海洋真菌产抗菌物质培养基及其性质研究

目的:海洋真菌Fy-04产抗菌物质培养基及性质研究.方法:管碟法测定抑菌效果确定海洋真菌Fy-04产抗菌物质的培养基及其性质.结果:海洋真菌Fy-04产抗菌物质对金黄色葡萄球菌抑菌最佳培养基组成为:马铃薯海水浸出液60%,葡萄糖2%,蛋白胨1%,酵母膏0.1%,抑菌圈直径34.6mm;对大肠杆菌抑菌最佳培养基组成为:葡萄糖2%,蛋白胨0.7%,酵母膏0.1%,FePO4 0.04%,抑菌圈直径21.6mm;抗菌物质80℃处理1h,残留活性60%以上,pH稳定范围在pH 6.0～10.0.结论:该菌株代谢产物对细菌有较好的抑菌效果,并能溶于乙醚、三氯甲烷中等极性的有机溶剂中.     

Leptographiumbhutanense提取物抑菌活性的初步分析

内生真菌是动植物体内的微生物类群,其多样性、复杂性和特殊性的特点使其能够产生化学结构新颖、具有多种生物活性的次生代谢产物。本研究采用纸片法检测Leptographium bhutanense发酵提取物对13种致病细菌的抑菌活性,并对抗菌化合物的最低抑菌浓度(MIC)、热稳定性、光稳定性进行检测;同时,采用OSMAC策略寻找产抗菌化合物的最佳培养基、最佳培养时间和最佳接种量。分析显示提取物对3种人体致病细菌(蜡样芽孢杆菌,副溶血性弧菌,溶血性葡萄球菌)具有抑菌活性;其MIC值分别为:12.5mg/m L、6.25 mg/m L和6.25 mg/m L;提取物具有较好的热稳定性和光稳定性。产抗菌化合物的最佳培养基为:MFS,最佳培养时间和最佳接种量分别为15 d和1.0 g/100 m L。本研究为昆虫内生真菌抗菌化合物的开发利用提供帮助。     

蒜头果内生真菌次生代谢产物抑制人类致病菌活性的研究

蒜头果是我国特有的单种属稀有树种,为了进一步开发利用蒜头果树皮内生真菌的抗菌活性化合物,该研究对来自蒜头果的植物内生真菌(白黄笋顶孢霉、哈茨木霉、大棘黑团孢、枝状枝孢菌、斑污拟盘多毛孢、赭绿青霉、淡紫紫孢菌、朱黄青霉、Xenoacremonium recifei、Xylaria feejeensis)进行液体培养,10 d后回收培养液并用乙酸乙酯萃取获得初提物,采用抑菌圈法检测蒜头果内生真菌初提物抑菌活性,同时测定了最低抑菌浓度(MIC)。结果表明:白黄笋顶孢霉、大棘黑团孢、枝状枝孢菌、斑污拟盘多毛孢、赭绿青霉、淡紫紫孢菌均有抑菌活性,大棘黑团孢、斑污拟盘多毛孢、淡紫紫孢菌的初提物均对缓慢芽孢杆菌、无乳链球菌和藤黄微球菌有明显抗菌活性,最低抑菌浓度在1.562 5~6.25 mg·m L~(-1)之间。这说明蒜头果树皮内生真菌的次生代谢产物具有抗菌活性,各内生真菌次生代谢产物的抗菌效果不同。     

杜仲内生真菌DZ05的鉴定及抗菌生物活性研究

结合菌株的形态学特征和ITS序列分析结果,对1株分离自杜仲茎部的内生真菌菌株DZ05进行鉴定,并对其在PDA液体培养基摇床培养3 d获得的发酵液对多种测试菌进行抗菌活性研究。结果显示:(1)分离自杜仲茎部的内生真菌菌株DZ05经形态学特征和ITS序列分析,被鉴定为淡紫色拟青霉。(2)其发酵液的乙酸乙酯提取物对6种测试细菌和9种测试植物病原菌均具有明显的抑菌活性,抑菌圈直径在13～45 mm之间,其中对番茄灰霉病菌、番茄叶霉病菌和苹果炭疽病菌抑菌圈直径＞40 mm。研究表明,杜仲内生真菌DZ05的代谢产物具有广谱的抗菌活性,在植物病原菌的生物防治领域具有较大的应用前景。     

牛樟芝发酵液提取物抗菌活性研究

牛樟芝作为一种珍稀食用和药用菌,具有极大的开发潜力。该研究以麦芽浸粉肉汤液体培养基(BD,美国BD公司)对牛樟芝菌丝体进行摇床培养60 d后,收获发酵液并用乙酸乙酯对其进行萃取,浓缩至干获得提取物;同时,采用抑菌圈法评价培养物对13种致病细菌抗菌活性(蜡样芽孢杆菌、缓慢芽孢杆菌、无乳链球菌、短小芽孢杆菌、福氏志贺氏菌、枯草芽孢杆菌、金黄色葡萄球菌、藤黄微球菌、副溶血性弧菌、溶血性葡萄球菌、铜尿假单胞菌、乙型副伤寒沙门氏菌、大肠埃希菌),并检测相应致病细菌的最低抑制浓度(MIC)。结果表明:牛樟芝麦芽浸粉肉汤发酵液提取物对供试的13种致病菌均有抑菌活性;在供试的13种致病菌中,提取物对缓慢芽孢杆菌、短小芽孢杆菌、枯草芽孢杆菌、副溶血性弧菌、藤黄微球菌5种致病菌的最低抑制浓度值均小于80μg·m L~(-1),其中对藤黄微球菌的最低抑制浓度最低为66.5μg·m~(-1);随着培养时间的增加,提取物的抗菌活性也增加。这说明牛樟芝菌丝体在液体培养条件下,能够产生广谱高效抑菌活性的次生代谢产物。该研究结果为牛樟芝进一步的有效利用开发奠定了理论基础。     

云南省4种蕨类植物提取液的抑菌活性

为了合理开发利用蕨类植物资源,本研究利用圆纸片法分析了云南省4种蕨类植物单芽狗脊蕨(Woodwardia unigemmata)、蜈蚣蕨(Pteris vittata)、鸡足山耳蕨(Polystichum jizhushanense)和灰绿耳蕨(Polystic-hum eximium)的提取液对金黄色葡萄球菌(Staphylococus aureus)、大肠杆菌(Escherichia coli)和枯草芽孢杆菌(Bacillus subtilis)的抑菌活性。抑菌试验结果表明,当4种蕨类植物提取液浓度分别为10%、8%、6%和4%时,对照组无抑菌圈出现,单芽狗脊蕨提取液对大肠杆菌和金黄色葡萄球菌无抑菌活性,灰绿耳蕨提取液对金黄色葡萄球菌无抑菌活性,其它的蕨类植物提取液对供试微生物都表现出不同程度的抑菌活性;并且这4种蕨类植物提取液至少对一种供试细菌具有抑菌活性,抑菌活性范围为8～14mm,平均抑菌圈直径为11.2mm,4种提取液对枯草芽孢杆菌抗菌活性效果最明显。由此,我们推测本研究中的4种蕨类植物均有抗菌物质存在,并将有望成为筛选抗菌新型药物的种质资源。     

越南槐内生真菌JXRPH-24的抗菌活性及其鉴定

以2株病原细菌金黄色葡萄球菌ATCC25923和耐甲氧西林金黄色葡萄球菌临床分离株为靶标菌,采用琼脂块法和改良的微量肉汤稀释法测定越南槐内生真菌JXRPH-24的抗菌活性;根据形态和分子特征鉴定该菌株的分类学地位。结果表明,该菌株的活菌块对金黄色葡萄球菌ATCC25923的抑菌圈直径与阳性对照相当;其代谢产物对这2株病原细菌的最小抑菌浓度均为10μg/mL,仅为阳性对照的16倍;基于形态和ITS序列分析未能鉴定该菌株。菌株JXRPH-24可能是一个潜在的新内生真菌,对参试金黄色葡萄球菌均具有强的抗菌活性,具有较大的开发应用潜力。     

多脂长喙壳菌次生代谢产物的抑菌活性研究

植物病原真菌是生态系统的重要组成部分,能够代谢产生多种生物活性物质。用纸片法检测多脂长喙壳菌(Ceratocystis adiposa)发酵提取物对13种致病细菌的抑菌活性,并对潜在抗菌活性化合物的热、酸碱和光稳定性进行检测,同时应用OSMAC策略寻找多脂长喙壳菌产抗菌化合物的最佳培养条件。结果表明:多脂长喙壳菌提取物对革兰阳性菌——蜡样芽胞杆菌(Bacillus cereus)、缓慢芽胞杆菌(Bacillus lentus)、藤黄微球菌(Micrococcus luteus)具有抗菌活性,MIC分别为6.25、3.125和1.562 5 mg/m L,抗菌化合物具有较好的耐热、耐酸碱和耐辐射性,培养基种类、培养时间和接种量会影响多脂长喙壳菌抗菌化合物的产生,为多脂长喙壳菌抗菌化合物的开发利用提供参考。     

濒危植物七子花内生真菌的分离鉴定及抗菌活性筛选

首先从濒危植物七子花中分离到38株内生真菌,综合运用形态特征和分子生物学技术,明确其分类地位。38株植物内生真菌分属于6个属和1个无孢子群,其中交链孢属和刺盘孢属为优势菌群,葡萄座腔菌属、镰刀菌属、赤霉菌属、拟茎点霉属和无孢子群为常见属。以大肠埃希菌、白色念珠菌、金黄色葡萄球菌为靶标菌对获得菌株发酵液粗提物进行抑菌试验,发现95%以上的菌株对试测细菌具有一定的抗菌活性,表明七子花内生真菌的抗菌活性十分普遍;其中QZHⅡ10对大肠埃希菌具有较好的抑菌活性,抑菌圈直径为12.0 mm;而QZHⅡ07则对金黄色葡萄球菌的抑菌活性较强,抑菌圈达到15.5 mm。这表明七子花内生真菌具有开发为微生物源杀菌剂的价值。     

二氢呋喃喹啉酮类化合物的抑菌效果

为了解二氢呋喃喹啉酮类化合物对四种细菌的抑制作用,选用大肠杆菌,金色葡萄球菌,绿脓杆菌,枯草杆菌作为测试菌;选用牛肉膏蛋白胨作为培养基;以化学合成的二氢呋喃喹啉酮化合物a～f为测试对象,采用抑菌圈试验测定样品抗菌活性,并根据所得抑菌率求得MIC50值。结果表明:对于大肠杆菌,金色葡萄球菌,枯草杆菌,六个化合物的抑菌效果均一般。但是化合物f(含甲氧基)对于绿脓杆菌的抑菌效果尤其出色,远高于对大肠杆菌、金葡杆菌、枯草杆菌的抑菌效果,具有明显的选择性。     

Antioxidant and antimicrobial activity of some lichen species

The aim of the research is to explore the overall in vitro antioxidant activity, total phenol content, reduction power and antimicrobial activity of the methanol extracts of the lichens Cetraria pinastri, Cladonia digitata, Cladonia fimbriata, Fulgensia fulgens, Ochrolechia parella and Parmelia crinita. The methanol extract of the Cetraria pinastri showed a strong antioxidant activity, whereas the extracts of the species Fulgenesi fulgens, Cladonia fimbriata and Parmelia crinita showed the moderate one and the extract of the species Ochrolechia parella and Cladonia digitata the weak one. The methanol extract of the lichen Cetraria pinastri had the biggest total phenol content (32.9 mg/g of the dry extract). A certain correlation was established between the antioxidant activity and the total phenol content for the researched lichen extracts. The work also explores the antimicrobial activity of the methanol extracts of the mentioned species of lichens against six bacterial and eleven fungi species by the disc-diffusion method and by establishing the minimum inhibitory concentration (MIC). The methanol extracts of the lichens Cetraria pinastri and Parmelia crinita showed the strongest both antibacterial and antifungal activity against most of the tested microorganisms. These researches suggest that the lichens Cetraria prunastri can be used as new sources of the natural antioxidants and the substances with antimicrobial features.     

Host use of a specialist lichen-feeder: dealing with lichen secondary metabolites

Host use by herbivores is largely determined by host properties such as nutrient content and chemical defence against foragers. The impacts of these attributes on a herbivore may largely depend on its life cycle stage. Lichen species are known to differ in nutritional quality and level of chemical defence and, consequently, vary as fodder for herbivores. The aim of this study was to explore the impact of several lichen species and the presence of their secondary metabolites on their use as hosts by a specialist lichen-feeder, Cleorodes lichenaria. This study also addressed, for the first time, how a specialist lichen-feeder deals with different lichen secondary metabolites. In the beginning of their development, larvae grew better on Xanthoria parietina than on the other host lichens, whereas older larvae grew best on Ramalina fraxinea. Lichen secondary chemicals in R. fraxinea and Parmelia sulcata hindered larval growth in the beginning but after 75 days lichen secondary chemicals had no impact on the mass of larvae. Physodic acids in Hypogymnia physodes were lethal to larvae. In general, larvae metabolized 70–95% of ingested lichen secondary chemicals and the rest of these were excreted in frass. Lichen secondary metabolites in P. sulcata restrict and in H. physodes prevent their use as a host for C. lichenaria larvae. Our main finding, the ability of larvae to metabolize several lichen secondary metabolites, indicates digestive adaptation to these chemicals. No signs of sequestration of these chemicals were found.     

Screening of antioxidant potential of Arctic lichens

Antioxidants are compounds that scavenge the free radicals produced in living organisms. The antioxidant potential of eight Arctic lichen species was evaluated in vitro using free radical scavenging activity (FRS), inhibition of lipid peroxidation (ILP), and Trolox equivalent antioxidant capacity assay (TEAC). FRS activities of lichen species in various organic solvents such as methanol, ethanol, acetone, and dimethyl sulphoxide (DMSO) were in the range 9.6–51.77%, while ILP activities in these solvents ranged from 32.5 to 82.43%. Pseudophebe pubescens showed the highest ILP (82.43%) and FRS (51.77%) activities as compared to other lichen species and the standard antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). The TEAC value was also found to be higher in all species compared to the standard water soluble vitamin E analog Trolox (3.9 mM). The order of antioxidative activities in lichen species was Pseudophebe pubescens > Cladonia amaurocraea > Cladonia mediterranea > Physcia caesia > Flavocetraria nivalis > Cetraria fastigata > Xanthoria elegans > Umbilicaria hyperborea. This is the first report of the measurement of antioxidant potential in Arctic lichens.     

UHPLC‐HRMS/MS Based Profiling of Algerian Lichens and Their Antimicrobial Activities

Lichens are complex symbiotic organisms able to produce a vast array of compounds. The Algerian lichen diversity has only prompted little interest even given the 1085 species listed. Herein, the chemodiversity of four Algerian lichens including Cladonia rangiformis, Ramalina farinaceae, R. fastigiata, and Roccella phycopsis was investigated. A dereplication strategy, using ultra high performance liquid chromatography‐high resolution‐electrospray ionization‐mass spectrometry (UHPLC‐HRMS/MS), was carried out for a comprehensive characterization of their substances including phenolics, depsides, depsidones, depsones, dibenzofurans, and aliphatic acids. Some known compounds were identified for the first time in some species. Additionally, the lichenic extracts were evaluated for their antifungal and antimicrobial activities on human pathogenic strains (Candida albicans, C. glabrata, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli). Cyclohexane extracts were found particularly active against human pathogenic fungi with MIC₈₀ values ranging from 8 to 62.5 μg/mL, without cytotoxicity. This study highlights the therapeutic and prophylactic potential of lichenic extracts as antibacterial and antifungal agents.     

The phenolic compounds in <Emphasis Type="Italic">Cladonia</Emphasis> lichens are not antimicrobial in soils

According to classic text books on lichen biology, the phenolic secondary chemicals in lichens have antibiotic effects on soil microorganisms and mycorrhizal fungi in ecosystems. However, the experimental evidence for this under natural conditions is still relatively scarce. We examined some of the assumptions behind the concept of antimicrobial effects of lichen secondary substances: (1) the secondary substances of Cladonia stellaris, usnic and perlatolic acids, are leached out from the lichens by rainwater; (2) these substances inhibit the microbial activity of soil, and; (3) since they are extremely resistant to microbial decomposition, the soil underneath a continuous lichen mat is enriched in usnic and perlatolic acids. Our results did not support any of these assumptions. The evidence for the antimicrobial activity of lichen secondary substances seems to be weak in comparison to other suggested functions such as light filtering and herbivore protection. We suggest that it is time to re-evaluate the evidence for the antimicrobial ecological role of lichen secondary substances in natural systems.     

Lichen secondary metabolites affect growth of <Emphasis Type="Italic">Physcomitrella patens</Emphasis> by allelopathy

Lichen secondary metabolites can function as allelochemicals and affect the development and growth of neighboring bryophytes, fungi, vascular plants, microorganisms, and even other lichens. Lichen overgrowth on bryophytes is frequently observed in nature even though mosses grow faster than lichens, but there is still little information on the interactions between lichens and bryophytes.In the present study, we used extracts from six lichen thalli containing secondary metabolites like usnic acid, protocetraric acid, atranorin, lecanoric acid, nortistic acid, and thamnolic acid. To observe the influence of these metabolites on bryophytes, the moss Physcomitrella patens was cultivated for 5 weeks under laboratory conditions and treated with lichen extracts. Toxicity of natural mixtures of secondary metabolites was tested at three selected doses (0.001, 0.01, and 0.1 %). When the mixture contained substantial amounts of usnic acid, we observed growth inhibition of protonemata and reduced development of gametophores. Significant differences in cell lengths and widths were also noticed. Furthermore, usnic acid had a strong effect on cell division in protonemata suggesting a strong impact on the early stages of bryophyte development by allelochemicals contained in the lichen secondary metabolites.Biological activities of lichen secondary metabolites were confirmed in several studies such as antiviral, antibacterial, antitumor, antiherbivore, antioxidant, antipyretic, and analgetic action or photoprotection. This work aimed to expand the knowledge on allelopathic effects on bryophyte growth.     

Induction of a complete secondary-product pathway in a cultured lichen fungus

Experimental studies on the secondary metabolism characteristic of lichens have been impeded by the slow growth of the fungi and by the inconsistent results of many attempts to induce the pathways in the fungi isolated from their photosynthetic partners. In the present study, a lichen-specific secondary pathway was consistently induced in a lichen fungus (Cladonia grayi) grown in the absence of the alga. The depside (4-O-demethylsphaerophorin) and two depsidones (grayanic and 4-O-demethylgrayanic acids) found in the natural lichen began to accumulate a few days after the transfer of lightly fragmented mycelia from liquid to solid medium. Induction was enhanced on drier substrates and was correlated with the proliferation of aerial hyphae, where the major product (grayanic acid) accumulated in extracellular patches visible by fluorescence microscopy. The time course was analyzed by quantitative high-performance liquid chromatography of extracts from small cultures grown on nylon filters. Induction was rapid in view of the slow growth of the fungus, and secondary productivity was comparable to that of some nonlichen fungi. These results confirm that the alga is not needed for catalysis in lichen depside and depsidone biosynthesis and suggest instead that the characteristic secondary metabolism of natural lichen is linked to their aerial habit of growth.     

Cytotoxic activity of some lichen extracts on murine and human cancer cell lines

Eight lichens were extracted successively with n-hexane, diethyl ether and methanol using a Soxhlet process. The cytotoxic activity of the 24 lichen extracts was evaluated in vitro using two murine (the L1210: lymphocytic leukaemia, and the 3LL: Lewis lung carcinoma) and four human (the K-562: chronic myelogenous leukaemia, the U251: glioblastoma, the DU145: prostate carcinoma, and the MCF7: breast adenocarcinoma) cancer cell lines and non-cancerous cells, the Vero cell line (African green monkey kidney cell line). The MTT assay revealed significant cytotoxicity (IC50 < or = 20 microg/ml) on one of the tested cancer cell lines for at least one extract of each lichen species. Some extracts of Cladonia convoluta, Cladonia rangiformis, Parmelia caperata, Platismatia glauca and Ramalina cuspidata demonstrated interesting activities particularly on human cancer cell lines as good selectivity indices were recorded (SI > 3).     

Antimicrobial activity of extracts of the lichens <Emphasis Type="Italic">Cladonia furcata,Parmelia caperata,Parmelia pertusa,Hypogymnia physodes</Emphasis> and <Emphasis Type="Italic">Umbilicaria polyphylla</Emphasis>

Antimicrobial features of acetone, methanol and aqueous extracts of lichens of Cladonia furcata, Parmelia caperata, Parmelia pertusa, Hypogymnia physodes and Umbilicaria polyphylla were investigated by two different methods at the same time. Testing of antimicrobial activities of extracts from five species of lichens was performed by disc diffusion test in relation to Gram-positive and Gram-negative bacteria and fungal organisms, and through determination of minimal inhibitory concentration (MIC) by Broth Tube Dilution method. The obtained results indicated that acetone and methanol extracts of all investigated lichens in different concentrations manifested selective antibacterial and antifungal activity. That activity was more evident in relation to Gram-positive, than Gram-negative bacteria and fungal organisms. Acetone and methanol extracts of lichens Parmelia pertusa, Hypogymnia physodes and Umbilicaria polyphylla inhibited the growth of all tested microorganisms, most of all of lichens Cladonia furcata and Parmelia caperata. Although, the methanol extracts were generally the most active against the test organisms, the lowest MIC value was measured for acetone extract of species Cladonia furcata 0.39 mg/mL in relation to bacterium Bacillus subtilis. Aqueous extracts of investigated lichens were inactive against all tested organisms.     

Studies on nutritional requirement for the culture of lichen <Emphasis Type="Italic">Ramalina nervulosa</Emphasis> and <Emphasis Type="Italic">Ramalina pacifica</Emphasis> to enhance the production of antioxidant metabolites

In the present report, nutritional requirement for the culture of two lichen species Ramalina nervulosa and Ramalina pacifica were studied in order to enhance their growth rate and antioxidant metabolite production. Extract of R. nervulosa cultured in Bold’s basal medium (BBM) showed higher antioxidant activity than R. pacifica cultured in Murashige and Skoog (MS) medium. The lichen species were sub-cultured in standardized nutrient media. R. nervulosa in BBM (1% glucose, 50 ppb asparagines, pH 6.5) yielded 2.76 g biomass with 26.18 mg sekikaic acid, 24.32 mg usnic acid/g dry biomass in a period of 60 days. R. pacifica in MS media (3% sucrose, 100 ppb thiamine, pH 5.9) yielded 3.54 g biomass and 58.92 mg salazinic acid, 40.16 mg usnic acid in the same time period. The standardized culture conditions implemented on bioreactor, R. nervulosa yielded 17.7 g biomass with the production of sekikaic acid 122.8 mg, usnic acid 75.4 mg in 4.5 days. R. pacifica produced 10.3 g biomass along with salazinic acid 200 mg and usnic acid 136.8 mg in the same duration. Lichen secondary metabolites produced in bioreactor showed moderate antioxidant activity; sekikaic acid 42% to 56.4%; salazinic acid 33.6% to 41.9% and usnic acid 19.9% to 29.5%.