In vitro protective effect of topical nanoemulgels containing Brazilian red propolis benzophenones against UV-induced skin damage.

The overexposure of the skin to ultraviolet (UV) radiation may lead to oxidative stress, resulting in severe damage. The prevention of skin injuries through the topical application of natural compounds rich in antioxidants, such as propolis extracts, has shown promising results. In Brazil, the "red propolis" extract has stood out due to its complex constitution, based mainly on polyprenylated benzophenones (BZP). However, although the use of red propolis extracts has been shown to be encouraging, their addition in topical formulations is limited by the low solubility of BZP. For this reason, this study aimed to develop topical nanoemulgels containing Brazilian red propolis (BRP) extract to increase the potential of topical application, and the evaluation of skin protection against UVA/UVB radiation damage by means of protein carbonylation, protein thiol content and TBARS assays. The nanoemulgels were obtained by adding gelling polymer to nanoemulsions that were previously prepared by spontaneous emulsification. In this sense, a nanoemulgel containing BRP extract-loaded nanoemulsions (H-NE) and a nanoemulgel containing BRP extract-loaded nanoemulsions with DOTAP (H-NE/DT) were prepared. The physicochemical characterization of nanoemulgels showed monodisperse populations of 200-300 nm. The H-NE zeta potential was -38 mV, while that of H-NE/DT was +36 mV. BZP content in the formulations was around 0.86 mg g-1. These parameters remained stable for 90 days under cold storage. H/NE and H-NE/DT presented a non-Newtonian pseudoplastic rheological behavior. Permeation/retention studies, through porcine ear skin, showed the highest BZP retention (18.11 µg cm-2 after 8 h) for H-NE/DT, which also demonstrated, in an in vitro study, the highest ability to protect skin against oxidative damage after UVA/UVB radiation exposure. The results concerning the antioxidant activity revealed that formulations containing the BRP n-hexane extract were the most promising in combating oxidative stress, probable due to the presence of polyprenylated BZP. Altogether, the outcomes of this study suggest that nanoemulgels have suitable characteristics for topical application, and may be an alternative for the prevention of oxidative skin damage caused by UVA/UVB radiation.

A stability-indicating ultra-fast liquid chromatography method for the assay of the main flavonoids of Achyrocline satureioides (Marcela) in porcine skin layers and nanoemulsions.

INTRODUCTION: Achyrocline satureioides (marcela or macela) is a plant widely used in folk medicine in South America. Recently, there has been increasing interest for the development of skin care products containing A. satureoides extracts, due to its well-documented antioxidant, antiherpetic, and wound healing properties. OBJECTIVES: The present study aimed to develop and validate a yet unexplored stability-indicating and robust ultra-fast liquid chromatography (UFLC) method for the simultaneous quantification of the main flavonoids of A. satureioides in extracts, nanoemulsions, and porcine skin layers. MATERIAL AND METHODS: The chromatographic separation of flavonoids quercetin, luteolin, and 3-O-methylquercetin was performed on a Luna C18 analytical column (100 mm × 2.0 mm i.d.; particle size 2.5 µm) using isocratic elution with methanol/phosphoric acid 1% (48:52 v/v) with a flow rate of 0.3 mL/min at 40°C. RESULTS: The method was found to be specific, linear (R > 0.998), precise, accurate, and robust for all flavonoids assayed in A. satureioides extract, nanoemulsions, and porcine ear skin. A low matrix effect was noted for all complex matrices. The stability-indicating UFLC method was evaluated by submitting isolated flavonoids, a mixture of standards, and A. satureioides extract to acidic, alkaline, oxidative, UV-A/UV-C light, and thermal stress conditions. No peaks were found co-eluting with the flavonoids of interest in all matrices. The robustness of the method was confirmed using Plackett-Burman experimental design. CONCLUSION: The short run time (8 min) and reliability of the method could be useful for the determination of A. satureioides flavonoids in topical product development since extracts of this medicinal plant have been used to treat various skin disorders.

Skin Permeation and Oxidative Protection Effect of Soybean Isoflavones from Topical Nanoemulsions-a Comparative Study of Extracts and Pure Compounds.

Soybean isoflavone-rich extracts have been considered as promising skin antiaging products due to their antioxidant activity. This study investigates the effect of soybean isoflavone forms on porcine ear skin permeation/retention from topical nanoemulsions and their potential in protecting skin against oxidative damage caused by UVA/UVB light. Soybean non-hydrolyzed (SNHE) and hydrolyzed (SHE) extracts, mainly composed of genistin and genistein, were produced. Nanoemulsions containing SNHE (NESNHE) and SHE (NESHE) were prepared by spontaneous emulsification procedure and yielded monodispersed nanoemulsions. A delay of isoflavone release was observed after extracts incorporation into nanoemulsions when compared to a propyleneglycol dispersion of pure compounds. An increase of isoflavone skin retention from nanoemulsions was also achieved. However, from extracts, a higher amount of genistin (NESNHE) and a lower amount of genistein (NESHE) were detected in the skin in comparison to pure isoflavones. Finally, the protection of porcine ear skin by formulations against UVA/UVB oxidative stress was evaluated. Extract-loaded nanoemulsions offered better skin protection than pure isoflavones. Skin lipids were similarly protected by NESHE and NESNHE, whereas skin proteins were more protected by NESNHE. Overall, nanoemulsions containing isoflavone-rich soybean extracts may be considered a better topical formulation aiming skin protection from UVA/UVB oxidative damage.

Effect of Hydrogel Containing Achyrocline satureioides (Asteraceae) Extract-Loaded Nanoemulsions on Wound Healing Activity.

Achyrocline satureioides (Lam.) DC extract-loaded nanoemulsions have demonstrated potential for wound healing, with promising effects on keratinocyte proliferation. We carried out the first in vivo investigation of the wound healing activity of a hydrogel containing A. satureioides extract-loaded nanoemulsions. We prepared hydrogels by adding the gelling agent (Carbopol® Ultrez) to extract-loaded nanoemulsions (~250 nm in diameter) obtained by spontaneous emulsification. The final flavonoid content in formulation was close to 1 mg/mL, as estimated by ultra-fast liquid chromatography. Permeation/retention studies using porcine ear skin showed that flavonoids reached deeper layers of pig ear skin when it was damaged (up to 3.2 µg/cm² in the dermis), but did not reach the Franz-type diffusion cell receptor fluid. For healing activity, we performed a dorsal wound model using Wistar rats, evaluating the lesion size, anti-inflammatory markers, oxidative damage, and histology. We found that extract-loaded formulations promoted wound healing by increasing angiogenesis by ~20%, reducing inflammation (tumor necrosis factor α) by ~35%, decreasing lipid damage, and improving the re-epithelialization process in lesions. In addition, there was an increase in the number of blood vessels and hair follicles for wounds treated with the formulation compared with the controls. Our findings indicate that the proposed formulation could be promising in the search for better quality healing and tissue reconstruction.

Achyrocline satureioides (Lam.) DC (Asteraceae) Extract-Loaded Nanoemulsions as a Promising Topical Wound Healing Delivery System: In Vitro Assessments in Human Keratinocytes (HaCaT) and HET-CAM Irritant Potential.

Achyrocline satureioides (Lam.) DC Asteraceae extracts (ASEs) have been investigated for the treatment of various skin disorders. This study reports the effects of ASE-loaded nanoemulsions (NEASE) on the cellular viability, death by necrosis, and migration of immortalized human keratinocytes (HaCaT cell line), as well as the irritant potential through the hen's egg chorioallantoic membrane test (HET-CAM). NEASE exhibited a polydispersity index above 0.12, with a droplet size of 300 nm, ζ-potential of -40 mV, and content of flavonoids close to 1 mg/mL. No cytotoxicity of the ASE was observed on HaCaT by MTT assay (up to 10 µg/mL). A significant increase of HaCaT viability was observed to NEASE (up to 5 µg/mL of flavonoids), compared to treatment with the ASE. The necrosis death evaluation demonstrated that only NEASE did not lead to cell death at all the tested concentrations. The scratch assay demonstrated that NEASE was able to increase the cell migration at low flavonoid concentrations. Finally, the HET-CAM test proved the non-irritative potential of NEASE. Overall, the results indicate the potential of the proposed formulations for topical use in wound healing, in view of their promising effects on proliferation and migration in keratinocytes, combined with an indication of the absence of cytotoxicity and non-irritating potential.

Hydrogels containing soybean isoflavone aglycones-rich fraction-loaded nanoemulsions for wound healing treatment - in vitro and in vivo studies.

Soybean isoflavone aglycones have been investigated as potential wound healing compounds for topical application. The aim of this study was to evaluate the wound healing properties of a soybean isoflavone aglycones-rich fraction (IAF) when incorporated into lipid nanoemulsions dispersed in acrylic-acid hydrogels. Formulations exhibited a mean droplet size in the sub 200 nm range, negative ζ-potential (-60 mV), and displayed non-Newtonian pseudoplastic behavior. The addition of a gelling agent decreased the IAF release from formulations and improved the retention of these compounds in intact porcine ear skin when compared with a control propylene glycol solution. No IAF were detected in receptor fluid of Franz-type diffusion cells. However, increasing amounts of IAF were noticed in both skin layers and the receptor fluid when the tissue was partially injured (tape stripping), or when the epidermis was completely removed. In vitro studies showed that IAF elicits an increased proliferation and migration of keratinocytes (HaCaT cell line). Subsequently, the healing effect of the formulations was evaluated in a model of dorsal wounds in rats, by assessing the size of the lesions, histology, inflammatory markers, and antioxidant activity. Overall findings demonstrated the potential of IAF-loaded formulations to promote wound healing by increasing angiogenesis by ∼200 %, reducing the lipid oxidation (TBARS) by ∼52 % and the inflammation (TNFα) by ∼35 %, while increasing re-epithelialization by ∼500 %, visualized by the epithelium thickness.