Análise das propriedades físico-químicas da glucosamina sulfato comercial, uma matéria- prima utilizada em formulações farmacêuticas / Physicochemical properties analysis of commercial glucosamine sulfate, a raw material used in pharmaceutical formulations

Introdução: A qualidade dos produtos em farmácias de manipulação é determinada pela Agência Nacional de Vigilância Sanitária (ANVISA), mas os métodos descritos podem não ser adequados para analisar seus aspectos físico-químicos. Objetivo: Comparar aspectos físico-químicos da glucosamina sulfato de dois diferentes fornecedores com análises realizadas na farmácia de manipulação. Métodos: As características organolépticas, pH, solubilidade e densidade da glucosamina (n=50) dos fornecedores foram analisadas conforme descrito na Farmacopeia Brasileira e Compêndio Oficial e comparados aos laudos técnicos dos produtos adquiridos. Usaram-se os testes de Kolmogorov-Smirnov, coeficiente de correlação intraclasse (CCI) e Bland-Altman. Resultados: A análise de CCIevidenciou baixa reprodutibilidade para o teste de pH e densidade, e a análise de Bland-Altman demonstrou que os fornecedores subestimavam ou superestimavam os valores de pH e densidade em relação à farmácia. Conclusão: Os aspectos físico-químicos estão adequados conforme orientações da Anvisa, mas novas técnicas mais sensíveis devem ser utilizadas para garantir a qualidade da glucosamina nas formulações.

Introduction: The raw materials quality in the manipulation pharmacies are determined by Brazilian Health Surveillance Agency (ANVISA), but the protocols described may not be appropriate to analyze their physicochemical properties. Objective: To compare the physicochemical properties of glucosamine sulfate from two different suppliers with results obtained in the manipulation pharmacy. Methods: The organoleptic characteristics, pH, solubility and density of glucosamine samples (n=50) were analyzed according to the Brazilian Pharmacopoeia and Official Compendium and compared the technical reports of the suppliers. The results were analyzed by the Kolmogorov-Smirnov test, intraclass correlation coefficient (CCI) and Bland-Altman. Results: CCI analyses showed low reproducibility for pH and density test in the samples tested. In addition, Bland-Altman analysis indicated pH values and density of suppliers were underestimated or overestimated compared to the pharmacy. Conclusion: Physicochemical properties of glucosamine are appropriate according to Anvisa specifications, but new more sensitive techniques should be employed to ensure the glucosamine quality in the formulations.

Glucosamine and chondroitin sulfate association increases tibial epiphyseal growth plate proliferation and bone formation in ovariectomized rats

OBJECTIVE: The growth plate consists of organized hyaline cartilage and serves as a scaffold for endochondral ossification, a process that mediates longitudinal bone growth. Based on evidence showing that the oral administration of glucosamine sulfate (GS) and/or chondroitin sulfate (CS) is clinically valuable for the treatment of compromised articular cartilage, the current study evaluated the effects of these molecules on the tibial epiphyseal growth plate in female rats. METHOD: The animals were divided into two control groups, including vehicle treatment for 45 days (GC45) and 60 days (GC60) and six ovariectomized (OVX) groups, including vehicle treatment for 45 days (GV45), GS for 45 days (GE45GS), GS+CS for 45 days (GE45GS+CS), vehicle for 60 days (GV60), GS for 60 days (GE60GS) and GS+CS for 60 days (GE60GS+CS). At the end of treatment, the tibias were dissected, decalcified and processed for paraffin embedding. Morphological and morphometric methods were employed for analyzing the distal tibial growth plates using picrosirius red staining and the samples were processed for histochemical hyaluronan detection. Morphometric analyses were performed using the 6.0ProPlus¯ Image system. RESULTS: Notably, after 60 days of treatment, the number of proliferative chondrocytes increased two-fold, the percentage of remaining cartilage increased ...

In vitro oxidant effects of D-glucosamine reduce adhesión and biofilm formation of Staphylococcus epidermidis / Los efectos oxidantes de la D-glucosamina in vitro reducen la adhesión y formación de biofilms de Staphylococcus epidermidis

Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenic factor is the ability to form adherent biofilms. In this work, three S. epidermidis strains isolated from infected catheters were chosen with the objective of investigating the effect of D-glucosamine (D-Glu) on reactive oxygen species (ROS) production, adhesion and biofilm formation. The chemiluminescence and nitroblue tetrazolium reduction assays were used to determine ROS production by planktonic S. epidermidis and the microtiter plate assay to quantify in vitro biofilm formation. D-Glu generated a dose-dependent increase in ROS in planktonic cells with maximum stimuli at a concentration of 0.05 mM, and reduced adhesion and biofilm formation. On the other hand, glucose showed an antioxidative stress action and promoted biofilm adhesion and growth. This study suggests a potential application of D-Glu against infections associated with indwelling medical devices, since the oxidative stress caused by this hexosamine in planktonic S. epidermidis contributed to reducing biofilm formation.

Staphylococcus epidermidis es un patógeno común en infecciones asociadas a dispositivos médicos. Su factor de patogenicidad más importante es la capacidad para formar biofilms. Se trabajó con tres cepas de S. epidermidis aisladas de catéteres, con las que se efectuaron ensayos de quimioluminiscencia y de reducción de azul de nitrotetrazolio, para determinar la producción de especies reactivas del oxígeno (ERO) en S. epidermidis planctónico, y ensayos dirigidos a cuantificar la formación de biofilm in vitro, empleando placas multipocillos. La D-glucosamina generó un aumento dependiente de la dosis en la producción de ERO en las células planctónicas, con un estímulo máximo a una concentración de 0,05 mM. Este aumento condμlo a la reducción de la adhesión y de la formación de biofilm. La adición de glucosa, en cambio, mostró un efecto anti estrés oxidativo y promovió la adhesión y el crecimiento de biofilm. Este estudio sugiere una posible aplicación de la D-glucosamina contra las infecciones asociadas a dispositivos médicos, ya que el estrés oxidativo provocado por esta hexosamina contribuyó a una menor formación de biofilm.

Glucosamine induces cell death via proteasome inhibition in human ALVA41 prostate cancer cell

Glucosamine, a naturally occurring amino monosaccharide, has been reported to play a role in the regulation of apoptosis more than half century. However the effect of glucosamine on tumor cells and the involved molecular mechanisms have not been thoroughly investigated. Glucosamine enters the hexosamine biosynthetic pathway (HBP) downstream of the rate-limiting step catalyzed by the GFAT (glutamine:fluctose-6-phosphate amidotransferase), providing UDP-GlcNAc substrates for O-linked beta-N-acetylglucosamine (O-GlcNAc) protein modification. Considering that O-GlcNAc modification of proteasome subunits inhibits its activity, we examined whether glucosamine induces growth inhibition via affecting proteasomal activity. In the present study, we found glucosamine inhibited proteasomal activity and the proliferation of ALVA41 prostate cancer cells. The inhibition of proteasomal activity results in the accumulation of ubiquitinated proteins, followed by induction of apoptosis. In addition, we demonstrated that glucosamine downregulated proteasome activator PA28gamma and overexpression of PA28gamma rescued the proteasomal activity and growth inhibition mediated by glucosamine. We further demonstrated that inhibition of O-GlcNAc abrogated PA28gamma suppression induced by glucosamine. These findings suggest that glucosamine may inhibit growth of ALVA41 cancer cells through downregulation of PA28gamma and inhibition of proteasomal activity via O-GlcNAc modification.

Rol de la glucosamina en el tratamiento de la osteoartritis / Role of glucosamine in the treatment of osteoarthritis

La osteoartritis es una enfermedad degenerativa del cartílago que produce disminución del espacio articular y cambios en el hueso subyacente. Se postula que la administración de glucosamina exógena estimularía la síntesis de matriz cartilaginosa y protegería el hueso. A pesar de esto, no hay evidencia sólida para sostener el uso de glucosamina en la osteoartritis leve.

Effects of glucosamine on the tibial epiphyseal disk of ovariectomized rats: morphologic and morphometric analysis

OBJECTIVE: The aim of this study was to analyze the effect of glucosamine sulfate on the tibial epiphyseal disk of the ovariectomized rats. METHODS: After ovariectomy (OVx), 28 female rats were randomly divided into 4 experimental groups with 7 animals each, treated as follows: OVx 21 - vehicle (NaCl 0.9 percent) 0.5 mL/day) for 21 days; OVx GS21 - 230 mg/kg/day glucosamine sulphate for 21 days; OVx 45 - treated with NaCl 0.9 percent as above for 45 days; and OVx - GS45230 mg/kg/day glucosamine sulphate for 45 days. Seven intact animals in the proestrous phase were used as controls (CG). Upon treatment completion, the animals were sacrificed and the left knee joint was dissected and prepared for histological analysis. RESULTS: The percentage of remaining cartilage in new bone of the CG; that found in THE OVx GS45 group was significantly less than that of the OVx 21, OVx GS21, and OVx 45 groups. The percentage of trabecular bone in proestrous animals was the highest. The OVx GS45 group showed higher values compared with the other ovariectomized groups. These results were paralleled by the findings regarding the cells of the proliferative zone, since the CG had the highest values, and the values of the OVx GS45 group were greater than those of the OVx 21, OVx GS21, and OVx 45 groups. CONCLUSION: Our studies suggested that glucosamine may stimulate tibial cartilage and bone growth after ovariectomy in rats.

OBJETIVO: O alvo deste estudo foi analisar o efeito do sulfato de glicosamina no disco epifisário da tíbia em ratas ooforectomizadas. MÉTODOS: Após a ooforectomia (OVx), 28 ratas foram aleatoriamente divididas em 4 grupos experimentais de 7 animais cada, tratados da seguinte maneira: OVx 21 - veículo (0,5ml de NaCl 0.9 por cento ip uma vez ao dia) por 21 dias; OVx GS21 230 - mg/kg peso corporal por dia de sulfato de glicosamina, 21 dias; OVx 45 - tratados com NaCl 0.9 por cento igual ao grupo OVx 21, por 45 dias; e OVx GS45 - 230 mg/kg peso corporal por dia com sulfato de glicosamina, 45 dias. Sete animais intactos, na fase de proestro, foram usados como controle (CG). Ao completar o tratamento, os animais foram sacrificados e a articulação do joelho esquerdo foi dissecada e preparada para análise histológica. RESULTADOS: A porcentagem de cartilagem remanescente no novo osso do CG foi a menor. Os achados no grupo OVx GS45 foi significantemente menor do que no grupo OVx 21, OVx GS21 e OVx 45. A porcentagem de osso trabecular nos animais em pró-estro foi a maior. O grupo OVx GS45 mostrou valores maiores em relação aos outros grupos ooforectomizados. Esses resultados foram correspondentes aos achados em relação às células da zona proliferativa, desde que o CG teve os maiores valores e os valores do grupo OVx GS45 foram superiores aos dos grupos OVx 21, OVx GS21 e OVx 45. CONCLUSÃO: Nossos estudos sugerem que a glicosamina pode estimular o crescimento da cartilagem e do osso tibial após a ooforectomia em ratas.

Efectos de Diclofenac, Celecoxib y sulfato de glucosamina sobre marcadores inflamatorios en cultivo de condrocitos de pacientes con osteoartritis / Articular cartilage in osteoarthritic patients: effects of diclofenac, celecoxib and glucosamine sulfate on inflammatory markers

La osteoartritis (OA) es una enfermedad articular crónica, progresiva que se instala como consecuencia de un proceso complejo que involcra alteraciones mecánicas y biológicas del sistema músculo-esquelético, siendo resultante de múltiples interacciones entre factores genéticos e injurias extrínsecas. La patogenia de esta enfermedad se ralaciona con alta y desviada producción de citokinas flogógenas y de enzimas proteolíticas, que degradan y destruyen la matriz extracelular en tejidos articulares y peri-articulares. Se estudiaron 20 casos con OA, de los cuales se obtuvo cartílago durante intervenciones quirúrgicas programadas. El cartílago se cultivó en medio Dulbecco-Eagle, con o sin agregado de AINEs o condromodulares. En los sobrenadantes se determinaron óxido nítrico por reacción de Giess y la medición espectrofotométrica; y colagenasa por ELISA doble sándwich en presencia de anticuerpos monoclonales. En ausencia de AINEs, los cultivos de condrocitos produjeron 1950 ± 665ng/ml de MMP-1, La adición de Diclofenac redujo esa cifra a 1140 ± 155 ng/ml, aunque esta diferencia no fue estadisticamente significativa, (p<0.60). Por el contrario, Celecoxib redujo el nivel de la enzima a 760 ± 75ng/ml (p<0,01) y la Glucosamina también provocó un descenso (950 ± 89 ng/ml) significativo (p<0.05). Los niveles de ON en ausencia de AINEs llegaron a 47,3 ± 4,9 µM. Su producción no varió significativamente con la adición de Diclofenac, Ceecoxib o Glucosamina (p=ns). Los resultados indicarían la incapacidad de Diclofenac para modificar la generación de enzimas proteolíticas, mientras que Celecoxib y Glucosamina disminuyen su producción significativamente. Ninguno de los fármacos utilizados en nuestro trabajo ha logrado alterar la concentración de ON. Muchos integrantes quedan aún sin resolver y todavía se carece de fármacos de eficacia comprobada para alterar el curso natural de la enfermedad.

Osteoarthritis is a chronic and progressive joint disease. It is established by a complex process involving mechanical and biological alterations of the musculoskeletal system, which are generated by a great variety of interactions between genetic factors and extrinsic injuries. The pathogenesis of this disease is related to an increased and divergent production of inflammatory markers and proteolytic enzymes that promote the degradation and destruction of the extracellular matrix of articular and periarticular tissues. Cartilage samples were taken from 20 osteoarthritic patients during programmed surgical interventions. The cartilage samples were cultured in Dulbecco-Eagle medium, with or without the addition of NSAIDs or modulators of chondrocyte metabolism. The content of nitric oxide in the supernatant was quantified using the Griess reaction; the concentration of MMP-1 was quantified via double-sandwich ELISA. Untreated chondrocyte cultures produced 1950 +/- 665 ng/ml MMP-1. With the addition of Diclofenac this value decreased to 1140 +/- 155 ng/ml, although this difference was not statistically significant (p < 0.06). However, in the presence of Celecoxib the level significantly dropped to 760 +/- 75 ng/ml (p < 0.01). Although the addition ofglucosamine did not produce such a noticeable reduction in the level of MMP-1 (950 +/- 89 ng/ml), it was statistically significant (p < 0.05). On the contrary, none of the drugs (Diclofenac, Celecoxib, Glucosamine) modified the level of nitric oxide which had a mean value of 47.3 +/- 4.9 microM in the control samples. This investigation evidenced the inability of Diclofenac to significantly modify the production of proteolytic enzymes in osteoarthritic chondrocyte cultures. However, both Celecoxib and Glucosamine significantly reduced the production of MMP-1. On the contrary, none of the drugs used in this study managed to modify the concentration of nitric oxide. To the present day, no drugs have been found to be...

Isolation of ethanol-induced genes in pancreatic beta-cells by representational difference analysis (RDA)

Recent epidemiological studies suggest that alcohol consumption is one of the risk factors leading to type 2 diabetes, but the direct effect of ethanol on beta-cell gene expression is not known. Here, using cDNA RDA method, we isolated 43 ethanol-induced genes in pancreatic beta-cells, and confirmed their differential expression by Northern blot or semi-quantitative RT-PCR. These genes were further categorized by the functional criteria based on the published data; Translation, Transcription, Metabolism, Signal transduction, Transport, Structure, Cytoskeleton, Regulation, or Putative/Unknown genes. The effects of each gene on beta-cell function need to be further investigated, however, the present data strongly suggest that these genes might be related to the metabolic alterations caused by ethanol as indicated in earlier study. In particular, RPS3 gene expression was increased by ethanol, glucosamine, and cytokines, implying that ethanol might decrease the metabolic activity by oxidative stress in beta-cells. Therefore, cloning of these genes in full-length and the detailed studies of each gene on beta-cell functions might provide clues on the pathophysiology of type 2 diabetes caused by alcohol.

Effect of sugars on the association between cowpea vicilin (7S storage proteins) and fungal cells

Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. In the present work vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia. Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the vicilins to the surface of cells of S. cerevisiae and F. oxysporum treated with this protein. After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of vicilins associated to chitinous structures present in yeast cells. Our results showed that vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine.