Rubia Casagrande

Vitexin Inhibits Inflammatory Pain in Mice by Targeting TRPV1, Oxidative Stress, and Cytokines

The flavonoid vitexin (1) is a flavone C-glycoside (apigenin-8-C-β-D-glucopyranoside) present in several medicinal and other plants. Plant extracts containing 1 are reported to possess antinociceptive, anti-inflammatory, and antioxidant activities. However, the only evidence that 1 exhibits antinociceptive activity was demonstrated in the acetic acid-induced writhing model. Therefore, the analgesic effects and mechanisms of 1 were evaluated. In the present investigation, intraperitoneal treatment with 1 dose-dependently inhibited acetic acid-induced writhing. Furthermore, treatment with 1 also inhibited pain-like behavior induced by phenyl-p-benzoquinone, complete Freunds adjuvant (CFA), capsaicin (an agonist of transient receptor potential vanilloid 1, TRPV1), and both phases of the formalin test. It was also observed that inhibition of carrageenan-, capsaicin-, and chronic CFA-induced mechanical and thermal hyperalgesia occurred. Regarding the antinociceptive mechanisms of 1, it prevented the decrease of reduced glutathione levels, ferric-reducing ability potential, and free-radical scavenger ability, inhibited the production of hyperalgesic cytokines such as TNF-α, IL-1β, IL-6, and IL-33, and up-regulated the levels of the anti-hyperalgesic cytokine IL-10. These results demonstrate that 1 exhibits an analgesic effect in a variety of inflammatory pain models by targeting TRPV1 and oxidative stress and by modulating cytokine production.

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.

Evaluation of the antioxidant activity of different flavonoids by the chemiluminescence method.

The objective of the present investigation was to study the antioxidant action of different flavonoids (quercetin, glabridin, red clover, and Isoflavin Beta, an isoflavones mixture) in order to determine if they could be added to a topical formulation used to treat damage caused by free radicals. Samples of 10 μL of the test compounds at different concentrations were mixed with 0.1 M phosphate buffer, pH 7.4, and a luminol solution was added to yield a final concentration of 0.113 mM. Hydrogen peroxide was then added at a final concentration of 0.05 mM. The reaction was started by introducing the horse-radish peroxidase enzyme at a final concentration of 0.2 IU/mL, in a final volume of 1.0 mL. Chemiluminescence was measured for 10 minutes at room temperature, and dimethylsulfoxide (DMSO) was used as a control. All samples showed marked inhibition of oxidative stress, with a concentration-dependent action for quercetin and Isoflavin Beta. The highest inhibition was observed with glabridin and the dry red clover extract. All flavonoids proved to be adequate for addition to topical formulations because of their high antioxidant activity.

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.

Kaurenoic acid from Sphagneticola trilobata Inhibits Inflammatory Pain: effect on cytokine production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway.

Kaurenoic acid [ent-kaur-16-en-19-oic acid (1)] is a diterpene present in several plants including Sphagneticola trilobata. The only documented evidence for its antinociceptive effect is that it inhibits the writhing response induced by acetic acid in mice. Therefore, the analgesic effect of 1 in different models of pain and its mechanisms in mice were investigated further. Intraperitoneal and oral treatment with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid. Oral treatment with 1 also inhibited overt nociception-like behavior induced by phenyl-p-benzoquinone, complete Freunds adjuvant (CFA), and both phases of the formalin test. Compound 1 also inhibited acute carrageenin- and PGE(2)-induced and chronic CFA-induced inflammatory mechanical hyperalgesia. Mechanistically, 1 inhibited the production of the hyperalgesic cytokines TNF-α and IL-1β. Furthermore, the analgesic effect of 1 was inhibited by l-NAME, ODQ, KT5823, and glybenclamide treatment, demonstrating that such activity also depends on activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that 1 exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of cytokine production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway.

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.

Acetic acid- and phenyl-p-benzoquinone-induced overt pain-like behavior depends on spinal activation of MAP kinases, PI3K and microglia in mice

The acetic acid and phenyl-p-benzoquinone are easy and fast screening models to access the activity of novel candidates as analgesic drugs and their mechanisms. These models induce a characteristic and quantifiable overt pain-like behavior described as writhing response or abdominal contortions. The knowledge of the mechanisms involved in the chosen model is a crucial step forward demonstrating the mechanisms that the candidate drug would inhibit because the mechanisms triggered in that model will be addressed. Herein, it was investigated the role of spinal mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated kinase), JNK (Jun N-terminal Kinase) and p38, PI(3)K (phosphatidylinositol 3-kinase) and microglia in the writhing response induced by acetic acid and phenyl-p-benzoquinone, and flinch induced by formalin in mice. Acetic acid and phenyl-p-benzoquinone induced significant writhing response over 20 min. The nociceptive response in these models were significantly and in a dose-dependent manner reduced by intrathecal pre-treatment with ERK (PD98059), JNK (SB600125), p38 (SB202190) or PI(3)K (wortmannin) inhibitors. Furthermore, the co-treatment with MAP kinase and PI(3)K inhibitors, at doses that were ineffective as single treatment, significantly inhibited acetic acid- and phenyl-p-benzoquinone-induced nociception. The treatment with microglia inhibitors minocycline and fluorocitrate also diminished the nociceptive response. Similar results were obtained in the formalin test. Concluding, MAP kinases and PI(3)K are important spinal signaling kinases in acetic acid and phenyl-p-benzoquinone models of overt pain-like behavior and there is also activation of spinal microglia indicating that it is also important to determine whether drugs tested in these models also modulate such spinal mechanisms.

Protective effects of the flavonoid hesperidin methyl chalcone in inflammation and pain in mice: role of TRPV1, oxidative stress, cytokines and NF-κB.

Cytokines and reactive oxygen species are inflammatory mediators that lead to increased sensitivity to painful stimuli, and their inhibition represents a therapeutic approach in controlling acute and chronic pain. The water-soluble flavonone hesperidin methyl chalcone (HMC) is used in the treatment of venous diseases, but its bioactivity as anti-inflammatory and analgesic is poorly understood. The present study evaluated the protective effects of HMC in widely used mouse models of acute and prolonged inflammation and pain. Male Swiss mice were treated with HMC (3-100 or 30 mg/kg, intraperitoneally) or vehicle (saline) 1h before inflammatory stimuli. In overt pain-like behavior tests, HMC inhibited acetic acid- and phenyl-p-benzoquinone-induced writhing, and capsaicin-, Complete Freunds Adjuvant (CFA)- and formalin-induced paw flinching and licking. HMC also inhibited carrageenan-, capsaicin- and CFA-induced mechanical and thermal hyperalgesia. Mechanistically, HMC inhibited carrageenan-induced cytokine (TNF-α, IL-1β, IL-6, and IL-10) production, oxidative stress and NF-κB activation. Furthermore, HMC did not cause gastric or hepatic injury in a 7 days treatment protocol. Thus, this is the first report that HMC reduces inflammation and inflammatory pain by targeting TRPV1 (transient receptor potential vanilloid type 1) receptor activity, oxidative stress, cytokine production, and NF-κB activity, which suggests its potential applicability in inflammatory diseases.

Analgesic activity of piracetam: effect on cytokine production and oxidative stress.

Piracetam is a prototype of nootropic drugs used to improve cognitive impairment. However, recent studies suggest that piracetam can have analgesic and anti-inflammatory effects. Inflammatory pain is the result of a process that depends on neutrophil migration, cytokines and prostanoids release and oxidative stress. We analyze whether piracetam has anti-nociceptive effects and its mechanisms. Per oral pretreatment with piracetam reduced in a dose-dependent manner the overt pain-like behavior induced by acetic acid, phenyl-p-benzoquinone, formalin and complete Freunds adjuvant. Piracetam also diminished carrageenin-induced mechanical and thermal hyperalgesia, myeloperoxidase activity, and TNF-α-induced mechanical hyperalgesia. Piracetam presented analgesic effects as post-treatment and local paw treatment. The analgesic mechanisms of piracetam were related to inhibition of carrageenin- and TNF-α-induced production of IL-1β as well as prevention of carrageenin-induced decrease of reduced glutathione, ferric reducing ability and free radical scavenging ability in the paw. These results demonstrate that piracetam presents analgesic activity upon a variety of inflammatory stimuli by a mechanism dependent on inhibition of cytokine production and oxidative stress. Considering its safety and clinical use for cognitive function, it is possible that piracetam represents a novel perspective of analgesic.

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.