黄曲霉素产毒真菌ver-1、verB及ITS基因序列多重PCR体系的优化

目的 对黄曲霉素产毒真菌的多重PCR体系进行优化，确定最佳PCR体系。方法 以黄曲霉素产生过程中的主要调控基因ver-1、verB及通用基因片段ITS序列为目的片段，设计多重PCR引物，采用单因素和正交优化试验，对DNA模板、Mg²⁺浓度、引物用量、dNTPs用量、退火温度等因素进行考察。结果 最佳多重PCR体系为：50 µL体系中含有引物(5 µmol·L^-1)3 mL，dNTPs(2.5 mmol·L^-1)5 µL，Mg²⁺(2.5 mmol·L^-1)4 µL，DNA浓度10 ng·µL^-1，退火温度56 ℃。结论 建立的体系可用于黄曲霉素产毒真菌的多重PCR鉴定，对黄曲霉产毒菌的源头鉴定具有一定的意义。     

山胡椒内生真菌Trichoderma sp. SHJN1和Perenniporia sp. SHJG1的抗肿瘤活性成分研究

目的 从民族药山胡椒内生真菌Trichoderma sp.SHJN1和Perenniporia sp.SHJG1的代谢物中寻找活性先导化合物。方法 采用正相硅胶、反相硅胶、Sephadex LH-20凝胶及制备型HPLC等对Trichoderma sp.SHJN1和Perenniporia sp.SHJG1发酵物进行分离纯化，再通过NMR、ESI-MS等鉴定化合物结构，同时采用人乳腺癌细胞（MCF-7）和人肺癌细胞（A549）对这些化合物的抗肿瘤活性进行初步评价。结果 从2株内生真菌次级代谢产物中共分离鉴定了12个化合物：alantrypinone （1）、oryzalactam （2）、phomoindene A （3）、cis-gregatin B （4）、huaspenone B （5）、stigmasta-7，22-dien-3β，5α，6α-triol （6）、ergosterol （7）、1-deoxy-2-demethylviridiol （8）、viridiol （9）、trichodermamides A （10）、chromone （11）、对-羟基苯乙酸（12）。抗肿瘤活性评价结果显示，化合物3 抑制MCF-7细胞增殖活性IC₅₀为（62.9±1.02）μmol·L^-1[顺铂（cisplatin，DDP） IC₅₀为（30.1±1.67）μmol·L^-1]；化合物8 和9 抑制A549细胞增殖活性的IC₅₀分别为（34.6±1.57）μmol·L^-1和（44.9±1.74）μmol·L^-1[DDP IC₅₀为（20.6±1.42）μmol·L^-1]。结论 化合物3 ，8 和9 具有潜在抗肿瘤活性。     

基于斑马鱼模型的中药来源化合物血管抑制活性筛选

目的 利用斑马鱼高通量模型，对10种中药来源化合物进行筛选。方法 选取受精后转基因血管荧光Fli-1品系斑马鱼对10个中药来源的化合物进行筛选，用不同的药物浓度分别作用于斑马鱼，观察对斑马鱼体血管的影响。以斑马鱼肠下血管面积为主要指标，斑马鱼肠下血管出芽数为次要指标进行定量分析，考察各化合物对斑马鱼血管生成的抑制作用。结果 木犀草素(87.34 μmol·L^–1)、洋川芎内酯A(52.02 μmol·L^–1)、盐酸小檗碱(537.91 μmol·L^–1)、和厚朴酚(9.39 μmol·L^–1)、齐墩果酸(10.95 μmol·L^–1)组斑马鱼肠下血管面积与正常对照组相比肠下血管面积显著减少(P<0.01或P<0.001)；木犀草素、洋川芎内酯A、盐酸小檗碱、齐墩果酸、和厚朴酚对肠下血管面积的抑制作用分别为12%，17%，18%，15%，24%；各化合物与正常对照组相比斑马鱼肠下血管出芽数无显著性差异；阳性对照组(索拉非尼5 μmol·L^–1)斑马鱼肠下血管面积与正常对照组比较显著性减少(P<0.05)，其对血管新生的抑制作用为22%，肠下血管出芽数与正常对照组相比显著性减少(P<0.05)。结论 木犀草素、洋川芎内酯A、盐酸小檗碱、齐墩果酸、和厚朴酚具有抑制血管新生作用。     

2',4'-二羟基-3'-甲基-3-甲氧基查耳酮上调p21抑制人肝癌HepG2细胞的生长

目的 探讨2'',4''-二羟基-3''-甲基-3-甲氧基查耳酮(C20)对人肝癌HepG2细胞的体外抗肿瘤作用及其潜在的作用机制。方法 通过CCK-8法、集落形成实验、5-乙炔基-2''-脱氧尿苷(EdU)染色法检测C20对人肝癌HepG2细胞增殖的影响;通过彗星实验检测C20(10 μmol·L^-1)对HepG2细胞DNA损伤的影响;通过流式细胞术检测C20(5、10 μmol·L^-1)对HepG2细胞周期阻滞的影响;通过Hoechst染色和流式细胞术检测C20(5、10 μmol·L^-1)对HepG2细胞凋亡的影响。借助Western blotting法检测C20(5、10 μmol·L^-1)处理对HepG2细胞中与凋亡、DNA损伤、细胞周期阻滞相关蛋白表达水平的调控作用。结果 与对照组比较,C20显著抑制HepG2细胞的活力(P<0.001),给药48 h的半数抑制浓度(IC₅₀)为7.937 μmol·L^-1;5 μmol·L^-1 C20能够显著抑制HepG2细胞的集落形成能力(P<0.01);EdU染色结果显示5、10 μmol·L^-1的C20能够抑制人肝癌HepG2细胞的增殖能力;5、10 μmol·L^-1的C20显著诱导HepG2细胞G₂/M期阻滞(P<0.001);5、10 μmol·L^-1的C20显著促进HepG2细胞凋亡(P<0.001),并显著上调Caspas-3、Caspase-9以及PARP的剪切水平(P<0.01);10 μmol·L^-1的C20能够诱导HepG2细胞发生DNA损伤,并且5、10 μmol·L^-1的C20显著上调γH2AX、p21的蛋白水平(P<0.01)。结论 C20能够造成HepG2细胞发生DNA损伤,上调p21蛋白水平,导致细胞G₂/M期阻滞,并进一步诱发凋亡,发挥体外抗肝癌作用。     

松果菊苷抑制卵巢癌SKOV3细胞的增殖、侵袭和干细胞样特性

目的 探讨松果菊苷对卵巢癌SKOV3细胞的作用。方法 选择不同浓度的松果菊苷处理卵巢癌SKOV3细胞,24 h后CCK8检测细胞存活率;选择不同浓度松果菊苷浓度(0,20,40,80μmol·L^-1)进行后续实验,EdU染色检测细胞增殖;Transwell检测细胞侵袭;免疫印迹法检测VEGF、E-cadherin、Vimentin的表达及p38 MAPK的磷酸化;显微观察细胞肿瘤球形态;流式细胞仪检测CD133和CD44的含量。结果 SKOV3细胞的存活率与松果菊苷呈浓度依赖性降低,松果菊苷浓度≥40μmol·L^-1时,SKOV3细胞的存活率较松果菊苷0μmol·L^-1时均明显降低(P<0.05)。与松果菊苷0μmol·L^-1组比较,松果菊苷40μmol·L^-1组和80μmol·L^-1组EdU阳性细胞数和侵袭细胞数均减少(P<0.05);VEGF和Vimentin蛋白表达水平及p38 MAPK磷酸化水平均降低(P<0.05);E-cadherin蛋白表达水平升高(P<0.05);肿瘤干细胞成球直径减小(P<0.05),成球数减少(P<0.05);CD133和CD44表达均降低(P<0.05)。结论 松果菊苷可以抑制卵巢癌SKOV3细胞的增殖、侵袭、干细胞样特性及p38 MAPK的磷酸化。     

细胞色素P450介导的氯沙坦钾对瑞格列奈在大鼠肝微粒体中代谢的影响

目的 研究瑞格列奈在大鼠肝微粒体中的酶促反应动力学，并考察氯沙坦钾对其在大鼠肝微粒体中代谢的影响。方法 建立大鼠肝微粒体体外孵育体系对瑞格列奈的代谢进行研究；以洛伐他汀为内标，应用UPLC测定大鼠肝微粒体中瑞格列奈的浓度。采用底物减少法，通过GraphPad Prism 5.0软件计算瑞格列奈的酶促反应动力学常数V_max和K_m；分别以系列浓度氯沙坦钾（2.5~50μmol·L^-1）与瑞格列奈（44 μmol·L^-1）于37℃水浴中共同孵育，并测定肝微粒体中瑞格列奈的减少量，考察氯沙坦钾对瑞格列奈的抑制作用。结果 瑞格列奈在大鼠肝微粒体的最佳孵育时间为40 min，最佳蛋白质量浓度为1 mg·mL^-1；瑞格列奈酶促反应动力学参数V_max=47.29μmol·min^-1·（mg·protein）^-1，K_m=51.41 μmol·L^-1；氯沙坦钾对瑞格列奈在体外肝微粒体抑制作用的IC₅₀值为17.89 μmol·L^-1。结论 氯沙坦钾对瑞格列奈在大鼠肝微粒体中的代谢具有较强的抑制作用，两药联合应用可能发生相互作用，具有诱发低血糖的风险。     

紫茎泽兰中的萜类成分研究

目的 对紫茎泽兰中的萜类成分及其药理活性进行研究。方法 采用D101、HP20大孔树脂、硅胶柱色谱、葡聚糖凝胶色谱、高效液相色谱等多种分离分析手段，MS、¹H-NMR和¹³C-NMR等多种波谱技术以及ECD计算鉴定化合物结构，并采用MTT法对分离得到的单体化合物进行细胞毒活性的筛选。结果 从紫茎泽兰95%乙醇提取物的石油醚和乙酸乙酯萃取部位中分离得到10个化合物，分别鉴定为泽兰酮D (1)、thymoquinol 5-O-β-glucopyranoside (2)、thymoquinol2-O-β-glucopyranoside (3)、2R^*,3S^*-toxol-7-O-β-D-glucopyranoside (4)、6-甲氧基-山柰酚-7-O-β-D-葡萄糖苷(5)、万寿菊素-4’-甲氧基-7-O-β-D-葡萄糖苷(6)、6-hydroxykaempferol-7-O-β-D-glucopyranoside (7)、eupatonriochromene (8)、demethoxyencecalin (9)、encecalin (10)。化合物10对食管癌细胞的IC₅₀值为178.4 μmol·L^-1。结论 其中化合物1为新化合物，化合物6为首次从泽兰属中分离得到，化合物2，3，7为首次从紫茎泽兰中分离得到。化合物10对食管癌细胞Eca-109具有一定的细胞毒活性。     

丹参水溶性成分拮抗脂多糖诱导HTR8/SVneo滋养层细胞凋亡的作用机制

目的 探讨丹参水溶性成分拮抗脂多糖(lipopolysaccharide,LPS)诱导HTR8/SVneo细胞凋亡的相关作用机制。方法 以不同浓度(0,100,200,400,800,1 600 ng·mL^-1)LPS处理HTR8/SVneo细胞,采用实时定量聚合酶链式反应验证HTR 8/SVneo细胞炎症模型的建立;细胞活力检测试剂盒检测各组细胞活力;划痕试验评估各组细胞向损伤区域迁移能力;流式细胞术检测各组细胞凋亡率并分析各组细胞线粒体膜电位。结果 200 ng·mL^-1 LPS干预后HTR8/SVneo细胞的NLRP3炎症小体指标及炎症因子表达量增高(P<0.05)。与LPS组相比,丹酚酸B(0.08 μmol·L^-1)、丹酚酸C(0.4 μmol·L^-1)、紫草酸(0.08 μmol·L^-1)干预后HTR8/SVneo细胞活性明显升高(P<0.01)。与LPS组比较,丹参水溶物质组凋亡率显著降低(P<0.05或P<0.01),线粒体膜电位水平显著升高。结论 丹参水溶物质能够提高LPS干预后HTR8/SVneo细胞活力,改善细胞迁移能力,提高细胞线粒体膜电位,进而抑制细胞凋亡。     

二茂铁杂环类化合物的合成及抗三阴性乳腺癌活性研究

目的 设计、合成杂环二茂铁衍生物,并研究其抗三阴性乳腺癌活性。方法 以二茂铁查耳酮为先导化合物,对其进行结构改造,合成了一系列含有杂环的二茂铁衍生物,并通过CCK8试剂盒测试化合物抗乳腺癌活性。结果 合成了28个二茂铁衍生物,其结构均通过¹H-NMR和MS加以确证。初步的生物活性测试结果表明,所合成的二茂铁衍生物对三阴性乳腺癌MDA-MB-231细胞有较强的选择性和抑制活性,其中咪唑杂环化合物抗肿瘤活性强于相应的吡唑类和嘧啶化合物。尤其是28a[IC₅₀=（1.6±0.23）μmol·L^-1]对MDA-MB-231的抑制活性分别是先导化合物3[IC₅₀=（10.7±1.41）μmol·L^-1]和他莫昔芬[IC₅₀=（13.7±1.17）μmol·L^-1]的6和10倍,同时这些二茂铁衍生物对正常乳腺上皮细胞MCF-10A均没有毒性。结论 本研究为开发具有抗三阴性乳腺癌活性的化合物提供了信息和依据。     

含有芳基脲结构的4H-吡喃类化合物的设计、合成及抗肿瘤活性研究

目的 设计并合成一系列含有芳基脲结构的4H-吡喃类化合物，评价该类化合物的体外抗肿瘤活性。方法 以间硝基苯甲醛、丙二腈和丙酮二羧酸二甲酯为原料，通过“一锅法”合成含有硝基的吡喃中间体，该中间体的硝基经铁粉还原为氨基，再与取代异氰酸苯酯反应得到一系列目标化合物。以人大细胞肺癌细胞H460、人肺癌细胞A549和人结肠癌细胞HT-29 3种肿瘤细胞为测试细胞株，采用MTT法评价了目标化合物的抗肿瘤活性。结果 合成了11个含有芳基脲结构的4H-吡喃类化合物。体外抗肿瘤活性试验表明，11个化合物对3种肿瘤细胞株均具有很好的抑制活性。其中化合物7c活性突出，对H460和A549细胞的IC₅₀值分别为0.82，0.98 μmol·L^-1, 优于阳性对照药索拉非尼(IC₅₀=3.20， 2.83 μmol·L^-1)。结论 含有芳基脲结构的4H-吡喃类化合物具有很好的抗肿瘤活性，可作为抗肿瘤化合物的结构骨架进一步研究。     

Neural anti-inflammatory sesquiterpenoids from the endophytic fungus Trichoderma sp. Xy24

Three new sesquiterpenoids trichoacorenols B–C and cyclonerodiol B (1–3), along with three known ones (4–6), were isolated from the mangrove plant endophytic fungus Trichoderma sp. Xy24 using various column chromatography techniques. The structures of these compounds were determined on the basis of extensive spectroscopic data analyses. Compounds 1, 2, 4, and 5 were four acorane sesquiterpenes, 3 and 6 were two monocyclic sesquiterpenediols. Compounds 3 and 5 exhibited significant neural anti-inflammatory activity by inhibiting LPS-induced NO production in BV2 cells with the inhibitory rates of 75.0% and 39.2% at 0.1 μM, respectively, which are more potent than curcumin, a positive control with the inhibitory rate of 21.1% at 0.1 μM.     

Fusaripeptide A: new antifungal and anti-malarial cyclodepsipeptide from the endophytic fungus Fusarium sp.

From the culture of the endophytic fungus Fusarium sp. isolated from the roots of Mentha longifolia L. (Labiatae) growing in Saudi Arabia, a new cyclodepsipeptide, namely fusaripeptide A (1), along with three known compounds adenosine (2), 2[(2-hydroxypropionyl)amino]benzamide (3), and cyclopentanol (4), have been isolated. Their structures were determined, using extensive 1D and 2D NMR and HRESI and GC mass spectral data. That is the first report for the isolation of compound 4 from natural source. In addition, compounds 2 and 3 are reported here for the first time from Fusarium sp. The absolute configuration of the amino acid residues of 1 was assigned by chiral GCMS and Marfey’s analysis after acid hydrolysis. Fusaripeptide A differs from the reported ones from Fusarium sp. in the length of fatty acidic alkyl chain. Compound 1 was evaluated for its antifungal, anti-malarial, and cytotoxic activities. It exhibited potent antifungal activity toward C. albicans, C. glabrata, C. krusei, and A. fumigates with IC₅₀ values of 0.11, 0.24, 0.19, and 0.14 μM, respectively. Furthermore, it had significant anti-malarial activity toward P. falciparum (D6 clone) with IC₅₀ value of 0.34 μM. However, it showed cytotoxic activity toward the tested cell lines.     

(+)-Cyclopenol,a new naturally occurring 7-membered 2,5-dioxopiperazine alkaloid from the fungus Penicillium sclerotiorum endogenous with the Chinese mangrove Bruguiera gymnorrhiza

One new naturally occurring 7-membered 2,5-dioxopiperazine alkaloid named (+)-cyclopenol (1), along with nine known compounds including viridicatol (2), 3-(dimethylaminomethyl)-1-(1,1-dimethyl-2-propenyl)indole (3), anacine (4), aurantiomide C (5), viridicatin (6), 3-O-methylviridicatin (7), verrucosidin (8), ergosterol (9), and ergosterol peroxide (10), was isolated from the EtOAc extract of fungus Penicillium sclerotiorum, an endophytic fungal strain isolated from Chinese mangrove Bruguiera gymnorrhiza. The chemical structure of the new compound 1 was elucidated on the basis of detailed spectroscopic analysis. The absolute configuration of 1 was determined by single-crystal X-ray analysis with Cu Kα radiation (λ = 1.54178 Å). To our knowledge, (+)-cyclopenol (1) represents the first example of 7-membered 2,5-dioxopiperazine isolated from mangrove endophytic fungus.     

丰肉结海绵相关青霉菌HLS-216次级代谢产物研究

目的 研究我国南海水域丰肉结海绵相关青霉菌Penicillium sp.HLS-216的次级代谢产物的提取分离方法 、结构鉴定及其抗肿瘤、抗炎活性.方法 采用大米固体发酵培养,乙酸乙酯提取后,经硅胶柱色谱、Sephadex LH-20凝胶柱色谱、高级液相色谱等手段进行分离,并对分离得到的单体化合物应用质谱、核磁共振等技术进行结构鉴定;采用噻唑蓝(MTT)法和Griess法对分离得到的单体化合物进行抗肿瘤、抗炎活性筛选.结果 分离得到7个化合物,分别鉴定为:黑麦酮酸F(secalonic acid F,1)、黑麦酮酸D(secalonic acid D,2)、meleagrin(3)、oxaline(4)、对羟基肉桂酰胺(4-hydroxycinnamamide,5)、对羟基苯乙酸甲酯(methyl 4-hydroxyphenylacetate,6)、对羟基苯乙醛(4-hydroxyphenylacetonitrile,7).结论 化合物5和7为首次从青霉菌中分离得到;化合物1和2显示出较强的抗肿瘤活性,化合物4表现出一定的抑制小鼠腹腔巨噬细胞一氧化氮生成的作用.     

Activity against Plasmodium falciparum of Lactones Isolated from the Endophytic Fungus Xylaria sp.

Three lactones were isolated from the culture medium of the endophytic fungus Xylaria sp. Grev. (Xylariaceae). The major compound, which showed weak activity (13 μ g/mL) against a chloroquine-resistant strain of Plasmodium falciparum, was identified as (+)-phomalactone (1). The others were 6-(1-propenyl)-3,4,5,6-tetrahydro-5-hydroxy-4H-pyran-2-one (2) and 5-hydroxymellein (3). Compounds 1 and 2 are reported for the first time as constituents of Xylaria. Also, this is the first report of the activity of the compounds 1–3 against a chloroquine-resistant Plasmodium falciparum strain.     

A β-resorcylic macrolide from the seagrass-derived fungus <Emphasis Type="Italic">Fusarium</Emphasis> sp. PSU-ES73

A new β-resorcylic macrolide, 5′-hydroxyzearalenone (1), and six known β-resorcylic macrolides were isolated from the seagrass-derived fungus Fusarium sp. PSU-ES73. Their structures were established by analysis of spectral data. All of the isolated compounds were evaluated for their antibacterial activity against Staphylococcus aureus, both standard and methicillin-resistant strains, as well as their antifungal activity against Cryptococcus neoformans. Only the known compound zearalenone (2) displayed weak antibacterial and antifungal activities.     

Novel chaetospirolactone and orsellide F from an endophytic fungus Chaetomium sp.

Chaetospirolactone (1), a novel spiro-lactone bearing a rare 1-oxaspiro [4.4] non-7-ene-2,6-dione skeleton, and orsellide F (2), together with six known compounds (3–8), were isolated from an endophytic fungus Chaetomium sp. NF00754. Their structures were determined by interpretation of spectroscopic data. The absolute configurations of 1 and 2 were established by analysis of single X-ray crystallographic data and CD spectra. Compounds 3, 4, and 6 showed moderate acetylcholinesterase inhibitory activity with IC₅₀ values of 7.34, 5.19, and 7.67 μM, respectively.     

Chemical constituents of <Emphasis Type="Italic">Melandrium firmum</Emphasis> Rohrbach and their anti-inflammatory activity

In our ongoing search for anti-inflammatory agents originating from Korean medicinal plants, we found that the hexane and BuOH fractions of the MeOH extract from the whole plants of Melandrium firmum Rohrbach inhibited 5-lipoxygenase (5-LOX) activity. By activity-guided fractionation, eleven compounds, α-spinaterol (1), ursolic acid (2), ergosterol peroxide (3), α-spinaterol glucoside (4), 2-methoxy-9-β-D-ribofuranosyl purine (5), aristeromycin (6), ecdysteron (7), polypodoaurein (8), (-)-bornesitol (9), mannitol (10) and cytisoside (11) were isolated from the hexane and BuOH fractions using column chromatography. Compounds 2, 5, 6, 8, 9, 10 and 11 were isolated for the first time from this plant. Compounds 1, 3, 4 and 7 inhibited 5-LOX activity with IC₅₀ values of 21.04 μM, 42.30 μM, 32.82 μM, and 17.18 μM, respectively. Ming Shan Zheng and Nam Kyung Hwang contributed equally to this work.     

Metabolites from endophytic fungus A12 of Dracaena cambodiana

A new drimane sesquiterpene, 1α,7α-dihydroxyconfertifolin (1), along with two known compounds 2 and 3, was isolated from endophytic fungus A12 of Dracaena cambodiana. The new compound was elucidated by HR-ESI-MS and spectroscopic techniques (IR, UV, 1D, and 2D NMR). Compound 2 showed inhibitory bacterial activity against Staphylococcus aureus with diameter of the inhibition zone of 13.5 mm.     

Three new tetralol analogs from soil-derived fungus Myrothecium verrucaria with anti-inflammatory activity

Three new tetralol analogs, myrochromanols A-C (1–3), together with 11 known trichothecenes (4–14), were isolated from a soil fungus Myrothecium verrucaria HL-P-1. The structures of the three new compounds were elucidated by extensive spectroscopic analysis including HRESIMS, NMR, and ECD calculation. All of the new compounds were tested for their anti-inflammatory activity and cytotoxicity. Compounds 1 and 3 inhibited lipopolysaccharide (LPS)-induced NO production in BV2 cells with IC₅₀ values of 26.04 and 25.80 μM, respectively.