Maria José Vieira Fonseca

Quercetin in w/o microemulsion: In vitro and in vivo skin penetration and efficacy against UVB-induced skin damages evaluated in vivo

The present study evaluated the potential of a w/o microemulsion as a topical carrier system for delivery of the antioxidant quercetin. Topical and transdermal delivery of quercetin were evaluated in vitro using porcine ear skin mounted on a Franz diffusion cell and in vivo on hairless-skin mice. Skin irritation by topical application of the microemulsion containing quercetin, and the protective effect of the formulation on UVB-induced decrease of endogenous reduced glutathione levels and increase of cutaneous proteinase secretion/activity were also investigated. The w/o microemulsion increased the penetration of quercetin into the stratum corneum and epidermis plus dermis at 3, 6, 9 and 12h post-application in vitro and in vivo at 6h post-application. No transdermal delivery of quercetin occurred. By evaluating established endpoints of skin irritation (erythema formation, epidermis thickening and infiltration of inflammatory cells), the study demonstrated that the daily application of the w/o microemulsion for up to 2 days did not cause skin irritation. W/o microemulsion containing quercetin significantly prevented the UVB irradiation-induced GSH depletion and secretion/activity of metalloproteinases.

Quercetin Reduces Inflammatory Pain: Inhibition of Oxidative Stress and Cytokine Production

Quercetin (1) is known to have both antioxidant and antinociceptive effects. However, the mechanism involved in its antinociceptive effect is not fully elucidated. Cytokines and reactive oxygen species have been implicated in the cascade of events resulting in inflammatory pain. Therefore, we evaluated the antinociceptive mechanism of 1 focusing on the role of cytokines and oxidative stress. Intraperitoneal and oral treatments with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid and phenyl-p-benzoquinone and also the second phase of formalin- and carrageenin-induced mechanical hypernociception. Compound 1 also inhibited the hypernociception induced by cytokines (e.g., TNFalpha and CXCL1), but not by inflammatory mediators that directly sensitize the nociceptor such as PGE2 and dopamine. On the other hand, 1 reduced carrageenin-induced IL-1beta production as well as carrageenin-induced decrease of reduced glutathione (GSH) levels. These results suggest that 1 exerts its analgesic effect by inhibiting pro-nociceptive cytokine production and the oxidative imbalance mediation of inflammatory pain.

Protective effect of Calendula officinalis extract against UVB-induced oxidative stress in skin: Evaluation of reduced glutathione levels and matrix metalloproteinase secretion

BACKGROUND AND PURPOSE Calendula officinalis flowers have long been employed time in folk therapy, and more than 35 properties have been attributed to decoctions and tinctures from the flowers. The main uses are as remedies for burns (including sunburns), bruises and cutaneous and internal inflammatory diseases of several origins. The recommended doses are a function both of the type and severity of the condition to be treated and the individual condition of each patient. Therefore, the present study investigated the potential use of Calendula officinalis extract to prevent UV irradiation-induced oxidative stress in skin. METHODS Firstly, the physico-chemical composition of marigold extract (ME) (hydroalcoholic extract) was assessed and the in vitro antioxidant efficacy was determined using different methodologies. Secondly, the cytotoxicity was evaluated in L929 and HepG2 cells with the MTT assay. Finally, the in vivo protective effect of ME against UVB-induced oxidative stress in the skin of hairless mice was evaluated by determining reduced glutathione (GSH) levels and monitoring the secretion/activity of metalloproteinases. RESULTS AND CONCLUSIONS The polyphenol, flavonoid, rutin and narcissin contents found in ME were 28.6 mg/g, 18.8 mg/g, 1.6 mg/g and 12.2mg/g, respectively and evaluation of the in vitro antioxidant activity demonstrated a dose-dependent effect of ME against different radicals. Cytoxicity experiments demonstrated that ME was not cytotoxic for L929 and HepG2 cells at concentrations less than or equal to of 15 mg/mL. However, concentrations greater than or equal to 30 mg/mL, toxic effects were observed. Finally, oral treatment of hairless mice with 150 and 300 mg/kg of ME maintained GSH levels close to non-irradiated control mice. In addition, this extract affects the activity/secretion of matrix metalloproteinases 2 and 9 (MMP-2 and -9) stimulated by exposure to UVB irradiation. However, additional studies are required to have a complete understanding of the protective effects of ME for skin.

Quercetin inhibits UV irradiation-induced inflammatory cytokine production in primary human keratinocytes by suppressing NF-κB pathway

BACKGROUND Topical flavonoids, such as quercetin, have been shown to reduce ultraviolet (UV) irradiation-mediated skin damage. However, the mechanisms and signaling pathways involved in this protective effect are not clear. UV irradiation leads to activation of two major signaling pathways, namely nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) pathways. Activation of NF-κB pathway by UV irradiation stimulates inflammatory cytokine expression, whereas activation of AP-1 pathway by UV irradiation promotes matrix metalloproteinase (MMP) production. Both pathways contribute to UV irradiation-induced skin damage, such as photoaging and skin tumor formation. OBJECTIVE To elucidate the underlying mechanism, we examined the effect of quercetin on UV irradiation induced activation of NF-κB and AP-1 pathways. METHODS Primary human keratinocytes, the major skin cell type subjected to physiological solar UV irradiation, were used to study the effects of quercetin on UV irradiation-induced signal transduction pathways. RESULTS Quercetin decreased UV irradiation-induced NF-κB DNA-binding by 80%. Consequently, quercetin suppressed UV irradiation-induced expression of inflammatory cytokines IL-1β (∼60%), IL-6 (∼80%), IL-8 (∼76%) and TNF-α (∼69%). In contrast, quercetin had no effect on UV irradiation activation of three MAP kinases, ERK, JNK, or p38. Accordingly, induction of AP-1 target genes such as MMP-1 and MMP-3 by UV irradiation was not suppressed by quercetin. CONCLUSION Our data indicate that the ability of quercetin to block UV irradiation-induced skin inflammation is mediated, at least in part, by its inhibitory effect on NF-κB activation and inflammatory cytokine production.

Efficacy of topical formulations containing Pimenta pseudocaryophyllus extract against UVB-induced oxidative stress and inflammation in hairless mice.

Plants rich in antioxidant substances may be a promising strategy for preventing UV-induced oxidative and inflammatory damage of the skin. Pimenta pseudocaryophyllus is native to Brazil and presents flavonoids and other polyphenolic compounds in high concentration. Thus, the present study evaluated the possible effects of topical formulations containing P. pseudocaryophyllus ethanolic extract (PPE) at inhibiting UV-B irradiation-induced oxidative stress and inflammation. PPE was administered on the dorsal skin of hairless mice using two formulations: F1 (non-ionic emulsion with high lipid content) and F2 (anionic emulsion with low lipid content) before and after UV-B irradiation. The following parameters were evaluated in skin samples: edema, myeloperoxidase activity, cytokines levels, matrix metalloprotease-9 (MMP-9) secretion/activity, reduced glutathione (GSH), superoxide anion and lipid peroxidation levels, and mRNA expression for glutathione reductase and gp91phox. The UV-B irradiation increased all parameters, except for IL-10 levels and glutathione reductase mRNA expression, which were not altered, and GSH levels, which were reduced by exposure to UV-B light. Treatments with F1 and F2 containing PPE inhibited UV-B-induced edema formation (89% and 86%), myeloperoxidase activity (85% and 81%), IL-1β production (62% and 82%), MMP-9 activity (71% and 74%), GSH depletion (73% and 85%), superoxide anion (83% and 66%) and TBARS (100% and 100%) levels, increased glutathione reductase (2.54 and 2.55-fold) and reduced gp91phox (67% and 100%) mRNA expression, respectively. F2 containing PPE also increased IL-10 levels. Therefore, this study demonstrates the effectiveness of topical formulations containing PPE in inhibiting UV-B irradiation-induced inflammation and oxidative stress of the skin.

Enhanced in vitro and in vivo antioxidant activity and mobilization of free phenolic compounds of soybean flour fermented with different β-glucosidase-producing fungi.

Aims:  To evaluate the soybean polyphenol glucosides bioconversion to aglycone forms by different β‐glucosidases‐producing filamentous fungi to enhance their antioxidant activity.

Comparison of antioxidant activities of tocopherols alone and in pharmaceutical formulations

The objective of the present investigation was to compare the antioxidant effect of different forms of Vitamin E (DL-alpha-tocopherol, Mixed Tocopherols, Ronoxan MAP and alpha-tocopherol acetate) and of topical formulations containing these active pharmaceutical ingredients, using chemiluminescence and the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays. Inhibition of the intensity of chemiluminescence, using the H2O2-luminol-horseradish peroxidase (HRP) enzyme system, was measured for 10 min at room temperature in 10 microl samples of each vitamin at different concentrations, and of formulations containing these vitamins. H-donor ability in the DPPH assay, was measured in 10 microl samples at different concentrations of each vitamin, as well as in formulation in ethanol solution; the decrease of absorbency was read at 517 nm. DL-alpha-tocopherol, Mixed Tocopherols and Ronoxan MAP alone or in formulations, markedly inhibited chemiluminescence intensity and decreased absorbency in the DPPH assay in a concentration-dependent manner. Alpha-tocopherol acetate and formulations containing this vitamin did not show antioxidant activity in either assay. Other components of the formulations did not interfere with the measurements, indicating that the methods employed can be used to evaluate antioxidant activity in topical formulations.

Evaluation of functional stability of quercetin as a raw material and in different topical formulations by its antilipoperoxidative activity

The present study evaluates the antioxidant activity of the flavonol quercetin, and its functional stability as a raw material and when added in formulations. The iron-chelating activity was determined using the bathophenanthroline assay, and the functional stability was evaluated with the antilipoperoxidative assay. Raw material presented concentration-dependent antilipoperoxidative and iron-chelating activities. The initial antilipoperoxidative activity of the raw material, cream and gel-cream were 63%, 78%, and 69%, respectively. There was no detectable loss of activity during 182 days (6 months) of storage at all tested temperatures (4°C, room temperature [RT], 37°C, and 45°C) for the raw material. Considering the method variability of 10%, activity loss greater than 10% for nonionic cream was detected after 126 days at 4°C (20.1%), decreasing thereafter to 22.2% after 182 days. At 45°C, the loss of activity started after 182 days (13.2%). For the anionic gel-cream, activity loss started after 84 days (28.4%, 45°C), decreasing after 182 days to 40.3% at 45°C. At 37°C, activity loss was detected after 182 days (12%). In conclusion, the results suggest that the activity of quercetin depends on iron chelation, and its posible usefulness as a topical antioxidant to prevent oxidative stress-induced skin damage depends on maintaining its antilipoperoxidative activity stored at RT, which avoids special storage conditions.

Chapter 9 - Flavonoids as Anti-Inflammatory and Analgesic Drugs: Mechanisms of Action and Perspectives in the Development of Pharmaceutical Forms

Abstract Flavonoids are polyphenolic compounds with very diverse roles. In this chapter, we examined the bioactivity and potential of flavonoids as anti-inflammatory and analgesic drugs and the perspectives on their applicability and pharmaceutical development. Further, the mechanisms of action of flavonoids are addressed, for example, their effects on cytokine production, oxidative stress, and inhibition of protein kinases. In addition, the role of these mechanisms in inflammation and pain, and the structure–activity relationship are discussed.

Evaluation of Protective Effect of a Water-In-Oil Microemulsion Incorporating Quercetin Against UVB-Induced Damage in Hairless Mice Skin

PURPOSE In the present study, histological aspects were considered in order to evaluate the in vivo photoprotective effect of a w/o microemulsion containing quercetin against UVB irradiation-induced dermal damages. The toxicity in cell culture and the potential skin irritation resulting from topical application of this formulation were also investigated. METHODS Mouse dorsal surfaces were treated topically with 300 mg of the unloaded and quercetin-loaded (0.3%, w/w) microemulsions before and after exposure to UVB (2.87 J/cm2) irradiation. The untreated control groups irradiated and non-irradiated were also evaluated. UVB-induced histopathological changes as well as the photoprotective effect of this formulation were evaluated considering the parameters of infiltration of inflammatory cells, epidermis thickening (basale and spinosum layers) and collagen and elastic fiber contents. The cytotoxicity of the reported formulation was evaluated in L929 mice fibroblasts by MTT assay and the skin irritation was investigated after topical application of both unloaded and quercetin-loaded microemulsions once a day for 15 days. RESULTS The results demonstrated that the w/o microemulsion containing quercetin reduced the incidence of histological skin alterations, mainly the connective-tissue damage, induced by exposure to UVB irradiation, this allows the suggestion that protective effects of this formulation against UV-induced responses are not secondary to the interference of UV transmission (i.e., blocking the UVB radiation from being absorbed by the skin), as is usually done with UVB absorbers and sunscreens, but is instead due to different biological effects of this flavonoid. Furthermore, by evaluating the cytotoxic effect on L929 cells and histological aspects such as infiltration of inflammatory cells and epidermis thickness of hairless mice, the present study also demonstrated no toxicity of the proposed system. CONCLUSIONS Therefore, based on these mouse models, a detailed characterization of the w/o microemulsion incorporating quercetin effects as a photochemoprotective agent on human skin is thus indicated.