蛹虫草缓解新型冠状病毒感染研究进展

严重急性呼吸综合征冠状病毒2（SARS-CoV-2）引起的COVID-19在全球范围内大流行,危害了人类健康和公共安全,随着对SARS-CoV-2的结构、功能和致病过程的了解,越来越多的潜在药物被开发。蛹虫草（Cordyceps militaris）是我国传统的药用真菌,具有显著的抗病毒作用,虫草素作为蛹虫草的主要活性成分能够与SARS-CoV-2刺突蛋白、主蛋白酶（Mpro）相结合,抑制病毒RNA依赖的RNA聚合酶（RDRP）活性,阻断SARS-CoV-2在机体内复制。蛹虫草还具有提升机体免疫力、修复受损组织的作用。本文概述虫草素抗SARS-CoV-2的机制和蛹虫草其他相关药理作用,以期为蛹虫草用于新型冠状病毒感染的辅助治疗提供参考。     

蝉虫草（蝉花）来源的化合物鉴定与抗菌活性检测

蝉虫草（蝉花）是我国重要的传统中药材之一,用于治疗慢性肾炎等,但对其活性成分的研究仍不充分。本文采用硅胶柱、凝胶柱、ODS柱分离,以及HPLC等色谱技术,对蝉虫草子实体样品的乙酸乙酯提取物进行分离纯化,得到3个化合物。根据波谱学数据,化合物1-3分别鉴定为(E,E)-9-酮-10,12-十八碳二烯酸(E,E)-9-oxooctadeca-10,12-dienoic acid（1）、麦角甾醇ergosterol（2）和白僵菌酮bassiatin（3）,其中化合物1为首次从蝉虫草中分离获得。抑菌实验表明,3个化合物均对革兰氏阴性大肠杆菌Escherichia coli和革兰氏阳性枯草芽孢杆菌Bacillus subtilis有显著的抑菌活性,化合物1和3还对条件致病真菌白色念珠菌Candida albicans有明显的抑菌活性。本文的研究结果丰富了蝉虫草的活性化合物组成。     

通过化学结构修饰提高虫草素抗菌活性的试验研究

本文以我国传统药用真菌蛹虫草(Cordyceps militaris)的主要活性成分虫草素为先导化合物,合成了4个虫草素的正构烷酰胺同系物(Cn-AAC),即N-丙酰基虫草素(C3-AAC)、N-正辛酰胺虫草素(C8-AAC)、N-月桂酰基虫草素(C12-AAC)和N-硬脂酰基虫草素(C18-AAC),并通过抑菌试验对比测定了虫草素和4个Cn-AAC的最低抑菌浓度(MIC)。在此基础上,用SPSS10.0对MIC和Cn-AAC烷酰基侧链中碳原子数目(n)之间的关系进行了定量分析。结果表明:通过化学结构修饰的手段将虫草素改变成Cn-AAC同系物后,其抗菌活性会发生有规律的变化。Cn-AAC的MIC和n之间的关系可以用一个一元二次方程进行定量描述,其中抗菌活性最强的为C8-AAC;和虫草素相比,C8-AAC对枯草杆菌的MIC降低5倍,并可对虫草素本身不能产生抗菌活性的金黄色葡萄球菌和白色念珠菌产生抗菌活性。     

柞蚕蛹虫草继代培育其活性物质变化规律

柞蚕蛹虫草中含有虫草素、腺苷、多糖等多种活性成分,具有提高免疫力、抗疲劳、保护心脑血管、抗癌等方面的作用,是冬虫夏草的良好替代品。以柞蚕蛹虫草的继代培育为基础,分别检测蛹虫草菌在不同传代次数时柞蚕蛹虫草子实体生长状态、蛹虫草中腺苷、虫草素、虫草多糖含量及蛹虫草菌菌丝、分生孢子中活性氧的分布。第1代蛹虫草菌接种后柞蚕蛹出现腐烂现象;第2、3代培育的子实体生长量、腺苷及虫草素含量均较高;第4代子实体生长量、腺苷及虫草素含量均出现大幅下降的现象。柞蚕蛹虫草中虫草多糖含量在传代过程中也逐渐降低,但降低幅度较腺苷和虫草素缓慢。第2代培育的柞蚕蛹虫草子实体生长状态优于第3代,故第2代蛹虫草菌更适合应用于批量生产。蛹虫草菌退化后含有活性氧的分生孢子比例增大,这可能是发生退化的表象之一。     

不同培养条件和前体对蛹虫草液体发酵产虫草素的影响

蛹虫草能产生虫草素等多种活性物质。为考察不同液体发酵方式及添加前体物质对虫草素积累的影响,选用蛹虫草08Y1菌株,通过光照振荡、光照静置、黑暗振荡、黑暗静置四种培养条件和添加前体物质（腺嘌呤1g/L+甘氨酸16g/L）,发酵16d后检测虫草素和腺嘌呤含量。结果表明：08Y1菌株在黑暗振荡培养条件下,虫草素积累达1,015.0mg/L,腺嘌呤利用率达98.5%,说明黑暗振荡培养并添加前体物质是提高虫草素产量的有效方法。     

虫草素对四氯化碳所致小鼠急性肝损伤的保护作用

本文探究蛹虫草活性成分虫草素对四氯化碳（CCl₄）造成的小鼠急性肝损伤的保护作用及其分子机制。首先建立四氯化碳致小鼠急性肝损伤的动物模型,通过检测血清生化指标、肝功指标的变化及HE染色观察组织切片病理的病变情况,评价虫草素的保肝效果,进一步通过Western blot检测虫草素能否通过激活Nrf-2/Keap1信号通路及其下游抗氧化因子（HO-1、NQO-1）的表达来提高机体抗氧化损伤能力以及抑制炎症因子（TNFα、TNFβ、IL-6、IL-10）的表达。对比模型组结果显示,虫草素能极显著降低（P<0.01）小鼠血清中ALT、AST及肝脏中MDA水平,并能极显著提高肝脏中SOD水平（P<0.01）;HE染色结果显示虫草素能有效降低改善受损肝组织中的炎细胞浸润及纤维组织增生;Western blot结果表明虫草素能够通过激活Nrf-2信号通路,促进下游抗氧化因子及抗炎因子的表达,从而降低炎症反应。虫草素对CCl₄致小鼠急性肝损伤具有一定的保护作用,其机制与Nrf-2信号通路相关,实验结果为后续蛹虫草及虫草素的开发应用奠定基础。     

生物磁效应对蛹虫草活性物质含量的影响

利用场强为0.1、0.25、0.4T恒定磁场,以流速为1 m·s^-1,分别对普通水进行9次处理,串联（SC）磁场处理3次的磁处理水,进而用于栽培蛹虫草,并研究了生物磁效应对蛹虫草虫草素、虫草酸、多糖含量及生物学转化效率的影响。结果表明,0.4T处理有利于蛹虫草虫草酸含量及生物学效率的提高,相比对照组分别提高了7.29%和15.37%;0.25T处理组有利于虫草素的积累,比对照组提高了11.31%;0.1T处理有利于多糖的积累,相比对照组提高了14.80%。方差分析表明,经不同场强处理的磁化水对蛹虫草子实体生长和活性物的代谢影响差异显著。但磁处理水对于不同物质含量的影响规律存在一定的差异性。     

蛹虫草培养液成分研究Ⅱ

目的：研究蛹虫草人工培养液的化学成分。方法：利用硅胶柱层析、制备薄层、SephadexLH-20、重结晶等方法分离、纯化单体化合物,并根据理化性质及光谱数据鉴定其化学结构。结果：分离并鉴定了8个化合物,分别为3,7,8-三甲基-异咯嗪（1）;琥珀酸（2）;胸腺嘧啶（3）;虫草素（4）;腺苷（5）;2吲哚乙丁二酰胺（6）;1（-5-羟甲基）-呋喃-3-羧基-β-咔啉（7）;5（-1-甲基丙基）-3,6-氧代-2-哌嗪乙酰胺（8）。结论：化合物1,6,7为首次从虫草属真菌中分离得到。     

虫草素产量不同的蛹虫草菌株代谢组差异

为探究虫草素高产和低产的两株蛹虫草菌代谢差异及其与虫草素代谢的关联性,本研究采用UPLC-QTOF-MS广泛靶向代谢组学技术和多元统计分析方法,结合相同蛹虫草菌不同发酵时间的菌丝体差异代谢物相对含量的变化情况,比较了两株蛹虫草菌相同发酵时间的菌丝体代谢差异。结果表明：蛹虫草CICC 14014菌株发酵第20和10天的菌粉（H20和H10）及其发酵液的虫草素含量都显著高于蛹虫草CGMCC 3.4655菌株（其菌粉为L20和L10）（P<0.01）。主成分模型显示H10、L10、H20和L20间分散明显;通过正交偏最小二乘法分析,以t检验P<0.05并且VIP>1为标准,从H10 vs L10和H20 vs L20分别识别出190和158种差异代谢物,其中具有生物活性的亚精胺为首次在蛹虫草菌中被检出;以P<0.01为标准,从H10 vs L10、H20 vs L20中筛选出2条显著富集的通路,分别为烟酸-烟酰胺和精氨酸-脯氨酸代谢通路。对H10 vs L10和H20 vs L20进行代谢调控网络分析,获得7条显著互作的通路,相同的互作通路为碱基切除修复。代谢网络分析结果表明抑制三羧酸循环、核黄素代谢、赖氨酸降解、鞘脂类代谢通路以及促进碱基切除修复、β-丙氨酸代谢、精氨酸-脯氨酸代谢、脂质代谢、嘌呤代谢通路有利于虫草素的生成;虫草素高产蛹虫草菌甘油三酯代谢和合成腺苷的能力高于虫草素低产蛹虫草菌;在嘌呤代谢途径中,腺嘌呤通过黄嘌呤代谢反馈促进虫草素的生物合成。本研究为蛹虫草菌生物活性成分的挖掘提供理论基础,为深入解析蛹虫草菌虫草素的代谢机制提供参考。     

人工蛹虫草不同菌株菌丝形态与其虫草素含量关系

对4个不同来源人工蛹虫草菌株进行平板试验,在培养条件相同情况下,观察比较其生长情况;对相同菌龄不同菌株菌丝体所含虫草素进行超声波提取。结果表明：峰面积与虫草素含量呈良好的线性关系,精密度高,其中菌株YJ11 3菌丝体虫草素含量最高,为0.505%,其次为菌株YJ11 1、YJ11 4,最低的是YJ11 2,仅0.097%;菌丝湿重依次为YJ11 3〉YJ11 1〉YJ11 4〉YJ11 2;不同蛹虫草菌株菌丝湿重与其虫草素含量呈正相关关系。     

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in medicinal fungus Cordyceps militaris

Cordyceps militaris is an insect-born fungus with various biological and pharmacological activities. The mutant library of C. militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation (ATMT), for the ultimate identification of genes involved in isolate degeneration during fruiting body production. Successful transformation of C.?militaris JM4 by A. tumefaciens AGL-1 carrying vector pATMT1 was performed, with efficiency in the range of 30-600 transformants per 1×10(5) conidia. Acetosyringone (AS) supplement in C. militaris ATMT was not necessary during either precultivation or cocultivation. The transformation procedure was optimised based on the ratios between donor A. tumefaciens and recipient conidia, and pH value of cocultivation media. The integration of the hyg gene into C. militaris genome was determined by PCR and Southern blot analysis, suggesting that 67-88% resulting transformants in cultivation conditions with or without AS were inserted by T-DNA and 55-80% were single-copy. Special mutants with altered phenotypes and growth potentials were characterised. The efficient TAIL-PCR approach was established for identifying T-DNA flanking sequences from C. militaris mutants. The successful construction of the mutant library indicated the usefulness of this approach for functional genetic analysis in this important fungus.     

Molecular cloning, expression, and characterization of the Cu,Zn superoxide dismutase (SOD1) gene from the entomopathogenic fungus Cordyceps militaris

We describe the molecular characterization of the Cu,Zn superoxide dismutase (SOD1) gene of Cordyceps militaris, which is one of the entomopathogenic fungi called a vegetable wasp and plant worm. The SOD1 gene of C. militaris spans 922 bp and consisted of three introns and four exons coding for 154 amino acid residues. The deduced amino acid sequence of the C. militaris SOD1 cDNA showed 88% identity to Claviceps purpurea SOD1, 82% to Neurospora crassa SOD1, and 75-64% to SOD1 sequences from other fungi. The C. militaris SOD1 possesses the typical metal binding ligands of six histidines and one aspartic acid common to fungal SOD1s. The cDNA encoding C. militaris SOD1 was expressed as a 17-kDa polypeptide in the baculovirus-infected insect Sf9 cells. The enzyme activity of the purified recombinant C. militaris SOD1 was approximately 568 U per mg(-1) . Southern blot analysis of the genomic DNA suggested the C. militaris SOD1 was a single gene. Northern and Western blot analysis and enzyme activity assays indicated SOD1 was expressed constitutively. This is the first report of an SOD1 gene from any entomopathogenic fungus.     

Biosynthesis of 3'-deoxyadenosine by Cordyceps militaris. Mechanism of reduction.

The biosynthesis of 3'-deoxyadenosine (cordycepin) by Cordyceps militaris has been investigated using [U-14C]adenosine and [3-3H]ribose. Crystallization of the resulting radioactive 3'-deoxyadenosine to a constant specific activity showed incorporation of both labeled compounds. A control showed that the 3H:14C ratio of the AMP isolated from the RNA was the same as the 3H:14C ratio in the 3'-deoxyadenosine. The 14C ratio in the adenine: ribose of the [U-14C]adenosine added to the 3'-deoxyadenosine producing cultures of C. militaris and of the isolated 3'-deoxyadenosine was the same, e.g. 50:50. These data provide strong evidence that adenosine in converted to 3'-deoxyadenosine without hydrolysis of the N-riboside bond. Degradation of the 3-deoxyribose from 3'-deoxyadenosine showed that the 3H was retained on carbon-3. These results suggest that the formation of 3'-deoxyadenosine may proceed by a reductive mechanism similar to that for the formation of 2'-deoxynucleotides.     

滇牡丹内生真菌Alternaria sp.PR-14的代谢产物研究

从滇牡丹内生真菌交链孢霉属(Alternaria sp.)菌株PR-14的发酵液提取物中分离得到6个化合物,通过波谱技术分别鉴定为交链孢酚(1)、djalonensone(2)、脑苷酯B(3)、脑苷酯C(4)、(2S,3S,4R,2’R)-2-(2’-hydroxytetracosanoylamino)octadecane-1,3,4-triol(5)和甘露糖醇(6),以上化合物均为首次从滇牡丹内生真菌中分离得到。     

Chemistry and weak antimicrobial activities of phomopsins produced by mangrove endophytic fungus Phomopsis sp. ZSU-H76

Three metabolites named phomopsin A (1), B (2) and C (3), together with two known compounds cytosporone B (4) and C (5), were isolated from the mangrove endophytic fungus, Phomopsis sp. ZSU-H76 obtained from the South China Sea. Their structures were elucidated by spectroscopic methods, mainly by 1D and 2D NMR spectroscopic techniques. The medium-sized cyclic phenol ether based on 1 or 2 is rare in natural products. In bioassays, compounds 1, 2, and 3 had no significant antibiotic activities, but compounds 4 and 5 inhibited two fungi Candida albicans and Fusarium oxysporum with an MIC ranging from 32 to 64 microg/ml.     

蛹虫草退化菌株的特征研究

蛹虫草作为虫草资源和健康食品之一业已产业化人工栽培。但是,蛹虫草菌种在继代培养过程中的频繁退化不仅导致其产量的急剧下降,而且成为影响行业发展的核心技术问题。作者研究了蛹虫草退化菌株与正常菌株在交配型、dsRNA感染、色度和脱氢酶活性的特征差异性。结果表明,退化菌株的脱氢酶活性和在色度培养基中的脱色能力都明显弱于正常菌株;而在交配型和dsRNA感染方面,两者未表现出差异。这一结果为蛹虫草人工栽培过程中退化菌种的早期检测提供了快速的方法。     

蛹虫草金属硫蛋白分离纯化及性质研究

研究探讨锌离子胁迫下蛹虫草Cordyceps militaris金属硫蛋白的产生及性质。蛹虫草菌丝体以15g/L Zn2+在10L发酵罐中诱导培养56h后收集,产率为每升发酵液收集12.021g菌丝体（干重）,细胞破碎取上清液通过两次凝胶柱层析,冷冻干燥得到蛹虫草金属硫蛋白纯品。利用Bradford法进行蛋白质含量测定,用银饱和分析法结合原子吸收光谱（AAS）测定MT含量,发酵终点处金属硫蛋白含量为12.876mg/g菌丝体（湿重）。用电喷雾质谱法测得金属硫蛋白的分子量为7 390Da,用Ellman’s方法和火焰原子吸收法分别测得每分子蛋白质含有14个巯基、结合5个Zn原子。氨基酸组成分析结果显示,每分子蛋白质共含57个氨基酸,其中含有13个半胱氨酸,疏水氨基酸占29.8%,且含有组氨酸。以上表明,研究中的蛹虫草金属硫蛋白与哺乳动物金属硫蛋白结构差异较大,但与酵母菌金属硫蛋白结构组成类似。     

Highly oxygenated and unsaturated metabolites providing a diversity of hispidin class antioxidants in the medicinal mushrooms Inonotus and Phellinus

Three new highly oxygenated and unsaturated metabolites named interfungins A (1), B (2), and C (3), which provide a diversity of hispidin class compounds in the fungi Inonotus and Phellinus, were isolated from the methanolic extract of the fruiting body of the fungus Inonotus xeranticus (Hymenochaetaceae). Their structures were established by spectroscopic methods. The existence of these functionalized metabolites implies that inoscavin A, davallialactone, and phelligridin F, which were previously isolated from the fungi Inonotus and Phellinus spp., are derived from 1. Compound 1 is derived from the condensation of hispidin and hispolon. Inoscavins B and C previously isolated from the fungus I. xeranticus are most probably derived from 2 which stemmed from the oxidative coupling of 3,4-dihydroxybenzalacetone and hispidin. This class of compounds exhibited significant free radical scavenging activity against the superoxide radical cation, ABTS radical anion, and DPPH radical.