Vanessa Vitoriano da Silva

In Vitro and In Vivo Inhibition of Skin Matrix Metalloproteinases by Pothomorphe umbellata Root Extract

Abstract Exposure to UV radiation up-regulates the synthesis of matrix metalloproteinases (MMPs), a group of matrix-degrading enzymes. MMPs are regarded as promising therapeutic targets and the development of effective inhibitors is an important research focus. The plant Pothomorphe umbellata has been shown to exert a potent antioxidant activity on the skin and to delay the onset and reduce the incidence of UVB-induced chronic skin damage. The aim of the present study was to determine the effect of P. umbellata ethanolic root extract on MMP-2 and MMP-9. The in vitro inhibition of MMP-2 and MMP-9 was measured by gelatin zymography in the presence of different concentrations of P. umbellata extract, as well as in the presence of its isolated active principle 4-nerolidylcatechol (4-NC). The inhibitory effect of the P. umbellata extract was stronger than that of 4-NC. Gelatin zymography and histological analysis revealed that P. umbellata was able to inhibit constitutive MMP-9 activity in vivo in mice sacrificed 2 h after UVB irradiation. The intensity of the MMP-2 band was unchanged. Our data contribute to the elucidation of the mechanism of prevention of photoaging by P. umbellata and may provide a rational basis for the use of this plant in prophylaxis against and treatment of skin cancer.

Pothomorphe umbellata Extract Prevents α-Tocopherol Depletion After UV-irradiation

Abstract In this work we evaluated the influence of topical application of P. umbellata root extract gel, containing 0.1% of 4-nerolidylcathecol, on the antioxidant network in UV-induced oxidative damage in hairless mouse skin. The UV-irradiation had no influence on ascorbic acid levels or on the antioxidant enzyme (superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase) activities, but topical P. umbellata treatment protected α-tocopherol from being depleted after UV-irradiation. α-Tocopherol concentration decreased significantly (≈40%, P < 0.01) in the irradiated control groups, whereas in the P. umbellata–treated group, α-tocopherol was totally preserved (≈100%, P > 0.05). These data demonstrate that P. umbellata may be successfully used as a topical photoprotective agent.

Simultaneous determination of chlorogenic acid, caffeic acid and caffeine in hydroalcoholic and aqueous extracts of Ilex paraguariensis by HPLC and correlation with antioxidant capacity of the extracts by DPPH· reduction

Um novo metodo de cromatografia liquida de alta eficiencia foi desenvolvido para a determinacao simultânea de acido clorogenico, acido cafeico e cafeina no extrato hidroalcoolico e aquoso de Ilex paraguariensis. As curvas de calibracao mostraram boa regressao linear nas faixas de concentracao 0,49-7,8 µg/mL para o acido clorogenico, 0,25-3,9 µg/mL para acido cafeico e 0,244-7,8 µg/mL para cafeina. A reducao do radical DPPH· foi usada para determinar a capacidade antioxidante dos extratos. Nosso metodo para a determinacao simultânea de acido cafeico, acido clorogenico e cafeina foi altamente sensivel, possuindo limites de deteccao e de quantificacao menores do que em outros trabalhos que empregaram metodologias semelhantes.

Dose-dependent in vitro inhibition of rabbit corneal matrix metalloproteinases by an extract of Pothomorphe umbellata after alkali injury

The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 microg protein, gels were incubated with 50, 100, or 250 microg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09%). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50% after incubation with 50 microg/mL and was completely abolished at 100 and 250 microg/mL of the extract. After incubation with 50 microg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50%. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 microg/mL of the extract. For MMP-2 the incubation with 100 or 250 microg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.

Assessment of acute and subchronic oral toxicity of ethanolic extract of Pothomorphe umbellata L. Miq (Pariparoba)

Existe uma grande preocupacao quanto ao uso seguro de extratos vegetais e, por esta razao, a necessidade de estudos toxicologicos pre-clinicos e clinicos destes extratos. O objetivo deste trabalho foi o de avaliar a toxicidade aguda e subcronica do extrato hidroalcoolico liofilizado de Pothomorphe umbellata L. Miq., administrado por via oral para animais de laboratorio. O potencial mutagenico do extrato foi tambem avaliado pelo teste do micronucleo. Os resultados dos estudos a curto e medio prazo demonstraram que o extrato nao apresenta propriedades toxicas.

Antioxidant status of dog aqueous humor after extracapsular lens extraction

We determined the antioxidant status of the aqueous humor after extracapsular lens extraction in 14 mongrel dogs weighing about 10 kg. The animals were examined by slit lamp biomicroscopy, applanation tonometry and indirect ophthalmoscopy. One eye was submitted to conventional extracapsular lens extraction and the other was used as control. Samples of aqueous humor were obtained by anterior chamber paracentesis before and at days 1, 2, 3, 7 and 15 after surgery. Total antioxidant status was determined as the capacity of aqueous humor to inhibit free radical generation by 2,2-azobis(2-amidopropane) chlorine. Ascorbic acid concentration was measured by HPLC with UV detection. Protein content was determined with the biuret reagent. Statistical analysis was performed by ANOVA followed by the Tukey-Kramer test. Protein concentration increased from 0.61 to 22 mg/ml 24 h after surgery. These levels were maintained and returned to normal at day 7. Total antioxidant capacity was reduced from 50 to about 30 min until day 3 and at day 7 it was equal to control. Ascorbic acid levels were reduced from 252 to about 110 microM and then returned to control values at day 15. Considering the importance of ascorbic acid concentration in aqueous humor for the maintenance of the antioxidant status of the anterior segment of the eye, the decrease of antioxidant defenses suggests that the surgical procedures promote an oxidative stress condition in the eye.

Blood and aqueous humour antioxidants in cataractous poodles

BACKGROUND Cataract is an important cause of blindness in dogs and frequently develops in young animals of certain breeds, such as the English cocker spaniel and the poodle. Protein oxidation is one of the mechanisms involved in lens opacification and may be causally related to depleted or diminished endogenous antioxidant defences. We evaluated the levels of enzymatic and nonenzymatic antioxidants in blood and aqueous humour of cataractous poodles in comparison to noncataractous poodles. METHODS We studied 35 cataractous poodles aged 2 to 11 years, 14 noncataractous poodles and 15 noncataractous mixed-breed dogs. The activity of erythrocyte antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase [G6PD]) was evaluated in 18 cataractous poodles and 14 noncataractous poodles. We evaluated ascorbic acid levels in plasma of all animals and in aqueous humour of cataractous poodles and mixed-breed dogs. The dogs were deprived of food for 12 hours before sampling. Blood samples were obtained from the jugular vein before and during anesthesia. Aqueous humour samples were obtained just before the anterior chamber was opened. RESULTS The activity of superoxide dismutase, G6PD and catalase was significantly higher in noncataractous poodles than in cataractous poodles (p < or = 0.05). The activity of glutathione peroxidase was lower in noncataractous poodles than in cataractous poodles, but not significantly so. There was no difference in mean plasma ascorbic acid concentration between cataractous poodles (21.3 microM [standard deviation (SD) 7.4 microM]), noncataractous poodles (21.6 microM [SD 7.4 microM]) and non-cataractous mixed-breed dogs (25.8 microM [SD 7.5 microM]). Similarly, there was no difference in mean aqueous humour ascorbic acid concentration between cataractous poodles (191.7 microM [SD 62.0 microM] and noncataractous mixed-breed dogs (215.7 microM [SD 91.8 microM]). INTERPRETATION The results indicate that, at least in the population studied here, no correlation exists between the onset of cataract and ascorbic acid concentration in blood and aqueous humour. The decreased activity of antioxidant enzymes may explain in part the onset of cataract in poodles.

Photoprotective effect of Pothomorphe umbellata on UVB radiation-induced biomarkers involved in carcinogenesis of hairless mouse epidermis

In this study we assessed the protective effect of topical application of Pothomorphe umbellata extract on ultraviolet B (UVB)-induced skin lesion parameters in hairless mouse epidermis. A single dose of UVB irradiation (0.23 kJ/m2) resulted in a significant decrease in thymine dimer-positive cells and apoptotic sunburn cells, with an increase in p53 and proliferating cell nuclear antigen-positive cells in the epidermis. After 5 weeks (total dose 13.17 kJ/m2) and 15 weeks (total dose 55.51 kJ/m2) of irradiation, P. umbellata treatment inhibited the hyperplasic response and induced an increase in p53-positive cells. These findings suggest that P. umbellata extract affords protection against UVB-induced skin lesions.

Padronização e determinação da fotoestabilidade do extrato de folhas de Pothomorphe umbellata L. Miq (pariparoba) e avaliação da inibição in vitro de metaloproteinases 2 e 9 na pele

Most researches that have been done until today about the beneficial effects of pariparoba (Pothomorphe umbellata L. Miq) have been done with root extract of this species, but the use in large scale would compromise the sustainable exploration of this natural resource. In this sense, the utilization of pariparoba leaves, substituting the roots, in the cosmetic industry does not put in risk the existence of the species. In this work the concentration of 4-nerolidylcathecol (4-NC) in leaf extract was determined by the analytical methodology validated in our laboratory. The concentration of 4-NC in leaf extract was around 30% less than that of root extract, obtained in the same way. Concerning the study of the photostability of a leaves extract solution containing 4-NC did not demonstrate meaningful alterations in the spectrometry profile after 2 hours of exposure under UVB radiation, showing its stability under this conditions. Metalloproteinases (MMPs) are endopeptidases that are zinc-dependent, involved in remodeling extracellular matrix (ECM), that are important in the appearance of typical photoaging wrinkles. In this work the capacity of leaf extract of P. umbellata to inhibit MMP-2 and 9 activities of hairless mouse skin in vitro by zymography gel was also evaluated. The leaf extract (0,1 mg/mL) inhibit in 80% activity of this enzymes, according to the densitometric zymography evaluation.