细脚拟青霉代谢产物清除自由基和抑制单胺氧化酶活性的研究

用二苯代苦味肼基自由基(DPPH)-TLC法和酶标仪法对一株细脚拟青霉RCEF0394发酵液甲醇提取物的清除自由基活性进行了定性和定量测定,发现该提取物具有较强的清除自由基活性,在浓度为5.0mg/mL,于37℃下保温10min时,它对0.4mg/mL的DPPH自由基的清除率可达75.4%。以大鼠肝脏线粒体单胺氧化酶为靶标的体外实验发现供试细脚拟青霉发酵液有较强的抑制单胺氧化酶活性,且其活性和浓度呈量效关系。该发酵液冻干品对单胺氧化酶的半数抑制浓度IC50为118.1μg/mL。其三氯甲烷提取物对单胺氧化酶的抑制活性明显强于发酵液冻干品,表明该抑制剂可能为极性较低的化合物。进一步的分型试验表明该三氯甲烷提取物对A型单胺氧化酶呈混合抑制,对B型呈竞争性抑制,其Km值分别为0.44mg/L,0.34mg/L。     

干酪乳杆菌JH-23中单胺氧化酶抑制剂的初步分离

采用丙酮抽提法得到了干酪乳杆菌JH-23中具有抑制单胺氧化酶作用的无细胞提取物,并利用硅胶薄层色谱从中分离出4组活性片段,其中片段Ⅰ对单胺氧化酶的抑制效果最佳,反应浓度在120μg/mL时抑制率为55.4%。该活性片段经国家新药筛选中心确证有抑制单胺氧化酶的效果,具有潜在的抗衰老作用。     

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。     

藏药穹代尔中内生真菌的分离及其次级代谢物的生物活性研究

从藏药穹代尔(攀茎钩藤,Uncaria scandens)的内生真菌中筛选对单胺氧化酶及乙酰胆碱酯酶具有抑制活性的提取物,并对高活性提取物进行化学成分的鉴定。采用组织切块法分离内生菌,发酵、萃取、甲醇回流提取法得到待测提取物,酶标法测定酶活性,波谱解析法鉴定结构。从穹代尔组织中共分离到9株内生真菌,检测发现3株内生真菌发酵提取物具有一定乙酰胆碱酯酶抑制活性,其抑制率在50%以上。通过分子生物学方法及形态学分析对活性菌株QDE-6进行了菌种鉴定,鉴定为黑曲霉属(Aspergillus sp.)真菌。从内生真菌QDE-6的次级代谢产物中分离鉴定了5个化合物,分别是Aspergiketone(1)、Gliotoxin(2)、Pseurotin A2(3)、Brevianamide F(4)、Pseurotin A1(5)。相比其他化合物,化合物3抑制率为51. 67%,具有较高的抗单胺氧化酶活性,化合物4抑制率为42. 76%,具有较高的抗乙酰胆碱酯酶活性。可作为进一步研究藏药穹代尔中内生真菌及开发药物的基础。     

单胺氧化酶抑制剂在临床方面的应用

单胺氧化酶(monoamine oxidase,MAO)是人体内天然存在的一种酶,催化单胺类物质氧化脱氨反应的酶。人体内含有两种单胺氧化酶:单胺氧化酶A和单胺氧化酶B。单胺氧化酶A主要分布在儿茶酚胺能神经元中;单胺氧化酶B主要分布在5-羟色胺能神经元、组胺能神经元和神经胶质细胞中,这两种亚型都均可以使单胺类神经递质失活。而单胺氧化酶抑制剂则能够通过抑制单胺氧化酶的对单胺类物质的氧化活性,从而达到减轻或者消除由各种原因引起的单胺类物质减少或单胺氧化酶活性过高导致的疾病。本文主要总结了近几年单胺氧化酶抑制剂在临床上用于治疗帕金森病、抑郁症和幽门螺旋杆菌方面的最新进展。     

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。     

单胺氧化酶抑制剂在临床方面的应用

单胺氧化酶（monoamine oxidase, MAO）是人体内天然存在的一种酶,催化单胺类物质氧化脱氨反应的酶。人体内含有两种单胺氧化酶：单胺氧化酶A 和单胺氧化酶B。单胺氧化酶A 主要分布在儿茶酚胺能神经元中;单胺氧化酶B 主要分布在5- 羟色胺能神经元、组胺能神经元和神经胶质细胞中,这两种亚型都均可以使单胺类神经递质失活。而单胺氧化酶抑制剂则能够通过抑制单胺氧化酶的对单胺类物质的氧化活性,从而达到减轻或者消除由各种原因引起的单胺类物质减少或单胺氧化酶活性过高导致的疾病。本文主要总结了近几年单胺氧化酶抑制剂在临床上用于治疗帕金森病、抑郁症和幽门螺旋杆菌方面的最新进展。     

黄蜻幼虫肠道菌QTYC01除草活性代谢产物的分离和鉴定

【目的】从黄蜻幼虫肠道中分离出具有除草活性的真菌,并在其代谢产物中寻找具有除草活性的先导化合物。【方法】采用涂布平板法对黄蜻幼虫肠道共生真菌进行分离,通过形态学观察和5.8S r RNA序列分析初步确定目标菌株QTYC01的分类地位。利用离体的方法测定菌株发酵液及其乙酸乙酯提取物的除草活性以及粗提物对常见农作物的安全性;利用盆栽方法测定发酵液对稗草幼苗的活性。运用重结晶的方法对发酵产物进行分离、纯化,利用质谱和核磁共振谱分析鉴定出化合物的结构。【结果】菌株QTYC01被鉴定为Curvularia crepinii。离体活性测试发现QTYC01发酵液可显著抑制稗草和反枝苋幼根的生长,其抑制率分别可达95.0%和90.1%,并可使经喷施发酵液的稗草幼苗的受害率达到71.1%。发酵液的乙酸乙酯提取物对稗草和反枝苋幼根具有很好的抑制效果,在100μg/m L的浓度条件下,粗提物对稗草和反枝苋的抑制活性分别为56.8%和71.2%,且在该浓度条件下对一些常见农作物具有很好的安全性,其抑制率均低于32.6%。进一步从其乙酸乙酯提取物中分离得到化合物(5Z)-7-oxozeaenol,活性测试表明化合物具有较好的抑制反枝苋活性,其IC_(50)为4.8μg/m L。【结论】菌株QTYC01具有开发为新型微生物除草剂的潜力。     

一种被毛孢菌丝体中抗肿瘤活性组分的分离纯化和液质联用分析

在一次多菌株的生物活性测定中,发现一株被毛孢菌株RCEF0851菌丝体提取物具有较强的抗肿瘤CHO细胞的活性。本研究用不同溶剂对活性组分进行了提取试验,发现乙酸乙酯能最大程度地提取出该活性成分。分离试验表明该提取物较适于用反相柱Synergi Hydro进行分离。活性指导下的分离得到一活性组分的纯品Hh-1。该组分在40μg/mL时对CHO细胞的抑制率达到83.61%。高分辨质谱分析表明该活性化合物的可能分子式是C30H22O10。该分子为首次从虫生真菌中发现。     

球毛壳菌H6次级代谢产物及其α-葡萄糖苷酶抑制活性

采用体外α-葡萄糖苷酶抑制模型对一株球毛壳菌H6的发酵液和菌丝体两种乙酸乙酯提取物进行活性评价,结果表明,发酵液乙酸乙酯提取物对α-葡萄糖苷酶具有较强的抑制活性,其IC₅₀值为(510.76±23.46)μg/mL。采用硅胶、Sephadex LH-20、半制备高效液相等色谱技术从其发酵液乙酸乙酯提取物中分离得到12个化合物。通过各种光谱分析,依次鉴定为chaetoviridins A-B(1-2),chaetoglobosins A-D(3-6),chaetoglobosin J(7),chaetoglobosin Q(8),prochaetogobosinsⅠ-Ⅲ(9-11),vibratilicin(12),其中化合物12为首次从毛壳属中分离得到。对化合物进行α-葡萄糖苷酶抑制活性测定发现,化合物12显示弱的抑制活性,其IC₅₀为(1 182.75±19.14)μg/mL。     

Interaction of N-(2-Nitro-4-Azidophenyl)-Serotonin with Two Types of Monoamine Oxidase in Rat Brain

The effects of N-(2-nitro-4-azidophenyl) serotonin (NAP-5-HT) on types A and B monoamine oxidase (MAO) in rat brain cortex were studied. In the dark this compound acted as a competitive inhibitor for both types A and B MAO (Ki values of 0.19 microM and 0.21 microM for types A and B MAO, respectively). Upon photolysis, NAP-5-HT became an irreversible inhibitor for only type B MAO. A 50% inhibition was obtained by irradiation of the enzyme in the presence of 35 nM NAP-5-HT. Furthermore the inhibition of type B MAO could be protected by including its substrate phenylethylamine during the irradiation. Under the same photolytic conditions photodependent inhibition of type A MAO by NAP-5-HT was not clearly observed. These results provide further evidence that there is a fundamental difference in the active site of the two types of MAO in brain. NAP-5-HT may be a useful photoaffinity probe for characterizing the active site of type B MAO.     

薏苡仁多菌发酵液的菌种优选及其抑制黑色素生成的作用研究

以薏苡仁作为发酵基质,确定利于提高发酵液体外活性的较优乳酸菌种,并分析优势乳酸菌种薏苡仁发酵液对斑马鱼胚体黑色素生成的抑制作用。通过比较分析乳酸乳球菌（Lactococcus lactis）、嗜热链球菌（Streptococcus thermophilus）和保加利亚乳杆菌（Lactobacillus bulgaricus）3种单一乳酸菌和三者复合乳酸菌的薏苡仁发酵液的还原糖、总酚、游离氨基酸、蛋白、总酸和乳酸含量等理化指标及体外羟自由基清除能力和酪氨酸酶活抑制率确定较优发酵菌种,采用高通量测序测定发酵过程中微生物菌群结构;利用斑马鱼模型研究发酵液对黑色素生成的抑制作用。研究结果表明,采用乳酸乳球菌、嗜热链球菌和保加利亚乳杆菌3种乳酸菌复合发酵比单一乳酸菌发酵更具优势。使用以上菌种复合发酵薏苡仁过程中,乳酸乳球菌和嗜热链球菌为发酵前期优势菌群,发酵中后期则以保加利亚乳杆菌为优势菌群。经复合乳酸菌发酵后,薏苡仁发酵液的羟自由基清除率和酪氨酸酶活抑制率分别提高了20.82%和87.26%;斑马鱼模型实验结果表明,薏苡仁发酵液可以显著减少斑马鱼体表黑色素分布,当使用含量为2.0%时,对黑色素生成抑制率达到59.45%。研究结果为利用薏苡仁多菌发酵液开发为具美白肌肤性能的功能性新原料提供了科学数据支撑,并希望进一步推进薏苡产业的升级。     

中国被毛孢发酵液中一种镇静催眠活性物质的分离纯化和结构鉴定

本研究对中国被毛孢Hirsutella sinensis菌株RCEF0273发酵液的乙酸乙酯粗提物中的镇静催眠活性物质进行了分析.通过活性指导下的色谱分离,从乙酸乙酯提取物中得到了一种白色晶体化合物,质谱(MS)和核磁共振(NMR)的鉴定结果表明该化合物为茶碱-9-葡萄糖苷.活性试验显示该化合物可抑制小鼠的自发活动,缩短小鼠入睡潜伏期,延长小鼠睡眠持续时间,具有镇静催眠活性.     

Antagonism between long acting Monoamine Oxidase Inhibitors (MAOI) and MD780515, a new specific and reversible MAOI

The inhibition of type A and B monoamine oxidase (MAO A and B) in rat brain, liver and heart by MD780515, 3-[4-(3 cyanophenylmethoxy) phenyl]-5-(methoxymethyl)-2-oxazolidinone, has been investigated ex vivo with 5-hydroxytryptamine (5-HT) and β-phenylethylamine (PEA) as substrates. MAO A was strongly inhibited for four hours after oral administration of 10 mg/kg MD780515 (maximum inhibition : 72%, 86% and 83% in brain, liver and heart respectively. In contrast, in heart where PEA is deaminated by type A MAO, the predominant form of MAO in that tissue, the inhibition was 68% 30 minutes after administration of the compound. In all cases, MAO activities reached control values 24 hours after drug administration (10 mg/kg), whereas some inhibitory activity was still present 24 hours after oral administration of higher doses. The strong MAO A inhibition (68 to 83%) remaining in the three tissues 24 hours after oral administration of clorgyline (5 mg/kg) was completely removed by pretreatment with MD780515 (10 mg/kg). In the same conditions, MD780515 protected against the inhibition (53%) by clorgyline of PEA deamination in heart. Oral pretreatment with increasing doses of MD780515 (2.6 to 84 mg/kg) gradually removed brain MAO A inhibition caused by clorgyline (92%, 28.2 mg/kg) or tranylcypromine (88%, 4.8 mg/kg), the complete removal being observed at the dose of 21 mg/kg of MD780515 for clorgyline, and at 42 mg/kg for tranylcypromine. Inhibition of brain MAO B by tranylcypromine (96%) was not modified by pretreatment with the same range of oral doses of MD780515. The results are consistent with a specific and reversible inhibition of MAO A activity by MD780515 which can protect against long acting MAO A inhibitory effects of clorgyline and tranylcypromine. MD780515 enhances the selectivity of tranylcypromine.     

Inhibition of Monoamine Oxidase Selectively in Brain Monoamine Nerves Using the Bioprecursor (E-β-Fluoromethylene-m-Tyrosine (MDL 72394), a Substrate for Aromatic L-Amino Acid Decarboxylase

(E)-beta-Fluoromethylene-m-tyrosine (FMMT) is a dual-enzyme-activated inhibitor of monoamine oxidase (MAO). The compound is not an inhibitor per se but is decarboxylated by aromatic L-amino acid decarboxylase (AADC) to yield a potent enzyme-activated irreversible inhibitor of MAO, (E)-beta-fluoromethylene-m-tyramine, which shows some selectivity for inhibition of MAO type A. Decarboxylation of FMMT was demonstrated in vitro using hog kidney AADC and in vivo in rats by the ability of alpha-monofluoromethyldopa (MFMD), a potent inhibitor of AADC, to prevent MAO inhibition produced by FMMT. In isolated synaptosomes, FMMT was decarboxylated by AADC, and, furthermore, the compound was actively transported into these isolated nerve endings. An active transport into the CNS has also been demonstrated in vivo by performing competition experiments with leucine. To demonstrate that FMMT is preferentially decarboxylated within monoamine nerves of the CNS, the nigrostriatal 3,4-dihydroxyphenylethylamine (dopamine) pathway of rats was unilaterally lesioned with 6-hydroxydopamine or infused with MFMD. Under these conditions, MAO inhibition produced by orally administered FMMT in the striatum ipsilateral to the lesion or infusion was markedly attenuated. Combination of FMMT with an inhibitor of extracerebral AADC, such as carbidopa, protected peripheral organs against the MAO inhibitory effects and concomitantly enhanced MAO inhibition in the CNS. Such combinations had a greatly reduced propensity to augment the cardiovascular effects of intraduodenally administered tyramine, when compared with FMMT given alone or with clorgyline, a selective inhibitor of MAO type A. The results obtained with FMMT suggest the possibility of achieving selective inhibition of MAO within monoamine nerves of the CNS and, further, suggest that combination of FMMT with an inhibitor of extracerebral AADC will reduce the propensity of this inhibitor to produce adverse interactions with tyramine.     

Selective Inhibition of Monoamine Oxidase B by Aminoethyl Substituted Benzyl Ethers

Aminoethyl 3-chlorobenzyl ether was shown previously (Ding, C.Z. and Silverman, R.B. (1993). Bioorg. Med. Chem. Lett., 3, 2077-2078) to be a potent and selective time-dependent, but reversible inhibitor of monoamine oxidase B (MAO B). Based on this result, a series of novel aminoethyl substituted benzyl ethers was synthesized and the compounds were examined as potential inhibitors of both isozymic forms of MAO. Each compound in the series inhibits both MAO A and MAO B competitively, and IC(50) values for each compound were determined. In general, the B isozyme is much more sensitive to these inhibitors than the A isozyme (except for the o- and p-substituted nitro analogues), in some cases by more than two orders of magnitude. The selectivity in favor of MAO B inhibition is relatively high for all of the meta-substituted analogues and quite low for all of the ortho-substituted analogues. Having the substituent at the ortho-position is most favorable for MAO A inhibition. With MAO B the meta-analogues were, in general, more potent than the corresponding ortho- and para-analogues with respect to their reversible binding constants. The meta-iodo analogue is the most potent analogue.     

Selective inhibition of monoamine oxidase B by aminoethyl substituted benzyl ethers

Aminoethyl 3-chlorobenzyl ether was shown previously (Ding, C.Z. and Silverman, R.B. (1993). Bioorg. Med. Chem. Lett., 3, 2077-2078) to be a potent and selective time-dependent, but reversible inhibitor of monoamine oxidase B (MAO B). Based on this result, a series of novel aminoethyl substituted benzyl ethers was synthesized and the compounds were examined as potential inhibitors of both isozymic forms of MAO. Each compound in the series inhibits both MAO A and MAO B competitively, and IC50 values for each compound were determined. In general, the B isozyme is much more sensitive to these inhibitors than the A isozyme (except for the o- and p-substituted nitro analogues), in some cases by more than two orders of magnitude. The selectivity in favor of MAO B inhibition is relatively high for all of the meta-substituted analogues and quite low for all of the ortho-substituted analogues. Having the substituent at the ortho-position is most favorable for MAO A inhibition. With MAO B the meta-analogues were, in general, more potent than the corresponding ortho- and para-analogues with respect to their reversible binding constants. The meta-iodo analogue is the most potent analogue.     

Detection of microbial contamination in fermentation processes: mass spectrometric determination of gram-negative bacteria in Leuconostoc mesenteroides cultures

Gas chromatography/mass spectrometry was used to detect the presence of Enterobacter cloacae in cultures of Leuconostoc mesenteroides, an organism used in an industrial process for production of dextrane. The penta-fluorobenzoyl-methyl ester derivative of 3-hydroxy-myristic acid, a characteristic compound of gram-negative bacteria, was used as the analyte. By using gas chromatography/mass spectrometry with selected ion monitoring, E. cloacae was determined over the range of 1 ppm to 1% in cultures of L. mesenteroides. The proposed analytical approach represents a useful alternative to conventional methods for determining contaminating organisms in industrial fermentation processes.     

Guinea Pig Striatum as a Model of Human Dopamine Deamination: The Role of Monoamine Oxidase Isozyme Ratio, Localization, and Affinity for Substrate in Synaptic Dopamine Metabolism

The kinetic properties of type A and type B monoamine oxidase (MAO) were examined in guinea pig striatum, rat striatum, and autopsied human caudate nucleus using 3,4-dihydroxyphenylethylamine (dopamine, DA) as the substrate. MAO isozyme ratio in guinea pig striatum (28% type A/72% type B) was similar to that in human caudate nucleus (25% type A/75% type B) but different from that in rat striatum (76% type A/24% type B). Additional similarities between guinea pig striatum and human caudate nucleus were demonstrated for the affinity constants (Km) of each MAO) isozyme toward DA. Endogenous concentrations of DA, 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid were also measured in guinea pig and rat striatum following selective type A (clorgyline-treated) and type B (deprenyl-treated) MAO inhibition. In guinea pig, DA metabolism was equally but only partially affected by clorgyline or deprenyl alone. Combined treatment with clorgyline and deprenyl was required for maximal alterations in DA metabolism. By contrast, DA metabolism in rat striatum was extensively altered by clorgyline but unaffected by deprenyl alone. Finally, the deamination of DA in synaptosomes from guinea pig striatum was examined following selective MAO isozyme inhibition. Neither clorgyline nor deprenyl alone reduced synaptosomal DA deamination. However, clorgyline and deprenyl together reduced DA deamination by 94%. These results suggest that the isozyme localization and/or isozyme affinity for DA, rather than the absolute isozyme content, determines the relative importance of type A and type B MAO in synaptic DA deamination. Moreover, based on the enzyme kinetic properties of each MAO isozyme, guinea pig striatum may serve as a suitable model of human DA deamination.