致癌病毒逆向转录酶的抑制剂:Revistin

本文报导,用抑制小鼠白血病病毒逆向转录酶(RT)的方法,从140株链霉菌和200株担子菌中,分别筛选得64株及23株的发酵液具有抑制 RT 活性的作用,抑制率为50%～100%。其中链霉菌 E397-1的发酵液抑制率为90～100%,经100℃加热5分钟不丧失活力,因此它不属蛋白酶,命名为 Revis-tin。其他菌株的发酵液经加热后,对 RT 的抑制作用明显降低。筛选方法是基于抑制~3H-dTMP 掺入致癌 RNA 病毒逆向转录酶合成的 DNA 中去。     

血清淀粉酶和脂肪酶联合检测对急性胰腺炎的诊断价值

目的探讨血清淀粉酶和脂肪酶联合检测对急性胰腺炎的诊断价值。方法对33例型胰腺炎患者、7例重型胰腺炎患者及40例健康体检者进行血清淀粉酶和脂肪酶检测。结果胰腺炎患者的血清淀粉酶和脂肪酶水平明显高于健康人(P <0.05),重型胰腺炎明显高于轻型胰腺炎(P <0.05)。血清脂肪酶诊断急性胰腺炎的灵敏度、特异度、准确度分别为62.3%、60.2%、61.9%;淀粉酶分别为63.5%、64.4%、65.1%,联合检测分别为85.3%、85.3%、85.3%。结论在急性胰腺炎的检测中,联合血清脂肪酶、淀粉酶可以提高诊断的准确性,为临床提供更为科学的依据。     

絮凝法生产胰酶

目的研究壳聚糖对胰酶的絮凝效果。方法探讨壳聚糖用量、pH值、温度对壳聚糖絮凝效果的影响,并按欧洲药典标准测定酶活,计算酶活收率。结果壳聚糖的适宜用量为0.045g/g蛋白,絮凝的最适pH值为5-6。蛋白酶、脂肪酶和淀粉酶的酶活分别为:1307U/g,74.65kU/g,12.9kU/g,酶活收率分别为18.0%.36.6%和7.0%。结论壳聚糖对胰酶有较好的絮凝效果,脂肪酶的酶活和收率较高。     

缓释醋酸亮丙瑞林微球对大鼠子宫异位内膜的抑制作用

目的 :观察国内研制的醋酸亮丙瑞林微球(LE ms)对异位子宫内膜的抑制作用 ,并初步探讨原料药的作用机制。方法 :以手术方法制备子宫内膜异位症 (endometriosis ,EMT)模型SD大鼠 6 0只 ,随机分组给药 ,并计算各用药组对异位内膜的抑制率。另取SD大鼠 30只 ,随机分为假手术组、EMT模型组、用药组。 3周后 ,测血清E2 水平 ;异位内膜体积 ;子宫、卵巢、胸腺和脾脏重量 ;NK细胞活性。结果 :醋酸亮丙瑞林 (LE) 2 0 μg·kg-1、进口LE ms2 0 μg·kg-1·d-1,抑制率依次为 87.2 %和 78.3%。国产LE ms 2、2 0、2 0 0 μg·kg-1·d-1对大鼠异位内膜抑制率依次为 5 7.3%、89.0 %和 94 .7%。LE可使EMT大鼠动情周期消失 ;血清E2 水平降低 ;子宫重量明显减轻 ;胸腺重量明显增加和NK细胞活性明显提高。结论 :国内研制的醋酸亮丙瑞林微球疗效与相同剂量的进口同类产品及原料药的作用相当 ,其作用机制可能是抑制大鼠性腺轴系并通过拮抗雌激素作用来提高EMT大鼠的免疫功能。     

氨基酸的抑菌作用研究

目的研究 19种氨基酸对大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌的抑菌作用。方法用氨基酸饱和液滤纸片分别对上述菌的每个混菌液培养基平板做定性抑菌实验 ;将有抑菌作用的氨基酸不同浓度对此三种菌的LB培养基混菌液做定量抑菌实验。结果半胱氨酸对金黄色葡萄球菌有较强的抑制作用 ,最佳抑菌时间为 6h ,最适抑菌浓度为 0 .6 2 5 % ,此条件下抑菌率为 92 .6 2 %。结论半胱氨酸可以抑制金黄色葡萄球菌。     

老鼠簕生物碱A及其衍生物对α-淀粉酶活性的影响

目的研究老鼠簕生物碱A及其衍生物对-淀粉酶活性的影响,以期为-淀粉酶抑制剂的进一步深入研究提供实验基础及结构依据,并拓展老鼠簕生物碱A及其衍生物的应用范围。方法以老鼠簕生物碱A及其衍生物为待测样品,阿卡波糖为阳性对照,采用Bernfeld法测定比较各化合物对-淀粉酶活性的影响。结果 a-1、a-2、a-3、a-4、a-5、b-7在样品浓度为0.625,1.25,2.5,5.0,10.0 g.L-1时均对-淀粉酶有抑制作用,且在实验浓度范围内随着浓度增加抑制率增大,浓度为10.0 g.L-1时抑制率分别为10.24%、71.52%、63.79%、19.71%、52.76%、38.96%。而a-7、b-3、b-1随浓度的增大,表现出增强-淀粉酶水解活性的趋势。结论推测老鼠簕生物碱A结构中的酚羟基是其对-淀粉酶起抑制作用的基本药效基团,对老鼠簕生物碱A进行结构修饰时,保留4-位酚羟基并在7-位引入氯原子、酰基或含羟基取代基均有利于抑制-淀粉酶的活性;而以乙酰氧基取代4-位酚羟基,并使7-位用含羧酸取代基取代或3-位无取代或以小分子基团取代时均有利于增强-淀粉酶的水解活性。     

天龙粉酶解工艺中最佳用酶的研究

目的探索天龙粉经过4种蛋白酶酶解后对肺腺癌A549细胞株的抑制作用,筛选出最佳用酶。方法天龙粉分别经过胰蛋白酶、胃蛋白酶、胰凝乳蛋白酶和弹性蛋白酶酶解,采用茚三酮比色法测定蛋白质的水解度,同时以天龙粉水提物作为对照组,用甲基噻唑蓝(MTT)法测定4种酶水解物对肺腺癌A549的抑制率。结果对照组不同药物浓度对肺腺癌A549细胞无明显抑制作用;弹性蛋白酶、胰蛋白酶对天龙粉的水解度分别为77.50%、28.02%,给药组不同药物浓度对肺腺癌A549细胞的抑制率明显高于对照组;胰凝乳蛋白酶和胃蛋白酶的水解度分别为34.63%、72.80%,给药组不同药物浓度对肺腺癌A549细胞无明显抑制作用。结论天龙粉酶解工艺的最佳用酶是弹性蛋白酶,其次是胰蛋白酶。     

新加沙参麦冬煎剂抑制肿瘤转移及其作用机制的实验研究

目的 探讨新加沙参麦冬煎剂防治肿瘤转移的机制。方法结合动物模型、免疫组化、细胞分子生物学技术，观察新加沙参麦冬煎剂对荷瘤小鼠抑瘤率、转移抑制率、生命延长率的影响，检测实验小鼠皮下移植瘤血管内等细胞生长因子（VEGF）、血管内皮细胞第Ⅷ因子（CD34）、黏附因子（CD44V6）、基质金属蛋白酶2（MMP2）、基质金属蛋白酶抑制酶（TIMP2）的表达情况。结果新加沙参麦冬煎剂治疗组的瘤重抑制率、肺转移抑制率、生命延长率分别为37.3%，58.3% 和20.8%；皮下移植瘤CD44V6 、VEGF表达、微血管密度（MVD）明显低于空白组（P＜0.01），TIMP2表达记分明显高出其他组（P＜0.01）。小鼠肺转移灶数与VEGF 、CD44V6、MVD呈线性相关，相关系数分别为0.490，0.398，0.455。结论①新加沙参麦冬煎剂对小鼠LA795高转移肺腺癌模型有较好抑制转移、抑制肿瘤生长和延长荷瘤小鼠生存时间的作用。②新加沙参麦冬煎剂可通过调控肿瘤转移过程中黏附、基质降解、血管生成相关分子的表达，多途径、多靶点防治肿瘤的转移。     

复合酶高产菌株选育的研究

出发菌株黑曲霉FJ008,经紫外线,紫外线和亚硝基胍复合诱变获得一株复合酶高产菌株SL2111,该菌在基础发酵培养基上,变温培养72h,每克鲜曲酸性蛋白酶酶活可达4525U/g、果胶酶7015U/g、纤维素酶4497U/g,分别比出发菌株提高97.9%,70.1%和13.4%,经斜面传代培养5代,产酶特性稳定,并运用相关分析法对17株变株所产的酸性蛋白酶酶活力。纤维素酶酶活力和果胶酶酶活力进行分析。结果表明,酸性蛋白酶酶活力与纤维素酶,果胶酶酶活力的变化呈正相关,因此可以选择酸性蛋白酶酶活力的高低作为复合酶菌种筛选及其特性研究的依据。     

几种虫草无性型及其相关真菌体外抑制单胺氧化酶活性研究

目的选择较强抑制单胺氧化酶(MAO)活性的菌株及初步确定活性部位。方法基于消耗底物犬尿胺原理,应用分光光度计对20个菌株进行初筛,在此基础上用酶标仪对10株细脚拟青霉进行比较研究,并由相对抑制率初步确定活性部位。结果RCEF1054、RCEF0943、RCEF1078和RCEF0440菌株活性较强。细脚拟青霉菌丝体不同溶剂先后提取所得产物MAO抑制率不同,氯仿组>甲醇组>水提组。氯仿组抑制率最高的菌株是Pt25(63.66%)。甲醇和水提物活性较低。10个菌株发酵液提取物均有一定活性,抑制率在15.5%~31.7%之间,较高的是pt12、pt02和pt57菌株。结论不同菌种或不同溶剂提取物的抑制MAO活性差异显著。细脚拟青霉RCEF0788(pt57)菌株提取物具有较高活性。     

高活力胰酶的生产

将酶激活剂及稳定剂用于高活力胰酶生产.酶活力为蛋白酶4.5ku/g,淀粉酶70ku/g,脂肪酶35ku/g.胰酶总收率为7.9～1O.8%.     

Effects of 3-tert-Butyl-4-hydroxyanisole and its hydroquinone and quinone metabolites on rat and human embryonic cells in culture

3-tert-Butyl-4-hydroxyanisole (BHA), tert-butyl hydroquinone (BHQ), 3-tert-butyl-4,5-dihydroxyanisole (BHAOH), tert-butylquinone (BQ) and 3-tert-butyl-5-methoxy-1,2-benzoquinone (BHAoQ) were assayed using cell-culture methods for assessing potential teratogenicity. In the rat embryonic cell differentiation assay, BQ had the greatest inhibitory effect on the differentiation of both limb-bud (LB) and midbrain (MB) cells. From the dose-response curves, the concentrations of BHA, BHQ, BHAOH, BHAoQ and BQ that inhibited the production of differentiated foci by 50% (IC₅₀) in LB cells were 533, 105, 25, 21 and 16 μ , respectively, and the IC₅₀ values for MB cells were 466, 67, 24, 18 and 14 μ , respectively. BQ also showed the most potent inhibitory action in the human embryonic palatal mesenchymal (HEPM) cell-growth assay. The concentrations of BHA, BHQ, BHAOH, BHAoQ and BQ that inhibited cell growth by 50% (IC₅₀) were 244, 48, 23, 15 and 3.3 μ , respectively. Therefore in these assays, the quinone and hydroquinone metabolites of BHA showed greater teratogenic potential than did BHA itself. The inhibitory action of BHA on HEPM cell growth was greater in the presence of S-9 mix prepared using livers from rats treated with phenobarbital and 5,6-benzoflavone. In the HEPM cell-growth assay, rat (F344) S-9 was more effective in inducing the metabolic activation of BHA than was S-9 from hamsters or mice.     

The effect of methylbromophenvinfos (Polfos) on some enzymes in vivo and in vitro. I. In vivo studies

The experiment was carried out on Wistar rats receiving orally either oil or oily solution of methylbromophenvinfos (Polfos) either in a single dose of 0.5 LD50, or doses of 0.1 LD50 once daily for a period of 2, 4 or 6 weeks. The activities of cholinesterase (ChE), beta-glucuronidase (beta-glu), lipase and amylase were assayed in the blood serum, the activity of acetylcholinesterase (AChE)-in brain homogenates, and the activities of lipase and amylase-in homogenates of the pancreas. Cholinesterases were inhibited in the course of both acute and chronic poisoning with Polfos. During the acute poisoning a sharp increase in the activity of beta-glu in the blood serum, 1 and 2 h after the pesticide administration, was observed. Polfos inhibited lipase and amylase both after acute and chronic treatment.     

Territrems B and C metabolism in human liver microsomes: major role of CYP3A4 and CYP3A5

Human liver microsomes, supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms, and control and CYP3A4 cDNA-transfected V79 Chinese hamster cells were tested for their ability to metabolize territrem B (TRB) and territrem C (TRC). Two TRB metabolites, designated MB(2) and MB(4), and one TRC metabolite, designated MC, were formed by all of these preparations. Of the nine supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5), only those expressing CYP3A4 or CYP3A5 metabolized TRB and TRC. MB(2), MB(4), and MC were formed by CYP3A4 cDNA-transfected V79MZ Chinese hamster cells, but not by non-transfected cells. In order to investigate which CYP450 isoforms were responsible for MB(2), MB(4) and MC formation in human liver microsomal preparations, six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketoconazole, and diethyldithiocarbamate) and antibodies against CYP3A4 were used. MB(2), MB(4), and MC formation was markedly inhibited by ketoconazole, but less affected by quinidine and sulfaphenazole. Anti-CYP3A4 antibody markedly inhibited MB(2), MB(4), and MC formation and also 6 beta-hydroxytestosterone formation from testosterone. The CYP3A-dependent reaction of testosterone 6 beta-hydroxylation showed a high correlation with 4 beta-C hydroxylation of TRB (r(2)=0.97, P<0.0001), O-demethylation of TRB (r(2)=0.95, P<0.0001), and 4 beta-C hydroxylation of TRC (r(2)=0.99, P<0.0001). Immunoblotting and RT-PCR showed that CYP3A4 and CYP3A5 were expressed in all four human liver microsomal preparations tested (HLM1-HLM4). The amount of MB(2), MB(4), and MC formed using different HLM preparations was related to the 6 beta-testosterone hydroxylase activity of the preparations. However, the extent of MB(2), MB(4), and MC formation was not related to the age or gender of the person from whom the microsomal sample was prepared. It was therefore suggest that CYP3A4 and CYP3A5 are the major enzymes responsible for TRB and TRC metabolism by human liver microsomes.     

Pharmacokinetics and pharmacodynamics of single and multiple doses of prasugrel in healthy native Chinese subjects

Aim:

To characterize the pharmacokinetics (PKs), pharmacodynamics (PDs), and tolerability of different dose regimens of prasugrel in healthy Chinese subjects.

Methods:

This was a single-centered, open-label, parallel-design study. Subjects received a single loading dose (LD) of prasugrel followed by once-daily maintenance dose (MD) for 10 d. They were enrolled into 3 groups: 60 mg LD/10 mg MD; 30 mg LD/7.5 mg MD; 30 mg LD/5 mg MD. Blood samples were collected after the first and last dose. The serum concentration of the active metabolite of prasugrel was determined using a LC/MS/MS method. Platelet aggregation was assessed using the VerifyNow-P2Y₁₂ assay.

Results:

Thirty-six healthy native Chinese subjects (19 males) aged 18–45 were enrolled; mean age and body weight were similar across the treatment groups (n=12 for each). The metabolite AUC_0–4 and C_max increased dose-proportionally across the dose range of 5 mg to 60 mg. The median T_max was 0.5 h in all groups. The PD parameters, indicated by the inhibition of ADP-induced platelet aggregation, were met more rapidly in the 60 mg group than the 30 mg group after the LD (94%–98%). This high degree of inhibition of platelet aggregation was maintained following the 10 mg MD (87%–90%) and was lower in the 7.5 mg and 5 mg MD groups (79%–83% and 64%–67%, respectively). Prasugrel was well tolerated in healthy Chinese subjects for single doses up to 60 mg and a MD of 10 mg for 10 d.

Conclusion:

The PKs and PDs of the active metabolite of prasugrel were similar to those in Chinese subjects reported by a previous bridging study, which demonstrated that the exposure to the active metabolite in Chinese subjects was higher than in Caucasians.     

In vitro inhibitory effect of triterpenoidal saponins from platycodi radix on pancreatic lipase

In the process of investigating anti-obesity effect of Platycodi Radix, we found that aqueous extract of Platycodi Radix might inhibit intestinal absorption of dietary fat by inhibiting pancreatic lipase (PL) activity. In order to clarify the anti-obesity mechanism of Platycodi Radix, activity-guided isolation was performed to find active components. The total saponin fraction of Platycodi Radix appeared to have a potent inhibitory activity against the hydrolysis of triolein emulsified with phosphatidycholine by pancreatic lipase in vitro. Based on these results, further purification of active components yielded 10 known triterpenoidal saponins, among these compounds, platycodin A, C, D, and deapioplatycodin D exhibited significant inhibitory effects on PL at the concentration of 500 microg/mL with 3.3, 5.2, 34.8, and 11.67% pancreatic lipase activity vs control, respectively. Platycodin D was found to inhibit the PL activity in a dose-dependent manner. Therefore, the anti-obesity effect of Platycodi Radix might be due to the inhibition of pancreatic lipase by its saponins.     

唑来膦酸盐通过p38 MAPK信号通路调控高糖微环境下成骨细胞分化

目的 探讨唑来膦酸盐（ZOL）对高糖微环境下小鼠前成骨细胞MC3T3-E1成骨分化的影响及p38丝裂原活化蛋白激酶（p38 MAPK）通路的调节作用。方法 体外培养MC3T3-E1细胞并分为低糖（LG）组、LG+ZOL组、高糖（HG）组、HG+ZOL组。ZOL为0.1 μmol/L,LG、HG的葡萄糖浓度分别为5.5 mmol/L和16.5 mmol/L。采用四甲基偶氮唑蓝（MTT）法检测细胞增殖水平;鬼笔环肽染色观察细胞骨架;试剂盒检测细胞碱性磷酸酶（ALP）活性;茜素红染色检测细胞矿化结节生成情况;免疫荧光法检测Wnt5a、p38 MAPK的荧光表达强度。另设HG+ZOL+p38 MAPK通路抑制剂（SB203580）组,SB203580为10 μmol/L。Western blot检测5组细胞中Wnt5a、p38 MAPK、磷酸化p38丝裂原活化蛋白激酶（p-p38 MAPK）、骨形态发生蛋白2（BMP2）和Ⅰ型胶原蛋白（COLⅠ）的表达水平。结果 4组细胞增殖水平差异无统计学意义（P>0.05）。与LG组比较,LG+ZOL组细胞骨架清晰程度,ALP活性,茜素红矿化结节生成,Wnt5a、p38 MAPK蛋白荧光表达强度,Wnt5a、p38 MAPK、p-p38 MAPK、BMP2和COLⅠ蛋白表达水平明显升高;HG组上述指标变化相反（P<0.05）。与HG组相比,HG+ZOL组细胞骨架清晰程度,ALP活性,茜素红矿化结节生成,Wnt5a、p38 MAPK蛋白荧光表达强度,Wnt5a、p38 MAPK、p-p38 MAPK、BMP2和COL Ⅰ蛋白表达水平明显升高（P<0.05）。与HG+ZOL组相比,HG+ZOL+SB203580组Wnt5a、p38 MAPK、p-p38 MAPK、BMP2和COL Ⅰ的蛋白表达水平降低（P<0.05）。结论 高糖对细胞成骨分化起抑制作用,ZOL可有效改善其抑制作用,其机制可能与p38 MAPK通路相关。     

New 3-unsubstituted isoxazolones as potent human neutrophil elastase inhibitors: Synthesis and molecular dynamic simulation

Human neutrophil elastase (HNE) is a proteolytic enzyme belonging to the serine protease family and is involved in a variety of pathologies. Thus, compounds able to inhibit HNE represent promising therapeutics for the treatment of inflammatory diseases. Here, we report the further elaboration of our previously reported 3-methylisoxazolone derivatives, synthesizing a new series of 3-nor-derivatives bearing different substituents at the 4-phenyl ring. The most potent compounds 3a , 3g , and 3h , had IC₅₀ values of 16, 11, and 18 nM, respectively. Molecular modeling studies and molecular dynamic (MD) simulations demonstrated no substantial differences between the 3-methylisoxazole derivatives previously tested and the corresponding 3-unsubstituted derivatives in the snapshot conformations sampled during the MD simulations, which is consistent with their similar levels of HNE inhibitory activity. Thus, we conclude that the isoxazolone scaffold is a good scaffold for developing HNE inhibitors, as it tolerates several modifications when adhering to basic scaffold requirements, and the resulting derivatives are quite potent HNE inhibitors.     

伍用刺乌头碱对3-乙酰乌头碱毒性及镇痛作用的影响

刺乌头碱(0.5mg·kg~(-1))对3-乙酰乌头碱(0.07mg·kg~(-1))诱发大鼠心律失常有显著的对抗作用.二者伍用(剂量LA:AA=7:1)对镇痛作用无明显影响.但刺乌头碱可显著提高3-乙酰乌头碱小鼠的LD_(50),从而提高其治疗指数.扩大其安全范围。