Cristina Dislich Ropke

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.

Apoptosis induction by 4-nerolidylcatechol in melanoma cell lines.

Pothomorphe umbellata, a native Brazilian plant, is popularly known to be effective in the treatment of skin lesions. This benefit is attributed to 4-nerolidylcatechol (4-NC), a compound extracted from P. umbellata. Since melanomas show prominent resistance to apoptosis and exhibit extreme chemoresistance to multiple forms of therapy, novel compounds addressing induction of cell death are worth investigating. Here, we evaluated effects on cell cycle progression and possible cytotoxic activity of 4-NC in melanoma cell lines as well as human dermal fibroblasts. Inhibitory effects on cell invasion and MMP activity were also investigated. 4-NC showed cytotoxic activity for all melanoma cell lines tested (IC50=20-40 microM, 24h for tumoral cell lines; IC50=50 microM for fibroblast cell line) associated with its capacity to induce apoptosis. Furthermore, this is the first time that 4-NC is described as an inhibitor of cell invasiveness, due mainly to a G1 cell cycle arrest and inhibition of MMP-2 activity in melanoma cell lines.

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.

Optimization of Pothomorphe umbellata (L.) Miquel topical formulations using experimental design

Pothomorphe umbellata is a native plant widely employed in the Brazilian popular medicine. This plant has been shown to exert a potent antioxidant activity on the skin and to delay the onset and reduce the incidence of UVB-induced skin damage and photoaging. The aim of this work was to optimize the appearance, the centrifuge stability and the permeation of emulsions containing P. umbellata (0.1% 4-nerolidylchatecol). Experimental design was used to study ternary mixtures models with constraints and graphical representation by phase diagrams. The constraints reduce the possible experimental domain, and for this reason, this methodology offers the maximum information while requiring the minimum investment. The results showed that the appearance follows a linear model, and that the aqueous phase was the principal factor affecting the appearance; the centrifuge stability parameter followed a mathematic quadratic model and the interactions between factors produced the most stable emulsions; skin permeation was improved by the oil phase, following a linear model generated by data analysis. We propose as optimized P. umbellata formulation: 68.4% aqueous phase, 26.6% oil phase and 5.0% of self-emulsifying phase. This formulation displayed an acceptable compromise between factors and responses investigated.

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.

Hydrolysis Influence on Phytochemical Composition, Antioxidant Activity, Plasma Concentration, and Tissue Distribution of Hydroethanolic Ilex paraguariensis Extract Components

The infusion of aerial parts of Ilex paraguariensis is widely consumed. Its antioxidant activity suggests an important role of this plant in the treatment/prevention of oxidative stress related diseases. Plant extract active compounds are frequently found in esterified form that may be poorly absorbed. Hydrolysis of the extract is a possible approach to increase its bioavailability. The aim of this study was to perform a phytochemical analysis and evaluate in rats the plasma concentration and tissue distribution of antioxidant compounds in the hydroethanolic extract of Ilex paraguariensis, before and after enzymatic hydrolysis. Both extracts presented high antioxidant activity and phenolic content. Rats given single or repeated doses of the hydrolyzed extract showed increased plasma antioxidant activity and higher plasma levels of caffeic acid. However, no changes of endogenous antioxidants were observed. In conclusion, hydrolysis of the extract of Ilex paraguariensis is a strategy to improve its bioavailability and in vivo antioxidant activity.

Chlorogenic Acid UVA–UVB Photostability

Chlorogenic acid is a natural potent antioxidant. It can be used in cosmetics formulations, but for this purpose its photochemical stability should be determined to ensure that the compound will not be degraded after UV radiation exposure. To evaluate this possibility, the concentration of a chlorogenic acid solution was determined by HPLC before and after UVA and UVB irradiation. The results indicate that chlorogenic acid is not degraded under UVA or UVB irradiation.

Validação de metodologias analíticas para determinação quantitativa de ±-tocoferol e 4-nerolidilcatecol

Topical administration of antioxidants, such as ±-tocopherol (±-T), provides an efficient manner of enriching the endogenous cutaneous protection system, and it constitutes a successful strategy for diminishing the ultraviolet radiation-mediated oxidative damage. Besides ±-tocopherol the use of other natural occurring compounds with antioxidant activity has been proposed for the same purpose. The aim of this study was to develop a validated analytical method for the determination of a-tocopherol and 4-nerolidylcathecol (4-NC) concentrations in skin homogenates in a pharmaceutical formulations. We employed liquid chromatography with electrochemical detection. Chromatography was performed on a Supelcosil LC-8, 3 mm, 75x4.6 mm column (Supelco, Bellefonte, PA, USA) with a mobile phase of methanol:water (9:1) for 4-NC and (95:5) for a-T, both containing 20 mM LiClO4 and 2 mM KCl. The flow rate was set at 1.0 ml/min. We established validation parameters including sensitivity, precision, accuracy, stability and found a linear relationship between the concentrations ranges of 0.025 µg/mL to 0.1 µg/mL of ±-T and 0.15 mg/mL to 2.5 mg/mL of 4-NC. The recovery of ±-T from skin homogenates was 94.03, 111.2 and 80.7% for the concentrations of 0.5, 0.1 and 0.025 µg/mL respectively. The recovery for the following concentrations of 4-NC: 2.5, 0.625 and 0.156 µg/mL was 103.7, 91.7 and 91.7%. This analytical procedure has been successfully employed in cutaneous permeation studies, antioxidant activity studies and determinations of 4-NC in Pothomorphe umbellata root extracts.