Usnic Acid Content Research Articles

Wpływ bazy kosmetycznej na uwalnianie kwasu usninowego

Usnic acid is a type of lichen acid which exhibits a broad spectrum of biological activity, functioning as an antimicrobial, an anti-inflammatory and a UV-filter. It is used as an active ingredient in herbal medicines and cosmetics products. Its influence as a cosmetics base, including through properties such as emulsion type or degree of internal phase droplet size, on the release of usnic acid, was studied. Two types (O/W, W/O) of usnic acid – loaded, stable emulsions with varying degrees of dispersion of the dispersed phase were obtained. The concentration of usnic acid in all of the formulations studied was 0.05% w/w. The formulations’ physicochemical properties, including stability, viscosity, pH and the droplet size of the dispersed phase, were evaluated. The kinesis of the usnic acid’s release was performed in a thermostatic diffusion chamber, at a temperature of T = 32°C, using dialysis membranes of regenerated cellulose. A mixture of phosphate buffer (PBS) and ethanol (EtOH), in 90/10, PBS/EtOH, volume ratio, was used as an acceptor solution. The concentration of released usnic acid was analyzed by UV-Vis spectrophotometer. The results showed that the O/W emulsions, as compared to W/O emulsions, are a much more effective means of administering usnic acid to the skin. After 24 hours, approx. 45% of the initial acid concentration penetrated into the acceptor solution from O/W emulsion, while from the W/O emulsion the released amount was about 18%.

The Impact of Cadmium on Photosynthetic Performance and Secondary Metabolites in the Lichens Parmelia sulcata, Flavoparmelia caperata and Evernia prunastri

Abstract Lichens are one of the most common air quality bioindicators. Airborne heavy metal pollution causes various physiological changes in lichens, but sensitivity to metal pollution is species specific. In this research, three lichen species (Parmelia sulcata, Flavoparmelia caperata and Evernia prunastri) were exposed to cadmium (50 mg L−1) in laboratory conditions. Photosynthetic efficiency of photosystem II and content of secondary metabolites were determined after one, three and eight days of exposure. In all investigated species treatment of lichen thalli with cadmium significantly changed Fv/Fm and RFd only after eight days of exposure. Quantification of metabolites showed a decreased content of the medullary depsidones salazinic acid (in P. sulcata) and protocetraric acid (in F. caperata) but increased content of cortical depside atranorin (in P. sulcata) and dibenzofurane usnic acid (in F. caperata) after cadmium exposure. However, no changes in secondary metabolites were found in E. prunastri. Results show that investigated species are relatively resistant to short-term cadmium-exposure and that secondary metabolites could have an important role in the protection of primary metabolism from negative cadmium impacts, at least in some species.

Usnic Acid Activity on Oxidative and Nitrosative Stress of Azole-Resistant Candida albicans Biofilm

Several studies report that (+)-usnic acid, a lichen secondary metabolite, inhibits growth of different bacteria and fungi; however, the mechanism of its antimicrobial activity remains unknown. In this study, we explored the ability of usnic acid, obtained from Usnea amblyoclada, as an antibiofilm agent against azole-resistant and azole-sensitive Candida albicans strains by studying the cellular stress and antioxidant response in biofilms. The biofilm inhibitory concentration of usnic acid (4 µg/mL) exhibited a significant biofilm inhibition, 71.08 % for azole-resistant and 87.84 % for azole-sensitive C. albicans strains. Confocal scanning laser microscopy showed that the morphology of mature biofilm was altered (reduced the biomass and thickness) in the presence of usnic acid. The antifungal effect was mediated by an oxidative and nitrosative stress, with a significant accumulation of intracellular and extracellular reactive oxygen species detected by confocal scanning laser microscopy and by nitro blue tetrazolium, respectively. In fact, azole-resistant and azole-sensitive C. albicans biofilms treated at the biofilm inhibitory concentration of usnic acid presented 30-fold and 10-fold increased reactive oxygen species measurements compared to basal levels, respectively, and important nitric oxide generation, showing 25-fold and 60-fold increased reactive nitrogen intermediates levels with respect to the controls, respectively. Nonenzymatic and enzymatic antioxidant defenses were increased in both strains compared to biofilm basal levels as response to the increase of oxidant metabolites. The present study shows for the first time that usnic acid can alter the prooxidant-antioxidant balance, which may be the cause of the irreversible cell damage and lead to cell death. Our results suggest that usnic acid could be an alternative for the treatment of Candida infections, which deserves further investigation.

Usnic acid-incorporated alginate and gelatin sponges prepared by freeze-drying for biomedical applications

There is considerable interest in the pharmaceutical and biomedical areas in the use of sponges, produced with biopolymers, as matrices for the controlled release of drugs. The objective of this work was to evaluate the physicochemical characterization of alginate–gelatin (ALG–GEL) sponges containing usnic acid (UA), by thermogravimetric analysis (TG/DTG), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The macroscopic aspect showed a flexible structure, with variations in coloration depending on the concentration of UA in the sponges. The TG/DTG curves of ALG–GEL sponges and sponges containing 1 and 5 % of UA showed similar profiles and percentages of loss of mass. The DSC curves of both the sponges showed similar profiles, with two events involving a variation of enthalpy being observed, the first an endothermic event and the latter exothermic events, typical of decomposition. These curves show the absence of an endothermic peak of UA at 201 °C, indicating that the drug was present in the polymeric system. The SEM showed the ALG–GEL sponge to have the largest average pore diameter and a more heterogeneous arrangement of polymeric fibers, while sponges incorporated with UA showed more homogeneous arrangement of polymeric fibers. In conclusion, the incorporation of UA did not change the thermal stability of the sponges. Furthermore, the sponges incorporated with the drug showed a microstructural organization suitable for drug release systems.

Evaluation of mutagenic, recombinogenic and carcinogenic potential of (+)-usnic acid in somatic cells of Drosophila melanogaster

The main of this study was to evaluate the mutagenic and carcinogenic potential of (+) – usnic acid (UA), using Somatic Mutation and Recombination Test (SMART) and the test for detecting epithelial tumor clones (wts) in Drosophila melanogaster. Larvae from 72 ± 4 h from Drosophila were fed with UA (5.0, 10.0 or 20.0 mM); urethane (10.0 mM) (positive control); and solvent (Milli-Q water, 1% Tween-80 and 3% ethanol) (negative control). ST cross produced increase in total mutant spots in the individuals treated with 5.0, 10.0 or 20.0 mM of UA. HB cross produced spot frequencies in the concentration of 5.0 mM that were higher than the frequency for the same concentration in the ST cross. In the highest concentrations the result was negative, which means that the difference observed can be attributed, in part, to the high levels of P450, suggesting that increasing the metabolic capacity maximized the toxic effect of these doses. In the evaluation of carcinogenesis using the wts test, the results obtained for the same concentrations of UA show a positive result for the presence of tumors when compared to the negative control. We conclude that UA has recombinogenic, mutagenic and carcinogenic effects on somatic cells in D. melanogaster.

Antioxidant and Antimicrobial Potential, and HPLC Analysis of Stictic and Usnic Acids of Three Usnea Species from Uludag Mountain (Bursa, Turkey).

In this study, antioxidant and antimicrobial potentials of Usnea intermedia, U. filipendula, and U. fulvoreagens and their stictic and usnic acid contents were investigated. Antioxidant activity and total phenolic contents were evaluated in acetone, ethanol, and methanol extracts of these three species. Antioxidant activity was measured by ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] method and total phenolic contents were measured by Folin-Ciocalteu method. High-performance liquid chromatography (HPLC) was used for the determination of lichen acids. It can be concluded from stictic and usnic acids contents that the order of solvent efficiency is acetone > ethanol > methanol and acetone > methanol > ethanol, respectively. Broth microdilution method was performed to determine the minimum inhibitory concentration (MIC) of the methanol extracts of three Usnea species. The MIC values of all the extracts ranged from 64 µg/mL to 512 µg/mL for all the bacterial strains that were tested in this study, and all the Fluoro quinolone-resistant Escherichia coli isolates (except for E101) were sensitive to the methanol extracts of the three Usnea species. This paper is the first study to determine the stictic acid content in U. intermedia and U. filipendula. Our findings indicate that these three Usnea species could be used as antioxidant and antimicrobial agents.

Increased snow accumulation reduces survival and growth in dominant mat-forming arctic-alpine lichens

AbstractRelative growth rates (RGR) and carbon-based secondary compounds (CBSCs) were quantified in four dominant terricolous arctic-alpine mat-forming lichens with different preferences for snow cover. The aim was to evaluate the effects of snow depth, and thus snow cover duration, on lichen growth and performance. The species,Alectoria ochroleuca,Flavocetraria nivalis,Cladonia mitisandCetrariella delisei, are associated with increasing snow depth, respectively. They were transplanted for one year at five snow depths (0, 60, 120, 160 and 200 cm measured in early May) along each of four natural ridge to snow bed gradient transects in oceanic-alpine sites (western Norway). Snow slightly thicker than in source habitats caused negative RGR in the ridge top-dependentA. ochroleucaand the co-occurringF. nivaliswith somewhat higher snow tolerance. OnlyC. mitiswith the broadest ecological niche had positive RGR along most of the gradients (0–160 cm), even outside its natural range. The most snow-tolerant species,C. delisei, tolerant also to temporal inundation in water, had the lowest RGR. Nevertheless, it performed as well in places with little or no snow as in places where it grows naturally. Snow depth significantly affected total concentrations of CBSCs mainly inA. ochroleuca, which experienced substantial mass loss under snow. There was a highly significant increase in usnic acid concentration with increasing mass loss inA. ochroleuca, probably because usnic acid decays more slowly than other components. In conclusion, snow reduced lichen RGR, but in species-specific ways. Therefore, increasing snow depthper sealong spatial and/or temporal scales likely reduces abundance and distribution of dominant mat-forming lichens in the alpine ecosystems of Scandinavia.

Evaluation of Anticancer and Antioxidant Activity of a Commercially Available CO2 Supercritical Extract of Old Man's Beard (Usnea barbata).

There is a worldwide ongoing investigation for novel natural constituents with cytotoxic and antioxidant properties. The aim of this study was to investigate chemical profile and stated biological activities of the supercritical CO2 extract (SCE) of old man’s beard compared to the extracts obtained using the conventional techniques (Soxhlet extracts and macerate). The most abundant compound identified was usnic acid, which content was inversely proportional to the polarity of the solvent used and was the highest in the SCE, which was the sample revealing the highest cytotoxic activity in tested tumor cell lines (B16 mouse melanoma and C6 rat glioma), with lower IC50 values compared to pure usnic acid. Further investigations suggested both SCE and usnic acid to induce apoptosis and/or autophagy in B16 and C6, indicating higher cytotoxicity of SCE to be related to the higher degree of ROS production. A good correlation of usnic acid content in the extracts and their antioxidant capacity was established, extricating SCE as the most active one. Presented results support further investigations of SCE of old man’s beard as a prospective therapeutic agent with potential relevance in the treatment of cancer and/or in oxidative stress-mediated conditions.

СОДЕРЖАНИЕ ВТОРИЧНЫХ МЕТАБОЛИТОВ В ЛИШАЙНИКАХ СОСНОВЫХ ЛЕСОВ ЦЕНТРАЛЬНОЙ ЯКУТИИ

В результате проведенного исследования были выделены вторичные метаболиты эпигейных кустистых лишайников Cladonia amaurocraea, C. arbuscula, C. stellaris, C. rangiferina и Flavocetraria cucullata, произраставших в сосновых лесах Центральной Якутии, которые были идентифицированы как лишайниковые кислоты (дибензофураны, депсиды и α-метилен γ-лактоны). Показано, что содержание лишайниковых кислот в талломах лишайников зависело от типа соснового леса, из которого они были отобраны. Наибольшее содержание усниновой кислоты во всех изученных лишайниках, а также содержание лихестериновой, протолихестериновой кислот в талломах F. cucullata, наблюдалось в образцах, отобранных в бруснично-лишайниковых, толокнянково-бруснично-лишайниковых и шикшево-бруснично-лишайниковых сосняках. В то же время содержание перлатолиевой кислоты, орцинолового депсида и атранорина было, напротив, выше в образцах C. stellaris C. amaurocraea и C. rangiferina, отобранных в лишайниково-брусничном сосняке с примесью лиственницы. Предположено, что различное содержание вторичных метаболитов в лишайниках, произрастающих в изученных типах лесных сообществ Центральной Якутии, может быть обусловлено как действием абиотических факторов среды (свет, температура, влажность), так и конкуренцией со стороны других видов растений.

Pharmacological activities of lichens from Russia

Lichens are supposed to have nearly 600 unique chemicals. Usnic acid, one of them, is reported to possess a broad spectrum of pharmacological activities, related to its antioxidative properties. We characterized and screened different lichen extracts/fractions for their antioxidative, antimicrobial, and cytotoxic activity. The lichens Cladonia arbuscula (L.) Hoffm., Evernia prunastri (L.) Ac. and Usnea barbata (L.) Wigg. were collected in Mari El Republic of Russia (June 2011). Chloroform (CHCl3)-, hexane (Hex)-, dichloromethane (DCM) and acetonitrile (ACN) extracts were prepared and analysed by HPLC. As reference (+)-usnic acid was used. The total antioxidant activity was evaluated with phosphomolybdenum method. The total content of phenols was determined by Folin-Ciocalteau method. The antimicrobial activity was estimated by double microdilution method. Toxicity was evaluated (24 hrs) in human skin fibroblasts (HSKF) and in U-87 glioblastoma cells by resazurin assay. Phosphorylation of ERK1/2 was measured in U-87 cells by Western blot. The usnic acid content of the crude extracts (%) of C. arbuscula, E. prunastri and U. barbata were 8.91 ± 1.18, 5.68 ± 0.47, 74.49 ± 8.64 respectively. The tested antimicrobiobal activities (Staphylococcus aureus, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis) and the antioxidative capacity differed for the 3 extracts/fractions and were unrelated to the usnic acid content. In HSKFs the IC50 were 29.3 µg/mL, 78.6 µg/mL, 44.2 µg/mL and 15.5 µg/mL for the extracts of E. prunastri (DCM, ACN) and C. arbuscola (Hex, DCM) respectively. In U87 cells IC50 values ranged from 10.8 µg to 98.1 µg/mL. Phosphorylation of ERK1/2 was concentration dependently reduced. The tested pharmacological activities did not relate to the usnic acid content. The identification of further secondary metabolites with in depth investigations of different modes of action is required for drug development. Acknowledgement: A. Shcherbakova was supported by the DAAD.

ГОДОВАЯ ДИНАМИКА СОДЕРЖАНИЯ УСНИНОВОЙ КИСЛОТЫ В ТАЛЛОМАХ ЛИШАЙНИКОВ РОДОВ CLADONIA И FLAVOCETRARIA, ПРОИЗРАСТАЮЩИХ В ЦЕНТРАЛЬНОЙ ЯКУТИИ

Annual dynamics of the content of usnic acid, the most frequently encountered and well-studied representative of lichen substances thalli Cladonia stellaris , Cladonia arbuscula and Flavocetraria cucullata , growing in pine- crowberry-cowberry-lichen forest, located in the vicinity of Yakutsk was studied. Determination of usnic acid in lichen thalli analyze by high performance liquid chromatography showed that the highest content of usnic acid (1,6–1,.7% of dry weight) is typical for lichens, collected in the June, and the lowest (0,7–0,8% of dry weight) - December. A comparison of monitoring data of temperature on the soil surface and content of usnic acid in lichen thalli were found a direct positive correlation - C. stellaris (r = 0,88; p <0.01), C. arbuscula (r = 0,81; p <0,01 ) and F. cucullata (r = 0,68; p = 0,01), which confirms the important role of the temperature factor in the biosynthesis of a secondary metabolite. The generally show that the best period of the studied lichens collection growing in the conditions of Central Yakutia (sharply continental climate, permafrost) for isolate usnic acid is the beginning of the summer.

Abstract 3220: Lichen derived polyphenols as potential anticancer drugs

Abstract Polyphenols such as resveratrol, curcumin, (-)-epigallocatechin 3-gallate (EGCG) etc. have been shown to have anticancer properties. The anticancer activities of polyphenols include, but are not limited to, anti-oxidative effects, pro-apoptosis, DNA damage, anti-angiogenic effects, and immunostimulation. Lichens, symbiotic systems involving a fungus and an alga and/or cyanobacterium, are a rich source of polyphenols. Based on our previous research we have demonstrated that crude extracts of different lichen species affect cell proliferation, cell cycle dynamics, and cause apoptosis in Burkitt's lymphoma (Raji cells). This research examines the effects of a lichen-derived polyphenol compound, usnic acid, on human colon adenocarcinoma (HT-29) cells. We treated HT-29 cells with various concentrations (10, 30 and 50 µg/ml) of usnic acid for 24 hours. Although the viability was not affected by the treatment, there was a significant decrease in cell proliferation in dose-dependent manner. We also observed that after treatment, the morphology of treated cells was completely different from that of untreated control cells. Using transmission electron microscopy (TEM), we studied the morphological differences between control and treated HT-29 cells. The TEM micrographs clearly show that usnic acid damaged the mitochondria, reduced the amount of free ribosomes, and also distorted the shape of the nucleus. As the treatment of HT-29 with usnic acid reduced the rate of cell proliferation significantly, we investigated the effects of usnic acid on HT-29 cell cycle dynamics. Our results indicate that with the higher usnic acid concentrations (30 and 50 µg/ml) the cell cycle was stopped at the G0/G1 stage. These results further document the pattern of decreasing proliferation of HT-29 cells treated with usnic acid. With the distortion of the nucleus following exposure to usnic acid, we also investigated the effects of usnic acid on DNA the HT-29 cells. We performed a single cell gel electrophoresis (Comet Assay) to study the DNA damaging effects of various concentrations of usnic acid. Our results showed that there was a dose dependent increase in damage to the HT-29 cells' DNA resulting in 56.7% of the total DNA moving into the comet tail at 50 µg/ml concentration of usnic acid. These preliminary results suggest that lichen derived polyphenols could be a potential source of anticancer drug therapies. However, additional research is needed to further validate these findings. Citation Format: Gajendra Shrestha, Michael Xiao, Richard Robison, Larry L. St. Clair, Kim O'Neill. Lichen derived polyphenols as potential anticancer drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3220. doi:10.1158/1538-7445.AM2014-3220

Mycotoxins and usnic acid and their distribution in lichens belonging to the genera Cetraria, Flavocetraria, and Cladonia

The content and the pattern of distribution of mycotoxins and usnic acid in thalli of lichens belonging to the genera Cladonia (four species), Cetraria (two species), and Flavocetraria (two species) have been studied using the method of immunoenzymatic analysis. Statistically significant differences have been estimated between the upper and the lower parts of the thalli. The levels of the mycotoxins in the lower, older parts of the thalli have been higher than in the upper, younger ones. The comparison of lichen specimens belonging to the same species and collected in the same location at different years did not reveal statistically significant differences in the content of mycotoxins both in the upper and the lower parts of the thalli. Statistically significant differences in the usnic acid content have been revealed only in the case of C. stellaris. In the upper part of the podetia of this species, the usnic acid content has been higher than in the lower one.

In vitro risk assessment of usnic acid

Lichens are symbiotic organisms composed of fungi and algae and are very common in Turkey. Lichen secondary metabolites are mainly phenolic compounds produced by fungal partner of lichen symbiosis. Usnic acid (UA) is one of the most common lichen metabolites, and it was reported that to be effective for a wide range of pharmacological purposes including antiviral, antitumor, and antiprotozoal. However, there are limited data on the genotoxic and antioxidant effects of UA in cultured human peripheral blood cells. Therefore, the aim of this thesis study was to investigate the genetic and oxidative effects of UA in cultured human blood cells (n = 5). The UA was added into culture tubes at various concentrations (0-200 μg/ml). Chromosomal aberrations (CA) and micronuclei (MN) tests were performed for genotoxic damage influences estimation. In addition, biochemical parameters (total antioxidant capacity (TAC) and total oxidative status (TOS)) were examined to determine oxidative effects. In our in vitro test systems, it was observed that UA had no mutagenic effects on human lymphocytes. Furthermore, our results indicated that low concentrations (1 and 5 μg/ml) of UA caused increases of TAC levels in cultured human blood cells. And, the TOS levels were not changed (p > 0.05) when all the concentrations (except for 200 μg/ml) of UA were applied. In conclusion, UA can be a new resource of therapeutics as recognized in this study with their nonmutagenic and antioxidant features.

Water Soluble Usnic Acid-Polyacrylamide Complexes with Enhanced Antimicrobial Activity against Staphylococcus epidermidis

Usnic acid, a potent antimicrobial and anticancer agent, poorly soluble in water, was complexed to novel antimicrobial polyacrylamides by establishment of strong acidic-base interactions. Thermal and spectroscopic analysis evidenced a molecular dispersion of the drug in the polymers and a complete drug/polymer miscibility for all the tested compositions. The polymer/drug complexes promptly dissolved in water and possessed a greater antimicrobial activity against Staphylococcus epidermidis than both the free drug and the polymer alone. The best results were obtained with the complex based on the lowest molecular weight polymer and containing a low drug content. Such a complex showed a larger inhibition zone of bacterial growth and a lower minimum inhibitory concentration (MIC) with respect to usnic acid alone. This improved killing effect is presumably due to the reduced size of the complexes that allows an efficient cellular uptake of the antimicrobial complexes. The killing effect extent seems to be not significantly dependent on usnic acid content in the samples.

Assessment of the genotoxicity and antigenotoxicity of (+)-usnic acid in V79 cells and Swiss mice by the micronucleus and comet assays

Usnic acid is one of the most common and abundant metabolites found in various lichen genera, which are important sources of biologically active compounds. The aim of this study was to evaluate the genotoxic and antigenotoxic potential of (+)-usnic acid (UA) by the micronucleus and comet assays in V79 cell cultures and Swiss mice. For assessment of genotoxicity, V79 cells were treated with 15, 30, 60, and 120μg/mL UA, established based on clonogenic efficiency cytotoxic assay. Swiss mice were treated with UA doses of 25, 50, 100, and 200mg/kg body weight. The same concentrations of UA were combined with methyl methanesulfonate (MMS) for evaluation of antigenotoxicity. The in vitro results demonstrated that UA induced DNA damage at concentrations of 60 and 120μg/mL in the comet assay. However, no genotoxic effect was observed in the micronucleus test using V79 cells at the concentrations tested. No genotoxic effects were observed for the different UA treatments in in vivo test system. Combined administration of UA and MMS significantly reduced the frequencies of micronuclei and DNA damage in vitro and in vivo when compared to treatment with MMS alone. Although the mechanisms underlying the protective effect of UA are not completely understood, the antioxidant activity of this metabolite may explain its protective effect against MMS-induced genotoxicity.

Influence of different pre-treatment methods on isolation of extracts with strong antibacterial activity from lichen Usnea barbata using carbon dioxide as a solvent

Multiplication and spreading of multiresistant pathogenic bacteria that cause fatal infections in humans and animals have boosted research on implementation of newer technologies for producing natural substances with strong antibacterial activities. In this study, SFE from lichen Usnea barbata was investigated for different pre-treatment methods with respect to extracts’ yield, usnic acid recovery and antibacterial activity against more than thirty methicillin resistant staphylococci strains. Extraction of U. barbata with subcritical carbon dioxide was studied as well. Extraction yield, usnic acid content and antibacterial activity of the extracts were strongly affected by the pre-treatment method. Intensive pre-treatment methods such as flaking and cutting+grinding caused a considerable comminution of the lichen thallus and enabled high extraction yields. On the other hand, mild pre-treatment of the lichen such as impact+shearing enabled isolation of extracts with lower MIC values. Experimental results indicated that extraction with liquid carbon dioxide at 30MPa and 25°C was the most favorable condition at which a satisfactory high extraction yield was obtained (2.08%). Extract isolated with liquid carbon dioxide had a high usnic acid content in the extract (632g/kg) and a very strong antibacterial activity (average MIC of 19.11μg/mL). Antibacterial activity of all the extracts against tested strains was comparable to the pure usnic acid or even stronger. This study indicated a potential use of the supercritical extracts of U. barbata in pharmaceutical formulations for treatment of infections caused by staphylococci.

SFE as a superior technique for isolation of extracts with strong antibacterial activities from lichen Usnea barbata L.

The number of cases of fatal infections in humans and animals caused by multiresistant and panresistant bacterial strains has been dramatically increased over the past 10 years on almost every continent. Therefore, implementation of newer technologies in order to develop processes for isolation of substances with strong antibacterial activities from natural sources is of increasing interest. In this study, advantage of high pressure processing and supercritical fluid extraction with carbon dioxide in isolation of active principles from lichen Usnea barbata L. was presented. Supercritical extraction showed extremely higher selectivity toward usnic acid compared to the conventional techniques tested. In order to simulate the process and estimate mass transfer coefficients, the new mathematical model of Sovová (2011) was applied. Supercritical extract obtained at 40°C and 30MPa showed stronger antibacterial activity against the most of tested strains compared to pure usnic acid and extracts obtained using ethanol as a solvent. Further, supercritical extract with lower usnic acid content expressed stronger antibacterial activity against the majority of tested strains including MRSA strains compared to the pure usnic acid and the supercritical extract with the higher usnic acid content. The same extract was far more effective than ampicillin against Streptococcus uberis, all tested Enterococcus faecalis, MRSA and MRSA ATCC 33591. Against few tested strains, the supercritical extract was superior to erythromycin and penicillin as well. These findings justified possible benefits of inclusion of supercritical extracts into pharmaceutical and cosmetic formulations. Applied mathematical model described the SFE process with high accuracy.

Interactive toxicity of usnic acid and lipopolysaccharides in human liver HepG2 cells

Usnic acid (UA), a natural botanical product, is a constituent of some dietary supplements used for weight loss. It has been associated with clinical hepatotoxicity leading to liver failure in humans. The present study was undertaken to evaluate the interactive toxicity, if any, of UA with lipopolysaccarides (LPS), a potential contaminant of food, at low non-toxic concentrations. The human hepatoblastoma HepG2 cells were treated with the vehicle control and test agents, separately and in a binary mixture, for 24 h at 37°C in 5% CO2. After the treatment period, the cells were evaluated by the traditional biochemical endpoints of toxicity in combination with the toxicogenomic endpoints that included cytotoxicity, oxidative stress, mitochondrial injury and changes in pathway-focused gene expression profiles. Compared with the controls, low non-toxic concentrations of UA and LPS separately showed no effect on the cells as determined by the biochemical endpoints. However, the simultaneous mixed exposure of the cells to their binary mixture resulted in increased cytotoxicity, oxidative stress and mitochondrial injury. The pathway-focused gene expression analysis resulted in the altered expression of several genes out of 84 genes examined. Most altered gene expressions induced by the binary mixture of UA and LPS were different from those induced by the individual constituents. The genes affected by the mixture were not modulated by either UA or LPS. The results of the present study suggest that the interactions of low nontoxic concentrations of UA and LPS produce toxicity in HepG2 cells.

USNIC ACID CONTENT AND ANTIOXIDANT ACTIVITY OF THREE LICHEN EXTRACTS

s. PHYTOPHARM 2012 M 68 Obzory po kliniceskoj farmacologii i lekarstvennoj terapii [Reviews of clinical pharmacology and drug therapy] ТОМ 10/2012/2 (+)-USNIC ACID CONTENT AND ANTIOXIDANT ACTIVITY OF THREE LICHEN EXTRACTS © Koptina A. , Shcherbakova A. , Soldati F. , Gabdullin M. , Kanarskiy A. , Ulrich-Merzenich G. 3 Mari State Technical University, Yoshkar-ola, Russia Pharmaton SA, 6934 Bioggio, Switzerland Medical Clinic III, University of Bonn, Willhelmstr. 35–37, 53111 Bonn, Germany lichens produce a variety of secondary metabolites which belong to the depsides, depsidones, dibenzofurans and pulvinic acid derivative family. They proved to be a good source of natural antioxidants (1). Usnic acid uniquely found in many lichens has been reported to have analgesic, antimicrobial, anti-inflammatory, antiplatelet/antithrombotic, antiviral, cytotoxic/antiproliferative, gastroprotective, weight loss, and wound healing activity (2). We examined the (+)-usnic acid content and the anti-oxidative properties of the chloroform extract of lichen: Cladonia sylvatica (l.) hoffm., Evernia prunastri (l.) Ach. and Usnea barbata (l.) Wigg. (s. i.) were collected in Mari El Republic of Russian federation in June, 2011. Reverse phase hPlC analysis was carried out (Perkin Elmer Series 200 hPlC instrument; C18 column (C18; 25 cm ×4.6 mm, 5μm); UV/Vid detector; solvent: methanol-water-phosphoric acid (80:20:.9, v/v/v)). The sample injection volume was 20 μl and the flow rate 1.0 ml/min. A standard of (+)-usnic acid was used. The total antioxidant activity of the extracts was evaluated by the phosphomolybdenum method as described Manojlovic et al. (3). The chloroform extracts of C. sylvatica, E. prunastri and U. barbata showed contents of (+)-usnic acid of 8.91 ± 1.18, 5.68 ± 0.47 and 74.49 ± 8.64 % respectively. The antioxidant activities were: 277.1 ± 20.0, 171.1 ± 17.1 and 205.8 ± 7.2 mg of ascorbic acid (AA) per g of extract respectively; in comparison, the antioxidant activity of the (+)-usnic acid standard was determined as 154 ± 6.5 mg AA/g. We can report that U. barbata is a good source of (+)-usnic acid. Other secondary metabolites than (+)-usnic acid present in C. sylvatica and E. prunastri contribute to their high antioxidant activity and need to be further investigated.