋亡。     

CYP2C9 genotypes and diclofenac metabolism in Spanish healthy volunteers

OBJECTIVE: This study analyzed the frequency of CYP2C9 variant alleles and evaluated the impact of CYP2C9 genotype on diclofenac metabolism in a Spanish population. METHODS: Diclofenac hydroxylation capacity was studied in a population of 102 healthy volunteers. After a single oral dose of 50 mg diclofenac the 0- to 8-h urinary concentrations of diclofenac and its main metabolites, 4'-hydroxy (OH), 3'-OH and 5-OH diclofenac were analyzed by high-performance liquid chromatography. CYP2C9 genotyping for the variant alleles CYP2C9*2 and *3 was carried out with PCR-RFLP. RESULTS: The frequencies of CYP2C9*1, *2, and *3 alleles were 0.74 (95%CI: 0.68-0.80), 0.16 (95%CI: 0.11-0.21) and 0.10 (95%CI: 0.06-0.15), respectively, among the 102 Spaniards studied. The diclofenac/4'-OH diclofenac urinary ratio, but not the diclofenac/3'-OH diclofenac and diclofenac/5-OH diclofenac ratios, was related to CYP2C9 genotype. The diclofenac/4'-OH ratio was significantly higher among subjects with CYP2C9*1/*3 (0.83+/-0.4, n=14, 95% CI for the difference: 0.02-0.4) and CYP2C9*2/*3 (1.10+/-0.5, n=4, 95% CI for the difference: 0.16-0.8) genotypes compared to CYP2C9*1/*1 (0.62+/-0.3, n=59) and approximately threefold higher (1.8) in the only subject homozygous for CYP2C9*3 variant. CONCLUSIONS: The frequencies of CYP2C9*1, *2, and *3 alleles in the Spanish population reported here were similar to those found in the previously studied white European populations, and different of the previously reported in another Spanish population. CYP2C9*3 allele seems to influence the 4'-hydroxylation of diclofenac, although there is a large overlapping in the urinary metabolic ratio between the genotype groups studied