Marcela M. Baracat

Quercetin-Loaded Microcapsules Ameliorate Experimental Colitis in Mice by Anti-inflammatory and Antioxidant Mechanisms

Quercetin (1) is an anti-inflammatory and antioxidant flavonoid. However, the oral administration of 1 did not lead to beneficial effects in experimental animal colitis models, which involve cytokines and oxidative stress. A possible explanation is that the absorption profile of 1 prevents its activity. Therefore, it was reasoned that the controlled release of 1 would improve its therapeutic effect. Thus, the therapeutic effect and mechanisms of 1-loaded microcapsules in acetic acid-induced colitis in mice were evaluated. Microcapsules were prepared using pectin/casein polymer and 1. The oral administration of 1-loaded microcapsules decreased neutrophil recruitment, attenuated histological alterations, and reduced macroscopical damage, edema, and IL-1β and IL-33 production in the colon samples. Microcapsules loaded with 1 also prevented the reduction of anti-inflammatory cytokine IL-10 and the antioxidant capacity of the colon. These preclinical data indicate that pectin/casein polymer microcapsules loaded with 1 improved the anti-inflammatory and antioxidant effects of 1 compared to the nonencapsulated drug. Therefore, quercetin seems to be a promising active molecule in inflammatory bowel disease if provided with adequate controlled release.

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.

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.

Topical Formulation Containing Naringenin: Efficacy against Ultraviolet B Irradiation-Induced Skin Inflammation and Oxidative Stress in Mice.

Naringenin (NGN) exhibits anti-inflammatory and antioxidant activities, but it remains undetermined its topical actions against ultraviolet B (UVB)-induced inflammation and oxidative stress in vivo. The purpose of this study was to evaluate the physicochemical and functional antioxidant stability of NGN containing formulations, and the effects of selected NGN containing formulation on UVB irradiation-induced skin inflammation and oxidative damage in hairless mice. NGN presented ferric reducing power, ability to scavenge 2,2′-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS) and hydroxyl radical, and inhibited iron-independent and dependent lipid peroxidation. Among the three formulations containing NGN, only the F3 kept its physicochemical and functional stability over 180 days. Topical application of F3 in mice protected from UVB-induced skin damage by inhibiting edema and cytokine production (TNF-α, IL-1β, IL-6, and IL-10). Furthermore, F3 inhibited superoxide anion and lipid hydroperoxides production and maintained ferric reducing and ABTS scavenging abilities, catalase activity, and reduced glutathione levels. In addition, F3 maintained mRNA expression of cellular antioxidants glutathione peroxidase 1, glutathione reductase and transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2), and induced mRNA expression of heme oxygenase-1. In conclusion, a formulation containing NGN may be a promising approach to protecting the skin from the deleterious effects of UVB irradiation.

Naringenin Inhibits Superoxide Anion-Induced Inflammatory Pain: Role of Oxidative Stress, Cytokines, Nrf-2 and the NO−cGMP−PKG−KATPChannel Signaling Pathway

In the present study, the effect and mechanism of action of the flavonoid naringenin were evaluated in superoxide anion donor (KO2)-induced inflammatory pain in mice. Naringenin reduced KO2-induced overt-pain like behavior, mechanical hyperalgesia, and thermal hyperalgesia. The analgesic effect of naringenin depended on the activation of the NO−cGMP−PKG−ATP-sensitive potassium channel (KATP) signaling pathway. Naringenin also reduced KO2-induced neutrophil recruitment (myeloperoxidase activity), tissue oxidative stress, and cytokine production. Furthermore, naringenin downregulated KO2-induced mRNA expression of gp91phox, cyclooxygenase (COX)-2, and preproendothelin-1. Besides, naringenin upregulated KO2-reduced nuclear factor (erythroid-derived 2)-like 2 (Nrf2) mRNA expression coupled with enhanced heme oxygenase (HO-1) mRNA expression. In conclusion, the present study demonstrates that the use of naringenin represents a potential therapeutic approach reducing superoxide anion-driven inflammatory pain. The antinociceptive, anti-inflammatory and antioxidant effects are mediated via activation of the NO−cGMP−PKG−KATP channel signaling involving the induction of Nrf2/HO-1 pathway.

Vanillic Acid Inhibits Inflammatory Pain by Inhibiting Neutrophil Recruitment, Oxidative Stress, Cytokine Production, and NFκB Activation in Mice

Vanillic acid (1) is a flavoring agent found in edible plants and fruits. It is an oxidized form of vanillin. Phenolic compounds form a substantial part of plant foods used as antioxidants with beneficial biological activities. These compounds have received considerable attention because of their role in preventing human diseases. Especially, 1 presents antibacterial, antimicrobial, and chemopreventive effects. However, the mechanisms by which 1 exerts its anti-inflammatory effects in vivo are incompletely understood. Thus, the effect of 1 was evaluated in murine models of inflammatory pain. Treatment with 1 inhibited the overt pain-like behavior induced by acetic acid, phenyl-p-benzoquinone, the second phase of the formalin test, and complete Freunds adjuvant (CFA). Treatment with 1 also inhibited carrageenan- and CFA-induced mechanical hyperalgesia, paw edema, myeloperoxidase activity, and N-acetyl-β-D-glucosaminidase activity. The anti-inflammatory mechanisms of 1 involved the inhibition of oxidative stress, pro-inflammatory cytokine production, and NFκB activation in the carrageenan model. The present study demonstrated 1 presents analgesic and anti-inflammatory effects in a wide range of murine inflammation models, and its mechanisms of action involves antioxidant effects and NFκB-related inhibition of pro-inflammatory cytokine production.

The ruthenium NO donor, [Ru(bpy)2(NO)SO3](PF6), inhibits inflammatory pain: involvement of TRPV1 and cGMP/PKG/ATP-sensitive potassium channel signaling pathway.

The activation of nitric oxide (NO) production is an analgesic mechanism shared by drugs such as morphine and diclofenac. Therefore, the controlled release of low amounts of NO seems to be a promising analgesic approach. In the present study, the antinociceptive effect of the ruthenium NO donor [Ru(bpy)2(NO)SO3](PF6) (complex I) was investigated. It was observed that complex I inhibited in a dose (0.3-10mg/kg)-dependent manner the acetic acid-induced writhing response. At the dose of 1mg/kg, complex I inhibited the phenyl-p-benzoquinone-induced writhing response and formalin- and complete Freunds adjuvant-induced licking and flinch responses. Additionally, complex I also inhibited transient receptor potential cation channel subfamily V member 1 (TRPV1)-dependent overt pain-like behavior induced by capsaicin. Complex I also inhibited the carrageenin-induced mechanical hyperalgesia and increase of myeloperoxidase activity (MPO) in paw skin samples. The inhibitory effect of complex I in the carrageenin-induced hyperalgesia, MPO activity and formalin was prevented by the treatment with ODQ, KT5823 and glybenclamide, indicating that complex I inhibits inflammatory hyperalgesia by activating the cGMP/PKG/ATP-sensitive potassium channel signaling pathway. The present study demonstrates the efficacy of a novel ruthenium NO donor and its analgesic mechanisms.

DETERMINATION OF ALBENDAZOLE IN ORAL SUSPENSION

Albendazole is a potent benzimidazole anthelmintic used in the treatment of human intestinal helmintiasis as well as of hydatid cysts and neurocysticercosis. Two analytical methods were standardized for determination of albendazole in pharmaceutical preparations (oral suspension), one by UV direct spectrophotometry at 230 nm usingas solvent 2% sulfuric acid in methanol, and another by HPLC using a Nucleosil® C18 (5μ−4.6×150 mm) Phenomenex® column, and a mobile phase consistingof methanol : 0.05M phosphate buffer (70:30; v/v), UV detection at 254nm and ambient temperature. The methods were applied to simulated and commercially available samples and the results were statistically compared showingthat both methods were accurate and sensitive and there was no significant difference between them.

Sensory evaluation of albendazole suspensions

Sensory analysis was used in the albendazole suspension stability study. Three formulations were prepared and stored for 1, 3, and 6 months at 4 ±1, 26 ±1, 37, 50 and 65 oC. Samples were evaluated through the difference from control sensory test using 24 trained judges in individual cabins. Although albendazole content was not altered in the conditions studied, sensory test showed differences between control and stored samples, except for one of the formulations stored under refrigeration for one month. These results have shown that the sensory evaluation is an important tool for quality control of pharmaceutical preparations, in association with chemical, physicochemical and microbiological tests.

Trans-chalcone added in topical formulation inhibits skin inflammation and oxidative stress in a model of ultraviolet B radiation skin damage in hairless mice

Trans-chalcone (TC) is a common precursor of flavonoids. However, the pharmacological properties of TC remain to be fully understood. The present study investigated whether topical formulation containing TC (TFcTC) presents therapeutic effect in UVB radiation-induced skin damage using disease, enzyme activity, antioxidant activity, protein and mRNA parameters. Control topical formulation (CTF) and TFcTC were applied in hairless mice before and after exposure to UVB radiation. Dorsal skin samples were collected after UVB exposure to evaluate: i) skin edema (weight) was measured by punch biopsy; ii) spectrophotometric assays were used to measure myeloperoxidase (MPO) and catalase activities, ferric (FRAP) and ABTS cation reducing antioxidant power, superoxide anion production and levels of reduced glutathione (GSH); iii) enzymography was used to measure matrix metalloproteinase-9 (MMP-9) activity; iv) chemiluminescence was used to measure the lipid peroxidation (LPO); v) enzyme-linked immunosorbent assay (ELISA) was used to measure tumor necrosis factor alpha (TNF-α) levels; vi) reverse transcription quantitative PCR (RT-qPCR) was used to measure cyclooxygenase-2 (COX-2), gp91phox (NADPH oxidase sub-unity), glutathione peroxidase-1 (Gpx1), glutathione reductase (Gr), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) mRNA expression. TFcTC inhibited UVB-induced skin edema, MPO activity, MMP-9 activity, TNF-α production, and COX-2 mRNA expression. TFcTC inhibited UVB-induced LPO, down-regulated superoxide anion levels and gp91phox mRNA expression, and improved antioxidant potential and GSH skin levels. The mRNA expression of detoxification systems such as Nrf2, HO-1, Gpx1 and Gr, and catalase activity were also enhanced by treatment with TFcTC. In conclusion, TFcTC protects mice skin from UVB radiation by inhibiting inflammation, and improving antioxidant and detoxification systems. Therefore, topical treatment with TC is a novel therapeutic approach for the treatment of UVB radiation skin damages, which merits further pre-clinical and clinical investigation.