Graphene Conjugated Usnic Acid Nano-Formulation For The Treatment Of Topical Fungal Infection

Objective: The study aims to investigate the antifungal response of the dug usnic acid with the carrier graphene. Methods: Nano-precipitation method by sonication was adopted to formulate the conjugate. SEM test was performed to check the shape and average size of the conjugate. FTIR test was performed for the chemical interaction between the drug and the carrier. Ointment was prepared by the fusion method and the viscosity test was performed by Brookfield viscometer. Spreadability test was performed by slide method. Animal activity was performed to confirm the antifungal effect of the formulated nano-conjugate. Statistical analysis was done by Anova. Results: SEM study shows that the conjugate is in the nano range and possess a spherical shape. FTIR study shows no interaction between the drug and the carrier. The result of in vitro drug release study shows that the conjugate posses a higher drug release rate as compared to the drug alone. Topical drug administration is more suitable for the treatment of the fungal infection, so the nano-conjugate was incorporated into the ointment by geometric mixing. The viscosity and the spreadability test were performed on the different formulations of the ointment and the suitable one was selected for the topical administration. Anti-fungal study had been performed on the Wistar albino rats for 6 d. Skin culture of rats was performed for the formation of the fungal colonies. Statistical analysis by Anova gives p<0.001. It was found that the normal form of usnic acid, graphene and the nano form both possess anti-fungal activity as 3/6 and 2/6 experimental animals are cured by normal formulation and nano-formulation. Conclusion: The present anti-fungal study revealed that the nano-form of the conjugate possess higher anti-fungal activity than the normal formulation of usnic acid with graphene.

miR-185-5p response to usnic acid suppresses proliferation and regulating apoptosis in breast cancer cell by targeting Bcl2

BACKGROUND: Breast cancer is the most common cancer types among women. Recent researches have focused on determining the efficiency of alternative molecules and miRNAs in breast cancer treatment. The AIMof this study was to determine the effect of usnic acid response-miR-185-5p on proliferation in the breast cancer cell and to determine its relationship with apoptosis pathway. METHODS: The cell proliferation and cell apoptosis rate were significantly increased following the ectopic expression of miR-185-5p in BT-474 cells. Furthermore, the results of cell cycle assay performed by flow cytometry revealed that the transfection with miR-185-5p induced G1/S phase arrest. The apoptosis-related genes expression analysis was performed by qRT-PCR and the direct target of miR-185-5p in BT-474 cells was identified by western blot and luciferase reporter assay. RESULTS: Our data showed that miR-185-5p can cause significant changes in apoptosis-related genes expression levels, suggesting that cell proliferation was suppressed by miR-185-5p via inducing apoptosis in breast cancer cells. According to western blot results, miR-185-5p lead to decrease BCL2 protein level in BT-474 cells and direct target of miR-185-5p was identified as BCL by luciferase reporter assay. CONCLUSION: This study revealed that miR-185-5p may be an effective agent in the treatment of breast cancer.

Interaction study between vancomycin and liposomes containing natural compounds against methicillin-resistant Staphylococcus aureus clinical isolates

ABSTRACT The treatment of infections caused by resistant microorganisms is limited, and vancomycin (VAN) treatment failures for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia are not uncommon, even when MRSA clinical isolates are susceptible to VAN. Thus, this study proposed the association of VAN with usnic acid and ß-lapachone encapsulated into liposomes as a novel therapeutic option for infections caused by MRSA. Liposomes containing ß-lap (ß-lap-lipo) or usnic acid (UA-lipo) were prepared by the thin lipid film hydration method followed by sonication. Antimicrobial activity against MRSA clinical isolates was investigated by the microdilution method according to the Clinical and Laboratory Standards Institute (CLSI). The interaction studies were carried out using the checkerboard method and epsilometer test (Etest). The interaction between VAN and ß-lap or ß-lap-lipo was synergistic (FICI = 0.453 and FICI = 0.358, respectively). An additive interaction between VAN and UA (FICI = 0.515) was found. UA-lipo resulted in synergism with VAN (FICI = 0.276). The Etest reproduced the results obtained by the checkerboard method for approximately 82% of the analysis. Thus, the present study demonstrated that VAN in combination with UA-lipo, ß-lap or ß-lap-lipo synergistically enhanced antibacterial activity against MRSA

Pharmacokinetics and tissue distribution of usnic acid phospholipid complex in rats / 中草药

Objective: To study pharmacokinetics and tissue distribution of usnic acid phospholipid complex (UAPC) in rats by oral administration. Methods: HPLC was established for determination of usnic acid (UA) in the rat plasma and tissue. Rats were ig administrated UA suspension and UAPC (35.0, 17.5, and 11.7 mg/kg, with UA count). The concentrations of UA in plasma and tissue were determined by HPLC at the different time points. The pharmacokinetic parameters were calculated by the DAS 2.0 software. Results: Compared with the administration of UA, the main pharmacokinetic parameters of UAPC: Cmax, AUC0-t of UAPC increased significantly (P 0.05) after ig administrations of UAPC at a dose of 11.7 mg/kg, the relative bioavailability of UAPC was 109.67%. The tissue distribution of UAPC: UAPC was distributed more in the liver, spleen and brain at a dose of 35 mg/kg; UAPC was distributed more in the lung and brain at a dose of 17.5 mg/kg; And UAPC was distributed more in the liver and kidney at a dose of 11.7 mg/kg. Conclusion: Phospholipid complex improve the oral bioavailability of UA, and change the distribution in the tissue of rats.

Formulation Optimization and Quality Evaluation of Usnic Acid Self-microemulsion / 中国药房

OBJECTIVE: To prepare Usnic acid self-microemulsion, prescription optimization and evaluate its quality. METHODS: Usnic acid self-microemulsion was prepared by emulsification method using medium chain triglycerides-single linoleic acid glyceride (1:1, m/m) as oil phase, polyoxyethylene hydrogenated castor oil as emulsifier, diethylene glycol monoethyl ether as co-emulsifier. Based on the solubility test and pseudotemary phase diagram, using self-emulsifying time (t), light transmittance (T), drug equilibrium solubility (S), accumulative dissolution rate at 5 min and 60 min (Q5 min, Q60 min) as evaluation indexes, central composite design-response surface methodology was utilized to optimize oil phase ratio and ratio of emulsifier to co-emulsifier (Km) in microemulsion formulation. At the same time, the optimal formulation was verified, the morphology, particle size, drug-loading amount and dissolution of Usnic acid self-microemulsion were investigated. RESULTS: The optimal formulation was as follows as oil phase ratio of 25％, Km of 2. 0; ratio of mixed oil phase, emulsifier, co-emulsifier in the formulation was 25％, 50％, 25％. t, T, S, Q5 min and Q60 min of Usnic acid self-microemulsion prepared by optimal formulation were 1. 96 min, 87. 67％, 5. 67 mg/g, 66. 58％, 76. 73％ (all RSD<3％, n = 3),respectively. The relative errors of them to predicted values were all less than 4％ (n = 3). Usnic acid self-microemulsion was spherical droplet in shape after diluted with water. The average particle size was 39. 4 nm, average drug-loading amount was 4. 55 mg/g. Q90 min of Usnic acid self-microemulsion reached 99. 58％ in pH 6. 8 phosphate buffer (n = 3). CONCLUSIONS: Prepared Usnic acid self-microemulsion is in line with quality requirements.

Chemical and pharmacological progress on usnic acid and its derivatives / 中国中药杂志

Usnic acid and its derivatives, a group of organic molecules with great importance, are characteristic to lichens, possessing pharmacological activities such as anti-virus, anti-bacteria, anti-humor, anti-inflammatory, analgesic, and anaesthetic effects. Many of them have been widely used as medicine, but also bring side effects such as dermatitis and liver damages. In the past decades, great efforts by isolation, organic synthesis, and structure modification methods were put on discovery of UA derivatives with higher biological activities or less side effects. This paper describes herein the most progress on natural sources, isolation and structure elucidation, structural characteristics, synthesis and modification results, pharmacological activities and toxicities of UA and its derivatives, hopefully to provide valuable reference for further research.

In vitro antimicrobial activity of the organic extract of Cladonia substellata Vainio and usnic acid against Staphylococcus spp. obtained from cats and dogs / Atividade antimicrobiana in vitro do extrato orgânico de Cladonia substellata Vainio e ácido úsnico frente Staphylococcus spp. obtidos de cães e gatos

ladonia substellata Vainio is a lichen found in different regions of the world, including the Northeast of Brazil. It contains several secondary metabolites with biological activity, including usnic acid, which has exhibited a wide range of biological activities. The aim of this study was to evaluate the in vitro antimicrobial activity of the organic extract of C. substellata and purified usnic acid. Initially, Staphylococcus spp., derived from samples of skin and ears of dogs and cats with suspected pyoderma and otitis, were isolated and analyzed. In antimicrobial susceptibility testing against Staphylococcus spp., 77% (105/136) of the isolates were resistant to the antimicrobials tested. In the assessment of biofilm production, 83% (113/136) were classified as producing biofilm. In genetic characterization, 32% (44/136) were positive for blaZ, no isolate (0/136) was positive for the mecA gene, and 2% (3/136) were positive for the icaD gene. The in vitro antimicrobial activity of the organic extract of C. substellata and purified usnic acid against Staphylococcus spp. ranged from 0.25mg/mL to 0.0019mg/mL, inhibiting bacterial growth at low concentrations. The substances were more effective against biofilm-producing bacteria (0.65mg/mL-0.42mg/mL) when compared to non-biofilm producing bacteria (2.52mg/mL-2.71mg/mL). Usnic acid and the organic extract of C. substellata can be effective in the treatment of pyoderma and otitis in dogs and cats caused by Staphylococcus spp.(AU)

Cladonia substellata Vainio é um líquen encontrado em diversos continentes do mundo, inclusive no nordeste do Brasil, possui vários metabólitos secundários com atividade biológica, entre eles, o ácido úsnico, que tem apresentado uma vasta gama de atividades biológicas. O objetivo deste trabalho foi avaliar a atividade antimicrobiana in vitro do extrato orgânico de C. substellata e do ácido úsnico purificado. Para isto, foram isolados Staphylococcus spp. de amostras de pele e orelha de cães e gatos com suspeita de piodermatite e otite. No teste de sensibilidade aos antimicrobianos frente Staphylococcus spp., 77% (105/136) foram resistentes. Na avaliação da produção de biofilme 83% (113/136) foram classificadas como produtoras de biofilme. Na caracterização genotípica, 32% (44/136) foram positivos para o gene blaZ, nenhum isolado (n=136) foi positivo para o gene mecA, e 2% (3/136) foram positivos para o gene icaD. A atividade antimicrobiana in vitro do extrato orgânico de C. substellata e do ácido úsnico purificado para Staphylococcus spp. variou de 0,25mg/ml a 0,0019mg/ml, inibindo o crescimento bacteriano em baixas concentrações. Foram mais eficazes contra bactérias produtoras de biofilme (0,65mg/ml-0,42mg/ml) quando comparadas às não produtoras de biofilme (2,52mg/ml-2,71mg/ml). Viabilizando a utilização do ácido úsnico e do extrato orgânico de C. substellata, no tratamento de otite e piodermatite em cães e gatos com o envolvimento de Staphylococcus spp.(AU)

Effects of the encapsulation of usnic acid into liposomes and interactions with antituberculous agents against multidrug-resistant tuberculosis clinical isolates

Mycobacterium tuberculosis (Mtb) has acquired resistance and consequently the antibiotic therapeutic options available against this microorganism are limited. In this scenario, the use of usnic acid (UA), a natural compound, encapsulated into liposomes is proposed as a new approach in multidrug-resistant tuberculosis (MDR-TB) therapy. Thus the aim of this study was to evaluate the effect of the encapsulation of UA into liposomes, as well as its combination with antituberculous agents such as rifampicin (RIF) and isoniazid (INH) against MDR-TB clinical isolates. The in vitro antimycobacterial activity of UA-loaded liposomes (UA-Lipo) against MDR-TB was assessed by the microdilution method. The in vitro interaction of UA with antituberculous agents was carried out using checkerboard method. Minimal inhibitory concentration values were 31.25 and 0.98 µg/mL for UA and UA-Lipo, respectively. The results exhibited a synergistic interaction between RIF and UA [fractional inhibitory concentration index (FICI) = 0.31] or UA-Lipo (FICI = 0.28). Regarding INH, the combination of UA or UA-Lipo revealed no marked effect (FICI = 1.30-2.50). The UA-Lipo may be used as a dosage form to improve the antimycobacterial activity of RIF, a first-line drug for the treatment of infections caused by Mtb.

Major phenolic acids in Usneae Filum by UHPLC-Triple-TOF-MS / 中草药

Objective: To establish a quick method of ultra-performance liquid chromatography-quadrupole time-of-fight mass spectrometry (UHPLC-Triple-TOF-MS) for the identification of phenolic acids (diffractaic acid, usnic acid, orsellinic acid, etc.) in Usneae Filum. Methods: The separation was performed on the chromatographic column of Phenomenex Luna 3u C18 (150 mm × 2.0 mm, 3 μm), and the mobile phase was methanol (0.05% formic acid)-0.05% formic acid solution (4 mmol ammonium acetate), with a gradient elution at a flow rate of 0.3 mL/min. The column temperature was 30℃, and negative ion mode was used for TOF-MS. Results: Seventeen compounds were identified or tentatively characterized based on the retention time and MS spectra. They are depsides, two benzo furan, ant multi substituted single benzene as well. And the preliminary fragmentation rules are summarized. Conclusion: The results demonstrate that UHPLC-Triple-TOF-MS method is novel, quick, and efficient for the identification of the compounds in Usneae Filum.

Does usnic acid affect microtubules in human cancer cells? / O ácido úsnico pode afetar microtúbulos em células cancerosas humanas?

Usnic acid, a lichen metabolite, is known to exert antimitotic and antiproliferative activities against normal and malignant human cells. Many chemotherapy agents exert their activities by blocking cell cycle progression, inducing cell death through apoptosis. Microtubules, protein structure involved in the segregation of chromosomes during mitosis, serve as chemotherapeutical targets due to their key role in cellular division as well as apoptosis. The aim of this work was to investigate whether usnic acid affects the formation and/or stabilisation of microtubules by visualising microtubules and determining mitotic indices after treatment. The breast cancer cell line MCF7 and the lung cancer cell line H1299 were treated with usnic acid 29 µM for 24 hours and two positive controls: vincristine (which prevents the formation of microtubules) or taxol (which stabilizes microtubules). Treatment of MCF7 and H1299 cells with usnic acid did not result in any morphological changes in microtubules or increase in the mitotic index. These results suggest that the antineoplastic activity of usnic acid is not related to alterations in the formation and/or stabilisation of microtubules.

O ácido úsnico, um metabólito de liquens, é conhecido por sua atividade antimitótica e antiproliferativa em células humanas normais e malignas. Muitos quimioterápicos exercem suas atividades bloqueando a progressão do ciclo celular e induzindo morte celular por apoptose. Os microtúbulos, estruturas protéicas envolvidas na segregação dos cromossomos durante a mitose, servem como alvo quimioterapêutico devido ao seu importante papel tanto na divisão celular quanto nos mecanismos de morte celular por apoptose. O objetivo deste trabalho foi investigar se o ácido úsnico afeta a formação e/ou estabilização dos microtúbulos, a partir da visualização de microtúbulos e determinação de índices mitóticos após o tratamento. Células de câncer de mama MCF7 e de câncer de pulmão H1299 foram tratadas por 24 horas com 29 µM de ácido úsnico e dois controles positivos: vincristina (que impede a formação de microtúbulos) e taxol (que estabiliza microtúbulos). O tratamento das células MCF7 e H1299 com o ácido úsnico não resultou em aumento do índice mitótico. Os resultados sugerem que a atividade antineoplásica do ácido úsnico não está relacionada a alterações na formação e/ou estabilização de microtúbulos.

Validação de método analítico espectrofotométrico UV para determinação de ácido úsnico em lipossomas / Validation of a UV-spectrophotometric analytical method for the determination of usnic acid in liposomes

O ácido úsnico (AU) é um composto de origem liquênica e tem demonstrado importantes atividades biológicas, tais como: antitumoral, antimicrobiano, antiviral, antiproliferativo e antiinflamatório. Os lipossomas são vesículas lipídicas contendo espaço aquoso interno e têm sido utilizados como carreadores coloidais de fármacos, principalmente na terapêutica de câncer e infecções bacterianas e fúngicas. O objetivo desse trabalho foidesenvolver e validar um método espectrofotométrico UV para determinação de ácido úsnico em lipossomas. Os parâmetros de validação linearidade, precisão, exatidão, robustez, limites de detecção e quantificação foram determinados segundo diretrizes internacionais de padronização e Farmacopéia Americana. A faixa de linearidade foi de 3 a 15 μg.mL-1, a equação de regressão:absorbância = 0,070 x [AU] (μg.mL-1) + 0,013 e r = 0,9997. A repetibilidade (coeficiente de variação) do método foi 1,96% e a precisão intermediária indicou que a diferença entre as médiasfoi estatisticamente insignificante (P < 0,05). A exatidão revelou média percentual de recuperação de 100,4%. O método foi robusto apesar da variação de temperatura e solventes. Os limites de detecção e quantificação do ácido úsnico foram de 0,34 e 1,13 μg.mL-1, respectivamente. O doseamento do ácido úsnico nos lipossomas foi de 96,8% (± 0,2). O método proposto é exato, preciso e reprodutível sendo capaz de quantificar o ácido úsnicoem matéria-prima e em preparações farmacêuticas.

The secondary lichen metabolite usnic acid [2,6-diacetyl- 7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzofuran]has demonstrated pharmacological potential activities such as antitumor, antimicrobial, antiviral, antiproliferative, and anti-inflammatory. Liposomes arevesicles composed of phospholipid bilayers surrounding aqueous compartments and they have been used as colloidal drug carriers. The aim of this study was to develop and validate a quantitative UV spectrophotometric method for determination of usnicacid in liposomal formulations. The validation parameters were assessed according to The International Conference on Harmonization (ICH) and American Pharmacopoeia guidelines. The linearity range was of 3-15 μg.mL-1,regression equation: absorbance = 0.070 x UA concentration (μg.mL-1) + 0.013, and r = 0.9997. The repeatability (relative standard deviation) of the method was 1.96% and intermediate precision indicated that the difference among mean was statistically insignificant (P < 0.05). The accuracy revealed a mean percentage recovery of 100.4% of usnic acid. The method was robust for the variation of temperature and solvent. The detection and quantization limits were found to be 0.34 and 1.13 μg.mL-1, respectively. The content of usnic acid in liposomes was of 96.8% (± 0.2). The proposed method is accurate, precise and reproducible for estimation of usnic acid as raw material and in pharmaceutical dosage forms such as liposomes.

New Record of Lecanora muralis (Lichenized Fungus) in South Korea

Lecanora muralis was found on the rock along coastal line during the field trip in Jeju island in 2006. Thallus crustose, placodioid, closely adnate, forming orbicular patches; upper surface grayish green, glossy; central lobes areolate, marginal parts plane, edges thin pruinose; lower surface ecorticate; apothecia sessile, lecanorine type, exciple dense and intact when young, and disc plane, but when mature, exciple laciniate, disc protrudent, yellowish brown to orange, 0.5~1.5 mm in diameter; ascospores ellipsoid, simple, colorless, 12.5~15.0 x 5.0~7.5 microm. Usnic acid and zeorin contained in thallus. This is the first record of this species in South Korea.

EFFECT OF USNIC ACID ON TNF-α AND NO PRODUCTION IN LIPOPOLYSACCHARIDE-STIMULATED MACROPHAGES / 药物分析学报

Objective To investigate the molecular mechanisms that are responsible for anti-inflammatory effect of usnic acid (UA), the effects of UA from usnea longissm on tumor necrosis factor-α(TNF-α) and nitric oxide (NO) production in peritoneal macrophages has been examined. Methods The different concentrations of UA were added to peritoneal macrophages. The TNF-α and NO production in peritoneal macrophages were examined with mouse TNF-α ELISA kit and NO content by measuring the amount of nitrite (NO-2μmol/L) formed in the medium using Griess reaction. The activity of inducible nitric oxide synthase (i-NOS) was determined using i-NOS detection kit and the TNF-α mRNA expression was tested by reverse transcriptase polymerase chain reaction (RT-PCR). Results UA decreased the TNF-α and NO level in LPS-stimulated peritoneal macrophages in dose-dependent manner, the IC50 values were 12.8μmol/L and 5.7μmol/L respectively. RT-PCR analysis indicated that UA could inhibit TNF-α mRNA expression; the activity analysis of i-NOS indicated that UA could inhibit the activity of i-NOS. Conclusion UA could inhibit the TNF-α and NO production in peritoneal macrophages, it may be associated with the anti-inflammatory activity of UA.

Quality standard for Compound Songluo Granules / 中成药

AIM: To establish the quality standard for Compound Songluo Granule (Rhizoma Corydalis, Radix Angelicae Sinensis, Lichen Usneae). METHODS:Rhizoma Corydalis, Radix Angelicae Sinensis were identified by TLC. Usnic acid in Lichen Usneae was determined by TLCS. RESULTS: Rhizoma Corydalis, Radix Angelicae Sinensis could be detected by TLC. Usnic acid showed a good linear relationship at the range of 0. 50～2.50 ?g, r=~0.999 9. The average recovery was 97.5% and RSD was 2.61%, respectively. CONCLUSION:The method is available with a good reproducibility and can be used for the quality control of Compound Songluo Granule.