Stachys byzantina K. Koch (Lamiaceae) as a potential ingredient for delaying skin ageing and treating hyperpigmentation disorders in pharmaceutical products.

OBJECTIVES: This study aimed to investigate whether the plant species Stachys byzantina produces bioactives with the potential to delay the skin ageing process and treat hyperpigmentation conditions. METHODS: The antioxidant action was assessed by 2,2-diphenyl-1-picrylhydrazylradical scavenging, Griess reaction, oxygen radical absorption capacity, and ß-carotene bleaching assays. Inhibitory activities for tyrosinase, hyaluronidase, and elastase enzymes were tested. The antiglycation activity, the sun protection factor (SPF), and the toxicity to skin cells by MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay were also evaluated. KEY FINDINGS: The ethanolic extract of S. byzantina aerial parts and all fractions obtained by solvent partition inhibited the tyrosinase enzyme at different levels. The dichloromethane fraction (DF) demonstrated the highest inhibition (IC50 = 63.5 ± 10.9 µg/ml). DF also inhibited the hyaluronidase enzyme with IC50 = 369 ± 11.64 µg/ml and elastase by 40% at 500 µg/ml. This fraction showed prominent antioxidant and antiglycation activities, high SPF, and no cytotoxicity at concentrations lower than 50 µg/ml. The phenolic and flavonoid contents were 116.30 ± 6.7 (mgTAE/g) and 66.38 ± 13.5 (mgQE/g), respectively. Chlorogenic acid (23.54 ± 2.46 mg/g) and verbascoside (203.97 ± 19.8 mg/g) were identified and quantified. CONCLUSIONS: Stachys byzantina is a potential source of cosmetic and therapeutic ingredients to reduce hyperpigmentation and the impacts caused by free radicals, advanced glycation end products, and sun radiation in skin ageing.

New aspects on the hepatoprotective potential associated with the antioxidant, hypocholesterolemic and anti-inflammatory activities of Vernonia condensata Baker.

ETHNOPHARMACOLOGICAL RELEVANCE: Vernonia condensata Baker (Asteraceae) is traditionally used in South American Countries as an anti-inflammatory, analgesic and hepatoprotective. AIM OF THE STUDY: This study aimed to investigate the in vivo hepatoprotective and antioxidant, and the in vitro anti-inflammatory activities of the ethyl acetate partition (EAP) from the ethanolic extract of this medicinal plant leaves. MATERIALS AND METHODS: For the in vivo hepatoprotective activity, rats were pretreated orally for seven days with vehicle, silymarin 100mg/kg or EAP 50, 100 and 200mg/kg. Then, acetaminophen 3g/kg was also orally administrated. Animals were euthanatized 24h after the damage inducement. The levels of the serum enzymes ALT, AST and ALP were determined, as well as the triglycerides, total cholesterol and fractions. The antioxidant activity was evaluated by TBARS assay and by the measurement of glutathione reductase, superoxide dismutase and catalase activities in the rats liver tissue. The in vitro anti-inflammatory assay using Raw 264.7 cell line induced by lipopolysaccharide was conducted to verify EAP ability to inhibit pro-inflammatory cytokines. RESULTS: EAP was able to inhibit all the acute biochemical alterations caused by acetaminophen overdose. EAP inhibited malondialdehyde formation, maintained the catalase and increased the glutathione reductase activities. Also, EAP decreased NO, IL-6 and TNF-α levels at concentrations from 10 to 20µg/mL. 1,5-dicaffeoylquinic acid was isolated and identified as the major compound in EAP. Apigenin, luteolin, chlorogenic acid were also identified. EAP anti-inflammatory action may be due to its antioxidant activity or its capacity to inhibit the pro-inflammatory cytokines. CONCLUSION: These results strongly suggested that V. condensata may be useful as a possible therapy against liver damage.

Increase of glyoosaminoglycans and metalloprotenases 2 and 9 in liver extracelllular matrix on early stages of extrahepatic cholestaiis / Aumento de glicosaminoglicanos e metaloproteinases 2 e 9 na matriz extracelular de fígado em estágios iniciais de colestase extra-hepática

Context Cholestasis produces hepatocellular injury, leukocyte infiltration, ductular cells proliferation and fibrosis of liver parenchyma by extracellular matrix replacement. Objective Analyze bile duct ligation effect upon glycosaminoglycans content and matrix metalloproteinase (MMPs) activities. Methods Animals (6-8 weeks; n = 40) were euthanized 2, 7 or 14 days after bile duct ligation or Sham-surgery. Disease evolution was analyzed by body and liver weight, seric direct bilirubin, globulins, gamma glutamyl transpeptidase (GGT), alkaline phosphatase (Alk-P), alanine and aspartate aminotransferases (ALT and AST), tissue myeloperoxidase and MMP-9, pro MMP-2 and MMP-2 activities, histopathology and glycosaminoglycans content. Results Cholestasis caused cellular damage with elevation of globulins, GGT, Alk-P, ALT, AST. There was neutrophil infiltration observed by the increasing of myeloperoxidase activity on 7 (P = 0.0064) and 14 (P = 0.0002) groups which leads to the magnification of tissue injuries. Bile duct ligation increased pro-MMP-2 (P = 0.0667), MMP-2 (P = 0.0003) and MMP-9 (P<0.0001) activities on 14 days indicating matrix remodeling and establishment of inflammatory process. Bile duct ligation animals showed an increasing on dermatan sulfate and/or heparan sulfate content reflecting extracellular matrix production and growing mitosis due to parenchyma depletion. Conclusions Cholestasis led to many changes on rats’ liver parenchyma, as so as on its extracellular matrix, with major alterations on MMPs activities and glycosaminoglycans content. .

Contexto Colestase produz lesão hepatocelular, infiltração leucocitária, proliferação de células ductulares e fibrose do parênquima hepático por matriz extracelular. Objetivo Analisar os efeitos da ligação do ducto biliar sobre conteúdo de glicosaminoglicanos e atividade de metaloproteinases de matriz (MMP). Métodos Animais (6-8 semanas; n = 40) foram eutanasiados 2, 7 ou 14 dias após ligação do ducto biliar ou falsa ligação. A evolução da doença foi analisada por peso corporal e do fígado, concentrações séricas de bilirrubina direta, globulinas, gama glutamil transpeptidase (GGT), fosfatase alcalina (Alk-P), alanina e aspartato aminotransfesases (ALT e AST), alterações teciduais de mieloperoxidase e metaloproteinases (MMP-9, pro MMP-2 e MMP-2), histopatologia e conteúdo de glicosaminoglicanos. Resultados A colestase causou dano celular com elevação dos níveis séricos de globulinas, GGT, Alk-P, ALT e AST. Houve também infiltração leucocitária observada pelo aumento na atividade de mieloperoxidase nos grupos 7 (P = 0,0064) e 14 dias (P = 0,0002) o que leva ao aumento das lesões no tecido. Ligação do ducto biliar aumentou as atividades de pro MMP-2 (P = 0,0677), MMP-2 (P = 0,0003) e MMP-9 (P<0,0001) aos 14 dias indicando remodelamento da matriz e estabelecimento de processo inflamatório. Animais com ligação do ducto biliar mostraram um aumento do conteúdo de dermatam sulfato e/ou heparam sulfato refletindo a produção de matriz extracelular e aumento de mitose devido a depleção do parênquima hepático. Conclusões Colestase causou várias mudanças no parênquima hepático de ratos, bem como em sua matriz extracelular, com importantes alterações na atividade ...

Positive correlation between disease activity index and matrix metalloproteinases activity in a rat model of colitis.

CONTEXT: Inflammatory bowel disease, including ulcerative colitis and Crohn's disease, comprising a broad spectrum of diseases those have in common chronic inflammation of the gastrointestinal tract, histological alterations and an increased activity levels of certain enzymes, such as, metalloproteinases. OBJECTIVES: Evaluate a possible correlation of disease activity index with the severity of colonic mucosal damage and increased activity of metalloproteinases in a model of ulcerative colitis induced by dextran sulfate sodium. METHODS: Colitis was induced by oral administration of 5% dextran sulfate sodium for seven days in this group (n=10), whereas control group (n=16) received water. Effects were analyzed daily by disease activity index. In the seventh day, animals were euthanized and hematological measurements, histological changes (hematoxylin and eosin and Alcian Blue staining), myeloperoxidase and metalloproteinase activities (MMP-2 and MMP-9) were determined. RESULTS: Dextran sulfate sodium group showed elevated disease activity index and reduced hematological parameters. Induction of colitis caused tissue injury with loss of mucin and increased myeloperoxidase (P<0.001) and MMP-9 activities (45 fold) compared to the control group. CONCLUSIONS: In this study, we observed a disease activity index correlation with the degree of histopathological changes after induction of colitis, and this result may be related mainly to the increased activity of MMP-9 and mieloperoxidase.

Positive correlttion between disease activity idex and matrix metalloproteinasses activity in a rat model of colitis / Correlação positiva entre índice de atividade de doença e metaloproteinase de matriz em modelo de colite em ratos

Context Inflammatory bowel disease, including ulcerative colitis and Crohn’s disease, comprising a broad spectrum of diseases those have in common chronic inflammation of the gastrointestinal tract, histological alterations and an increased activity levels of certain enzymes, such as, metalloproteinases. Objectives Evaluate a possible correlation of disease activity index with the severity of colonic mucosal damage and increased activity of metalloproteinases in a model of ulcerative colitis induced by dextran sulfate sodium. Methods Colitis was induced by oral administration of 5% dextran sulfate sodium for seven days in this group (n=10), whereas control group (n=16) received water. Effects were analyzed daily by disease activity index. In the seventh day, animals were euthanized and hematological measurements, histological changes (hematoxylin and eosin and Alcian Blue staining), myeloperoxidase and metalloproteinase activities (MMP-2 and MMP-9) were determined. Results Dextran sulfate sodium group showed elevated disease activity index and reduced hematological parameters. Induction of colitis caused tissue injury with loss of mucin and increased myeloperoxidase (P<0.001) and MMP-9 activities (45 fold) compared to the control group. Conclusions In this study, we observed a disease activity index correlation with the degree of histopathological changes after induction of colitis, and this result may be related mainly to the increased activity of MMP-9 and mieloperoxidase. .

Contexto Doenças inflamatórias intestinais, entre elas colite ulcerativa e doença de Crohn, compreendem um amplo espectro de doenças que apresentam em comum inflamação crônica do trato gastrointestinal, alterações histológicas e um aumento de atividade de determinadas enzimas, tais como, metaloproteinases. Objetivos Avaliar possível correlação do índice de atividade de doença em modelo de colite ulcerativa induzida por dextran sulfato de sódio com o grau de severidade de danos na mucosa colônica e aumento de atividade de metaloproteinases. Métodos Colite foi induzida por administração oral de dextran sulfato de sódio 5% durante sete dias no grupo (n = 10), enquanto que o grupo controle (n = 16) recebeu água. Efeitos foram analisados diariamente pelo índice de atividade de doença. No sétimo dia, os animais foram sacrificados e as medições hematológicas, alterações histológicas (hematoxilina e eosina e coloração de azul Alcian), mieloperoxidase e atividades de metaloproteinases (MMP-2 e MMP-9) foram determinados. Resultados Grupo dextran sulfato de sódio mostrou elevação no índice de atividade de doença e redução dos parâmetros hematológicos. A indução da colite causa lesão no tecido, com perda de mucina e aumento da mieloperoxidase (P<0,001) e as atividades MMP-9 (45 vezes) em comparação com o grupo de controle. Conclusões Neste estudo, observamos uma correlação do índice de atividade de doença com o grau de alterações histopatológicas após indução da colite por dextran sulfato de sódio, podendo associar este resultado ao aumento principalmente da atividade de MMP-9 e de mieloperoxidase. .

Increase of glycosaminoglycans and metalloproteinases 2 and 9 in liver extracellular matrix on early stages of extrahepatic cholestasis.

CONTEXT: Cholestasis produces hepatocellular injury, leukocyte infiltration, ductular cells proliferation and fibrosis of liver parenchyma by extracellular matrix replacement. OBJECTIVE: Analyze bile duct ligation effect upon glycosaminoglycans content and matrix metalloproteinase (MMPs) activities. METHODS: Animals (6-8 weeks; n = 40) were euthanized 2, 7 or 14 days after bile duct ligation or Sham-surgery. Disease evolution was analyzed by body and liver weight, seric direct bilirubin, globulins, gamma glutamyl transpeptidase (GGT), alkaline phosphatase (Alk-P), alanine and aspartate aminotransferases (ALT and AST), tissue myeloperoxidase and MMP-9, pro MMP-2 and MMP-2 activities, histopathology and glycosaminoglycans content. RESULTS: Cholestasis caused cellular damage with elevation of globulins, GGT, Alk-P, ALT, AST. There was neutrophil infiltration observed by the increasing of myeloperoxidase activity on 7 (P = 0.0064) and 14 (P = 0.0002) groups which leads to the magnification of tissue injuries. Bile duct ligation increased pro-MMP-2 (P = 0.0667), MMP-2 (P = 0.0003) and MMP-9 (P<0.0001) activities on 14 days indicating matrix remodeling and establishment of inflammatory process. Bile duct ligation animals showed an increasing on dermatan sulfate and/or heparan sulfate content reflecting extracellular matrix production and growing mitosis due to parenchyma depletion. CONCLUSIONS: Cholestasis led to many changes on rats' liver parenchyma, as so as on its extracellular matrix, with major alterations on MMPs activities and glycosaminoglycans content.

Purificação e utilização das condroitinases B e AC de Flavobacterium heparinum no estudo dos proteoglicanos de matriz extracelular / Purification and utilization of chondroitinases AC, B and C from Flavobacterium in the study of proteoglycans the extracellular matrix

As condroitinases AC, B e C de Flavobacferium heparinum são importantes instrumentos para a identificação e a análise da estrutura de condroitim sulfatos e dermatam sulfatos, bem como de seus proteoglicanos, de diferentes origens. Entretanto, para que sejam úteis, preparações de enzimas puras e estáveis devem ser obtidas. No presente trabalho, descrevemos um procedimento simples, reprodutível e com alto rendimento para preparo dessas enzimas, baseado em cromatografia de troca iônica em Q-Sepharose FF e cromatografia de interação hidrofóbica em Pheny-Sepharose HP. Para análise quantitativa da purificação e do rendimento das enzimas purificadas, estabelecemos uma metodologia que se baseia na queda em metacromasia que acompanha a despolimerização dos glicosaminoglicanos. Este método, que utiliza a interação dos glicosaminoglicanos com azul de dimetilmetileno, permitiu a dosagem das condroitìnases presentes em extratos brutos induzidos de F. heparinum e nas preparações purificadas. A vantagem deste procedimento sobre outros métodos comumente empregados, que quantificam os produtos insaturados formados, é que a. presença de glicuronidases e sulfatares não interfere na determinação das atividades enzimáticas. As condroitinases AC e B assim preparadas foram empregadas na identificação e na análise estrutural de proteoglicanos e glicosaminoglicanos extraídos de diversos tecidos e fluidos biológicos, como córnea humana, urina, soro e diversos tecidos de gato, miométrio e leiomioma humanos. Além disso, uma nova metodologia foi desenvolvida para imunolocalizar proteoglicanos de condroitim sulfato ou dermatam sulfato em cortes de tecidos. Essa metodologia baseia-se na incubação de cortes de tecidos com condroitinase AC (para condroitim sulfato) ou condroitinase 8 (para dermatam sulfato). Em seguida, os resíduos insaturados gerados pelas enzimas são reconhecidos por anticorpos monoclonais específicos. Estudos de dupla marcação para as cadeias de condroitim sulfato ou dermatam sulfato e o esqueleto protéico (decorim ou versicam) ou queratam sulfato ou, ainda, filamentos de actina foram também realizados. As imagens de dupla marcação foram submetidas a análise morfométrica, utilizando a ferramenta MATLAB, para determinação do grau de co-localização...