Variation in the amounts of volatile constituents of Cymbopogon citratus (DC) Staf, Poaceae, collected in different regions of São Paulo State

The essential oil extracted from Cymbopogon citratus (DC) Staf, Poaceae, is rich in citral and has many applications in chemical and pharmaceutical industry. The aims of this study were evaluate the amount of citral in populations of C. citratus collected in two different regions (Ibiúna and Pindamonhangaba, São Paulo State). The essential oil extracted of a cultivated population in Pidamonhangaba-SP, showed a high content of citral (approximately 96.0%), while the essential oil from Ibiuna-SP, showed citral and high amounts of another monoterpene, geraniol. Additionally, the essential oils from this population of C. citratus were extracted and analyzed during 24 h (each 3 h), being the major concentration of citral in the night period, indicating that studies which evaluated the chemical differences associated to circadian changes. The components of essential oils were identified through 13C NMR, GC/MS, GC and Kovat's index.O óleo essencial extraído de Cymbopogon citratus (DC) Staf, Poaceae, é rico em citral sendo amplamente empregado na indústria química e farmacêutica. Os objetivos desse estudo foram avaliar o conteúdo de citral entre populações de C. citratus coletados em duas regiões diferentes (Ibiúna e Pindamonhangaba, Estado de São Paulo). O óleo essencial extraído de uma população cultivada em Pindamonhangaba-SP apresentou uma concentração alta de citral (aproximadamente 96,0%), enquanto que o óleo extraído da população de Ibiúna-SP apresentou, além de citral, proporções elevadas de outro monoterpeno, o geraniol. Adicionalmente, os óleos essenciais obtidos desta população de C. citratus foram extraídos e analisados ao longo de 24 h (a cada 3 h), sendo que o teor de citral foi maior no período da noite, o que mostra a importância de estudos que avaliam as diferenças químicas associadas às mudanças circadianas. Os componentes dos óleos essenciais foram identificados por RMN de 13C, CG/EM e CG através do índice de Kovats.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade de Taubaté Departamento de AgronomiaCentro Universitário Fundação Instituto Educacional de OsascoUniversidade Federal de São Paulo (UNIFESP) Departamento de PsicobiologiaUniversidade Federal de São Paulo (UNIFESP) Departamento de Ciências Exatas e da TerraUNIFESP, Depto. de PsicobiologiaUNIFESP, Depto. de Ciências Exatas e da TerraSciEL

Evaluation of the metabolism of glycosaminoglycans in patients with interstitial cystis

Introduction: Painful bladder syndrome/interstitial cystitis (PBS/IC) pathogenesis is not fully known, but evidence shows that glycosaminoglycans (GAG) of bladder urothelium can participate in its genesis. The loss of these compounds facilitates the contact of urine compounds with deeper portions of bladder wall triggering an inflammatory process. We investigated GAG in urine and tissue of PBS/IC and pure stress urinary incontinence (SUI) patients to better understand its metabolism. Materials and Methods: Tissue and urine of 11 patients with PBS/IC according to NIDDK criteria were compared to 11 SUI patients. Tissue samples were analyzed by histological, immunohistochemistry and immunofluorescence methods. Statistical analysis were performed using t Student test and Anova, considering significant when p < 0.05. Results: PBS/IC patients had lower concentration of GAG in urine when compared to SUI (respectively 0.45 ± 0.11 x 0.62 ± 0.13 mg/mg creatinine, p < 0.05). However, there was no reduction of the content of GAG in the urothelium of both groups. Immunofluorescence showed that PBS/IC patients had a stronger staining of TGF-beta, decorin (a proteoglycan of chondroitin/dermatan sulfate), fibronectin and hyaluronic acid. Conclusion: the results suggest that GAG may be related to the ongoing process of inflammation and remodeling of the dysfunctional urothelium that is present in the PBS/IC.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)University of São Paulo Medical Schoo Division of UrologyFederal University of São Paulo Division of UrologyFederal University of São Paulo Division of Molecular BiologyUNIFESP, Division of UrologyUNIFESP, Division of Molecular BiologySciEL

Sulfated glycosaminoglycans in human vocal fold lamina propria

Introduction: The distribution, concentration and function of glycosaminoglycans in the various vocal fold tissues are still unclear. Objective: To evaluate the distribution and concentration of sulfated glycosaminoglycans in different layers of the human vocal fold according to gender and age. Methods: We used 11 vocal folds obtained from cadavers (7 men and 4 women) with no laryngeal lesion, less than 12 h after death, and aged between 35 and 98 years. The folds underwent glycosaminoglycans extraction from the cover and ligament, and post-electrophoresis analysis. Data were compared according to the layer, age and gender. Results: The concentration of dermatan sulfate was significantly higher in all layers. No differences were observed in the total concentrations of glycosaminoglycans in layers studied according to gender. It is significantly lower in the cover of individuals aged below 60 years. Conclusion: Dermatan sulfate, chondroitin sulfate, and heparan sulfate were observed in the human vocal folds cover and ligament of both genders, with the concentration of dermatan sulfate being significantly higher in all layers. Glycosaminoglycans concentration on the cover is significantly lower in individuals below 60 years compared with elderly. (C) 2016 Associacao Brasileira de Otorrinolaringologia e Cirurgia Cervico-Facial. Published by Elsevier Editora Ltda.Univ Fed Sao Paulo UNIFESP, EPM, Dept Otorrinolaringol & Cirurgia Cabeca & Pescoco, Setor Laringe & Voz, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, EPM, Dept Bioquim, Div Biol Mol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, EPM, Dept Med, Div Endocrinol & Metab, Sao Paulo, SP, BrazilPUC SP, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, EPM, Dept Otorrinolaringol & Cirurgia Cabeca & Pescoco, Setor Laringe & Voz, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, EPM, Dept Bioquim, Div Biol Mol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, EPM, Dept Med, Div Endocrinol & Metab, Sao Paulo, SP, BrazilWeb of Scienc

Anti-Sporothrix spp. activity of medicinal plants

Cases of sporotrichosis in humans and animals without satisfactory clinical response have increased, a warning sign of strains resistant to conventional antifungal agents. The urgent search for alternative therapies was an incentive for research on medicinal plants with anti-Sporothrix spp. properties. A bibliographic survey was performed based on scientific papers about in vitro and in vivo antifungal activity of essential oils and extracts of plants in differents solvents against the fungal of the Sporothrix schenckii complex. The study methodology consisted of a literature review in Google Scholar, Science Direct, Pubmed, Bireme and Springer link with papers from 1986 to 2015. We found 141 species of plants that were investigated, of which 100 species were concentrated in 39 botanical families that had confirmed anti-Sporothrix activity. Combretaceae, Asteraceae and Lamiaceae represented the botanical families with the greatest number of plants species with antifungal potential, using different methodologies. However, there are few studies with medicinal plants in experimental infection in animals that prove their activity in the treatment of sporotrichosis. It reinforces the need for further research related to standardization of in vitro methodologies and in vivo studies related to safety and to toxicity potential of these plants with anti-Sporothrix spp. activity.Casos de esporotricose em humanos e animais sem resposta clínica satisfatória têm aumentado, sinal de alarme para o surgimento de cepas resistentes aos antifúngicos convencionais. A urgente busca por alternativas terapêuticas tem incentivado as pesquisas em plantas medicinais com atividade anti-Sporothrix spp. Um levantamento bibliográfico foi realizado com base em artigos científicos sobre a atividade antifúngica in vitro e in vivo de óleos essenciais e extratos de plantas preparados em diferentes solventes contra o complexo Sporothrix schenckii. A metodologia do estudo consistiu em uma revisão bibliográfica em Google Scholar, Science Direct, Pubmed, Bireme e Springer link com artigos desde 1986 até 2015. Foram encontradas 141 espécies de plantas já investigadas, das quais 100 espécies concentradas em 39 famílias botânicas apresentaram atividade anti-Sporothrix spp. confirmada. Combretaceae, Asteraceae e Lamiaceae representaram as famílias botânicas com maior número de espécies vegetais com potencial antifúngico, empregando diferentes metodologias. Entretanto, há poucos estudos com plantas medicinais em infecção experimental animal comprovando sua atividade no tratamento da esporotricose. Reforçase a necessidade de mais pesquisas relacionadas à padronização de metodologias in vitro e a estudos in vivo relacionados à segurança e potencial tóxico dessas plantas com atividade anti Sporothrix spp

Efeitos dos óleos essenciais de Rosmarinus officinalis Linn. e Origanum vulgare Linn. de diferentes origens em Sporothrix brasiliensis e complexo Sporothrix schenckii

Rosmarinus officinalis L. (rosemary) and Origanum vulgare L. (oregano) are known to have antimicrobial properties, but studies on sporotrichosis are scarce. This study aimed to evaluate the anti-Sporothrix spp. activity of essential oils from commercial products and oils extracted from aerial parts of these plants and analyze their chemical constituents. S. schenckii complex and S. brasiliensis (n: 25) isolated from humans, cats, dogs, and environmental soil were tested through M27-A3 guidelines of CLSI with modification for phytotherapics. The essential oils of R. officinalis L. were similar for MIC50 and MFC50 ≤2.25mg/mL for extracted oil; and 4.5mg/mL and 9mg/mL, respectively, for commercial oil. Both products showed MIC90 of 18mg/mL and MFC90 of 36mg/mL. In O. vulgare L., the extracted oil had better activity with MIC50 and MFC50 ≤2.25mg/mL, and MIC90 and MFC90 of 4.5mg/mL, whereas the commercial oil showed MIC50 and MFC50 of 9mg/mL and MIC90 18mg/mL, respectively, and MFC90 of 36mg/mL. Through gas chromatography (CG/FID), thymol and α-terpinene were majority for extracted oil of O. vulgare L., and carvacrol and γ-terpinene made up the majority of the commercial oil. Both essential oils of R. officinalis L. showed 1,8-cineole and α-pinene as major. The fungal isolates were susceptible to all tested essential oils, including in itraconazole-resistant S. brasiliensis isolates. The extracted and commercial oils of the plants presented in vitro anti-Sporothrix spp. activity, and they are promising for treatment of sporotrichosis, including in cases refractory to itraconazole. More studies should be performed about toxicity and in vivo efficacy for its safe use.Rosmarinus officinalis L. (alecrim) e Origanum vulgare L. (orégano) são conhecidos pelas propriedades antimicrobianas, entretanto seus estudos na esporotricose são escassos. Este trabalho objetivou avaliar a atividade anti-Sporothrix spp. de óleos extraídos e comerciais dessas plantas e analisar seus constituintes químicos. Isolados do complexo S. schenckii e S. brasiliensis (n: 25) de humanos, gatos, cães e solo, foram testados pela diretriz M27-A3 do CLSI com modificações para fitoterápicos. Os óleos de R. officinalis L. foram similares com CIM50 e CFM50 ≤2.25mg/mL para extraído; e 4.5mg/mL e 9mg/mL, respectivamente, para comercial. Ambos os produtos demonstraram CIM90 de 18mg/mL e CFM90 de 36mg/mL. Em O. vulgare L., o óleo extraído apresentou melhor atividade com CIM50 e CFM50≤2.25mg/mL e CIM90 e CFM90 de 4.5mg/mL, ao passo que o óleo comercial mostrou CIM50 e CFM50 de 9mg/mL; e CIM90 de 18mg/mL e CFM90 de 36mg/mL. Por meio da cromatografia gasosa (CG/FID), timol e α-terpineno foram majoritários para o óleo extraído de O. vulgare L., e carvacrol e γ-terpineno para o comercial. Ambos os óleos de R. officinalis L. apresentaram 1,8-cineol e α-pineno como prevalentes. Os isolados foram sensíveis a todos os óleos essenciais testados, inclusive S. brasiliensis, resistentes ao itraconazol. Os óleos extraídos e comerciais de R. officinalis L. e O. vulgare L. apresentaram atividade anti-Sporothrix spp. in vitro e são promissores para o tratamento da esporotricose, inclusive em casos refratários ao itraconazol. Mais estudos devem ser realizados sobre toxicidade e eficácia in vivo para seu uso seguro

HPLC-MS/MS METHOD FOR SIMULTANEOUS QUANTIFICATION OF VILDAGLIPTIN, METFORMIN, AND METFORMIN-RELATED COMPOUNDS IN TABLETS

Objective: The present study developed and validated a high-performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of vildagliptin, metformin, and metformin-related compounds (A, B, and C) in tablets. Methods: Chromatographic separations were achieved on a C8 column (150 × 4.6 mm, 5 µm particle size) maintained at 25°C. The isocratic mobile phase consisted of acetonitrile/water/formic acid (20:80:0.1, v/v/v), eluted at 800 μL/min. The volume of injection was 20 μL and the electrospray source was operated in positive ion mode. Results: Data from validation studies demonstrated that the new method was highly selective, sensitive (limits of detection ≤ 0.6 ng/mL), precise (RSD &lt; 5%), and accurate (RE &lt; 5%). All of the calibration curves showed excellent coefficients of correlation (r ³ 0.99) over the ranges of 5‑150 ng/mL for vildagliptin, 250‑2000 ng/mL for metformin, and 2.5‑25 ng/mL for metformin-related compounds A, B, and C. Quantification of these compounds in tablets showed that they contained from 95.2‑101.2% (vildagliptin) and 97‑104.4% (metformin) of the stated level. No metformin-related compounds were detected using this highly sensitive method. Conclusion: This new method is presented as an alternative approach to the quality control of tablets containing vildagliptin, metformin, and metformin-related compounds

Urinary glycosaminoglycans excretion and the effect of dimethyl sulfoxide in an experimental model of non-bacterial cystitis

PURPOSE: We reproduced a non-bacterial experimental model to assess bladder inflammation and urinary glycosaminoglycans (GAG) excretion and examined the effect of dimethyl sulfoxide (DMSO). MATERIALS AND METHODS: Female rats were instilled with either protamine sulfate (PS groups) or sterile saline (control groups). At different days after the procedure, 24 h urine and bladder samples were obtained. Urinary levels of hyaluronic acid (HA) and sulfated glycosaminoglycans (S-GAG) were determined. Also to evaluate the effect of DMSO animals were instilled with either 50% DMSO or saline 6 hours after PS instillation. To evaluate the effect of DMSO in healthy bladders, rats were instilled with 50% DMSO and controls with saline. RESULTS: In the PS groups, bladder inflammation was observed, with polymorphonuclear cells during the first days and lymphomononuclear in the last days. HA and S-GAG had 2 peaks of urinary excretion, at the 1st and 7th day after PS injection. DMSO significantly reduced bladder inflammation. In contrast, in healthy bladders, DMSO produced mild inflammation and an increase in urinary HA levels after 1 and 7 days and an increase of S-GAG level in 7 days. Animals instilled with PS and treated with DMSO had significantly reduced levels of urinary HA only at the 1st day. Urinary S-GAG/Cr levels were similar in all groups. CONCLUSIONS: Increased urinary levels of GAG were associated with bladder inflammation in a PS-induced cystitis model. DMSO significantly reduced the inflammatory process after urothelial injury. Conversely, this drug provoked mild inflammation in normal mucosa. DMSO treatment was shown to influence urinary HA excretion

Cytotoxicity and anti-Sporothrix brasiliensis activity of the Origanum majorana Linn. oil

The study aimed to evaluate the anti-Sporothrix sp. activity of the essential oil of Origanummajorana Linn. (marjoram), its chemical analysis, and its cytotoxic activity. A total of 18fungal isolates of Sporothrix brasiliensis (n: 17) from humans, dogs and cats, and a standardstrain of Sporothrix schenckii (n: 1) were tested using the broth microdilution technique (Clin-ical and Laboratory Standard Institute – CLSI M27-A3) and the results were expressed inminimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). TheMIC50and MIC90of itraconazole against S. brasiliensis were 2 g/mL and 8 g/mL, respectively,and the MFC50and MFC90were 2 g/mL and >16 g/mL, respectively, with three S. brasilien-sis isolates resistant to antifungal. S. schenckii was sensitive at MIC of 1 g/mL and MFC of8 g/mL. For the oil of O. majorana L., all isolates were susceptible to MIC of ≤2.25–9 mg/mLand MFC of ≤2.25–18 mg/mL. The MIC50and MIC90were ≤2.25 mg/mL and 4.5 mg/mL, respec-tively, and the MFC50/90values were twice more than the MIC. Twenty-two compounds wereidentified by gas chromatography with a flame ionization detector (CG-FID) and 1,8-cineoleand 4-terpineol were the majority. Through the colorimetric (MTT) assay, the toxicity wasobserved in 70–80% of VERO cells between 0.078 and 5 mg/mL. For the first time, the studydemonstrated the satisfactory in vitro anti-Sporothrix sp. activity of marjoram oil and furtherstudies are needed to ensure its safe and effective use

Macrocalcitonin Is a Novel Pitfall in the Routine of Serum Calcitonin Immunoassay

Context: Calcitonin (CT) is a sensitive marker of medullary thyroid carcinoma (MTC) and is used for primary diagnosis and follow-up after thyroidectomy. However, persistently elevated CT is observed even after complete surgical removal without evidence of a recurrent or persistent tumor. Objective: To investigate the presence of assay interference in the serum CT of MTC patients who are apparently without a structural disease. Patients and Methods: We studied three index MTC cases for CT assay interference and 14 patients with metastatic MTC. The CT level was measured using an immunofluorometric assay. Screening for assay interference was performed by determination of CT levels before and after serum treatment with polyethylene glycol. Additionally, samples were analyzed by chromatography on ultra-performance liquid chromatography and protein A-Sepharose. Results: Patients with biochemical and structural disease showed CT mean recovery of 84.1% after polyethylene glycol treatment, whereas patients suspected of interference showed recovery from 2-7%. The elution profile on UPLC showed that the immunometric CT from these three patients behaved like a high molecular mass aggregate (>300 kDa). Additionally, when these samples were applied to the protein A-Sepharose, CT immunoreactivity was retained on the column and was only released after lowering the pH. Conclusions: For the first time, our results show the presence of a novel pitfall in the CT immunoassay: "macrocalcitonin." Its etiology, frequency, and meaning remain to be defined, but its recognition is of interest and can help clinicians avoid unnecessary diagnostic investigations and treatment during the follow-up of MTC.Sao Paulo State Research Foundation-FAPESPFAPESPFederal Agency of Support and Evaluation of Postgraduate Education (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior)National Council for Scientific and Technological DevelopmentUniv Fed Sao Paulo, Escola Paulista Med, Div Endocrinol, Dept Med,Thyroid Dis Ctr, BR-04039032 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Div Endocrinol, Dept Med,Lab Mol & Translat Endocrinol, BR-04039032 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Biochem, Div Mol Biol, BR-04044020 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Div Endocrinol, Dept Med,Thyroid Dis Ctr, BR-04039032 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Div Endocrinol, Dept Med,Lab Mol & Translat Endocrinol, BR-04039032 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Biochem, Div Mol Biol, BR-04044020 Sao Paulo, SP, BrazilFAPESP: 2006/60402-1FAPESP: 2010/51547-1FAPESP: 2010/19478Web of Scienc

Activities and mechanisms of oregano, marjoram and rosemary essential oils against Malassezia pachydermatitis isolates from canine and feline otitis

Malassezia pachydermatis is an opportunistic yeast found in the ear canal of small animals; however, the current azole-based therapy applied to it has failed to achieve clinical success due to the antifungal resistance. This issue has encouraged the studies in natural products, such as Origanum vulgare (oregano), Origanum majorana (marjoram) and Rosmarinus officinalis (rosemary) essential oils, although their mechanism of action remains unclear. Malassezia pachydermatis specimens deriving from otitis cases in dogs (n = 22) and cats (n = 2) were subjected to CLSI M27-A3. Sorbitol protection and ergosterol effect were analyzed to investigate their mechanism of action. Fungistatic (MIC) and fungicidal (MFC) activities were observed for oregano (MIC90/MFC90: 0.625 mg/mL); marjoram (MIC90/MFC90: 2.5 mg/mL) and rosemary MIC90/MFC90 > 2.5 mg/mL). Oregano showed superior antifungal effect even at lower MIC and MFC values. All three oils acted on cell wall and at complexation to fungal ergosterol. By gas chromatography (GC-FID), carvacrol was the major compound found in oregano (73.9%); 1,8-cineole was for marjoram and rosemary (20.9% and 49.4%, respectively). These findings support the potential use of these essential oils to treat canine and feline otitis caused by Malassezia pachydermatis