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.     

Evaluation of antimicrobial activity of the lichens Lasallia pustulata, Parmelia sulcata, Umbilicaria crustulosa, and Umbilicaria cylindrica

The antimicrobial properties of acetone, methanol, and aqueous extracts of the lichens Lasallia pustulata, Parmelia sulcata, Umbilicaria crustulosa, and Umbilicaria cylindrica were studied comparatively in vitro. Antimicrobial activities of the extracts of different lichens were estimated by the disk diffusion test for Gram-positive bacteria, Gram-negative bacteria, and fungal organisms, as well as by determining the MIC (minimal inhibitory concentration). The obtained results showed that the acetone and methanol extracts of Lasallia pustulata, Parmelia sulcata, and Umbilicaria crustulosa manifest antibacterial activity against the majority of species of bacteria tested, in addition to selective antifungal activity. The MIC of lichen extracts was lowest (0.78 mg/ml) for the acetone extract of Lasallia pustulata against Bacillus mycoides. Aqueous extracts of all of the tested lichens were inactive. Extracts of the lichen Umbilicaria cylindrica manifested the weakest activity, inhibiting only three of the tested organisms.     

Evaluation of antimicrobial activity of the lichens <Emphasis Type="Italic">Lasallia pustulata,Parmelia sulcata,Umbilicaria crustulosa</Emphasis>, and <Emphasis Type="Italic">Umbilicaria cylindrica</Emphasis>

The antimicrobial properties of acetone, methanol, and aqueous extracts of the lichens Lasallia pustulata, Parmelia sulcata, Umbilicaria crustulosa, and Umbilicaria cylindrica were studied comparatively in vitro. Antimicrobial activities of the extracts of different lichens were estimated by the disk diffusion test for Gram-positive bacteria, Gram-negative bacteria, and fungal organisms, as well as by determining the MIC (minimal inhibitory concentration). The obtained results showed that the acetone and methanol extracts of Lasallia pustulata, Parmelia sulcata, and Umbilicaria crustulosa manifest antibacterial activity against the majority of species of bacteria tested, in addition to selective antifungal activity. The MIC of lichen extracts was lowest (0.78 mg/ml) for the acetone extract of Lasallia pustulata against Bacillus mycoides. Aqueous extracts of all of the tested lichens were inactive. Extracts of the lichen Umbilicaria cylindrica manifested the weakest activity, inhibiting only three of the tested organisms.     

Degradation of the disease-associated prion protein by a serine protease from lichens

The disease-associated prion protein (PrP(TSE)), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrP(TSE) inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrP(TSE). Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrP(TSE)-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrP(TSE) and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.     

Nitrate uptake by isolated bionts of the lichen <Emphasis Type="Italic">Parmelia sulcata</Emphasis>

It was found that bionts isolated from the lichen Parmelia sulcata Taylor had a marked difference with respect to nitrate assimilation. Isolated and purified photobiont, the green alga Trebouxia sp. showed no ability of nitrate absorption. Mycobiont and thallus fragments containing both bionts absorbed nitrate. Illumination had no essential influence on the rate of nitrate uptake. The respiratory inhibitor sodium azide decreased the rate of nitrate uptake by 80–100%, whereas the photosynthetic inhibitor dichlorophenyldimethylurea did not reduce it. Using mass-spectroscopic technique, it was shown that, in the intact thallus, nitrate was first absorbed by the mycobiont, and only later appeared in the photobiont. Probably such nutritional difference between bionts serves as one of the mechanisms by which the host fungi control the associated green algae and support their symbiosis.     

Melanogenesis inhibitory effects of methanolic extracts of Umbilicaria esculenta and Usnea longissima

The primary objective of this study was to assess the in vitro melanogenesis inhibitory effects of methanolic extracts of the edible and medicinal lichens, Umbilicaria (Gyrophora) esculenta and Usnea longissima. The quantities of the total phenolic compounds of methanolic extract of the two lichen extracts were determined to be 1.46% and 2.62%, respectively. In order to evaluate the antioxidative effects of the extracts, we also measured electron donating abilities (EDA) and lipid peroxidation rates. The EDA values measured by the reduction of 1.1'-diphenyl-2-picrylhydrazyl (DPPH) were 72.8% and 80.7% for the extracts, with SC50 (median scavenging concentration) values of 1.29+/-0.05 mg/ml and 1.03+/-0.06 mg/ml, respectively. The rates of inhibition of lipid peroxidation using linoleic acid were 92.1% and 97.3% for the extracts, with IC50 (median inhibitory concentration) values of 0.57+/-0.05 mg/ml and 0.53+/-0.06 mg/ml, respectively. The inhibitory rates of the extracts against tyrosinase were 67.4% and 84.8%, respectively. The extracts were shown to reduce melanin formation in human melanoma cells. Melanin contents in the samples treated with 0.01% and 0.1% U. esculenta were 47.1% and 31.2%, respectively, and those treated with 0.01% and 0.1% Usnea longissima were 51.1% and 34.9%, respectively, whereas a value of 54.0% was registered when ascorbic acid was utilized as a positive control. In addition to direct tyrosinase inhibition, it was determined that the lichen extracts affected the activity of tyrosinase via the inhibition of tyrosinase glycosylation. As a result, the methanolic extracts of U. esculenta and Usnea longissima evidenced melanogenesis inhibitory effects, which occurred via multiple routes.     

Purification and properties of phaseolamin, an inhibitor of alpha-amylase, from the kidney bean, Phaseolus vulgaris.

Kidney beans, Phaseolus vulgaris, contain a proteinaceous inhibitor of alpha-amylase, which we have named phaseolamin. The inhibitor has been purified to homogeneity by conventional protein fractionation methods involving heat treatment, dialysis, and chromatography on DEAE-cellulose, Sephadex G-100, and CM-cellulose. Phaseolamin is specific for animal alpha-amylases, having no activity towards the corresponding plant, bacterial, and fungal enzymes, or any other hydrolytic enzyme tested. Optimal inhibitory activity is expressed during preincubation of enzyme and inhibitor at pH 5.5 and 37 degrees. Substrate prevents inhibition. Measurement of the stoichiometry on inhibition showed that a 1:1 complex of alpha-amylase and inhibitor is formed. Complex formation was demonstrated by chromatography on Sephadex G-100. The phaseolamin-amylase complex is dissociated at low pH values, apparently as a result of destruction of the enzyme; the complex cannot be dissociated by other conditions unfavorable for inhibition (low temperature or high pH). Phaseolamin inhibits hog pancreatic alpha-amylase in a noncompetitive manner.     

Antimicrobial activity of extracts of the lichen Parmelia sulcata and its salazinic acid constituent

The antimicrobial activity of the acetone, chloroform, diethyl ether, methanol, and petroleum ether extracts of the lichen Parmelia sulcata and its salazinic acid constituent have been screened against twenty eight food-borne bacteria and fungi. All of the extracts with the exception of the petroleum ether extract showed antimicrobial activity against Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Proteus vulgaris, Yersinia enterocolitica, Staphylococcus aureus, Streptococcus faecalis, Candida albicans, Candida glabrata, Aspergillus niger, Aspergillus fumigatus, and Penicillium notatum. Salazinic acid did not show antimicrobial activity against L. monocytogenes, P. vulgaris, Y. enterocolitica, and S. faecalis but showed activity against Pseudomonas aeruginosa and Salmonella typhimurium as well. The MIC values of the extracts and the acid for the bacteria and fungi have also been determined.     

Hydrolytic activities of anaerobic fungi from wild blue bull (Boselaphus tragocamelus)

The anaerobic fungi play an active role in the plant fibre degradation by producing a wide array of potential hydrolytic enzymes in the rumen. In present study, 12 anaerobic fungal strains were isolated from the faecal samples of wild blue bull, and identified as species of Piromyces, Anaeromyces, Orpinomyces and Neocallimastix based on their morphological characteristics. Isolate WNG-12 (Piromyces sp.), showed maximum filter paper cellulase (23 mIU ml(-1)) and xylanase (127 mIU ml(-1)) activity, while WNG-5 (Piromyces sp.) showed maximum carboxymethyl cellulase activity (231 mIU ml(-1)). Based on the results obtained, it can be stated that Piromyces sp. WNG-12 may be a promising isolate in utilizing fibre rich diets in the rumen as evidenced by the production of hydrolytic enzymes in vitro.     

Chitinolytic and cellulolytic Pseudomonas sp. antagonistic to fungal pathogens enhances nodulation by Mesorhizobium sp. Cicer in chickpea.

Pseudomonas strains isolated from the rhizosphere of chickpea (Cicer arietinum L.) and green gram (Vigna radiata L.) were screened for the production of chitinases and cellulases. Five Pseudomonas strains were found to produce appreciable amounts of both enzymes in culture-free supernatants and showed growth inhibition of the two fungi Pythium aphanidermatum (Oomycete) and Rhizoctonia solani (Basidiomycete) in plates on potato dextrose agar medium. The fungal growth inhibition was not correlated with cell wall-degrading enzyme activity, which suggested that other antifungal compounds produced by these rhizobacteria were also involved in antagonism. Coinoculation of the Pseudomonas strains with the Mesorhizobium sp. Cicer strain Ca181 resulted in a significant increase in nodule biomass when grown under sterilized chillum jar conditions. The results suggest that hydrolytic enzymes produced by Pseudomonas sp. contribute to suppression of plant diseases by inhibiting growth of phytopathogenic fungi and also promote nodulation of legumes by rhizobia.     

Ribose 1,5-bisphosphate inhibits fructose-1,6-bisphosphatase in rat kidney cortex

Fructose-1,6-bisphosphatase is one of the regulatory enzymes of gluconeogenesis in kidney cortex. The effect of ribose 1,5-bisphosphate on fructose-1,6-bisphosphatase purified from rat kidney cortex was studied. Rat kidney cortex, fructose-1,6-bisphosphatase exhibited hyperbolic kinetics with regard to its substrate, but the activity was inhibited by ribose 1,5-bisphosphate at nanomolar concentrations. The inhibitory effect of ribose 1,5-bisphosphate on the fructose-1,6-bisphosphatase was enhanced in the presence of AMP, one of the inhibitors of fructose-1,6-bisphosphatase. Fructose-2,6-bisphosphate, which is an inhibitor of fructose-1,6-bisphosphatase, inhibited rat kidney cortex fructose-1,6-bisphosphatase activities at a low concentration of fructose-1,6-bisphosphate but a high concentration of fructose-1,6-bisphosphate relieved fructose-1,6-bisphosphatase from fructose-2,6-bisphosphate-dependent inhibition. On the contrary, fructose-1,6-bisphosphate was not effective for the recovery of fructose-1,6-bisphosphatase from ribose 1,5-bisphosphate-dependent inhibition. These results suggest that ribose 1,5-bisphosphate is a potent inhibitor and is involved in the regulation of fructose-1,6-bisphosphatase in rat kidney cortex.     

Lipid and Fatty Acid Composition of Tree-Growing and Terrestrial Lichens

A comparison of lipid and fatty acid composition was made of the tree-growing lichens Evernia prunastri (L.) Ach., Parmelia saxatilis (L.) Ach. and Hypogymnia physodes (L.) Ach. and the terrestrial species Cetraria islandica (L.) Ach. and Cladonia impexa Harm. In the terrestrial species the total lipid content varied strongly during spring, while the tree-growing species showed much less variation. Phospholipid and sterol content of all lichens was unusually low. Monoglycosyl diglyceride was absent from Parmelia saxatilis. Fatty acids common to higher plants as palmitic, stearic, oleic, linoleic and linolenic acid were invariably present in all lichen species. In addition large quantities of extra-long chain fatty acids like behenic acid, eicosadienoic acid and cyclic aliphatic lichen acids were present in the terrestrial species. The degree of (poly) unsaturation decreased in the order Evernia prunastri, Parmelia saxatilis. Fatty acids common to higher plants as palmitic, stearic, impexa, which decrease was compensated by an increase in extralong chain fatty acids and lichen acids. It is suggested that the lichen acids are of adaptive value for lichen species growing in the terrestrial habitats, which were characterized by extreme diurnal temperature variations. Just as the polyunsaturated fatty acids, lichen acids guarantee at lower temperatures a high flexibility of the membranes involved, at the same time as they are less susceptible to photo-oxidation at the high daytime temperatures.     

The antimicrobial activity of substances derived from the lichens Physcia aipolia, Umbilicaria polyphylla, Parmelia caperata and Hypogymnia physodes

In this study, in vitro antimicrobial activity of the physodic acid, usnic acid, atranorin and gyrophoric acid isolated from the lichens Hypogymnia physodes, Parmelia caperata, Physcia aipolia and Umbilicaria polyphylla, has been investigated. An antibiotic assessment was done against six bacteria (three Gram-positive and three Gram-negative) and eight fungi by determining the minimal inhibitory concentration (MIC) by the broth tube dilution method. The tested lichen substances inhibited growth of all the tested microorganisms. The bacteria showed a higher sensitivity against the tested fungi. The highest antimicrobial activity was found in the usnic acid of the Parmelia caperata lichen, where the lowest MIC was 0.0037 mg/ml against the Klebsiella pneumoniae (even lower than the one given by the streptomycin standard). The weakest antimicrobial activity was found in the physodic acid, which inhibited most of the microorganisms in the concentration of 1 mg/ml. Generally, all the components had relatively strong antimicrobial activity against the tested microorganisms, among which were human and animal pathogens. This could be of significance for their use for pharmaceutical purposes.     

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。     

Screening the antioxidant and antimicrobial properties of the lichens Parmelia saxatilis, Platismatia glauca, Ramalina pollinaria, Ramalina polymorpha and Umbilicaria nylanderiana.

The aim of this study is to investigate in vitro antimicrobial and antioxidant activities of the methanol extracts of Parmelia saxatilis (L) Ach., Platismatia glauca (L.) W.L. Club. & C.F. Culb., Ramalina pollinaria (Wesstr.) Ach., Ramalina polymorpha (Liljeblad) Ach. and Umbilicaria nylanderiana (Zahlbr.) H. Magn. Antioxidant activity was evaluated by two separate methods: scavenging of free radical DPPH and the inhibition of linoleic acid oxidation. Extracts of Parmelia saxatilis, Platismatia glauca., Ramalina pollinaria and Ramalina polymorpha did not exert any activity in both assays, whereas those of Umbilicaria nylanderiana provided 50% inhibition at 400.2 microg/ml concentration in the former and gave 53% inhibition at 2g/l concentration. Total phenolic constituents of extracts from lichen species tested (P. saxatilis, P. glauca, R. pollinaria, R. polymorpha and U. nylanderiana) were 1.0% (w/w), 1.1% (w/w), 1.0% (w/w), 0.8% (w/w) and (3.0% w/w), respectively (as gallic acid equivalent); implying that the observed activity could be related to the amount of polar phenolics. Extracts were also found to possess antimicrobial activity against some test bacteria and fungi and yeast.     

Effect of quinacrine treatment on the activity of acid hydrolases (phosphatase, N-acetyl-beta-glucosaminidase, glucosidase, galactosidase and esterase) in Tetrahymena.

The effect of phospholipase A2 (PLA2) inhibitor, quinacrine, on the activity of hydrolytic enzymes in Tetrahymena pyriformis homogenate, was investigated. The activity of all of the enzymes studied (acid phosphatase, N-acetyl-beta-glusosaminidase, glucosidase, galactosidase and esterase) was significantly reduced in the presence of quinacrine. Since there are no data on the inhibitory effect of PLA2 and PLA2 influenced metabolic pathways to the hydrolytic enzymes, the direct effect of quinacrine on the hydrolytic enzymes (of Tetrahymena) can be supposed. This is supported by the fact that the other PLA2 inhibitor, 4-bromophenacyl bromide, did not influence phosphatase activity.     

Simple method for fractionation of <Emphasis Type="Italic">Parmelia sulcata</Emphasis> lichen thalli

Three fractions were isolated from Parmelia sulcata lichen thalli: mycobiont-enriched fraction, highly purified preparation of Trebouxia sp. alga, and a photobiont-enriched fraction, in which the fungal component and symbiotic relations between the bionts were retained. The ratio between the fungal and algal components in all fractions was estimated from the photosynthesis/respiration and chlorophyll/dry weight ratios. Simple methods for lichen thallus fractionation are suggested to study symbiotic relationships of bionts.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 306–310.Original Russian Text Copyright © 2005 by Maslov, Pavlova.This revised version was published online in April 2005 with a corrected cover date.     

Production of enzymes and antimicrobial compounds by halophilic Antarctic <Emphasis Type="Italic">Nocardioides</Emphasis> sp. grown on different carbon sources

This study demonstrated the potential of microbial isolates from Antarctic soils to produce hydrolytic enzymes by using specific substrates. The results revealed potential of the strains to produce a broad spectrum of hydrolytic enzymes. Strain A-1 isolated from soil samples in Casey Station, Wilkes Land, was identified as Nocardioides sp. on the basis of morphological, biochemical, physiological observations and also chemotaxonomy analysis. Enzymatic and antimicrobial activities of the cell-free supernatants were explored after growth of strain A-1 in mineral salts medium supplemented with different carbon sources. It was found that the carbon sources favored the production of a broad spectrum of enzymes as well as compounds with antimicrobial activity against Gram-positive and Gram-negative bacteria, especially Staphylococcus aureus and Xanthomonas oryzae. Preliminary analysis showed that the compounds with antimicrobial activity produced by the strain A-1 are mainly glycolipids and/or lipopeptides depending on the used carbon source. The results revealed a great potential of the Antarctic Nocardioides sp. strain A-1 for biotechnological, biopharmaceutical and biocontrol applications as a source of industrially important enzymes and antimicrobial/antifungal compounds.     

拮抗临床致病菌的一株地衣内生真菌

本研究运用组织分离法对采自贵州省都匀市斗篷山景区的地衣进行内生真菌分离,乙酸乙酯萃取菌株液体发酵物,通过打孔法筛选地衣内生真菌提取物并对9株临床致病菌株及耐药菌株进行抑菌活性实验.结果表明一株分离自星点梅衣属地衣Puncteliasp.的内生真菌DPS-165-9对其中的6株显示抑制活性.此6株菌为金黄色葡萄球菌Sta...     

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).