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.

Topical formulation containing chitosan-chamomile microparticles in cutaneous wound healing in rats.

Objective: The aim of this research was to evaluate the efficacy of a topical formulation containing chitosan-chamomile microparticles in cutaneous healing in rats. Method: Male Wistar rats (n=57) were randomly distributed into three groups: treatment; vehicle; and control. Evaluations were performed on days 2, 7 and 14 after the surgical procedure using skin lesion photography, and histological and biochemical analyses. Results: The results showed that there was no difference in the healing index and in the histological analysis of the inflammatory infiltrate among groups. Fibrogenesis was more significant in the group treated with the test formulation at day 7, and angiogenesis was greater in the vehicle and chamomile groups at day 2. The quantification of hydroxyproline showed a higher amount of collagen in the group treated with chamomile, mainly at day 14, although the histological quantification of collagen showed no difference between the groups. Conclusion: From the results of this study, it can be concluded that the formulation, although it had no effect on the healing time, improved the quality of the cicatricial tissue formed with a greater quantity of fibroblasts and collagen.

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.

Nanoemulsions Based on Sunflower and Rosehip Oils: The Impact of Natural and Synthetic Stabilizers on Skin Penetration and an Ex Vivo Wound Healing Model.

Vegetable oils offer excellent biological properties, but their high lipophilicity limits their bioavailability. This work aimed to develop nanoemulsions based on sunflower and rosehip oils and to evaluate their wound-healing activity. The influence of phospholipids of plant origin on nanoemulsions' characteristics was investigated. A nanoemulsion prepared with a mixture of phospholipids and synthetic emulsifiers (Nano-1) was compared with another prepared only with phospholipids (Nano-2). The healing activity was evaluated in wounds induced in human organotypic skin explant culture (hOSEC) based on histological and immunohistochemical analysis. The hOSEC wound model was validated, showing that high nanoparticle concentration in the wound bed interferes with cell mobility and the ability to respond to the treatment. Nanoemulsions were 130 to 370 nm, with a concentration of 1013 particles/mL, and a low potential to induce inflammatory processes. Nano-2 was three times larger than Nano-1 but less cytotoxic and could target the oils to the epidermis. Nano-1 permeated intact skin to the dermis and showed a more prominent healing effect than Nano-2 in the hOSEC wound model. Changes in the lipid nanoemulsion stabilizers impacted the cutaneous and cellular penetration of the oils, cytotoxicity, and healing kinetics, resulting in versatile delivery systems.

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.

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.

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.

Leaf extract of Coffea arabica L. reduces lipid peroxidation and has anti-platelet effect in a rat dyslipidemia model

Abstract This study aimed to evaluate the antioxidant potential of the Coffea arabica Lineu (L.) leaf extract and its effects on platelet aggregation of dyslipidemic rats. The extract was obtained by the percolation of C. arabica L. leaves in hydroethanolic solution 70% (v/v). The mass spectrometry FIA-ESI-MS² suggested the presence of chlorogenic acid, rutin acid, and quinic acid. The DPPH• radicals scavenging capacity was demonstrated (IC50 = 0.06 mg/mL). The extract was administered to rats by gavage (300 mg/kg/day) for 56 days. Dyslipidemia was induced by administering Triton WR-1339 (300 mg/kg body weight) on the 54th day. On day 56, blood was collected by puncturing the abdominal aorta artery and the aortic artery was removed. Lipid profile, markers of renal and hepatic injury, lipid peroxidation, and platelet aggregation tests were carried out. The ingestion of extract reduced the lipid peroxidation (aorta and plasma) and platelet aggregation in dyslipidemic rats. The extract did not affect markers of renal and hepatic function as analyzed in this study, suggesting neither impaired liver nor kidney function in these animals. Therefore, our results demonstrate that the extract of leaves of C. arabica L. show antioxidant potential in vitro and in vivo as well as anti-platelet aggregation in dyslipidemic animals

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.

Coffee intake (Coffea arabica L. ) reduces advanced glycation end product (AGEs) formation and platelet aggregation in diabetic rats

Objectives: The study aimed to determine the effect of coffee intake on AGEs formation and platelet aggregation in diabetic Wistar rats. Methods: Coffee powder samples were used to prepare a 10% beverage. Diabetes mellitus was induced in the animals by administering 2% alloxan. All animal experiments were approved by the ethics committee for animal experiments under N°. 420/2012 and 536/2013. Diabetic and non-diabetic rats were divided into 6 groups treated and untreated with coffee (7.2 mL/Kg body weight) and aminoguanidine (AGE inhibiting agent) (100 mg/Kg body weight) for 50 days. After 50 days, the animals were fasted for 12 h and anesthetized (40 mg/Kg sodium pentobarbital) intraperitoneally. Blood samples were collected from the abdominal artery puncture. Hematological parameters (red cells, hemoglobin, hematocrit and leukocyte) and glycemic and HbA1c levels were measured. AGEs quantification (spectrofluorometric method) and the platelet aggregation test (aggregation of cuvettes in a four-channel platelet aggregometer) were also conducted. The rats' renal function was evaluated by measuring serum urea and creatinine. Results: Data showed that coffee intake had no effect on the hematological parameters. Fasting glucose and HbA1c dosage were significantly higher in diabetic animals compared to non-diabetic animals (confirmed the effectiveness of inducing and maintaining diabetic status). Results showed that coffee reduced AGE formation and platelet aggregation in our animal model, not altering the animals' renal function. Conclusions: These results suggest beneficial effects on vasculopathy, a common complication in diabetic patients.

Coffee beverage reduces ROS production and does not affect the organism's response against Candida albicans

Coffee is a mixture of substances with potential beneficial and adverse health effects. Several studies demonstrate the antioxidant effect of the phenolics compounds present in coffee. Neutrophils produce reactive oxygen species (ROS) by activating of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), which plays a key role in organism defenseagainst microbial pathogens. Diabetes mellitus patients are more susceptible to bacterial and fungal infections. The present study evaluated the influence of coffee beverage on NOX2 activity and ROS generation and the impact of this effect on phagocytosis and killing of Candida albicansby neutrophils from diabetic and non-diabetic animals. Diabetes mellitus was induced in male Wistar rats using 2% alloxan. Diabetic and non-diabetic animals were divided into groups treated and untreated with coffee drink (7.2 mL/kg/day) or apocyanine (16 mg/kg/day) for 50 days. After 50 days, the animals' glycemic profile was measured by blood glucose and glycated hemoglobin (HbA1c) tests. The generation of ROS in neutrophilic cells was measured by chemiluminescence and cytochrome C reduction assays. C. albicans phagocytosis and death were evaluated by optical microscopy using the May-Grunwald-Giemsa staining method. The coffee drink has not altered the glycemic profile and NOX2 activity of the animals. However, coffee reduced the ROS pool in non-diabetic and diabetic animals, but this activity did not harm the phagocytosis or killing of neutrophils. Treatment with apocyanin decreased ROS production and killing capacity of neutrophils from non-diabetic animals against C. albicans. We suggest that the coffee drink intake prevents oxidative damage and does not impair response of the organism against opportunistic microorganism.

Caffeinated instant coffee prevents an increase in exercise-mediated superoxide anion production in rat peritoneal neutrophils

The present study analyzed the in vivo effects of drinking caffeinated and decaffeinated instant coffee (8% w/v) by adult male Wistar rats submitted to high-intensity exercises. The parameters used in the evaluation were the determination of the activities of NADPH oxidase, myeloperoxidase and other antioxidant enzymes present in neutrophils of rats. It was observed that exercise-induced superoxide anion production depends on the NADPH oxidase activity (estimated by the cytochrome C reduction test) in peritoneal neutrophils (p < 0.05). The intake of caffeinated and decaffeinated instant coffee beverages and of a caffeine solution to 1.67% did not induced changes in the activities of the enzymes myeloperoxidase, superoxide dismutase and glutathione peroxidase (p < 0.05). But consumption of caffeinated instant coffee drink prevented an increase in NADPH oxidase-mediated superoxide production induced by highly intense exercise in rat neutrophils. While the decaffeinated instant coffee drink or caffeine solution alone did not affect NADPH oxidase-mediated superoxide production. We suggest that this activity is associated with the chemical composition and concentration of phenolic compounds and other antioxidant substances formed during roasting. From the obtained results, it was concluded that moderate intake of caffeinated instant coffee (equivalent to a daily human consumption of 4 50-mLcups of coffee) may have beneficial effects on health, contributing to a reduction in superoxide anion generation. Therefore, more research must be conducted to elucidate the mechanism of action of caffeinated coffee on NADPH oxidase in neutrophils.

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.