Enzyme immunoassay of the secondary metabolites of micromycetes as components of lichen substances

The composition of low-molecular biologically active metabolites typical of microscopic fungi has been studied in blastemas of fruticose lichens of the genera Cladonia, Cetraria, Evernia, Bryoria, and Usnes. The enzyme immunoassay method showed the presence of sterigmatocystin, emodin, mycophenolic acid, citrinin, alternariol, and diacetoxyscirpenol, which occurred regularly and, in most cases, at a frequency of 55 to 100%. The highest levels of accumulation were 0.001–0.003% for emodin, 0.0002% for alternariol and citrinin, 0.0001% for sterigmatocystin and mycophenolic acid, and 0.00005% of the weight of air-dry material for diacetoxyscirpenol. Other metabolites (cyclopiazonic acid, ergot alkaloids, ochratoxin A, PR toxin, deoxynivalenol, zearalenone, and fumonisins) were detected in these lichens less frequently (sometimes only upon the expansion of the territory of sampling), and their content was no more than 0.00005%. The peculiarities of the component composition and the levels of accumulation of fungal metabolites in lichens of different taxonomic affiliation were discussed.     

The ecological role of secondary lichen substances

Considerable evidence, both direct and indirect, supports the hypothesis that many lichen secondary substances have significant ecological roles. Thus secondary chemistry may be important in determining the relative ecological success of individual lichen species and thus the structure and diversity of natural lichen communities. Primary ecological roles of secondary lichen substances can be classified into four groups: light-screen compounds, chemical weathering compounds, allelopathic compounds, (including antibiotic compounds), and anti-herbivore defense compounds.     

地衣型真菌次级代谢产物抗菌活性初步研究

地衣是一种传统的民族药物,能产生多种具有活性的物质。该研究对地衣型真菌(Xanthoria elegans,Myelochroa indica,Ramalina peruviana,Cladonia macilenta,Nephromopsis pallescens,Cladonia coccifera)进行液体培养,2个月后,培养液用乙酸乙酯萃取后获得初提物。该研究采用抑菌圈法评价地衣型真菌初提物对7种致病细菌(Bacillus subtilis,Bacillus cereus,Vibrio parahaemolyticus,Straphylococcus haemolyticus,Pseudomonas aeruginosa,Staphylococcus aureus,Micrococcus luteus)的抗菌活性,并测定最低抑菌浓度(MIC)。结果表明:6种地衣型真菌的初提物均具有一定的抗菌活性,且不同培养基对地衣型真菌产生抗菌物质有显著影响。其中,R.peruviana在MY液体培养基中所产生的次级代谢产物对金黄色葡萄球菌、藤黄微球菌、溶血性葡萄球菌、铜尿假单胞菌具有抑制效果,但在YMG培养基中所得初提物对供试7种致病细菌不具有抑菌效果。X.elegans在YMG培养基中所得初提物对枯草芽孢杆菌具有明显抗菌活性,其抑菌圈直径可达17.77 mm。该研究证实不同地衣型真菌液体培养初提物具有抗菌活性,不同的培养基也直接影响地衣型真菌抗菌效果。该研究结果为地衣型真菌的进一步研究及民族药的开发利用奠定了基础。     

Preservation of secondary fungal metabolites in herbarium lichen specimens

Fresh picked and herbarium thalli of Cladonia stellaris, C. rangiferina, Allocetraria nivalis, A. cucullata, Cetraria islandica, Peltigera canina, and Nephroma articum epigene lichens were studied using the immune-enzyme analysis. No big difference was observed in the contents of mycotoxin secondary metabolites, i.e., deoxynivalenol, diacetoxyscirpenol, zearalenone, alternariol, citrinin, sterigmatocystin, cyclopiazonic acid, mycophenolic acid, emodin, and PR-toxin. The discovery of these substances in the specimens preserved for several decades shows that lichens have an effective system of conservation of metabolic exchange products.     

Bioactivity of secondary metabolites and thallus extracts from lichen fungi

Fungal secondary compounds and total extracts are known to affect growth of bacteria, fungi, and plants. This study tested the effects of purified compounds and total extracts from three lichens on the growth of two plant pathogens, Ophiostoma novo-ulmi ssp. americana and Sclerotinia sclerotiorum. Usnic acid showed no reduction in relative growth rates (RGR), whereas vulpinic acid reduced RGR for both fungi and atranorin reduced RGR of S. sclerotiorum only. However, purified vulpinic acid showed stronger effects than total extracts on fungal growth. The results suggest that these lichens show further promise as a source for bioactive compounds against fungi.     

Enzyme immunoassay measurement of the urinary metabolites of thromboxane A2 and prostacyclin

We have used a recently developed enzyme immunoassay (EIA) method for measuring urinary concentrations of TXB2, 6-keto PGF1 alpha, 2,3-dinor-TXB2, 2,3-dinor-6-keto PGF1 alpha and 11-dehydro-TXB2 using acetylcholinesterase from Electrophorus Electricus coupled to TXB2, 6-keto PGF1 alpha and 11-dehydro-TXB2. Urinary PGI2 and TXA2 breakdown products and their metabolites were extracted from 3-40 ml of urine corresponding to 100 mumoles creatinine. Measurements were performed after Sep-Pak extraction and thin layer chromatography separation in a system that allows separation between dinor- and parent derivatives. Because of the relatively high cross reactivity (10-15%) of the anti-TXB2 serum with 2,3-dinor TXB2 and the anti-6-keto PGF1 alpha serum with 2,3-dinor-6-keto PGF1 alpha, measurements were done using 3 antisera (anti-TXB2 and anti-6-keto PGF1 alpha diluted 1/50,000, anti 11-dehydro-TXB2 diluted 1/200,000). The reproducibility of the technique was assessed by measuring the same urine stored frozen in aliquots together with each series of samples (Coefficient of variation 6-12% (n = 20), depending on the compound). In addition, the use of a different solvent system for the thin layer chromatography did not affect the results although the migration of the compounds was modified significantly. Determination of the urinary excretion of TXB2 and prostacyclin metabolites in 17 healthy individuals by this method provided results in agreement with those obtained by other methodologies. In addition, comparisons made between EIA and gas chromatography/mass spectrometry analysis showed good correlation between the urinary metabolites as determined by each technique (r = 0.98).     

Use of lichen secondary metabolites as antifeedants to protect higher plants from damage caused by slug feeding

Extracts of 15 species of lichen were tested for antifeedant properties against the field slug Deroceras reticulation (Müller) (Mollusca: Pulmonata). All but three showed some activity and two of these were from endolithic species. The most effective extract was from Letharia vulpina. The major active compound from this species, vulpinic acid, was tested for dose response, applied as a foliar spray to turnip plants and as a dressing to wheat seeds in laboratory experiments. It provided effective protection against slug feeding under these conditions.     

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.     

红色红曲霉Mr-1的次级代谢产物生物活性成分分析

【背景】红曲霉(Monascus)是一种重要的药食同源性真菌,其自身产生的次级代谢产物具有多种生理活性功能,然而红曲霉中的生物活性成分却鲜有报道。利用红曲霉发酵液进行药效物质成分追溯,对了解红曲霉药效物质基础具有十分重要的意义。【目的】对红色红曲霉(M.ruber)Mr-1次级代谢产物中的生物活性成分和生物学功能进行研究。【方法】采用硅胶柱、SephadexLH-20凝胶柱等色谱技术对活性成分进行分离纯化,通过核磁共振和高分辨质谱技术对化合物结构进行解析;对鉴定的化合物进行体外抗氧化、抑菌和酶活性测定。【结果】从红色红曲霉Mr-1次级代谢产物中分离得到4个活性化合物,鉴定为3个黄酮类化合物Luteolin(1)、Hesperetin(2)、Glycitein(3)和1个萜类化合物Ursolic acid(4)。化合物1、2、4为首次从红曲菌科中分离得到。在抗氧化试验中,化合物1对ABTS⁺、DPPH和OH^-自由基具有较强的清除能力,IC₅₀分别为13.36、8.74和32.75μg/mL;在抑菌试验中,化合物4对金黄色葡萄球菌(Staphylococcus aureus)和李斯特菌(Listeria monocytogenes)表现出中等强度的抑菌能力,抑菌圈直径分别为13.4 mm和11.9 mm;在α-葡萄糖苷酶抑制活性试验中,化合物4表现出很强的抑制能力,IC₅₀为21.34μg/mL。【结论】红色红曲霉Mr-1是宝贵的微生物种质资源,其产生的次级代谢产物生物活性成分多样,具有开发成功能性食品原料的潜能。     

石果衣菌藻次级代谢产物的初步研究

荒漠地衣石果衣 Endocarpon pusillum中未能检测出任何次级代谢产物,然而,其共生菌基因组中却含有14个PKS基因和2个NRPS基因。通过激活培养后从中分离出3个次级代谢产物,4,6,9-三羟基-7-甲基-1H,3H-萘并[1,8-cd]吡喃-1,3-二酮（lamellicolic anhydride）、石果衣菌素A（endocarpin A）及石果衣菌素B（endocarpin B）,后二者为新结构化合物。从其共生藻柯氏复球藻 Diplosphaera chodatii中分离出3个脱镁叶绿素类化合物,即脱镁叶绿素甲酯酸b（phaeophorbide b）、13-表-脱镁叶绿素甲酯酸a（13- epi-phaeophorbide a）及脱镁叶绿素甲酯酸a（phaeophorbide a）。其中脱镁叶绿素甲酯酸b具有中等的清除DPPH活性。     

Bioactive lichen metabolites: alpine habitats as an untapped source

Lichens are fungal and algal/cyanobacterial symbioses resulting in the production of specific metabolites. Some of these are forming an available biomass for phytochemical investigations, including the assessment of biological activities of the isolated compounds. The alpine or polar region are characterised by highly stressful environmental conditions for many organisms, but lichens are among the dominating organisms in these habitats. In the performant mutual protective system, lichen fungi often accumulate high amounts of metabolites with specific physicochemical properties (UV absorbents, hydrophobicity) which help the lichens to survive. Unique secondary metabolites and polysaccharides have been isolated and tested from these organisms. Even though this has been tested until now only with a low number of compounds so far, interesting activities have been recorded. We review here some of the antimicrobial, anti-inflammatory, antiproliferative and antioxidant activities properties described. Solutions with axenic biotechnological cultivation of each symbiotic partner and particularly the mycobiont to obtain the lichen secondary metabolites are challenging to overcome the limitations for the supply of these rare compounds. Additionally, these lichens appear to harbour a diversity of culturable microorganisms from which active compounds have also been isolated recently.     

Interaction between lichen secondary metabolites and antibiotics against clinical isolates methicillin-resistant Staphylococcus aureus strains

The in vitro antimicrobial activities of five compounds isolated from lichens, collected in several Southern regions of Chile (including the Chilean Antarctic Territory), were evaluated alone and in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC₉₀, MIC₅₀, as well as MBC₉₀ and MBC₅₀, for the lichen compounds were evaluated. The MIC₉₀ was ranging from 32 µg/ml for perlatolic acid to 128 µg/ml for α-collatolic acid. MBC₉₀ was ranging from onefold up to twofold the MIC₉₀ for each compound. A synergistic action was observed in combination with gentamicin, whilst antagonism was observed for some lichen compounds in combination with levofloxacin. All combinations with erythromycin were indifferent, whilst variability was observed for clindamycin and oxacillin combinations. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. These could mainly be explained by the failure of FIC approach, being too much subjective and sensitive to experimental errors. These findings suggest, however, that the natural compounds from lichens are good candidates for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.     

Pyrenomycetes and Loculoascomycetes as sources of secondary metabolites

Over 400 secondary metabolites have been reported from members of the Pyrenomycetes and the Loculoascomycetes. Among these, members of the Hypocreaceae, the Clavicipitaceae, the Xylariaceae, the Melanosporaceae, and the Sordariaceae in the Pyrenomycetes, and those of the Pleosporaceae and the Sporormlaceae in the Loculoascomycetes have been explored frequently; and representative secondary metabolites produced by these fungi are illustrated. Many of them are reported to be phytotoxic and some of them have antibacterial or antifungal activities. Only recently were the compounds tested in screens targeted for specific enzyme inhibitors or receptoragonists/antagonists. This group of fungi are attractive for screening for novel natural products because of the diversity of species and physiology.     

Biocidal effect of lichen secondary metabolites against rock-dwelling microcolonial fungi,cyanobacteria and green algae

The use of commercial biocides in outdoor environments is increasingly discouraged because of their ecotoxicity, new methods being thus invoked to control patinas of biological origin on the stone cultural heritage. The effects of secondary metabolites (usnic acid, norstictic acid, parietin) produced by saxicolous lichens, natural competitors of rock dwelling microorganisms, were investigated in vitro against microcolonial fungi (MCF: Coniosporium apollinis, Coniosporium perforans, Coniosporium uncinatum, Phaeococcomyces-like sp.), coccoid cyanobacteria (Chroococcus minutus) and green algae (Scenedesmus ecornis) which commonly occur on stonework. An acetone/water 10/90 vol/vol mixture was screened as suitable to solubilise the lichen metabolites and to not affect the bioassay results. Benzalkonium chloride 1% was used as positive control.All the three metabolites (approx. 10⁻² mM) inhibited the growth of the assayed MCF species, displaying the same effect of benzalkonium chloride. Chroococcus and Scenedesmus exhibited sensibility to the lichen metabolites when exposed to high incubation temperatures (35 °C), chemicals and temperature synergically yielding percentage decreases of intact cells with red chlorophyll epifluorescence. These findings suggest lichen secondary metabolites as allelopathic agents against rock dwelling microorganisms and as potential natural sources for their control on stone materials in restoration and conservation programmes. In this perspective, the detection of a negligible chromatic alteration (ΔE<0.5) caused by LSM to the white Carrara marble is reported as the first step of the necessary extensive evaluation of the LSM-stone material interactions.     

Insights into physiological responses of mosses Physcomitrella patens and Pohlia drummondii to lichen secondary metabolites

Protoplasma - It is widely accepted that allelopathy among mosses and lichens do exist due to its similar ecological needs, though it is rarely documented. With an aim to test whether there is an...     

Effect of volatile and gaseous metabolites of germinating pea seeds on micromycetes

Differences in the effect of volatile and gaseous metabolites of germinating pea seeds on the germination of spores of Mucor racemosus and macroconidia of Fusarium oxysporum are described. Germination of spores of M. racemosus was inhibited by seed metabolites whereas germination of macroconidia of F. oxysporum was stimulated during the first two days and inhibition occurred only after further two days of germination of the seeds. A pronounced inhibition of germination of spores of both micromycetes took place due to absorption of CO2 from volatile and gaseous metabolites. Absorption of some components of seed metabolites in a KMnO4 solution led to a decrease of the inhibitory effect on germination of spores of M. racemosus and stimulatory effect on germination of macroconidia of F. oxysporum.     

Lichen secondary metabolites from the cultured lichen mycobionts of Teloschistes chrysophthalmus and Ramalina celastri and their antiviral activities

Lichens and spore-derived cultured mycobionts of Teloschistes chrysophthalmus and Ramalina celastri were studied chemically, and results indicated that they produced, respectively, parietin and usnic acid as major secondary metabolites, which were purified and identified. Identification of the compounds was performed by high performance liquid chromatography and structural elucidation by nuclear magnetic resonance (1H) and electron impact mass spectrometry. Usnic acid exhibited antiviral activity whereas parietin had a virucidal effect against the arenaviruses Junin and Tacaribe.