大黄蒽醌衍生物对酪氨酸酶的抑制作用

大黄素对酪氨酸酶有显著的竞争性抑制作用,K_i值为1.51×10~(-4)mol,50％抑制的药物浓度为36.6μg/ml;大黄酸的抑制作用较弱,芦荟大黄素几乎无抑制作用。氯化铜(3.3×10~(-7)mol/L)、半胱氨酸(3.3×10~(-7)mol/L)和牛血清白蛋白(1.0mg/ml)对大黄素抑制酪氨酸酶有较强的拮抗作用,恢复率分别为60.0％、45.7％和61.1％。大黄素能与牛血清白蛋白非特异性结合形成复合物,引起吸收光谱红移55毫微米。大黄蒽醌衍生物对酪氨酸酶的抑制作用可能是大黄抗黑色素瘤的作用机制之一。     

波叶大黄多糖对胰腺五种酶的抑制作用

 本文报道波叶大黄多糖(Rheum hotaoense polysaccharide,RHP)对胰腺五种酶的抑制作用。结果表明。(1)RHP对胰蛋白酶、胰脂肪酶、胰淀粉酶、胰弹性蛋白酶和胰激肽释放酶均有很明显的抑制作用。IC_(50)分别为250μg/mL、21μg/mL、18μg/mL,189μg/mL和300μg/mL。(2)动力学研究表明,RHP对胰蛋白酶和胰脂肪酶的抑制均是非竞争性的,Km值分别为1.2×10~(-4)μmol/mL和1.2mg/mL,Ki值分别为355.0μg/mL和63.85μg/mL。(3)牛血清白蛋白(BSA)对RHP抑制胰蛋白晦和胰脂肪酶具有拮抗作用。当BSA浓度达72.0mg/mL时,对胰蛋白酶活性的恢复率为71.54％。当BSA浓度达20.0mg/mL时,对胰脂肪酶活性的恢复率为63.64％。上述结果表明,RHP对胰腺五种酶的抑制作用,可能是大黄治疗急性胰腺炎作用的生化机制之一,预示RHP在临床上将有重要应用前景。     

不同菌株白僵菌代谢产物对大鼠肝线粒体单胺氧化酶的抑制作用研究

对8株白僵菌[Beauveriasp.]代谢产物的抑制单胺氧化酶的活性进行测定,发现菌株Ba02和Ba05的乙酸乙酯提取物在终浓度为55μg/mL时,对单胺氧化酶A型(MAO＿＿A)有较强的抑制活性,其抑制率分别为95.9%和83.4%;Ba02及Ba03菌株脂溶性成分在终浓度为55μg/mL时对单胺氧化酶B型(MAO＿B)有较强的抑制活性,其抑制率分别为94.8%和84.1%。浓度和抑制活性关系研究表明在一定浓度范围内Ba02菌株脂溶性成分对MAO的抑制活性呈量效关系,经计算得出其对MAO＿A、MAO＿B抑制的IC50分别为18.3μg/mL、28.2μg/mL;抑制特征曲线表明Ba02菌株脂溶性成分对MAO＿A呈竞争型抑制,对MAO＿B为混合型抑制,Km值分别为0.47×10-5mol/L,0.11×10-5mol/L。     

中药抗厌氧菌的系列研究：Ⅲ.大黄抗厌氧菌的实验研究

本研究进一步用大黄水煎液,大黄醇提物和大黄蒽醌衍生物(芦荟大黄素、大黄酸和大黄素),对临床分离的100株厌氧菌进行MIC测定,并对部分菌株进行MBC测定和亚抑菌浓度(Sub-MIC)下细菌形态观察。结果表明,大黄水煎液在1600μg/ml浓度时能抑制74％厌氧菌生长,大黄醇提物的MIC约为水煎液的1/15。三种蒽醌衍生物在8μg/ml时能抑制76～91％厌氧菌生长,这与国际公认的抗厌氧菌药甲硝唑相近。对部分菌株的MBC测定表明,大黄的MBC要大于MIC几倍以上,说明大黄抗厌氧菌主要是抑菌不是杀菌。从Sub-MIC下厌氧菌形态改变提示,大黄主要是抑制细胞壁的合成。     

单宁酸对甜菜夜蛾幼虫生长发育及酚氧化酶活性的抑制作用

采用摄食毒力法研究了单宁酸对甜菜夜蛾[Spodoptera exigua (Hübner)]幼虫生长发育的影响及毒力作用,并分析了单宁酸对甜菜夜蛾酚氧化酶的抑制动力学.结果表明,不同质量浓度单宁酸对甜菜夜蛾初孵幼虫具有较强的毒杀作用,对3龄幼虫的体质量以及蛹质量、化蛹及羽化过程也均有明显的抑制作用(P<0.05).随单宁酸质量浓度的提高或处理时间的延长,甜菜夜蛾初孵幼虫的校正死亡率逐渐增大,单宁酸的抑制生长效应逐渐增强,处理第7天,单宁酸对试虫的半致死浓度(LC50)为3.772 3 g·L-1;8 g·L-1单宁酸对3龄幼虫体质量增长的抑制率最高(35.09%);16 g·L-1单宁酸对蛹质量和羽化率的影响最明显.单宁酸能明显抑制甜菜夜蛾酚氧化酶的活力,且随单宁酸浓度的提高,酚氧化酶相对活力下降趋势明显趋缓,单宁酸对甜菜夜蛾酚氧化酶活性的半抑制浓度(IC50)为0.234 3 mmol·L-1;在加入不同浓度单宁酸的反应体系中,酚氧化酶的活力与酶液量呈明显的线性关系;不同浓度单宁酸对甜菜夜蛾酚氧化酶抑制作用的Lineweaver-Burk双倒数图为1组横轴截距不变的直线;另外,单宁酸和半胱氨酸均可将多巴醌还原为多巴,且还原量均随反应时间的延长而增加.研究结果说明,单宁酸对甜菜夜蛾酚氧化酶的抑制作用是可逆的,为典型的非竞争性抑制剂,抑制常数(Ki)为0.193 8 mmol·L-1;单宁酸对甜菜夜蛾酚氧化酶二酚酶的抑制作用与L-多巴氧化产物的还原有关.     

苦瓜素Ⅰ对亚洲玉米螟幼虫酚氧化酶活性的抑制作用

为了探讨苦瓜素类化合物抑制昆虫生长发育的作用机制,测定了苦瓜叶乙酸乙酯萃取物和苦瓜素Ⅰ对亚洲玉米螟Ostrinia furnacalis幼虫体内酚氧化酶活性的抑制作用以及苦瓜素Ⅰ、苦瓜素Ⅱ和苦瓜皂苷元L对亚洲玉米螟幼虫离体酚氧化酶活性的抑制作用。结果表明,苦瓜叶乙酸乙酯萃取物和苦瓜素Ⅰ均能明显抑制亚洲玉米螟幼虫酚氧化酶的活性。用含有不同浓度苦瓜叶乙酸乙酯萃取物和苦瓜素I的人工饲料饲喂亚洲玉米螟3龄幼虫48 h后,苦瓜叶乙酸乙酯萃取物和苦瓜素Ⅰ对亚洲玉米螟幼虫体内酚氧化酶活性的抑制中浓度IC_(50)分别为0.17%和0.34%。苦瓜素Ⅰ和苦瓜素Ⅱ对亚洲玉米螟离体酚氧化酶的活力也表现出明显的抑制作用,抑制中浓度IC_(50)分别为169.28μg/mL和1182.08μg/mL。苦瓜素I的抑制作用明显强于苦瓜素Ⅱ。苦瓜叶提取物及其活性成分苦瓜素Ⅰ对亚洲玉米螟幼虫酚氧化酶活性的抑制作用可能是其抑制昆虫生长发育的主要机制之一。     

刚竹叶提取物对斜纹夜蛾酚氧化酶抑制作用研究

采用酶标仪法测定了刚竹叶提取物对斜纹夜蛾酚氧化酶(PO)活性的影响。结果表明:6种不同溶剂(极性大小:去离子水乙醇丙酮乙酸乙酯氯仿石油醚)获得的竹叶提取物均对PO表现为抑制作用(三氯甲烷提取物在0-300 mg/L范围内,对PO表现为激活作用),且随提取物浓度的升高,抑制作用有逐渐增强的趋势。丙酮提取物对PO的抑制效果最显著(IC50为550 mg/L),其次为乙酸乙酯提取物和乙醇提取物(IC50分别为575 mg/L和700 mg/L);丙酮提取物对斜纹夜蛾酚氧化酶的抑制效果属于可逆过程,抑制类型为非竞争性抑制,抑制常数Ki为158.2 mg/L。     

改良内皮抑素对人脐静脉内皮细胞抑制作用的实验研究

目的:探讨改良内皮抑素(RGDRGD-ES)对人脐静脉内皮细胞(HUVEC)的抑制作用,摸索RGDRGD-ES对HUVEC细胞抑制作用的相对最佳作用浓度和时间。方法:通过快速定点诱变PCR方法获得含有RGDRGD膜序的改良人内皮抑素基因,并构建其原核表达载体。表达、纯化改良内皮抑素(RGDRGD-ES),运用MTT法和流式细胞仪检测RGDRGD-ES对人脐静脉内皮细胞的抑制作用。结果:1.诱变了ES基因,获得了改良的RGDRGD-ES基因,并成功构建其原核表达载体。2.获得了RGDRGD-ES蛋白。3.改良的RGDRGD-ES能够有效抑制人脐静脉内皮细胞的生长(P<0.01);抑制率随着药物浓度(10μg/ml、20μg/ml、30μg/ml)的增加和作用时间(24 h、48 h、72 h)的延长而逐渐增加,具有浓度和时间依赖性(P<0.01);而30μg/ml与40μg/ml、50μg/ml组间、72 h与96 h组间无明显差异(P>0.05)。4.细胞凋亡率(作用24 h)具有药物浓度(10μg/ml、20μg/ml、30μg/ml)依赖性(P<0.01),30μg/ml与40μg/ml、50μg/ml组间凋亡率无明显差异(P>0.05)。结论:成功构建了改良RGDRGD-ES基因的原核表达载体,RGDRGD-ES蛋白在30μg/ml浓度作用72小时条件下能够有效抑制人脐静脉内皮细胞的生长,改良内皮抑素(RGDRGD-ES)对HUVEC的抑制作用较ES明显提高。     

过量表达NADH氧化酶加速光滑球拟酵母合成丙酮酸

[目的]进一步提高光滑球拟酵母(Torulopsis glabrata)发酵生产丙酮酸的生产强度.[方法]将来源于乳酸乳球菌(Lactococcus lactis)中编码形成水的NADH氧化酶noxE基因过量表达于丙酮酸工业生产菌株T. glabrata CCTCC M202019中,获得了一株NADH氧化酶活性为34.8 U/mg蛋白的重组菌T. glabrata-PDnoxE.[结果]与出发菌株T. glabrata CCTCC M202019相比,细胞浓度、葡萄糖消耗速率和丙酮酸生产强度分别提高了168%、44.9%和12%,发酵进行到36 h葡萄糖消耗完毕.补加50 g/L葡萄糖继续发酵20 h,则使丙酮酸浓度提高到67.2 g/L.葡萄糖消耗速度和丙酮酸生产强度增加的原因在于形成水的NADH氧化酶过量表达,导致NADH和ATP含量分别降低了18.1%和15.8%.而NAD<' 增加了11.1%.[结论]增加细胞内NAD<' 含量能有效地提高酵母细胞葡萄糖的代谢速度及目标代谢产物的生产强度.     

豇豆初生叶多胺氧化酶的催化特性

从豇豆幼苗 (6d苗龄 )初生叶提纯得到的多胺氧化酶 (EC 1 .4.3 .6 )属于二胺氧化酶 ,最有效的底物是 1 ,4 二胺丁烷 (腐胺 )、1 ,5 二胺戊烷 (尸胺 )、1 ,6 二胺己烷、1 ,1 0 二胺癸烷等α 二胺 ,其催化活性随二胺类底物碳链的增长而相应减弱。豇豆多胺氧化酶对亚精胺和精胺也具有较高的催化活性。另外 ,底物腐胺和尸胺的浓度超过 2mmol/L或亚精胺和精胺浓度超过 3mmol/L时会对酶活性有抑制效应。以腐胺和尸胺为底物时 ,酶的最适 pH约为7.0 ,而以亚精胺和精胺为底物时其最适pH为 6 .5。该酶的催化活性还随反应介质的离子强度增加而降低。K ,Ca2 和Mg2 (皆为 1 0mmol/L)对酶活性无明显抑制作用 ,而同样浓度的Mn2 ,Zn2 ,Fe2 ,Co2 和Cd2 则对酶活性有不同程度的抑制作用。金属螯合剂EDTA(1 0mmol/L)和腺苷蛋氨酸脱羧酶抑制剂甲基乙二醛 双脒腙 (0 .1mmol/L)可抑制酶活性约 80 % ,而铜结合剂KCN(1 .0mmol/L)、羰基试剂羟胺 (0 .1mmol/L)和氨基胍 (0 .1mmol/L)可导致该酶完全失活     

Design,Synthesis, Molecular Docking,and Biological Evaluation of New Emodin Anthraquinone Derivatives as Potential Antitumor Substances

The emodin anthraquinone derivatives are generally used in traditional Chinese medicine due to their various pharmacological activities. In the present study, a series of emodin anthraquinone derivatives have been designed and synthesized, among which 1,3‐dihydroxy‐6,8‐dimethoxyanthracene‐9,10‐dione is a natural compound that has been synthesized for the very first time, and 1,3‐dimethoxy‐5,8‐dimethylanthracene‐9,10‐dione is a compound that has never been reported earlier. Interestingly, while total seven of these compounds showed neuraminidase inhibitory activity in influenza virus with inhibition rate more than 50 %, specific four compounds exhibited significant inhibition of tumor cell proliferation. The further results demonstrate that 1,3‐dimethoxy‐5,8‐dimethylanthracene‐9,10‐dione showed the best anticancer activity among all the synthesized compounds by inducing highest apoptosis rate to HCT116 cancer cells and arresting their G0/G1 cell cycle phase, through elevation of intracellular level of reactive oxygen species (ROS). Moreover, the binding of 1,3‐dimethoxy‐5,8‐dimethylanthracene‐9,10‐dione with BSA protein has thoroughly been investigated. Altogether, this study suggests the neuraminidase inhibitory activity and antitumor potential of the new emodin anthraquinone derivatives.     

Antioxidant,anti‐inflammatory,and enzyme inhibitory activity of natural plant flavonoids and their synthesized derivatives

The synthesized flavonoid derivatives were examined for their antioxidant, anti‐inflammatory, xanthine oxidase (XO), urease inhibitory activity, and cytotoxicity. Except few, all the flavonoids under this study showed significant antioxidant activity (45.6%–85.5%, 32.6%–70.6%, and 24.9%–65.5% inhibition by DPPH, ferric reducing/antioxidant power, and oxygen radical absorption capacity assays) with promising TNF‐α inhibitory activity (42%–73% at 10 μM) and IL‐6 inhibitory activity (54%–81% at 10 μM) compared with that of control dexamethasone. The flavonoids luteolin, apigenin, diosmetin, chrysin, O^3?, O⁷‐dihexyl diosmetin, O^4?, O⁷‐dihexyl apigenin, and O⁷‐hexyl chrysin, showed an inhibition with IC₅₀ values (4.5‐8.1 μg/mL), more than allopurinol (8.5 μg/mL) at 5 μM against XO and showing more than 50% inhibition at a final concentration (5 mM) with an IC₅₀ value of ranging from 4.8 to 7.2 (μg/mL) in comparison with the positive control thiourea (5.8 μg/mL) for urease inhibition. Thus, the flavonoid derivatives may be considered as potential antioxidant and antigout agents.     

Discovery and biological evaluation of some (1H-1,2,3-triazol-4-yl)methoxybenzaldehyde derivatives containing an anthraquinone moiety as potent xanthine oxidase inhibitors

A series of (1H-1,2,3-triazol-4-yl)methoxybenzaldehyde derivatives containing an anthraquinone moiety were synthesized and identified as novel xanthine oxidase inhibitors. Among them, the most promising compounds 1h and 1k were obtained with IC₅₀ values of 0.6 μM and 0.8 μM, respectively, which were more than 10-fold potent compared with allopurinol. The Lineweaver-Burk plot revealed that compound 1h acted as a mixed-type xanthine oxidase inhibitor. SAR analysis showed that the benzaldehyde moiety played a more important role than the anthraquinone moiety for inhibition potency. The basis of significant inhibition of xanthine oxidase by 1h was rationalized by molecular modeling studies.     

Inhibition of plasma membrane NADH oxidase activity and growth of HeLa cells by natural and synthetic retinoids

Several retinoids, both natural and synthetic, were evaluated for their ability to modulate NADH oxidase activity of plasma membranes of cultured HeLa cells and the growth of HeLa cells in culture. Both NADH oxidase activity and the growth of cells were inhibited by the naturally-occurring retinoids all trans-retinoic acid (tretinoin) and retinol as well as by the synthetic retinoids, trans-acitretin, 13-cis-acitretin, etretinate and arotonoid ethylester (Ro 13-6298). For all retinoids tested, inhibition of NADH oxidase activity and inhibition of growth were correlated closely. With tretinoin, etretinate and arotonoid ethylester, NADH oxidase activity and cell growth were inhibited in parallel in proportion to the logarithm of retinoid concentration over the range of concentrations 10-8 to 10-5 M. Approximately 70% inhibition of both NADH oxidase activity and growth was reached at 10 µM. With retinol, trans-acitretin and 13-cis-acitretin, inhibition of NADH oxidase activity and growth also were correlated but maximum inhibition of both was about 40% at 10 µM. The possibility is suggested that inhibition of the plasma membrane NADH oxidase activity by retinoids may be related to their mechanism of inhibition of growth of HeLa cells in culture. (Mol Cell Biochem 166: 101-109, 1997)     

木榄内生真菌菌株ZD6及其代谢产物的抑菌活性

根据形态学特征和ITS序列分析结果,将一株从木榄茎中分离到的内生真菌菌株ZD6鉴定为桔青霉Penicillium citrinum Thom.,该菌株在优化后的发酵培养基(1%麦芽糖,2%甘露醇,1%谷氨酸钠,0.5%蛋白胨,0.15%酵母膏,200g/L土豆汁,pH6.5)中28℃、160r/min振荡培养7d后的发酵液具有明显的抑菌活性。利用柱层析及重结晶等方法从该发酵液的乙酸乙酯和正丁醇提取物中分离得到4个化合物:大黄素、环(丙氨酸-甘氨酸)、赤藓醇和甘露醇,其中的大黄素和赤藓醇为首次在桔青霉中发现,它们对枯草芽孢杆菌Bacillus subtilis的生长具有明显的抑制作用,最低抑菌浓度(MIC)分别为25μg/mL和50μg/mL;大黄素对绿脓杆菌Pseudomonas aeruginosa的生长也具有一定的抑制作用,MIC为100μg/mL。     

Stimulation of NADH oxidation by xanthine oxidase and polyvanadate in presence of some dehydrogenases and flavin compounds

The rates of NADH oxidation in presence of xanthine oxidase increase to a small and variable extent on addition of high concentrations of lactate dehydrogenase and other dehydrogenases. This heat stable activity is similar to polyvanadate-stimulation with respect to pH profile and SOD sensitivity. Isocitric dehydrogenase (NADP-specific) showed heat labile, SOD-sensitive polyvanadate-stimulated NADH oxidation activity. Polyvanadate-stimulated SOD-sensitive NADH oxidation was also found to occur with riboflavin, FMN and FAD in presence of a non-specific protein, BSA, suggesting that some flavoproteins may possess this activity.     

Direct Inhibition of Plant Mitochondrial Respiration by Elevated CO2

Doubling the concentration of atmospheric CO2 often inhibits plant respiration, but the mechanistic basis of this effect is unknown. We investigated the direct effects of increasing the concentration of CO2 by 360 [mu]L L-1 above ambient on O2 uptake in isolated mitochondria from soybean (Glycine max L. cv Ransom) cotyledons. Increasing the CO2 concentration inhibited the oxidation of succinate, external NADH, and succinate and external NADH combined. The inhibition was greater when mitochondria were preincubated for 10 min in the presence of the elevated CO2 concentration prior to the measurement of O2 uptake. Elevated CO2 concentration inhibited the salicylhydroxamic acid-resistant cytochrome pathway, but had no direct effect on the cyanide-resistant alternative pathway. We also investigated the direct effects of elevated CO2 concentration on the activities of cytochrome c oxidase and succinate dehydrogenase (SDH) and found that the activity of both enzymes was inhibited. The kinetics of inhibition of cytochrome c oxidase were time-dependent. The level of SDH inhibition depended on the concentration of succinate in the reaction mixture. Direct inhibition of respiration by elevated CO2 in plants and intact tissues may be due at least in part to the inhibition of cytochrome c oxidase and SDH.     

Induction and activation of the alternative oxidase of potato tuber mitochondria

Potato tubers ( Solanum tubersum L. cv. Grata) were stored for atleast 1 week at room temperature and then incubated with an equal amount of apples ( Malus domestica L.) for 2 days. After this treatment, intact tuber mitochondria isolated by Percoll gradient centrifugation showed a high degree of induction of the alternative oxidase, measured as cyanide-resistant, salicylhydroxamic acid-sensitive respiration. With succinate as substrate an activity of more than 130 nmol O₂(mg protein) ¹ min ^t was obtained. An assay of the alternative oxidase using duroquinol as an electron donor was developed. To become reliable the assay required the presence of defatted bovine serum albumin (BSA) and catalase (EC 1. 11. 1. 6). Furthermore, a lowering of the assay temperature to 15°C improved the stability of the duroquinol-based activity. One remarkable finding was that with duroquinol (or external NADH) as substrate the alternative oxidase was synergistically activated by succinate (as well as by malate) even in the presence of the succinate dehydrogenase inhibitor malonate. Our interpretation is that succinate and malate (indirectly) activate the alternative oxidase and that this activation is part of a physiological mechanism for regulation of the alternative oxidase.     

Cooperative Substrate Oxidation by Mitochondria from Castor Bean Hypocotyls

Cooperative oxidation of succinate and exogenous NADH was followed in the mitochondria from five- to six-day-old castor bean (Ricinus communisL.) seedlings. Although succinate was oxidized at a much higher rate than NADH, the former inconsiderably (less than 15%) inhibited the oxidation of the latter substrate in state 4, while, in state 3 (in the presence of ATP), the two substrates did not compete and were jointly oxidized. When two substrates were oxidized by the mitochondria with the alternative CN-resistant oxidase (AO) inhibited with salicylhydroxamic acid, the rate of NADH oxidation in state 4 dropped by over 40% as compared to the initial rate. Meanwhile, the rate of succinate oxidation was not considerably affected by AO inhibition. We believe that one of the AO functions in the mitochondria is to provide for noncompeting oxidation of two (or more) substrates by employing two (or several) dehydrogenases of the respiratory chain.