水果中抗病原真菌物质成分的提取分离方法及应用前景

诱发因子如草酸、拮抗菌产生的抗菌素等,以及采后热处理、紫外线处理可以诱导水果产生抗病性,此外有的水果具有先天性抗病成分。对这些活性成分进行分析,并用之于水果采后病害的生物防治,为水果的防腐保鲜提供了新的方法。追踪这些抗病物质的产生和分布,需要采用一些专门的分析方法。目前采用的方法是将果实组织破碎,离心,通过萃取、层析或电泳技术进行纯化,然后用核磁共振、X_射线衍射及各种波谱分析技术进行结构鉴定。抗真菌物质的活性可以用薄层层析生物学方法、孢子萌发法或纸碟法进行鉴定。而抗真菌活性物质的定位则用组织化学法。电子显微术、放射自显影技术和免疫印迹技术也广泛用于组织化 学分析。     

海洋分离芽胞杆菌抗白念珠菌活性物质的理化性质及类别

为寻找新型抗真菌活性物质,采用管碟法对7株分离自海洋的芽胞杆菌在不同Na Cl浓度下产生抗白念珠菌活性物质特性、活性物质的耐热性及不同p H值条件下的活性进行了比较,八大溶剂系统纸层析法对活性物质的类别进行了初步鉴定。结果表明,随着Na Cl浓度的变化产生活性物质的量也在变化,Na Cl浓度达7%时均不能产生,但在正常海洋环境盐浓度(Na Cl含量2%~3%)下都产生;活性物质有很强的耐热性和耐酸碱性,说明其较稳定;7株菌产生的抗白念珠菌活性物质均为碱性水溶性抗生素。由于目前临床上抑制人体病原真菌活性物质绝大多数为脂溶性,因而这些芽胞杆菌产生的抗白念珠活性物质有可能为新型物质,此外本研究结果为这些菌株所产生活性物质的分离纯化提供了依据。     

海洋真菌活性代谢产物最新研究进展

海洋微生物因其特殊的生存环境而具有产生新型生物活性物质的巨大潜力.近年来,从海洋真菌中分离鉴定了许多结构新颖的次级代谢产物,这些化合物显示了良好的抗肿瘤、抗细菌或抗真菌等生物活性.本文对近两年来海洋真菌所产生的活性物质的研究进展进行综述,并对其发展进行展望.     

唐菖蒲球茎中抗真菌物质的部分性质研究

本文报道唐菖蒲球茎中抗真菌活性物质的成分及其分子量测定方法和结果。用沸水提取,经过超滤、透析、柱层析,纯化到一种有抗真菌活性的物质。排除了它是蛋白质、核酸、脂类及小分子物质的可能性,确定这是一种多糖,定名为唐菖蒲抗真菌多糖（Gladiolus hntifungal Polysaccharide,简称GAFS）。真空旋转蒸发干燥后,用苯酚-硫酸法测得干燥后的样品糖含量为44．08％,用FPLC（快速蛋白质液相层析仪）测其分子量为15750。     

蝉花抗真菌活性成分的分离纯化研究

通过抗真菌实验进行活性跟踪,采用NKA大孔吸附树脂和硅胶两步柱色谱对蝉花中抗真菌活性成分进行分离纯化;并通过红外、质谱进行结构鉴定。结果表明,分离得到的抗真菌活性成分为多球壳菌素,对真菌的最小抑制浓度为0.02 mg/mL。本研究为进一步研究蝉花及制备医药工业所需的多球壳菌素提供了依据。     

培养调控激活卡伍尔氏链霉菌NA4产frontalamides类物质的研究

微生物(尤其是链霉菌)是农用抗生素的主要来源之一。前期基因筛选发现卡沃尔氏链霉菌NA4具有产生多种PKS和NRPS类型化合物的潜能,但只分离到PKS I型的抗植物病原真菌的物质bafilomycins,为了挖掘更多的抗真菌先导化合物,采用Hiseq2500高通量测序技术获得其基因组扫描图,经生物信息学分析和文献检索发现,其除了能产生bafilomycins,还具有产生polycyclic tetramate macrolactam(PTM)、alkylresorcinol和valinomycin类型抗真菌活性物质的潜力。为了激活这些基因簇,采用基于OSMAC策略中的培养调控为手段,通过改变培养基碳氮源组分,添加氨基酸前体及不同金属离子,使用活性追踪和HPLC-UV发现用麸皮替代NM2培养基中甘油的培养条件能激活PTM类型frontalamides合成基因簇,经LC-MS分析有7个主要的化合物是PTM类化合物,其中的3个可能为新化合物。通过培养调控激活了PTM基因簇,为新型PTM类化合物进一步的分离鉴定提供参考。     

吸水链霉菌BS-112产生的抗真菌活性物质的分离纯化与结构鉴别

【目的】分离纯化吸水链霉菌(Streptomyces hygroscopicus)BS-112产生的抗真菌活性物质,究明各活性组分的结构,测定其对黄曲霉的抑制作用,为该菌株及其产生的抗真菌活性物质的应用提供依据。【方法】通过大孔吸附树脂柱层析、硅胶柱层析及制备HPLC等方法,对该菌株产生的抗真菌活性物质进行分离纯化;利用质谱(MS)和核磁共振谱(NMR)解析各活性组分的结构;采用微量液体稀释法测定各活性组分对黄曲霉的最小抑菌浓度(MIC)和最小杀菌浓度(MFC)。【结果】从BS-112菌株发酵液中分离获得4个抗真菌活性组分,利用波谱技术确定其结构分别为Tetrins A和B、Tetramycins A和B。96孔板法测得这4个化合物对黄曲霉的MIC分别为3.13μg/mL、12.56μg/mL、1.56μg/mL、6.25μg/mL,MFC分别为6.25μg/mL、25.0μg/mL、3.13μg/mL、12.56μg/mL。【结论】BS-112菌株产生的抗真菌活性物质由Tetrins A和B、Ttramycins A和B 4个化合物组成,它们对黄曲霉均具有良好的抑制作用。     

链霉菌IMS002的分类鉴定及其抗真菌活性物质解析

【目的】对一株分离自植物根际土壤的具有抗真菌活性的链霉菌IMS002进行菌株分类鉴定,通过活性追踪分离纯化并鉴定有机相中的活性物质。【方法】通过16S rDNA和5个不同基因(atpD,gyrB,recA,rpoB,trpB)串联聚类分析以及生理生化实验分析,对链霉菌IMS002进行菌株分类鉴定,用扫描电子显微镜观察该株链霉菌的菌丝及孢子形态,以尖孢镰刀菌(Fusarium oxysporum)为指示菌进行生物活性追踪,通过硅胶柱层析、凝胶柱层析及高压液相色谱(HPLC)对活性物质进行分离和纯化,使用液质联用高分辨质谱仪、500 MHz核磁共振波谱仪以及圆二色光谱仪确定该物质的化学结构。【结果】IMS002经初步鉴定与产二素链霉菌(Streptomycesambofaciens)具有较近的亲缘关系,其发酵液对尖孢镰刀菌具有良好的抑菌效果,经分离和纯化以及现代波谱技术分析,确定有机相中的抑菌活性组分为Borrelidin。【结论】链霉菌IMS002能够产生化合物Borrelidin,该化合物对尖孢镰刀菌具有抑制活性。     

一种被毛孢(Hirsutella sp.)培养液中自由基清除剂的分析、分离和制备

[目的]在一次对虫生真菌代谢物进行大规模的清除自由基活性物质筛选中,发现一种被毛孢(Hirsutella sp.)菌株RCEF0881发酵液中存在有较强的清除自由基活性物质.本研究目的是初步搞清这些活性成分的具体组成,并制备出一定量的纯品用于进一步的结构鉴定.[方法]用有机溶剂法提取活性成分;用二苯基苦基苯肼自由基(DPPH)酶标仪法和薄层色谱法进行活性测定;用高分辨液质联用方法进行活性成分初步分析和鉴定;用反相制备色谱法制备活性组分.[结果]提取实验结果表明具清除自由基活性的物质能较好地被乙酸乙酯提取出来;液相色谱-质谱-活性测定分析表明提取物中活性组分的可能分子式分别为C7H6O4、C8H8O3和C12H14N2O.结合色谱特性、紫外光谱特征、质谱碎片和数据库查询可初步推断它们分别为二羟基苯甲酸、羟基甲基苯甲酸和生物碱类物质,但具体结构还有待于进一步确认.从高效液相色谱和质谱离子流的峰面积可知上述3种活性物质中C12H14N2O的含量最高.本研究成功地用反相制备色谱制备出该天然活性组分的纯品.该3种清除自由基活性物质都是首次发现存在于虫生真菌的代谢物中.     

一种Zalerion arboricola发酵产物的分离、纯化和结构鉴定

抗真菌药物卡泊芬净是以丝状真菌Zalerion arboricola的菌体代谢产物纽莫康定B0为中间体经化学合成得到。该菌在代谢产纽莫康定B0的同时还会产生多种具有相似结构的代谢物。为了从这些结构类似物中寻找潜在的抗真菌药物中间体,对Zalerion arboricola菌株HCCB30111的代谢产物进行了分离纯化和结构鉴定。本实验成功的从该菌株的发酵液中分离到一个化合物,经结构鉴定证明其为纽莫康定B0的结构类似物。该发现为进一步质量研究,结构修饰和抗真菌活性物质的筛选工作奠定了基础。     

Antifungals and acetylcholinesterase inhibitors from the stem bark of Croton heliotropiifolius

The ethanolic extract of the stem bark of Croton heliotropiifolius Kunth (Euphorbiaceae) showed significant in vitro inhibition of acetylcholinesterase using a dilution spectrophotometric assay and antifungal activity against Candida albicans with a thin layer chromatography (TLC) bioautographic assay. In order to isolate the active compounds, bioassay-guided fractionation was undertaken using HPLC to localize the active compounds. Different zones of the HPLC-UV chromatogram were linked to acetylcholinesterase inhibition or to antifungal activities. In parallel to this HPLC-based activity profiling, HPLC-PDA-ESI-MS and HPLC-TOF-HRMS were used for the early identification of some of the compounds present. The targeted isolation of the active compounds was performed by medium pressure liquid chromatography (MPLC-UV) and further semi-preparative HPLC. Using this approach, nine compounds were isolated, one of them being a new indole alkaloid derivative. The structures of the isolated compounds were elucidated by spectroscopic methods including UV, NMR, MS and HRMS.     

Evaluation of Some Trypanostatic Agents by Means of Herpetomonas culicidarum

SYNOPSIS. A rapid quantitative method for measuring trypanostatic activity by means of the nonpathogenic flagellate Herpetomonas culicidarum is described. Of the known trypanocidal agents tested, pentamidine was the most active; stilbamidine and propamidine somewhat less active. H. culicidarum is more resistant to these agents than are some pathogenic hemoflagellates. Two new antifungal antibiotics, nystatin and heptamycin (a candicidin-like antibiotic), had powerful trypanostatic activity in vitro. The potential trypanostatic activity of antifungal agents is noted.     

New antifungal agents for the systemic mycoses

The azoles are the prominent broad spectrum oral antifungal agents in use or under clinical investigation for the systemic mycoses. This class of antifungal agents is represented by the marketed drug ketoconazole (Nizoral) and the experimental triazoles furthest along in clinical trials in the United States, itraconazole and fluconazole. Ketoconazole use is limited by its side effect profile and activity spectrum. Itraconazole appears to be better tolerated and less toxic to liver function, does not cause adrenal suppression and is more active against Aspergillus and Sporothrix schenckii. Fluconazole appears to be a highly promising agent due its highly favorable pharmacokinetic profile; it is water soluble, is well tolerated, is not metabolized to inactive constituents, it has a long half-life and, unlike the other azoles, high cerebrospinal fluid levels are readily attained for consideration in meningeal mycoses. It remains to be determined what place these new triazoles have in managing immunosuppressed patients including those with acquired immune deficiency syndrome known as AIDS. Other experimental antifungal agents, including ambruticin, amphotericin B methyl ester and saramycetin are also described. Sales figures are presented of drugs marketed in the United States for the systemic mycoses and reflect the growing problem of fungal diseases in the population.Presented as part of the Everett S. Beneke Symposium in Mycology, May 27, 1988.     

Antifungal activities of the essential oils in Syzygium aromaticum (L.) Merr. Et Perry and Leptospermum petersonii Bailey and their constituents against various dermatophytes

This study was carried out in order to investigate the potential of using plant oils derived from Leptospermum petersonii Bailey and Syzygium aromaticum L. Merr. Et Perry as natural antifungal agents. The antifungal effects of essential oils at concentrations of 0.05, 0.1, 0.15, and 0.2 mg/ml on the dermatophytes Microsporum canis (KCTC 6591), Trichophyton mentagrophytes (KCTC 6077), Trichophyton rubrum (KCCM 60443), Epidermophyton floccosum (KCCM 11667), and Microsporum gypseum were evaluated using the agar diffusion method. The major constituents of the active fraction against the dermatophytes were identified by gas chromatography-mass spectrometry and high-performance liquid chromatography analysis. The antifungal activities of S. aromaticum oil (clove oil) against the dermatophytes tested were highest at a concentration of 0.2 mg/ml, with an effectiveness of more than 60%. Hyphal growth was completely inhibited in T. mentagrophytes, T. rubrum, and M. gypseum by treatment with clove oil at a concentration of 0.2 mg/ml. Eugenol was the most effective antifungal constituent of clove oil against the dermatophytes T. mentagrophytes and M. canis. Morphological changes in the hyphae of T. mentagrophytes, such as damage to the cell wall and cell membrane and the expansion of the endoplasmic reticulum, after treatment with 0.11 mg/ml eugenol were observed by transmission electron microscopy (TEM). At a concentration of 0.2 mg/ml, L. petersonii oil (LPO) was more than 90% effective against all of the dermatophytes tested, with the exception of T. rubrum. Geranial was determined to be the most active antifungal constituent of L. petersonii oil. Taken together, the results of this study demonstrate that clove and tea tree oils exhibited significant antifungal activities against the dermatophytes tested in this study.     

Cooperative Anti-Candida Effects of Lactoferrin or Its Peptides in Combination with Azole Antifungal Agents

The effects of lactoferrin (LF), an antimicrobial protein secreted in body fluids, and its peptides in combination with azole antifungal agents were investigated by the micro-broth-dilution method in a study of Candida albicans. In the case of LF, its pepsin hydrolysate (LFhyd) or the LF-derived antimicrobial peptide Lactoferricin® B (LF-B), the concentrations required to inhibit the growth of Candida decreased in the presence of relatively low concentrations of clotrimazole (CTZ). The minimum inhibitory concentration (MIC) of all azole antifungal agents tested was reduced by 1/41/16 in the presence of a sub-MIC level of each of these LF-related substances. Polyene and fluoropyrimidine antifungal agents did not show such a combined effect with these LF-related substances. The anti-Candida activity of LF or LF-B in combination with CTZ was shown to be synergistic by checkerboard analysis. These results indicate that LF-related substances function cooperatively with azole antifungal agents against C. albicans.     

Antifungal activity of essential oils of Croton species from the Brazilian Caatinga biome

Aims: To find new antifungal agents among essential oils from Brazilian Croton species. Methods and Results: Plant leaves were steam distilled and the obtained essential oils were analyzed by gas chromatography/mass spectroscopy. The main constituents were estragole and anethole for Croton zehntneri, methyl-eugenol and bicyclogermacrene for Croton nepetaefolius and spathulenol and bicyclogermacrene for Croton argyrophylloides. The antifungal activity of essential oils was evaluated against Candida albicans, Candida tropicalis and Microsporum canis by the agar-well diffusion method and the minimum inhibitory concentration (MIC) by the broth microdilution method. Essential oils of Croton species demonstrated better activity against M. canis. Among the three plants C. argyrophylloides showed the best results, with MIC ranging from 9 to 19 μg ml⁻¹. The acute administration of the essential oil up to 3 g kg⁻¹ by the oral route to mice was devoid of overt toxicity. Conclusions: The studied essential oils are active in vitro against the dermatophyte M. canis and present relative lack of acute toxicity in vivo. Significance and Impact of the Study: Because of its antifungal activity and low toxicity, the essential oils of studied Croton species are promising sources for new phytotherapeutic agents to treat dermatophytosis.     

Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens

Aims: A Bacillus amyloliquefaciens strain, surviving epiphytically on the surface of fruit, was isolated while searching for naturally occurring biological control agents. This bacterial strain was characterized for its antifungal activity against seven selected fungal postharvest pathogens of citrus. Methods and Results: To understand the antifungal activity, seven postharvest fungal pathogens were screened for growth inhibition by B. amyloliquefaciens strain. Assays using B. amyloliquefaciens lipopeptide extracts showed a strong inhibitive activity. The inhibitory effect was observed in abnormal conidial germination and germ tube development when conidia were treated with different lipopeptide extract concentrations. Further analysis using PCR and chromatography confirmed the presence of fengycin, iturin and surfactine, of which iturin A showed the strongest and most common inhibitory effect. The results are supported by site‐directed mutagenesis analysis, targeted to suppress the biosynthesis of iturin A production. Fruit trials confirmed disease development inhibition when the antagonist was applied 1 day prior to or 1 day after fungal application. Conclusions: We conclude that the iturin family of lipopeptides are vital in the antagonism of B. amyloliquefaciens against the seven citrus postharvest pathogenic fungi tested. Significance and Impact of the Study: We elucidated the principal mechanism used by B. amyloliquefaciens PPCB004 to suppress postharvest disease development on stored fruits.     

In Vitro Antifungal Susceptibility of Oral <Emphasis Type="Italic">Candida</Emphasis> Isolates from Patients Suffering from Caries and Chronic Periodontitis

Caries and chronic periodontitis are common oral diseases where a higher Candida colonization is reported. Antifungal agents could be adjuvant drugs for the therapy of both clinical conditions. The aim of the current study has been to evaluate the in vitro activities of conventional and new antifungal drugs against oral Candida isolates from patients suffering from caries and/or chronic periodontitis. In vitro activities of amphotericin B, fluconazole, itraconazole, miconazole, nystatin, posaconazole and voriconazole against 126 oral Candida isolates (75 Candida albicans, 18 Candida parapsilosis, 11 Candida dubliniensis, six Candida guilliermondii, five Candida lipolytica, five Candida glabrata, four Candida tropicalis and two Candida krusei) from 61 patients were tested by the CLSI M27-A3 method. Most antifungal drugs were highly active, and resistance was observed in less than 5% of tested isolates. Miconazole was the most active antifungal drug, being more than 98% of isolates susceptible. Fluconazole, itraconazole, and the new triazoles, posaconazole and voriconazole, were also very active. Miconazole, fluconazole and voriconazole have excellent in vitro activities against all Candida isolates and could represent suitable treatment for a hypothetically adjunctive therapy of caries and chronic periodontitis.     

Purification and structural characterization of bacillomycin F produced by a bacterial honey isolate active against Byssochlamys fulva H25

Aims:  Isolate and characterize antifungal peptides exhibiting activity against Byssochlamys fulva H25, a spoilage mould associated with juices and beverages.
Methods and Results:  A bacterium (H215) isolated from honey showed high antifungal activity against B. fulva H25. The antifungal producer strain was identified as Bacillus subtilis using 16S rDNA sequencing. The antifungal peptide was purified by 20% ammonium sulfate precipitation of the bacterial culture supernatant, followed by Octyl-Sepharose CL-4B and reverse phase-high performance liquid chromatography. The five active fractions were lyophilized and subjected to mass, tandem mass spectrometry and amino acid analysis to deduce their corresponding molecular masses and structural characteristics. The five peaks were determined to be identical to bacillomycin F, varying in the length of the fatty acid chain moiety from C14 to C16.
Conclusions:  The broad-spectrum antifungal activity produced by a bacterium from honey was determined to be due to the production of bacillomycin F.
Significance and Impact of the Study:  The antifungal compound produced by a bacterial strain isolated from honey was determined to be stable over a broad pH range and was stable to heat treatments up to 100°C. This is the first report of honey microflora producing bacillomycin F or any antifungal compound.