被内生真菌侵染的禾草提取液对真菌的抑制作用

以带有与不带有Neotyphodium属内生真菌的醉马草Achnatherum inebrians、披碱草Elymus dahuricus和野大麦Hordeum brevisubulatum的草粉浸提液对细交链孢Alternaria alternata、根腐离蠕孢Bipolaris sorokiniana、燕麦镰孢Fusarium avenaceum和绿色木霉Trichoderma viride进行了抑菌活性研究。结果表明:披碱草、醉马草和野大麦草粉浸提液对细交链孢、根腐离蠕孢、燕麦镰孢和绿色木霉的菌落生长、孢子萌发率和芽管长度均有一定的抑制作用。而披碱草中的Neotyphodium可显著增强披碱草草粉浸提液对细交链孢、燕麦镰孢、绿色木霉菌落生长及对细交链孢和根腐离蠕孢孢子萌发及燕麦镰孢芽管长度的抑制作用;醉马草中的Neotyphodium显著增强了醉马草草粉浸提液对燕麦镰孢、绿色木霉菌落生长和芽管长度,以及细交链孢、根腐离蠕孢和燕麦镰孢孢子萌发的抑制作用;野大麦中的Neotyphodium显著增强了野大麦草粉浸提液对绿色木霉菌落生长、孢子萌发和芽管长度的抑制作用。     

Evaluation of the antifungal photodynamic activity of Thymophylla pentachaeta extracts against Candida albicans and its virulence factors

BackgroundCandida albicans is one of the most common causative of opportunistic infections. Treatment of candidiasis is challenging considering the few antifungal drugs available and the increase in resistance. Antimicrobial photodynamic therapy (aPDT) is a recently developed therapeutic option that combines a non-toxic photosensitizer (PS) and light to kill the microbial pathogens. Targeting virulence, defined as the ability of a pathogen to cause overt disease, represents another attractive target for the development of novel antifungal agents. Thymophylla pentachaeta (DC.) Small var. belenidium (DC.) is an endemic plant from Argentina in which the presence of thiophenes, biologically active compounds whose antifungal activity is enhanced by irradiation with Ultraviolet A (UVA), have been already described.PurposeThe purpose of this study was to evaluate the photodynamic antifungal activity of hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH) extracts from T. pentachaeta var. belenidium and their inhibitory effects on C. albicans virulence factors as well as biofilm formation and eradication.Study Design/MethodsAntifungal photodynamic activity of Hex, DCM, EtOAc and MeOH extracts from different parts of the plant were assessed with the microbroth dilution, bioautography and the time-kill assays, under light and darkness conditions. The capacities of the most active extracts of inhibiting Candida virulence factors (adherence to epithelial cells, germ tube and pseudomycelium formation and hydrolytic enzyme secretion) were assessed. In addition, the activity against biofilm formation and eradication has been investigated by reaction with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) that quantifies living cells in these structures.ResultsHex and DCM extracts from T. pentachaeta roots exhibited high photodynamic antifungal activity against C. albicans [Minimal fungicide concentrations (MFCs)= 7.8 µg/ml] under UVA light irradiation. Chemical analysis of active extracts (Hex and DCM from roots) revealed the presence of photoactive thiophenes. Both extracts generate reactive oxygen species through type I and II mechanisms. These extracts, at sub-inhibitory concentrations, under light conditions decreased the adherence of C. albicans to Buccal Epithelial Cells (BEC), inhibited germ tube formation and reduced esterase production. Finally, they demonstrated activity against preformed biofilms submitted to irradiation (MFCs= 3.91 µg/ml and 15.63 µg/ml for Hex and DCM extracts, respectively).ConclusionTaking together, results demonstrated the strong photodynamic effects of T. pentachaeta root extracts under UVA irradiation, making them valuable alternatives to the already established antifungal drugs against C. albicans.     

Antifungal activity of Apulia region propolis

A study was carried out to assess the in vitro antifungal activity of some natural Apulian propolis extracts of different origin. Their antifungal activity was compared to the antifungal activity of conifers and commercial propolis extracts. All extracts revealed antifungal activity against dermatophytes and Candida species. The antifungal activity differences found depended on the origin of the propolis and the solvent used for extraction. The best antifungal activity was given by the 'Orimini' propolis. The antifungal activity may have been influenced by the presence of different cinnamic and flavonoid components and their different concentration in the extracts. Further investigations are needed to validate this hypothesis.     

覆盆子提取物联合唑类药物抗真菌活性研究

目的 探讨中药覆盆子提取物联合唑类药物的体外抗真菌作用.方法 采用CLSI公布的M27-A方案微量液基稀释法和棋盘式微量稀释法,测定覆盆子提取物单用及联合唑类药物对不同念珠菌的MIC值和FICI指数.结果 覆盆子不同溶液提取物与氟康唑均表现出协同关系,以覆盆子醇提物为例,单用对念珠菌的MIC80测定值范围主要集中在0.16～1.25 mg/mL,与氟康唑合用后表现出协同关系(FICI≤0.5),且MIC80测定值范围降至0.01 ～0.04 mg/mL;合用后的氟康唑抗真菌活性也明显增强.另外,覆盆子醇提物与不同唑类药物合用后均有协同关系,其MIC80测定值由单用时大于10 mg/mL降至0.04 mg/mL.结论 覆盆子醇提物和唑类药物单用时对耐药念珠菌的抑菌作用较弱,但二者合用后表现出明显的协同关系,对耐药念珠菌的抑菌作用明显增强.     

海洋芽孢杆菌(Bacillus marinus)B-9987菌株抑制病原真菌机理

[目的]探讨海洋芽孢杆菌(Bacillus marinus)B-9987菌株的代谢产物BMME-1,对植物病原真菌茄链格孢菌的抑菌作用机理.[方法]分别使用分光光法、气相色谱-质谱GC-MS联用技术、红外光谱法等,检测了BMME-1处理病原真菌后,菌体渗透性、细胞壁及细胞膜成份的变化.[结果]BMME-1对茄链格孢菌的抑菌中浓度(MIC_(50))为6.2 mg/L,最小杀菌浓度(MFC)为50 mg/L,在MIC_(50)浓度或高于此浓度处理靶标菌,将导致菌体蛋白质、核酸等大分子物质的外流;处理菌株葡聚糖结构β-型糖苷键、碳-氧键(C-O)、碳-氢键(C-H)等基团的特征吸收强度降低,-OH、C=O的伸缩振动吸收强度升高;菌体细胞壁几丁质结构中酰胺I键吸收强度发生变化;与对照菌株的麦角甾醇含量(62.52±3.31%)相比,处理菌株麦角甾醇减少为(56.36±2.52)%,同时出现麦角固醇合成中间产物粪甾醇.[结论]BMME-1对病原真菌的抑制表现为:干扰细胞膜麦角甾醇的合成从而改变了细胞的通透性;对细胞壁葡聚糖结构的影响较大而几丁质次之.     

Structure,chemistry, and biological activity of pseudophomins A and B,new cyclic lipodepsipeptides isolated from the biocontrol bacterium Pseudomonas fluorescens

Pseudophomins A and B are cyclic lipodepsipeptides isolated from Pseudomonas fluorescens strain BRG100, a bacterium with potential application for biocontrol of plant pathogens and weeds. Their chemical structures were established by a combination of spectroscopic data, X-ray crystallography, and selective chemical degradation. This unique chemical degradation allowed the unambiguous determination of the absolute configuration of the amino acid residue Leu-1, due to gamma-lactam formation followed by selective cleavage of the adjacent N(8)-C(7) bond. To the best of our knowledge this is the first application of gamma-lactam formation to the determination of absolute configuration of an adjacent amino acid. Pseudophomin B showed higher antifungal activity against the phytopathogens Phoma lingam/Leptosphaeria maculans and Sclerotinia sclerotiorum than pseudophomin A, and is likely to be the main component responsible for the antifungal activity of EtOAc extracts of strain BRG100. By contrast, pseudophomin A showed stronger inhibition of green foxtail (Setaria viridis) root germination than pseudophomin B.     

<Emphasis Type="Italic">In vitro</Emphasis> antifungal activity of propolis samples of Czech and Slovak origin

Propolis has been used in traditional folk medicine for ages owing to a number of biological effects. Four propolis samples of Czech and one of Slovak origin were extracted using Soxhlet apparatus and analysed by thin-layer chromatography. Raw propolis samples and their extracts were tested by microdilution broth method to determine minimal inhibitory concentration (MIC) in eight strains of human pathogenic fungi. Raw propolis samples showed a lower in vitro antifungal activity than their extracts. In general, the petroleum ether extracts exhibited the highest in vitro antifungal activity (MIC range of 16–64 μg/ml). The content of flavonoids in the samples varied according to region. The highest amount of flavonoids was found in sample A that originated from Broumov (4%). The most susceptible to the propolis extracts were Trichophyton mentagrophytes and Candida albicans. The propolis samples of Czech and Slovak origin and their extracts showed a considerable in vitro antifungal effect which was associated especially with nonpolar petroleum ether and toluene extracts. There was only a partial correlation between flavonoids content and in vitro antifungal activity.     

The Relationship between Systemic Resistance Induced by Pruning and Accumulation of Antifungal Substances in Barley Seedlings

Infection by a compatible race of Erysiphe graminis f. sp. hordei on barley secondary leaves was significantly suppressed upon pruning of the primary leaves when E. graminis hordei was inoculated 3–12 h after the pruning, but it, was rather enhanced during 15–21 h. The accumulation of antifungal substances was detected in hot ethanol extracts of barley seedlings from 15–27 h after pruning the primary leaves. Taking the time of the infection process of a challenger (E. graminis, hordei) into consideration, timing of systemic resistance induced upon pruning coincided with the accumulation of antifungal substances.     

Antifungal activity of essential oils from leaves and flowers of Inula viscosa (Asteraceae) by Apulian region

Some essential oils from several plants (Artemisia verlotorum, Lavandula augustifolia, Ocimum gratissimum) have proved to have acaricidal, antifungal and antibacterial activity. Inula viscosa Ait. (Asteraceae), a plant growing spontaneously in the Mediterranean area, is currently used by popular medicine for its therapeutic effects. Flavonoids, azulenes, sesquiterpenes, and essential oils have been isolated and identified from its leaves. This paper reports the results of the composition and antifungal activity in vitro against dermatophytes and Candida spp. of the four essential oils obtained by steam distillation of the leaves, flowers, whole plant and whole plants without flower extracts of I. viscosa. All the extracts proved to have a significant antifungal activity against dermatophytes even at low concentrations (0.01 mg/ml). The leaf extracts exhibited the greatest antifungal efficacy. The high concentration of the sesquiterpene (carboxyeudesmadiene), occurring in the leaf extracts, may explain its greater antifungal activity.     

Deglucosidation of quercetin glucosides to the aglycone and formation of antifungal agents by peroxidase-dependent oxidation of quercetin on browning of onion scales

Outer scales of yellow onion bulbs turn brown during maturing. The brown outer scales contain an antifungal component, 3,4-dihydroxybenzoic acid. An aim of the present study is to elucidate the mechanism of formation of the benzoic acid. In a browning scale, the scale was divided into three areas; fleshy, drying and dried brown areas. Levels of quercetin glucosides in dried brown areas were less than 10% of the glucosides in fleshy and drying areas, whereas levels of quercetin were high in dried brown areas. This result suggests that quercetin was formed by deglucosidation of quercetin glucosides on the border between drying and dried brown areas. Peroxidase (POX) activity of dried brown areas was about 10% of those of fleshy and drying areas. Quercetin was oxidized by autooxidation, and cell-free extracts of drying areas and POX isolated from onion scales enhanced the oxidation even in the absence of externally added hydrogen peroxide. The enhancement of quercetin oxidation was suppressed by catalase. No tyrosinase-like activity was detected in the cell-free extracts and the POX preparation. These results suggest that, during the enhanced oxidation of quercetin, hydrogen peroxide is formed. 3,4-Dihydroxybenzoic acid and 2,4,6-trihydroxyphenylglyoxylic acid, which were the oxidation products of quercetin, were found in dried brown area. These results suggest that an antifungal agent 3,4-dihydroxybenzoic acid is formed by POX-dependent oxidation of quercetin on browning of onion scales.     

Exploring antifungal activities of acetone extract of selected Indian medicinal plants against human dermal fungal pathogens

A broad spectrum of medicinal plants was used as traditional remedies for various infectious diseases. Fungal infectious diseases have a significant impact on public health. Fungi cause more prevalent infections in immunocompromised individuals mainly patients undergoing transplantation related therapies, and malignant cancer treatments. The present study aimed to investigate the in vitro antifungal effects of the traditional medicinal plants used in India against the fungal pathogens associated with dermal infections. Indian medicinal plants (Acalypha indica, Lawsonia inermis Allium sativum and Citrus limon) extract (acetone/crude) were tested for their antifungal effects against five fungal species isolated from skin scrapings of fungal infected patients were identified as including Alternaria spp., Curvularia spp., Fusarium spp., Trichophyton spp. and Geotrichum spp. using well diffusion test and the broth micro dilution method. All plant extracts have shown to have antifungal efficacy against dermal pathogens. Particularly, Allium sativum extract revealed a strong antifungal effect against all fungal isolates with the minimum fungicidal concentration (MFC) of 50–100 μg/mL. Strong antifungal activity against Curvularia spp., Trichophyton spp., and Geotrichum spp. was also observed for the extracts of Acalypha indica, and Lawsonia inermis with MFCs of 50–800 μg/mL respectively. The extracts of Citrus limon showed an effective antifungal activity against most of the fungal strains tested with the MFCs of 50–800 μg/mL. Our research demonstrated the strong evidence of conventional plants extracts against clinical fungal pathogens with the most promising option of employing natural-drugs for the treatment of skin infections. Furthermore, in-depth analysis of identifying the compounds responsible for the antifungal activity that could offer alternatives way to develop new natural antifungal therapeutics for combating resistant recurrent infections.     

A new approach to drug discovery: high-throughput screening of microbial natural extracts against Aspergillus fumigatus using resazurin

Natural products are an inexhaustible source for drug discovery. However, the validation and selection of primary screening assays are vital to guarantee a selection of extracts or molecules with relevant pharmacological action and worthy of following up. The assay must be rapid, simple, easy to implement, and produce quick results and preferably at a low cost. In this work, we developed and validated a colorimetric microtiter assay using the resazurin viability dye. The parameters of the resazurin method for high-throughput screening (HTS) using natural extracts against Aspergillus fumigatus were optimized and set up. The extracts plus RPMI-1640 modified medium containing the spores and 0.002% resazurin were added per well. The fluorescence was read after 24 to 30 h of incubation. The resazurin proved to be as suitable as Alamar Blue for determining the minimal inhibitory concentration of different antifungals against A. fumigatus and effective to analyze fungicidal and fungistatic compounds. An HTS of 12 000 microbial extracts was carried out against two A. fumigatus strains, and 2.7% of the extracts displayed antifungal activity. Our group has been the first to use this methodology for screening a collection of natural extracts to identify compounds with antifungal activity against the medically important human pathogen A. fumigatus.     

Antifungal diterpenoids of Pseudolarix kaempferi, and their structure-activity relationship study

The in vitro antifungal activities of 19 structurally diversified analogues of pseudolaric acids tested against the major pathogenic fungus Candida albicans has led to the establishment of a very clear structure-activity relationship of pseudolaric acids derivatives. Pseudolaric acid A was first found to be a potent antifungal component comparable with pseudolaric acid B. Among the tested 19 diterpenoids, pseudolaric acids A2 (1), B2 (3), B3 (4) and methyl pseudolarate A2 (2) are new isolates of the root bark of Pseudolarix kaempferi, and their structures were elucidated mainly by 2D-NMR techniques and chemical methods. Compounds 12-19 were first semi-synthesized by efficient routines from pseudolaric acid B.     

Pn-AMPs,the hevein-like proteins from Pharbitis nil confers disease resistance against phytopathogenic fungi in tomato,Lycopersicum esculentum

The antifungal activity of hevein-like proteins has been associated with their chitin-binding activities. Pn-AMP1 and Pn-AMP2, two hevein homologues from Pharbitis nil, show in vitro antifungal activities against both chitin and non-chitin containing fungi. Purified Pn-AMPs retained antifungal activities only under non-reducing conditions. When Pn-AMP2 cDNA was constitutively expressed in tomato (Lycopersicon esculentum) plants under the control of CaMV35S promoter, the transgenic plants showed enhanced resistance against both the non-chitinous fungus Phytophthora capsici, and the chitin-containing fungus Fusarium oxysporum. Thus, the chitin component in the fungal cell wall is not an absolute requirement for Pn-AMP's antifungal activities. These results when considered together suggest that Pn-AMPs have the potential for developing transgenic plants resistant to a wide range of phytopathogenic fungi.     

Expression of the antimicrobial peptides in plants to control phytopathogenic bacteria and fungi

Three antimicrobial peptides exhibiting in vitro antifungal activity were expressed in Arabidopsis to compare their in planta activity. β-Purothionin, cecropin B, and phor21 were expressed under an endogenous promoter with moderate-level activity and excreted extracellularly. Expression of β-purothionin rendered the greatest antibacterial and antifungal resistance while cecropin B enhanced only antibacterial activity and phor21 did not improve antimicrobial resistance. The transgenic β-purothionin arrested fungal growth on leaf surfaces and infection of stomata. Leaf extracts from plants producing β-purothionin and cecropin B displayed membrane permeabilizing activity. The in planta antimicrobial activity of the tested peptides was consistent with previously reported in vitro experiments. The expression strategy allowed enhanced antifungal resistance without high-level transgene expression.Electronic Supplementary Material Supplementary material is available for this article at     

生防链霉菌ZM-16的发酵产物生物活性及其微波诱变育种

【目的】链霉菌ZM-16对多种细菌有良好的抑菌作用,其发酵产物的主要活性物质为放线菌素D。本研究旨在进一步探讨其抗真菌活性,并通过诱变育种的方法对菌种进行改良,以提高活性物质的产量,从而为其在植物病害生物防治领域的实践应用奠定基础。【方法】利用液体发酵的方法获取活性产物并对其进行粗提取;利用抑菌圈法检测菌株发酵产物对11种植物病原真菌的抑制作用;通过微波处理并利用抗生素抗性筛选的方法对菌株进行诱变育种。【结果】粗提液对11种植物病原真菌均具有抑菌效果,最为明显的3种病菌分别是苹果炭疽病菌、油菜菌核病菌和禾谷镰刀病菌;经微波诱变筛选到了一株耐利福平突变株,其放线菌素D的产量提高了36.75%;传代研究表明其经过10代选育,遗传稳定性较好。【结论】链霉菌ZM-16的发酵产物具有良好的抗植物病原真菌的活性,经过育种后其活性产物产量也有所提高,因此具有较高的实际应用价值。     

Antifungal constituents of Northern prickly ash, Zanthoxylum americanum Mill.

Leaf, fruit, stem, bark and root of Zanthoxylum americanum were investigated for antifungal activity with 11 strains of fungi representing diverse opportunistic and systemic pathogens, including Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. All extracts demonstrated a broad spectrum of antifungal activity and inhibited at least eight fungal species in a disk diffusion assay (600 microg/disk). Antifungal activity was light-dependent, with fruit and leaf extracts most active in general. The presence of light-mediated compounds, such as psoralen, 8-methoxypsoralen and imperatorin in extracts of different organs was confirmed by RP-HPLC. A high furanocoumarin content was detected in fruit and leaf and low furanocoumarin levels were found in bark and wood. A high positive correlation was observed between total furanocoumarin content and fungal inhibition zones (r2=0.902, p<0.001). The results provide a phytochemical basis for the very widespread use of Z. americanum in indigenous North American ethnomedical tradition for conditions that may be related to fungal infections.     

几种野生植物提取物抑菌作用研究

本文以乙醇为溶剂,分别提取了瓦松、泽漆及一组中草药配方的有效成分;室内测定了三种提取液对5种植物病原菌(棉花枯萎病菌Fusarium oxysporum f.sp.vasinfectum;小麦赤霉病菌Fusarium graminearum Schw;西瓜枯萎病菌Fusarium oxysporum f.sp.niveum;仙人掌软腐病菌Erwinia chrysanthemi pv.chrysanthemi;魔芋软腐病菌Erwiniacarotovora pv.carotovora)的抑菌作用.结果表明:三种提取物对镰刀菌均有不同程度的抑制作用,且三种提取物的抑菌效果差异显著,其中瓦松的抑菌效果最强.当在50 mL培养基中加入5mL不同提取液时,瓦松对不同镰刀菌的相对抑制率为91.39%～100%;中草药配方提取液对不同镰刀菌的相对抑制率分别为41.11%～85.37%;而泽漆提取液对镰刀菌的相对抑制率仅为12.86%～21.23%;当在50 mL培养基中加入提取液体积降低为2 mL、1 mL、0.5mL时,三种提取液对三种镰刀菌的抑制效果骤然下降,但下降的梯度不一.对大白菜软腐病菌与魔芋软腐病菌而言也是瓦松的抑菌效果最强,中草药配方次之,泽漆的抑制效果最差.     

Antifungal activity of C3a and C3a-derived peptides against Candida

Antimicrobial peptides are generated during activation of the complement system [Nordahl et al. Proc. Natl. Acad. Sci. U. S. A. 2004, 101:16879-16884]. Here we show that the anaphylatoxin C3a exerts antimicrobial effects against the yeast Candida. Fluorescence microscopy and electron microscopy analysis demonstrated that C3a-derived peptides bound to the cell surface of Candida, and induced membrane perturbations and release of extracellular material. Various Candida isolates were found to induce complement degradation, leading to generation of C3a. Arginine residues were found to be critical for the antifungal and membrane breaking activity of a C3a-derived antimicrobial peptide, CNY21 (C3a; Cys57-Arg77). A CNY21 variant with increased positive net charge displayed enhanced antifungal activity. Thus, C3a-derived peptides can be utilized as templates in the development of peptide-based antifungal therapies.