Photochemoprevention of topical formulation containing purified fraction of Inga edulis leaves extract.

Natural antioxidants have attracted attention for their therapeutic use as photochemopreventive agents. Inga edulis leaves extract and its purified fraction have high polyphenolic content and high antioxidant capacity. In addition, they presented UV photostability and low citotoxicity in fibroblast cells. In this context, this study first aimed at development of topical formulation containing purified fraction of I. edulis extract and the evaluation of skin penetration of the compounds. Moreover, the photoprotective/photochemopreventive potential of the formulation containing I. edulis purified fraction were investigated in vitro and in vivo. The topical formulation containing 1% of the purified fraction of I. edulis increased the endogenous antioxidant potential of the skin, and vicenin-2 and myricetin compounds were able to penetrate the epidermis and dermis. Additionally, the purified fraction (25 and 50 mg/mL) showed a photoprotective effect against UVA and UVB radiation in L929 fibroblast cells. In vivo studies have shown that the formulation added with purified fraction provided an anti-inflammatory effect on the skin of animals after UVB exposure, since it was observed a reduction in MPO activity, IL-1ß and TNF-α cytokines, and CXCL1/KC chemokine concentrations. In conclusion, the purified fraction of I. edulis, rich in phenolic compounds, when incorporated in topical formulation, appears as an alternative to prevent skin damages induced by UV radiation, due to its antioxidant and anti-inflammatory properties.

Photoprotective effect of solid lipid nanoparticles of rutin against UVB radiation damage on skin biopsies and tissue-engineered skin.

Solid lipid nanoparticles (SLNs) containing rutin were prepared to enhance their photochemopreventive effect on the skin. SLNs were produced by the hot melt microemulsion technique. Two 3D skin models: ex vivo skin explants and 3D tissue engineering skin were used to evaluate the photochemopreventive effect of topical formulations containing rutin SLNs, against ultraviolet B (UVB) radiation, inducing sunburn cells, caspase-3, cyclobutane pyrimidine dimers, lipid peroxidation, and metalloproteinase formation. The rutin SLNs presented average size of 74.22 ± 2.77 nm, polydispersion index of 0.16 ± 0.04, encapsulation efficiency of 98.90 ± 0.25%, and zeta potential of -53.0 ± 1.61 mV. The rutin SLNs were able to efficiently protect against UVB induced in the analysed parameters in both skin models. Furthermore, the rutin SLNs inhibited lipid peroxidation and metalloproteinase formation. These results support the use of rutin SLNs as skin photochemopreventive agents for topical application to the skin.

Soybean extract modified by Aspergillus awamori stimulates a greater collagen-I synthesis in the intracellular matrix of human fibroblasts.

Aglycone isoflavones are estrogen-like bioactive compounds found in low amounts in soybean, which are increased by biotransformation processes. This study investigated two biotransformation processes of soybean extracts with Aspergillus awamori fungus, evaluating aglycone content and capability of stimulation of collagen-I deposition. Isoflavones were quantified via HPLC; cytotoxicity of biotransformed extracts toward mouse and human fibroblasts was evaluated via NRU and apoptosis/necrosis assays; and collagen-I deposition was measured through Western blot, immunofluorescence, and immunoassay. BSE-2 was the biotransformed soybean extract with the highest aglycone content and did not decrease viability or demonstrated cytotoxicity to either L929 or HDFa cells. BSE-2, at the optimal concentration of 1.33 µg/mL, increased substantially collagen-I amount in HDFa intracellular matrix compared to non-biotransformed soybean extract (NBSE) and immunoassay demonstrated that the extracellular deposition was mostly inhibited by BSE-2 concentrations, except at 1.33 µg/mL. Hence, biotransformed soybean extract by the enzymatic filtrate of Aspergillus awamori fungus demonstrated a high nutricosmetic potential, showing safeness and effective collagen-I augmentation.

Evaluation of cell biomarkers as in vitro photoprotective assays for sunscreen formulations

Objectives: The aim of this study was to evaluate cellular indicators, which change with exposure to ultraviolet (UV) radiation and can be used as parameters for measuring sunscreens efficiency. Methods: Commercial strains of L929 and HaCaT cells (skin dermis and epidermis, respectively), from the cell bank of Rio de Janeiro, were exposed to different doses of UVA (350 nm) and UVB (309 nm) radiation. The evaluation of the photoprotective potential of sunscreens was analyzed with cell viability, lipid peroxidation and ROS generation tests. Samples of sunscreen with SPF values ranging from 15 to 60 were applied to a quartz plate superimposed on the top of a microplate containing the cell culture, and then the system was irradiated. Results: The viability and lipid peroxidation of the two cell lines remained unchanged after exposure to UVA radiation. When exposed to UVB radiation, the reduction in viability and the increase in lipid peroxides were dose-dependent, that is, they varied from 3.15% to 95.4%, and from 1.2 to 42.7 nM MDA/pg protein, respectively, both for the L929 strain. The dose of 0.5 J/cm2 reduced by 41.4%±1.67 the number of viable cells, and the dose of 30 J/cm2 promoted the oxidation of 42.7 nM of MDA/pg protein. These doses were selected to evaluate the photoprotective effectiveness of commercial sunscreens. Sunscreens exposed to UVB rays could prevent the loss of cell viability (viability remained around 100% for higher SPF) and the formation of lipid peroxides (30 to 80% reduction of peroxide levels). None of the two cell strains, submitted to UVB radiation, formed amounts of intracellular ROS in a dose-dependent manner. Under exposure to UVA radiation, only the HaCaT cell line produced the largest amounts of ROS in a dose-dependent manner. After treating these cells with photoprotective formulations (20 J/cm2), the researchers observed a reduction in the amount of ROS formed. Conclusions: The parameters of cell viability and lipid peroxidation were promising to evaluate the photoprotective capacity of sunscreens against UVB radiation. The generation of ROS expressed in the HaCaT strain can discriminate the photoprotective potential of formulations against UVA radiation, as sunscreens reduced the formation of ROS. These results suggest that in vitro tests that evaluate the damage caused to cells can predict cellular indicators of the photoprotective effectiveness of sunscreens and contribute to minimize these tests in the initial phase of product research and development.

Liquid crystalline nanodispersion functionalized with cell-penetrating peptides improves skin penetration and anti-inflammatory effect of lipoic acid after in vivo skin exposure to UVB radiation.

In this study, the development and the performance of a new targeted liquid crystalline nanodispersion (LCN) by the attachment of cell-penetrating peptides (CPP) onto their surfaces to improve skin delivery of lipoic acid (LA) were evaluated. For that, the synthesis and characterization of this new platform as well as its spatiotemporal analysis from in vitro and in vivo topical application were explored and extensively discussed in this paper. The TAT or D4 peptides were chosen as CPP due to specific target strategies by the charge grouping on the skin surface or target the overexpressed epidermal growth factor receptor (EGFR) of cell membrane of keratinocytes, respectively. Thus, the nanoparticle characterization results when taken together suggested that designed LCNs maintained their hexagonal phase structure, nanoscale particle size, and low polydispersity index even after drug, lipopolymers, and peptide additions, which are proved to be favorable for topical skin delivery. There were no statistical differences among the LCNs investigated, except for superficial charge of LCN conjugated with TAT which may have altered the LCN zeta potential due to cationic charge of TAT amino acid sequence compared with D4. The cumulative amounts of LA retained into the skin were determined to be even higher coming from the targeted LCNs. Moreover, the exogenous antioxidant application of the LA from the LCNs can prevent ROS damage, which was demonstrated by this study with the less myeloperoxidase (MPO) activity and decrease in cytokine levels (TNF-alpha and IL-1ß) generated by the oxidative stress modulation. Together, the data presented highlights the potential of these targeted LCNs, and overall, opens new frontiers for preclinical trials.

Apple Extract (Malus sp.) and Rutin as Photochemopreventive Agents: Evaluation of Ultraviolet B-Induced Alterations on Skin Biopsies and Tissue-Engineered Skin.

The skin is exposed to the solar ultraviolet B (UVB) radiation, which leads to the formation of several types of skin damage responsible for cancer initiation and aging. Malus sp. is a genus of apples, which are a good source of polyphenolic compounds. Malus sp. and more precisely one of its components, rutin, have preventive effects on many diseases caused by reactive oxygen species. In addition, previous studies have suggested the topical usage of the extract as a cosmetic product to prevent skin damage caused by oxidative stress. In this study, we evaluated the efficacy of two topical formulations containing 1.25% of Malus sp. extract and the equivalent amount of rutin (0.75%). The photochemopreventive effect was assessed on two three-dimensional (3D) skin models, that is, ex vivo skin explants and 3D tissue-engineered skin to compare the models. Both formulations protected against the UVB-induced increase in sunburn cell formation, as well as caspase-3 activation and cyclobutane pyrimidine dimer formation in both skin models. Furthermore, the formulations inhibited the lipid peroxidation and the metalloproteinase formation induced by UVB radiation. The tissue-engineered skins and the skin explants provided effective tools to assess the UVB-induced damages. These results support use of the Malus sp. extract and rutin as skin photochemopreventive agents for topical application.

Yangambin and Epi-yangambin Isomers: New Purification Method from Ocotea fasciculata and First Cytotoxic Aspects Focusing on In Vivo Safety.

Ocotea fasciculata presents yangambin (YAN) and its isomer epi-yangambin (EPI-YAN) as major lignans, which are employed as the plant markers for quality control purposes and as potential pharmacological compounds. However, a gap between the pure isomers and safety and efficacy protocols is faced by the scientific community. In this context, this work aimed to report (i) a new and advantageous purifying process in a semi-preparative scale for YAN and EPI-YAN isolation from Ocotea fasciculata, and (ii) an in vitro cytotoxicity study to estimate, for the first time, the LD50 values of the isolated epimers, as well as the influence of albumin concentration in cell culture medium. The best condition for epimers isolation was achieved in normal-phase liquid chromatography. The lignan fraction (LF), previously obtained from the plant ethanolic extract, was purified yielding 17% and 29% of YAN and EPI-YAN, respectively. The in vitro study demonstrated that YAN and EPI-YAN were safe, and only at the highest concentration studied, a decrease on cell viability was observed. The estimated LD50 value was higher than 1612 mg/kg for both epimers. The LF, on the other hand, demonstrated an estimated LD50 of 422 mg/kg. Lignan cytotoxicity studies also evidenced that the higher cell viability was related to the higher concentration of fetal bovine serum as a source of albumin in medium. This is the first time the LD50 and safety of the isolated epimers were estimated, opening up great perspectives of success in in vivo studies.

Pharmacokinetics, Pharmacodynamics and Dermal Distribution of 5-Methoxypsoralen Based on a Physiologically Based Pharmacokinetic Model to Support Phytotherapy Using Brosimum gaudichaudii.

The treatment of vitiligo includes the combination of psoralens and ultraviolet type A exposure. Psoralens belong to a group of natural furanocoumarins that cause the skin to become sensitive temporarily to ultraviolet type A. The aim of this study was to develop a physiologically based pharmacokinetic model of 5-MOP from Brosimum gaudichaudii to support psoralen and ultraviolet type A therapy. A study of rats was used to establish and validate rat tissue distribution. The same chemical-specific parameters used in the rat model were also employed in the human model to project human pharmacokinetics. The highest exposures in the rats were in the brain and skin. Following a single dose of 1.2 mg/kg 5-MOP in humans, the model predicted a maximum concentration of 20 ng/mL and an area under the curve of 125 ng.h/mL, matching clinical results. The half-maximum melanogenesis concentrations in B16F10 cells were 29.5, 18.5, 11.5, and 6.5 ng/mL for synthetic 5-MOP, synthetic 5-MOP with ultraviolet type A, B. gaudichaudii alone, and B. gaudichaudii plus ultraviolet type A, respectively. Physiologically based pharmacokinetic model prediction in humans supported a once-every-two-day regimen for optimal melanin production. This type of framework can be applied to support strategies for dose selection and to investigate the impact of drugs on melanocyte recovery.

Obtainment of an enriched fraction of Inga edulis: identification using UPLC-DAD-MS/MS and photochemopreventive screening.

Inga edulis is a fruit tree native from Central and South America. Different species of Inga have demonstrated high polyphenolic content and high antioxidant capacity. The oxidative stress significantly contributes to the development of several chronicle diseases, particularly age-related diseases. Therefore, photochemoprevention is an emerging interest in the use of naturally occurring antioxidants for their therapeutic use. A partial purification of the extract was carried out onto macroporous resin and the main components of I. edulis leaf extract and fraction were identified using UPLC-DAD-MS/MS as epicatechin, apigenin C-di-hexoside, myricetin-O-hexose-deoxyhexose, myricetin-O-deoxyhexose and vicenin-2, which was identified for the first time in I. edulis. Both extract and fraction presented high antioxidant capacity, the fraction exhibiting higher polyphenol and flavonoid contents and higher content of vicenin-2, suggesting that the fractioning process effectively resulted in the partial purification of the extract. The cytotoxicity and photostability were assessed in L929 fibroblast cells to evaluate whether I. edulis extract and fraction were suitable for topical application. The UVA and UVB irradiated extract and fraction did not decrease the antioxidant capacity and cell viability of L929 fibroblasts, demonstrating the photostability of the samples. The fractioning process successfully purified and enriched I. edulis extract, and both the extract and fraction are potential candidates to be safely incorporated in topical photochemopreventive formulations.

Cecropia obtusa extract and chlorogenic acid exhibit anti aging effect in human fibroblasts and keratinocytes cells exposed to UV radiation.

Cecropia obtusa is popularly used in the Amazonian region and exhibits antioxidant activity. Cosmetic formulations containing C. obtusa extract are commercially available for purchase; however, the chemical composition and the effects of the topical application of the extract are not described in the literature. Therefore, this study aimed to identify the main components of C. obtusa for the first time and to assess the anti aging effect in human fibroblasts and keratinocytes exposed to UVR. The main components in C. obtusa extract were identified by LC-DAD-MS/MS as chlorogenic acid (CGA), luteolin-C-hexoside, luteolin-C-hexose-O-deoxy-hexose, and apigenin-C-hexose-O-deoxy-hexose. C. obtusa extract and CGA decreased the metalloproteinase-1 and protein carbonyl levels and increased the collagen and hyaluronic acid contents. Overall, the extract exhibited better activity than CGA, and we demonstrated the ability of the extract to protect against the UV-induced increase in the pro inflammatory cytokines IL-1ß and IL-6, which are potential pathways of the antioxidant and anti aging effect. The chemical characterization added important data to broaden the knowledge related to C. obtusa, and the results suggest that the extract is a promising candidate to be incorporated in topical photochemoprotective formulations.

A novel research model for evaluating sunscreen protection in the UV-A1.

The use of a broad spectrum sunscreen is considered one of the main and most popular measures for preventing the damaging effects of ultraviolet radiation (UVR) on the skin. In this study we have developed a novel in vitro method to assess sunscreens efficacy to protect calcineurin enzyme activity, a skin cell marker. The photoprotective efficacy of sunscreen products was assessed by measuring the UV-A1 radiation-induced depletion of calcineurin (Cn) enzyme activity in primary neonatal human dermal fibroblast (HDFn) cell lysates. After exposure to 24J/cm2 UV-A1 radiation, the sunscreens containing larger amounts of UV-A1 filters (brand B), the astaxanthin (UV-A1 absorber) and the Tinosorb® M (UV-A1 absorber) were capable of preventing loss of Cn activity when compared to the sunscreens formulations of brand A (low concentration of UV-A1 filters), with the Garcinia brasiliensis extract (UV-B absorber) and with the unprotected cell lysate and exposed to irradiation (Irradiated Control - IC). The Cn activity assay is a reproducible, accurate and selective technique for evaluating the effectiveness of sunscreens against the effects of UV-A1 radiation. The developed method showed that calcineurin activity have the potential to act as a biological indicator of UV-A1 radiation-induced damages in skin and the assay might be used to assess the efficacy of sunscreens agents and plant extracts prior to in vivo tests.

Photochemoprotective effect of a fraction of a partially purified extract of Byrsonima crassifolia leaves against UVB-induced oxidative stress in fibroblasts and hairless mice.

Ultraviolet B (UVB) irradiation increases the risk of various skin disorders, leading to inflammation and oxidative stress and thereby increasing the risk of skin photoaging and carcinogenesis. The use of photochemoprotectors such as natural products with antioxidant and anti-inflammatory properties represents a strategy for preventing UVB-induced skin damage. We investigated the photochemoprotective effect of a fraction of a partially purified extract of Byrsonima crassifolia leaves (BCF) on fibroblasts and hairless mice exposed to UVB radiation. The mixture of phenolic compounds in BCF prevented the decrease in reduced glutathione (GSH) levels in fibroblast cultures induced by UVB radiation more than some of their individual standards ((+)-catechin (CAT), epigallocatechin gallate and quercetin 3-O-ß-d-glucopyranoside). Prepared gel formulations increased skin antioxidant activity, and BCF components and the CAT standard were retained in the HRS/J hairless mice epidermis 2h after application. Topical treatment with the BCF or CAT formulations (1%) significantly reduced the decrease in GSH levels and decreased myeloperoxidase activity and the secretion of pro-inflammatory cytokines IL-1ß and IL-6 induced by UVB radiation (P<0.05), indicating that both BCF and CAT had anti-inflammatory effects. BCF inhibited UVB-induced metalloproteinase (MMP)-9 secretion/activity, whereas CAT had no effect on MMP-9 activity in the skin of treated animals. These results therefore suggest that BCF can be used as a photochemoprotective agent and antioxidant in the prevention/treatment of inflammation and oxidative stress of the skin induced by UVB radiation.

The extended production of UV-induced reactive oxygen species in L929 fibroblasts is attenuated by posttreatment with Arrabidaea chica through scavenging mechanisms.

Ultraviolet radiation (UVR) exposure causes various injurious effects to human skin by generating reactive oxygen species (ROS). Excessive ROS production can lead to oxidative stress which may damage cellular components like lipids and proteins and causing photoaging. The use of natural photochemopreventive agents with antioxidant properties is an important alternative to improve the effectiveness of sunscreens and reduce skin photodamage. A crude extract (CE) from the leaves of Arrabidaea chica underwent partition by a liquid-liquid method. The hexane fraction (FH), chloroform fraction (FC), and ethyl acetate fraction (FEA) were obtained. The antioxidant capacity of the CE, FH, FC, and FEA was studied in a cell-free system using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and the xanthine/luminol/xanthine oxidase system. The FC had the best antioxidant activity. We also evaluated the photochemoprotective effect of A. chica in protecting L929 fibroblasts against UV-A- and UV-B-induced cell damage. A. chica inhibited the extended production of ROS up to 3h. Posttreatment with the CE and FC attenuated UV-induced cell damage through scavenging mechanisms, including the quenching of intracellular ROS and mitochondrial O2- and preventing lipid peroxidation. These results suggest that A. chica may be a promising non-sunscreen photoprotector that can improve the effectiveness of commercial sunscreens.

RNAi mediated IL-6 in vitro knockdown in psoriasis skin model with topical siRNA delivery system based on liquid crystalline phase.

Gene therapy by RNA interference (RNAi) is a post-transcriptional silencing process that can suppress the expression of a particular gene and it is a promising therapeutic approach for the treatment of many severe diseases, including cutaneous disorders. However, difficulties related to administration and body distribution limit the clinical use of small interfering RNA (siRNA) molecules. In this study, we proposed to use nanocarriers to enable siRNA application in the topical treatment of skin disorders. A siRNA nanodispersion based on liquid crystalline phase and composed of monoolein (MO), oleic acid (OA) and polyethylenimine (PEI) was developed and its physicochemical properties, efficiency of complexation and carrier/siRNA stability were assessed. Subsequently, cell viability, cellular uptake, in vitro skin irritation test using reconstructed human epidermis (RHE) and in vitro IL-6 knockdown in psoriasis skin model were evaluated. The results showed that the liquid crystalline nanodispersion is a promising topical delivery system for administration of siRNA, being able to overcome the limitations of the route of administration, as well those resulting from the characteristics of siRNA molecules. The formulation was effective at complexing the siRNA, presented high rate of cell uptake (∼90%), increased the skin penetration of siRNA in vitro, and did not cause skin irritation compared with Triton-X (a moderate irritant), resulting in a 4-fold higher viability of reconstructed human epidermis and a 15.6-fold lower release of IL-1α. A single treatment with the liquid crystalline nanodispersion carrying IL-6 siRNA for 6h was able to reduce the extracellular IL-6 levels by 3.3-fold compared with control treatment in psoriasis skin model. Therefore, liquid crystalline nanodispersion is a suitable nanocarrier for siRNA with therapeutic potential to suppress skin disease-specific genes. This study also highlights the applicability of reconstructed skin models in pharmaceutical field to evaluate the performance of delivery systems without the use of animal models.

Biotransformed soybean extract (BSE) inhibits melanoma cell growth and viability in vitro: involvement of nuclear factor-kappa B signaling.

Melanoma is recognized as one of the most aggressive cancers with a relatively high propensity for metastasis. The prognosis of melanoma remains poor in spite of treatment advances, emphasizing the importance of additional preventive measures. Isoflavonoids have become not only potential chemopreventive, but also important therapeutic natural agents. We evaluated the antiproliferative and proapoptotic properties of biotransformed soybean extract (BSE) in A375 melanoma cells. Previous analyses demonstrated that the concentration of daidzein, genistein and aminoacids/peptides present in BSE, fermented by Aspergillus awamori is much higher than in the non biotransformed extract (NBSE). Experiments comparing the efficacy of the extracts in preventing cancer cell growth showed that treatment (24 h) of aggressive melanoma cells (A375 and 451Lu) with BSE resulted in a dose-dependent inhibition of growth and viability. In contrast, treatment with similar doses of NBSE failed to inhibit melanoma cell viability. Further studies in A375 cells showed that decrease in cell viability with BSE treatment (1.5-1.9 mg/ml; 24 h) was associated with induction of apoptosis. Immunoblot analysis revealed that BSE treatment resulted in induction of PARP cleavage, activation of caspase-3, -7, and -8 and increased expression of TRAIL and its receptor DR4. BSE did not activate the intrinsic apoptotic pathway in A375 cells, as no change was observed in caspase-9 expression. The expression of Bcl-2 apoptotic proteins such as Bid and Bax remained unaffected with BSE treated cells. Interestingly, we also showed that BSE treatment increased the phosphorylation and activation of IKK, IκBα degradation and p65/NF-κB translocation to the nucleus, and that stimulation of the NF-???B pathway was required for BSE-induced apoptosis of A375 cells. Our findings indicate that the biotransformation of soybean plays a crucial role in the extract anti-cancer effect observed in melanoma cells. However, further studies are warranted to define the active anti-cancer agent(s) present in BSE.

Skin penetration and photoprotection of topical formulations containing benzophenone-3 solid lipid microparticles prepared by the solvent-free spray-congealing technique.

PURPOSE: Solid-lipid microparticles loaded with high amounts of the sunscreen UV filter benzophenone-3 were prepared by spray congealing with the objective of decreasing its skin penetration and evaluate whether the sunscreen's photoprotection were impaired by the microencapsulation process. METHODS: The microparticles were produced using the natural lipids carnauba wax or bees wax and three different concentrations of benzophenone-3 (30, 50 and 70%) using spray congealing technique. RESULTS: The microparticles presented properties suitable for topical application, such as spherical morphology, high encapsulation efficiency (95.53-102.2%), average particle sizes between 28.5 and 60.0 µm with polydispersivities from 1.2 to 2.5. In studies of in vitro skin penetration and preliminary stability, formulations of gel cream containing carnauba wax solid lipid microparticles and 70% benzophenone-3 when compared to the formulation added of bees wax solid-lipid microparticles containing 70% benzophenone-3, was stable considering the several parameters evaluated and were able to decrease the penetration of the UV filter into pig skin. Moreover, the formulations containing solid lipid microparticles with 70% benzophenone-3 increased the photoprotective capacity of benzophenone-3 under UV irradiation. CONCLUSION: The results show that spray-congealed microparticles are interesting solid forms to decrease the penetration solar filters in the skin without compromising their photoprotection.

In vitro and in vivo photoprotective/photochemopreventive potential of Garcinia brasiliensis epicarp extract.

The damaging effects of sunlight to the skin has triggered studies that involve the synthesis and extraction of organic compounds from natural sources that can absorb UV radiation, and studies on polyphenolic compounds with antioxidant and anti-inflammatory properties that can be used as photochemopreventive agents for reducing skin damage. We investigated the in vitro and in vivo photoprotective/photochemopreventive potential of Garcinia brasiliensis epicarp extract (GbEE). We evaluated the cell viability of L929 fibroblasts after UVB exposure using a quartz plate containing the extract solution or the GbEE formulation. The in vivo photoprotective effect of the GbEE formulation was evaluated by measuring the UVB damage-induced decrease in endogenous reduced glutathione (GSH), the increase in myeloperoxidase (MPO) activity and secretion of cytokines IL-1ß and TNF-α. The in vitro methodology using fibroblasts showed that the photoprotective properties of the GbEE solutions and 10% GbEE formulation were similar to the commercial sunscreen (SPF-15). In vivo results demonstrated of the GbEE formulation in decreasing UVB induced-damage such as GSH depletion, an increased in MPO activity and secretion of cytokines IL-1ß and TNF-α. The results showed that the extract has great potential for use as a sunscreen in topical formulations in addition to UV filters.

Zymographic and ultrastructural evaluations after low-level laser irradiation on masseter muscle of HRS/J strain mice.

Low-level laser therapy (LLLT) has been widely used in the treatment of the stomatognathic system dysfunction; however, its biological effect remains poorly understood. This study evaluated the effect of LLLT (GaAlAs, 780 nm, 20 J/cm(2), 40 mW) on masseter muscle of HRS/J mice after different numbers of laser irradiations (three, six, and ten) for 20 s in alternate days. Three experimental groups were defined according to the number of laser irradiations and three control groups (n=5) were used. On the third day after the last irradiation, all animals were killed and the masseter muscle was removed and processed for the following analysis: (a) transmission electron microscopy, (b) zymography, (c) immunohistochemistry for vascular endothelial growth factor (VEGF) and VEGFR-2. The results showed: (a) with six laser applications, a dilation of T tubules, and sarcoplasmic reticulum cistern, increased pinocytosed vesicles in the endothelium; with ten laser applications, few pinocytic vesicles in the endothelium and condensed mitochondria. (b) Under the conditions of this study, the synthesis of other matrix metalloproteinases was not observed, only the MMP-2 and -9. (c) After ten laser irradiations, immunostaining was observed only for VEGFR-2. We conclude that after six laser applications, ultrastructural changes may facilitate the Ca(+2) transfer to cytosol and increase the fluid transport from one surface to another. The ultrastructural changes and no immunostaining for VEGF with ten applications may decrease the metabolic activity as well as damage the angiogenic process, suggesting that an effective number of laser applications may be less than ten, associating to this therapy a better cost-benefit.

Effect of ultraviolet filters on skin superoxide dismutase activity in hairless mice after a single dose of ultraviolet radiation.

Organic sunscreens may decrease their protective capability and also behave as photo-oxidants upon ultraviolet radiation (UVR) exposure. The present study investigated the effect of a cream gel formulation containing the UV filters benzophenone-3, octyl methoxycinnamate, and octyl salicylate on skin superoxide dismutase (SOD) after a single dose of UVR (2.87 J/cm(2)). The retention of these UV filters was first evaluated in vivo using hairless mice to guarantee the presence of the filters in the skin layers at the moment of irradiation. The in vivo effect of the UV filters on skin SOD was then assayed spectrophotometrically via the reduction of cytochrome c. The cream gel formulation promoted the penetration of the three UV filters into the epidermis and the dermis at one hour post-application. A significant decrease in SOD activity was observed in irradiated animals treated with sunscreen formulation. However, no effect on SOD activity in skin was observed by the isolated presence of the sunscreens, the formulation components, or the exposure to UVR. The sunscreens may have formed degradation products under UVR that may have either inhibited the enzyme or generated reactive species in the skin.

Effects of low-level laser therapy on the oxidative metabolism and matrix proteins in the rat masseter muscle.

OBJECTIVE: This study aims to analyze the effects of low-level laser therapy (LLLT) on the oxidative activity and the expression/activity of metalloproteinases of the masseter muscle. BACKGROUND DATA: Currently in dentistry LLLT has been used on patients with muscular disorders, such as the temporomandibular disorders (TMDs) but its effect at the cellular level has not been fully elucidated. METHODS: Thirty male Wistar rats divided into 6 groups (n=5) received 10 laser irradiations (780 nm, 5 mmW, CW laser, illuminated area 0.04 cm(2), power density 125 mW/cm(2)), with different energy densities (group I-0; group II-0.5; group III-1.0; group IV-2.5; group V-5.0; and group VI-20 J/cm(2)). Muscles were processed for nicotinamide adenine dinucleotide diaphorase (NADH) and sucinate dehydrogenase (SDH) activities and zymography. The photomicrographs were evaluated by the point counting method using a test system and ImageJ software; and by the ANOVA statistical test. The proteinases' secretion/activity was qualitatively analyzed by zymography. RESULTS: LLLT significantly increased (p<0.05) masseter muscle oxidative metabolism shown by the increased area of intermediary fibers in the NADH (groups IV, V, and VI) and SDH (group V) reactions. The same metabolic pattern was observed among the groups in both reactions. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) zymography detected only the MMP-2 expression/activity for the untreated-control group (group I). The exposure to LLLT increased the activity of MPP-2 in group VI and the activity of MMP-9 in all groups exposed to different energy densities of laser irradiation (groups II, III, IV, V, and VI). CONCLUSIONS: Thus, LLLT stimulated the oxidative metabolism and the expression of matrix metalloproteinase (MMPs) of the masseter muscle, which may indicate a matrix remodeling process. However, group VI did not show the best results for oxidative metabolism, probably indicating that the dosage they were given was high for this protocol.