Effect of the metanolic extract from the leaves of Garcinia humilis Vahl (Clusiaceae) on acute inflammation.

Garcinia humilis is popularly used to treat digestive, intestinal and inflammatory illness. We investigated the in vivo and in vitro effects of the methanol extract of G. humilis leaves (MEGh) on inflammatory cells behavior (migration and chemical mediators release) and hypersensitivity. Anti-inflammatory activity was investigated using carrageenan-induced inflammation in the subcutaneous tissue of male Swiss mice treated orally with MEGh (0.1-30 mg/kg). Leucocyte migration, chemical mediators secretion (TNF, IL-1ß, IL-6 and CXCL1) and protein exudation were quantified in the exudate. The adhesion molecules expression (CD62L and CD18), chemical mediators and chemotaxis was evaluated using neutrophils or macrophages RAW.264.7 previously treated with the extract (1-100 µg/mL) and activated with LPS. The anti-inflammatory activity of the isolated compounds friedelin, canophyllol, amentoflavone and 3-desmethyl-2-geranyl-4-prenylbellidypholine xanthone (10 µM) was evaluated in macrophages nitric oxide (NO) and TNF release. MEGh, given orally (30 mg/kg), significantly reduced neutrophil migration and decreased TNF, IL-1ß and CXCL1 levels, without interfering with protein exudation and IL-6. In vitro, the extract significantly reduced IL-1ß and IL-6 levels but did not alter TNF and CXCL1. The MEGh also reduced the expression of CD62L and CD18 and consequently neutrophil chemotaxis. The compounds friedelin, amentoflavone and 3-demethyl-2-geranyl-4-prenylbellidypholine xanthone decreased the secretion of NO and TNF by RAW264.7. The MEGh effects were extended to the pain-like behaviour induced by carrageenan in the mice hindpaw. MEGh presented important anti-inflammatory effects probably due to its activity on neutrophil migration and on important chemical mediator release, scientifically reinforcing its use as medicinal plant.

Cellular and Molecular Mechanisms of Environmental Pollutants on Hematopoiesis.

Hematopoiesis is a complex and intricate process that aims to replenish blood components in a constant fashion. It is orchestrated mostly by hematopoietic progenitor cells (hematopoietic stem cells (HSCs)) that are capable of self-renewal and differentiation. These cells can originate other cell subtypes that are responsible for maintaining vital functions, mediate innate and adaptive immune responses, provide tissues with oxygen, and control coagulation. Hematopoiesis in adults takes place in the bone marrow, which is endowed with an extensive vasculature conferring an intense flow of cells. A myriad of cell subtypes can be found in the bone marrow at different levels of activation, being also under constant action of an extensive amount of diverse chemical mediators and enzymatic systems. Bone marrow platelets, mature erythrocytes and leukocytes are delivered into the bloodstream readily available to meet body demands. Leukocytes circulate and reach different tissues, returning or not returning to the bloodstream. Senescent leukocytes, specially granulocytes, return to the bone marrow to be phagocytized by macrophages, restarting granulopoiesis. The constant high production and delivery of cells into the bloodstream, alongside the fact that blood cells can also circulate between tissues, makes the hematopoietic system a prime target for toxic agents to act upon, making the understanding of the bone marrow microenvironment vital for both toxicological sciences and risk assessment. Environmental and occupational pollutants, therapeutic molecules, drugs of abuse, and even nutritional status can directly affect progenitor cells at their differentiation and maturation stages, altering behavior and function of blood compounds and resulting in impaired immune responses, anemias, leukemias, and blood coagulation disturbances. This review aims to describe the most recently investigated molecular and cellular toxicity mechanisms of current major environmental pollutants on hematopoiesis in the bone marrow.

Development of an Inflamed High Throughput Stem-cell-based Gut Epithelium Model to Assess the Impact of Annexin A1.

OBJECTIVE AND DESIGN: Annexin A1 (ANXA1) plays a role in maintaining intestinal hemostasis, especially following mucosal inflammation. The published data about ANXA1 was derived from experimental animal models where there is an overlapping between epithelial and immune cells. There is no in vitro gut epithelial model that can assess the direct effect of ANXA1 on the gut epithelium. METHODS: We developed high-throughput stem-cell-based murine epithelial cells and bacterial lipopolysaccharides (LPS) were used to induce inflammation. The impact of ANXA1 and its functional part (Ac2-26) was evaluated in the inflamed model. Intestinal integrity was assessed by the transepithelial electrical resistance (TEER), and FITC-Dextran permeability. Epithelial junction proteins were assessed using confocal microscopy and RT-qPCR. Inflammatory cytokines were evaluated by RT-qPCR and ELISA. RESULTS: LPS challenge mediated a damage in the epithelial cells as shown by a drop in the TEER and an increase in FITC-dextran permeability; reduced the expression of epithelial junctional proteins (Occludin, ZO-1, and Cadherin) and increased the expression of the gut leaky protein, Claudin - 2. ANXA1 and Ac2-26 treatment reduced the previous damaging effects. In addition, ANXA1 and Ac2-26 inhibited the inflammatory responses mediated by the LPS and increased the transcription of the anti-inflammatory cytokine, IL-10. CONCLUSION: ANXA1 and Ac2-26 directly protect the epithelial integrity by affecting the expression of epithelial junction and inflammatory markers. The inflamed gut model is a reliable tool to study intestinal inflammatory diseases, and to evaluate the efficacy of potential anti-inflammatory drugs and the screening of new drugs that could be candidates for inflammatory bowel disease.

Development of Ac2-26 Mesoporous Microparticle System as a Potential Therapeutic Agent for Inflammatory Bowel Diseases.

Introduction: Inflammatory bowel diseases (IBDs) disrupt the intestinal epithelium, leading to severe chronic inflammation. Current therapies cause adverse effects and are expensive, invasive, and ineffective for most patients. Annexin A1 (AnxA1) is a pivotal endogenous anti-inflammatory and tissue repair protein in IBD. Nanostructured compounds loading AnxA1 or its active N-terminal mimetic peptides improve IBD symptomatology. Methods: To further explore their potential as a therapeutic candidate, the AnxA1 N-terminal mimetic peptide Ac2-26 was incorporated into SBA-15 ordered mesoporous silica and covered with EL30D-55 to deliver it by oral treatment into the inflamed gut. Results: The systems SBA-Ac2-26 developed measurements revealed self-assembled rod-shaped particles, likely on the external surface of SBA-15, and 88% of peptide incorporation. SBA-15 carried the peptide Ac2-26 into cultured Raw 264.7 macrophages and Caco-2 epithelial cells. Moreover, oral administration of Eudragit-SBA-15-Ac2-26 (200 µg; once a day; for 4 days) reduced colitis clinical symptoms, inflammation, and improved epithelium recovery in mice under dextran-sodium sulfate-induced colitis. Discussion: The absorption of SBA-15 in gut epithelial cells is typically low; however, the permeable inflamed barrier can enable microparticles to cross, being phagocyted by macrophages. These findings suggest that Ac2-26 is successfully delivered and binds to its receptors in both epithelial and immune cells, aligning with the clinical results. Conclusion: Our findings demonstrate a simple and cost-effective approach to delivering Ac2-26 orally into the inflamed gut, highlighting its potential as non-invasive IBD therapy.

Effect of nanocapsules containing docosahexaenoic acid in mice with chronic inflammation.

BACKGROUND: Omega 3 fatty acids, such as docosahexaenoic acid (DHA) have been widely consumed as supplements to control chronic inflammation. Nanocapsules containing DHA (MLNC-DHA-a1) were developed and showed excellent stability. Thus, our objective was to evaluate the effect of MLNC-DHA-a1 nanocapsules on biomarkers of chronic inflammation. METHODS: Cells viability was determined by flow cytometry. The uptake of MLNC-DHA-a1 nanocapsules by macrophages and their polarization were determined. In vivo, LDLr(-,-) mice were fed a Western diet to promote chronic inflammation and were treated with MLNC-DHA-a1 nanocapsules, intravenously injected via the caudal vein once a week for 8 weeks. RESULTS: MLNC-DHA-a1 nanocapsules decreased the concentration of TNFα (p = 0.02) in RAW 264.7 cells compared to the non-treated group (NT), with no changes in IL-10 (p = 0.29). The nanocapsules also exhibited an increase in the M2 (F4/80+ CD206) phenotype (p < 0.01) in BMDM cells. In vivo, no difference in body weight was observed among the groups, suggesting that the intervention was well tolerated. However, compared to the CONT group, MLNC-DHA-a1 nanocapsules led to an increase in IL-6 (90.45 ×13.31 pg/mL), IL-1ß (2.76 ×1.34 pg/mL) and IL-10 (149.88 ×2.51 pg/mL) levels in plasma. CONCLUSION: MLNC-DHA-a1 nanocapsules showed the potential to promote in vitro macrophage polarization and were well-tolerated in vivo. However, they also increased systemic pro-inflammatory cytokines. Therefore, considering that this immune response presents a limitation for clinical trials, further studies are needed to identify the specific compound in MLNC-DHA-a1 that triggered the immune response. Addressing this issue is essential, as MLNC-DHA-a1 tissue target nanocapsules could contribute to reducing chronic inflammation.

Role of Annexin A1 Secreted by Neutrophils in Melanoma Metastasis.

Annexin A1 (AnxA1) is highly secreted by neutrophils and binds to formyl peptide receptors (FPRs) to trigger anti-inflammatory effects and efferocytosis. AnxA1 is also expressed in the tumor microenvironment, being mainly attributed to cancer cells. As recruited neutrophils are player cells at the tumor sites, the role of neutrophil-derived AnxA1 in lung melanoma metastasis was investigated here. Melanoma cells and neutrophils expressing AnxA1 were detected in biopsies from primary melanoma patients, which also presented higher levels of serum AnxA1 and augmented neutrophil-lymphocyte ratio (NLR) in the blood. Lung melanoma metastatic mice (C57BL/6; i.v. injected B16F10 cells) showed neutrophilia, elevated AnxA1 serum levels, and higher labeling for AnxA1 in neutrophils than in tumor cells at the lungs with metastasis. Peritoneal neutrophils collected from naïve mice were co-cultured with B16F10 cells or employed to obtain neutrophil-conditioned medium (NCM; 18 h incubation). B16F10 cells co-cultured with neutrophils or with NCM presented higher invasion, which was abolished if B16F10 cells were previously incubated with FPR antagonists or co-cultured with AnxA1 knockout (AnxA1-/-) neutrophils. The depletion of peripheral neutrophils during lung melanoma metastasis development (anti-Gr1; i.p. every 48 h for 21 days) reduced the number of metastases and AnxA1 serum levels in mice. Our findings show that AnxA1 secreted by neutrophils favors melanoma metastasis evolution via FPR pathways, addressing AnxA1 as a potential biomarker for the detection or progression of melanoma.

Development of Annexin A1-surface-functionalized metal-complex multi-wall lipid core nanocapsules and effectiveness on experimental colitis.

Annexin A1 (AnxA1), a 37KDa protein, is secreted by inflammatory and epithelial cells and displays anti-inflammatory and wound healing activities in intestinal bowel diseases. Herein, we aimed to functionalize recombinant AnxA1 (AnxA1) on multi-wall lipid core nanocapsules (MLNC) and investigate its effectiveness on experimental colitis. MLNC were prepared by covering lipid core nanocapsules (LNC) with chitosan, which coordinates metals to specific protein chemisorption sites. Therefore, MLNC were linked to Zn2+ and AnxA1 was added to form MLNC-AnxA1. LNC, MLNC and MLNC-AnxA1 presented average size of 129, 152 and 163 nm, respectively, and similar polydispersity indexes (0.xx); incorporation of chitosan inverted the negative potential zeta; the coordination efficiency of AnxA1 was 92.22 %, and transmission electron microscope photomicrograph showed MLNC-AnxA1 had a spherical shape. The effectiveness of MLNC-AnxA1 was measured in Dextran Sulfate Sodium (DSS)-induced colitis in male C57BL/6 mice. DSS (2 % solution) was administered from days 1-6; saline, LNC, MLNC, MLNC-AnxA1 or AnxA1 were administered, once a day, by oral or intraperitoneal (i.p.) routes, from days 6-9. Clinical parameters of the disease were measured from day 0-10 and gut tissues were collected for histopathology, immunohistochemistry and flow cytometry analyses. Only i.p. treatment with MLNC-AnxA1 reduced weight loss, diarrhea and disease activity index, and prevented loss of colonic structure integrity; induced the switch of macrophages into M2 phenotype in the lamina propria; recovered the colonic histoarchitecture by decreasing dysplasia of crypts, inflammation and ulcerations; restored the expression of claudin-1 Zonna-occludens-1 tight junctions in the inflamed gut; and induced stem cell proliferation in intestinal crypts. Associated, data highlight the functionalization of MLNC with AnxA1 as a tool to improve the local actions of such protein in the inflamed gut by inducing resolution of inflammation and tissue repair.

Sambucus nigra: A traditional medicine effective in reducing inflammation in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Sambucus nigra L. is a plant of European origin and popularly known as elder, elderberry, black elder, European elder, European elderberry, and European black elderberry, being described in pharmacopoeia of several countries. Its flowers and berries have been used in folk medicine to treat feverish conditions, coughing, nasal congestion, and influenza besides its popular use as anti-inflammatory, analgesic, and diuretic agent. AIM OF THE STUDY: The aim of this investigation was to elucidate the anti-inflammatory and the relaxant effect of the lyophilized aqueous extract obtained from S. nigra's flowers on in vivo and in vitro inflammation assays and on the isolated rat vascular and airway smooth muscle tissue. MATERIAL AND METHODS: The anti-inflammatory activity of the extract was investigated using carrageenan-induced inflammation model in the subcutaneous tissue of male Swiss mice orally treated with S. nigra extract (30, 100, 300 or 600 mg/kg). Leukocyte influx and the secretion of chemical mediators were quantified in the inflamed exudate. Additionally, histological analysis of the pouches was performed. N-Formyl-methionine-leucine-phenylalanine-induced chemotaxis, lipopolysaccharide-induced TNF, IL-6, IL-1ß, IL-10 and NO production, and adhesion molecule expression (CD62L, CD49d and CD18, flow cytometry) were analyzed in vitro using oyster glycogen-recruited peritoneal neutrophils or macrophages (RAW 264.7) stimulated with LPS and treated with the extract (1, 10 or 100 µg/mL). The resolution of inflammation was accessed by efferocytosis assay, and the antinociceptive activity was investigated using carrageenan-induced mechanical hypersensitivity. Finally, the effect of the extract was evaluated in isolated rat aorta and trachea rings. RESULTS: The oral treatment with S. nigra promoted reduction in the neutrophil migration as well as the decrease of TNF, IL-1ß and IL-6 levels in the inflamed exudate. In vitro treatment with S. nigra decreased NO2-, TNF, IL-1ß and IL-6 and promoted increase of IL-10 in LPS-stimulated neutrophils. Similarly, the extract reduced the NO2-, TNF and IL-6 in LPS-stimulated macrophages. Rutin, the major constituent of S. nigra extract reduced NO2-, TNF, IL-1ß, and IL-6 and promoted the increase of IL-10 in LPS-stimulated neutrophils supernatant. The extract also shed CD62L and CD18 expressions. The extract was able to increase the efferocytosis of apoptotic neutrophils by increasing the IL-10 and decreasing the TNF levels. Additionally, the extract reduced the hypersensitivity induced by carrageenan and promoted a relaxant effect in isolated vascular and non-vascular rat tissue. CONCLUSIONS: S. nigra flowers extract presents anti-inflammatory effect by modulating macrophage and neutrophil functions including the production of inflammatory mediators and cell migration, by promoting efferocytosis and consequently the resolution of acute inflammation, besides exerting antinociceptive effects, scientifically proving its popular use as medicinal plant. Allied to the relaxant effect in both vascular and non-vascular smooth muscle tissue, S. nigra extract represents an important tool for the management of acute inflammation.

Anti-Inflammatory and Healing Activity of the Hydroalcoholic Fruit Extract of Solanum diploconos (Mart.) Bohs.

BACKGROUND: Solanum diploconos (Mart.) Bohs is a native Brazilian plant belonging to the Solanaceae family, popularly known as "tomatinho do mato" and poorly investigated. Herein, we presented for the first time evidence for the anti-inflammatory and wound healing activities of S. diploconos fruit hydroalcoholic extract. Material and Methods. In vitro fMLP-induced chemotaxis, LPS-induced inflammatory mediator levels (cytokines by ELISA and NO release by Griess reaction), and adhesion molecule expression (CD62L, CD49d, and CD18, by flow-cytometry) were assessed in neutrophils treated with different concentrations of the extract. Inflammation resolution was measured by the efferocytosis assay and the healing activity by in vivo and in vitro assays. The air pouch model of carrageenan-induced inflammation in Swiss mice was used to investigate the in vivo anti-inflammatory effects of the extract. Leukocyte influx (by optical microscopy) and cytokine release were quantified in the pouch exudates. Additionally, the acute and subacute toxic and genotoxic effects of the extract were evaluated. RESULTS: In vitro, the extract impaired neutrophil chemotaxis and its ability to produce and/or release cytokines (TNFα, IL-1ß, and IL-6) and NO upon LPS stimuli (p < 0.01). LPS-treated neutrophils incubated with the extract presented increased CD62L expression (p < 0.01), indicating a reduced activation. An enhanced efferocytosis of apoptotic neutrophils by macrophages was observed and accompanied by higher IL-10 and decreased TNFα secretion (p < 0.01). In vivo, similar results were noted, including reduction of neutrophil migration, protein exudation, and cytokine release (p < 0.01). Also, the extract increased fibroblast proliferation and promoted skin wound healing (p < 0.01). No signs of toxicity or genotoxicity were observed for the extract. CONCLUSION: S. diploconos fruit extract is anti-inflammatory by modulating neutrophil migration/activation as well macrophage-dependent efferocytosis and inflammatory mediator release. It also indicates its potential use as a healing agent. Finally, the absence of acute toxic and genotoxic effects reinforces its possible use as medicinal product.

Formyl Peptide Receptors and Annexin A1: Complementary Mechanisms to Infliximab in Murine Experimental Colitis and Crohn's Disease.

Non-responsiveness to anti-TNF-α therapies presents relevant rates in inflammatory bowel disease patients, presenting the need to find biomarkers involved in therapeutic efficacy. Herein, we demonstrate that higher levels of colonic formyl peptide receptor 1 and annexin A1 correlate with histological recovery in Crohn's disease patients under remission. Using the dextran sulfate sodium colitis model in mice, we suggest that infliximab induces annexin A1 expression and secretion in activated intestinal leukocytes. Conversely, this mechanism might stimulate epithelial formyl peptide receptors, inducing wound healing and consequent histological remission. Our data indicate that assessing intestinal expressions of formyl peptide receptors and annexin A1 might provide precious information on the disease activity and responsiveness to infliximab in inflammatory bowel disease patients.

Red light-emitting diode treatment improves tissue recovery in DSS-induced colitis in mice.

Inflammatory bowel diseases are debilitating illnesses characterized by severe inflammation of the gastrointestinal tract. Treatments currently available are expensive and ineffective. We here investigated the role of red-light emitting diode (LED) on dextran sodium sulfate (DSS)-induced colitis. DSS was added to the drinking water of male mice at days 0, 2, 4 and withdrawn at day 6. LED irradiation was performed daily for 90s from day 6 to 9 on the right and left sides of the ventral surface and beside the external anal region. LED treatment decreased the amount of crypt dysplasia/edema, inflammatory infiltrates and ulcers, attenuated apoptosis and increased proliferation of crypt cells. Also, LED treatment induced expression of annexin A1 in the damaged epithelium, preserved the organization of claudin-1 and skewed cytokine profiling towards a more anti-inflammatory status. Thus, LED treatment promotes structural protection and modulates the inflammatory response, constituting a potential non-invasive and low-cost combined therapy to help patients achieve disease remission.

Pioglitazone-Mediated Attenuation of Experimental Colitis Relies on Cleaving of Annexin A1 Released by Macrophages.

Ulcerative colitis and Crohn's disease are chronic inflammatory bowel diseases (IBDs) which burden health systems worldwide; available pharmacological therapies are limited and cost-intensive. Use of peroxisome proliferator activated-receptor γ (PPARγ) ligands for IBD treatment, while promising, lacks solid evidences to ensure its efficacy. Annexin A1 (AnxA1), a glucocorticoid-modulated anti-inflammatory protein, plays a key role on IBD control and is a potential biomarker of IBD progression. We here investigated whether effects of pioglitazone, a PPARγ ligand, rely on AnxA1 actions to modulate IBD inflammation. Experimental colitis was evoked by 2% dextran sodium sulfate (DSS) in AnxA1 knockout (AnxA1-/-) or wild type (WT) C57BL/6 mice. Clinical and histological parameters were more severe for AnxA-/- than WT mice, and 10 mg/kg pioglitazone treatment attenuated disease parameters in WT mice only. AnxA1 expression was increased in tissue sections of diseased WT mice, correlating positively with presence of CD68+ macrophages. Metalloproteinase-9 (MMP-9) and inactive 33 kDa AnxA1 levels were increased in the colon of diseased WT mice, which were reduced by pioglitazone treatment. Cytokine secretion, reactive oxygen species generation and MMP-9 expression caused by lipopolysaccharide (LPS) treatment in AnxA1-expressing RAW 264.7 macrophages were reduced by pioglitazone treatment, effects not detected in AnxA1 knockdown macrophages. LPS-mediated increase of AnxA1 cleaving in RAW 264.7 macrophages was also attenuated by pioglitazone treatment. Finally, pioglitazone treatment increased extracellular signal-regulated kinase (ERK) phosphorylation in AnxA1-expressing RAW 264.7 macrophages, but not in AnxA1-knockdown macrophages. Thus, our data highlight AnxA1 as a crucial factor for the therapeutic actions of pioglitazone on IBDs.

Protective effects of flavonoid composition rich P. subpeltata Ortega. on indomethacin induced experimental ulcerative colitis in rat models of inflammatory bowel diseases.

ETHNOPHARMACOLOGICAL RELEVANCE: Polyphenolics (flavonoid and phenolic) rich plants are the effective source for the treatment of acute and chronic degenerative diseases including inflammatory bowel disease. OBJECTIVE: This study was aimed to examine the effects of polyphenolics rich leaf acetone extract of P. subpeltata against the indomethacin induced ulcerative colitis in rats. MATERIALS AND METHODS: Two consecutive days administration of indomethacin produced chronic inflammation in GIT tissues of rats. Further, the plant extract 200 and 400 mg/kg treatment were continued until 11th day. Then hematological, enzymatic antioxidants, MPO and histological evaluations were analyzed. Moreover, the extracts were treated with RAW267.4 cells for the cytotoxicity, NO and TNF-α analysis. RESULTS: The obtained results revealed, that higher dose of the plant extract dropped neutrophil infiltration followed by inhibiting the MPO enzyme levels and controls the enzymatic antioxidants such as SOD, CAT, GSH and LPO. RAW cells study also proved that the plant extract effectively inhibits NO and TNF-α production. CONCLUSIONS: Thus, these results suggest that P. subpeltata extract may have therapeutic potential for the treatment of IBD although further clinical research is still warranted.

Effects of passion fruit peel flour (Passiflora edulis f. flavicarpa O. Deg.) in cafeteria diet-induced metabolic disorders.

ETHNOPHARMACOLOGICAL RELEVANCE: Passiflora edulis f. flavicarpa O. Deg. is a native Brazilian fruit known as sour or yellow passion fruit. From its peel, mainly in the northeast of Brazil, is produced a flour that is largely used as folk medicine to treat diabetes and other metabolic conditions. AIM OF THE STUDY: The aim of the study was to show the effects of P. edulis peel flour (PEPF) in metabolic disorders caused by cafeteria diet in mice. MATERIAL AND METHODS: The antioxidant activity in vitro of PEPF extract was determined by ferric reducing/antioxidant power, ß-carotene/linoleic acid system and nitric oxide scavenging activity assay. C57BL/6 mice divided in 3 groups: Control group, fed on a standard diet (AIN); Cafeteria diet (CAF) group, fed on a cafeteria diet, and PEPF group, fed on a cafeteria diet containing 15% of PEPF, during 16 weeks. The glucose tolerance and insulin sensitivity were evaluated through the glucose tolerance test (GTT) and the insulin tolerance test (ITT). After the intervention period, blood, hepatic, pancreatic and adipose tissues were collected for biochemical and histological analysis. Cholesterol, triglyceride, interleukins and antioxidant enzymes were measured in the liver tissue. RESULTS: PEPF extract presented antioxidant activity in the higher concentrations in the performed assays. The PEPF intake decreased the body weight gain, fat deposition, predominantly in the liver, improved the glucose tolerance and insulin sensitivity in metabolic changes caused by cafeteria diet. CONCLUSION: Together, the data herein obtained points out that P. edulis peel flour supplementation in metabolic syndrome condition induced by CAF-diet, prevents insulin and glucose resistance, hepatic steatosis and adiposity.

Safety assessment of Piper cernuum Vell. (Piperaceae) leaves extract: Acute, sub-acute toxicity and genotoxicity studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper cernuum Vell (Piperaceae) is a native species from Atlantic rain forest, popularly known as pariparoba. Its leaves have been commonly used by rural and urban communities from State of São Paulo, Brazil, to treat pain (orally and topically), and hepatic and renal complications. AIM OF THE STUDY: In this study we evaluated the acute and sub-acute toxicity, genotoxicity and mutagenicity of hydroalcoholic extract obtained from P. cernuum leaf using in vivo and in vitro methods. MATERIAL AND METHODS: In the acute toxicity study, mice were orally treated with P. cernuum extract (2000 mg/kg, p.o.). General behavior and mortality were observed for up to 14 days. In the sub-acute toxicity study, P. cernuum extract was given orally as a single administration to the rats at doses of 50 or 250 mg/kg/day, for 28 days. General behavior, body weight, biochemical and hematological parameters, organ coefficients and pathological morphology were analyzed. The P. cernuum mutagenicity was evaluated using mammalian cell micronucleus assay. Additionally, in vitro toxicity profile of the extract was assessed through cytotoxicity, hemolytic activity, and genotoxicity assay. RESULTS: Data from comet assay demonstrates that high concentrations of P. cernuum extract induce genotoxicity. However, no evidence of hemolytic, cytotoxic or mutagenicity activity was found. In addition, the acute and sub-acute toxicity studies did not show significant changes in body weight, general behavior, hematology and biochemical parameters, organ weight and liver and kidney histopathological analysis. CONCLUSIONS: Together, the results herein obtained indicate that P. cernuum leaves extract did not present significant toxicity when administered to male or female rats. Additionally, no significant alteration in hematological, biochemical and morphological parameters were observed. Data obtained in vitro shows that extract did not present cytotoxicity and mutagenicity. However, the extract induces in vitro genotoxicity, but in high concentration. Further studies are necessary to evaluate the safety of long-term exposure to P. cernuum leaves extract added to in vivo genotoxicity.

Effects of Tithonia diversifolia (Asteraceae) extract on innate inflammatory responses.

ETHNOPHARMACOLOGICAL RELEVANCE: Tithonia diversifolia (Helms.) A. Gray, popularly known in Brazil as "margaridão" or "mão-de-Deus" has been used in the folk medicine as anti-inflammatory and against other illnesses in several countries. Indeed, many studies show de effect of T. diversifolia in the inflammatory process, however, any of them have demonstrated the mechanism of cell migration. AIM OF THE STUDY: The aim of this investigation was to show the in vivo and in vitro effects of T. diversifolia leaves ethanol extract on neutrophil trafficking from the blood to the inflamed tissue and on cell-derived secretion of chemical mediators, as well as, the effects on inflammatory resolution and inflammatory pain. MATERIALS AND METHODS: Anti-inflammatory activity was investigated using carrageenan-induced inflammation in the subcutaneous tissue of male Swiss mice orally treated with the T. diversifolia extract (0.1, 1 or 3 mg/kg). The leukocyte influx (optical microscopy) and the secretion of chemical mediators (TNF, IL-6, IL-1ß and CXCL1, by enzyme-linked immunosorbent assay) were quantified in the inflamed exudate. Histological analysis of the pouches was performed. N-Formyl-methionine-leucine-phenylalanine-induced chemotaxis, lipopolysaccharide-induced TNF, IL-6, IL-1ß, CXCL1 and NO production, and adhesion molecule expression (CD62L and CD18, flow cytometry) were in vitro quantified using oyster glycogen recruited peritoneal neutrophils previous treated with the extract (1, 10, or 100 µg/mL). The resolution of inflammation was accessed by efferocytosis assay, and the antinociceptive activity was investigated using carrageenan-induced mechanical hypersensitivity. RESULTS: The oral treatment with T. diversifolia promoted reduction in the neutrophil migration as well as the decrease in total protein, TNF, IL-1ß and CXCL1 levels in the inflamed exudate. In vitro treatment with T. diversifolia shedding of ß2 integrin expressions, without alter CD62L expression. The extract was able to increase the efferocytosis of apoptotic neutrophils, and the increase of the IL-10 and the decrease of TNF secretion. Additionally, the extract reduced the hypersensitivity induced by carrageenan. CONCLUSIONS: Together, the data herein obtained showed that T. diversifolia extract presented anti-inflammatory activity by inhibiting the cytokine and NO production, and also the leukocyte migration. The mechanisms involved in the extract anti-inflammatory effects include the impairment in the leukocyte migration to the inflamed tissue, the pro-resolution activity, and consequently the anti-hypersensitivity.

Effects of Eugenia umbelliflora O. Berg (Myrtaceae)-leaf extract on inflammation and hypersensitivity.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Eugenia species are widely used in popular medicine to treat several diseases, such as arthritis, rheumatism and diabetes. Eugenia umbelliflora O. Berg is popularly known in Brazil as "baguaçu", name also conferred to Eugenia jambolana probably due to their apparent similarity. Although the popular use scientifically proved of E. jambolana as anti-diabetes and also as anti-inflammatory, there are only two scientific studies demonstrating anti-ulcer and bactericide activities of E. umbelliflora leaves extract, without reference to its possible anti-inflammatory activity. AIM OF THE STUDY: The aim of this study was to show the anti-oxidant and anti-inflammatory activity of the methanol extract obtained from E. umbelliflora leaves (EuL) using in vitro and in vivo protocols. MATERIALS AND METHODS: The total phenolic content was evaluated using the folin-Ciocalteu colorimetric method and phloroglucinols content by HPLC. The anti-oxidant activity was evaluated by ORAC, ABTS•+, DPPH, and metal chelation methods. The anti-inflammatory activity was investigated using carrageenan-induced inflammation in the subcutaneous tissue of male Swiss mice orally pre-treated with the EuL (0.3, 1 or 3 mg/kg). The leukocyte influx (optical microscopy) and secretion of chemical mediators (TNF, IL-6, IL-1ß and CXCL1, by enzyme-linked immunosorbent assay) were quantified in the inflamed exudate. Histological analysis of the pouches was also performed. The anti-hypersensitive activity was investigated using carrageenan-induced mechanical hypersensitivity and mice were then evaluated using the von Frey filaments. The Open Field test was used to evaluate possible interference of adverse effect of EuL on locomotor activity that could lead to misinterpretation of the hypersensitivity evaluation. RESULTS: The EuL demonstrated important and moderate reducing capacity on ABTS•+ and DPPH assays, respectively, but with slight activity in ORAC test. It reflects low protection against cell damage. The EuL also presented 30% of phenolic compounds. The phloroglucinols content of EuL was 25.9 mg/g, 18.4 mg/g and 16.6 mg/g of eugenial C, eugenial D and eugenial E, respectively. The in vivo analysis of the inflammatory exudate of EuL-treated mice demonstrated reduction in the polymorphonuclear cells (PMN) migration to the inflamed tissue, as well as the reduction of the pro-inflammatory cytokine IL-1ß. Histologically, it was observed evident decrease in the oedema, formed essentially by non-haemorrhagic fibrin exudate, as well as PMN infiltrate, when compared with control mice injected with carrageenan. Furthermore, the extract also presented effective reduction of the mechanical hypersensitivity induced by carrageenan without any interference in animal's locomotor and exploratory activity. CONCLUSIONS: Together, the results herein obtained show that EuL presented anti-inflammatory activity by decreasing the influx of PMN to the inflamed tissue, as well as the cytokine IL-1ß level. This anti-inflammatory activity was also accompanied by significant anti-hypersensitive effect. The effects presented by EuL seem not to be correlated with an antioxidant activity. However other extract chemical compounds could be responsible for its important anti-inflammatory effects.

Synthetic chalcones as potential tool for acute- and chronic-pain control.

The purpose of this study was to validate the potential anti-hypersensitive activity of two chalcones, (2E)-1-(4-aminophenyl)-3-(4-nitrophenyl)prop-2-en-1-one (ANCh) and N-{4-[(2E)-3-(4-nitrophenyl)prop-2-enoil]phenyl}acetamide (AcANCh), by different models of acute and persistent pain in mice, besides in silico analysis. Molecules computational investigation for prediction of Lipinki's and Veber's rules to determine solubility, % absorption, drug likeness and toxicity liabilities was performed. Male and female C57BL/6 mice (20-30 g, n = 6) were used. Firstly, mice were pre-treated with the compounds ANCh or AcANCh and then submitted to the models of acute hypersensitivity by the intraplantar injection of different phlogistic agents. The mechanical sensitivity was assessed using von Frey hairs (0.6 g). The obtained data shows that both compounds presented important inhibitory effects on mechanical hypersensitivity induced by carrageenan (with oral bioavailability). The anti-hypersensitive effect was also accompanied by the interference in leukocyte migration, interleukin-1ß (IL-1ß) and tumour necrosis factor (TNF) levels reduction and by the absence of unspecific effects. Added to the in vivo results, the in silico analysis presented none violation in Lipinski's or Veber's rules, good probability to cell membrane permeability and oral bioavailability, positive values of drug likeness and few risk of computational toxicity. ANCh partially reduced the hypersensitivity induced by IL-1ß and TNF, epinephrine and prostaglandin E2 (PGE2). AcANCh had similar effect, except for the absent of inhibition in PGE2-injected mice. Both compounds were capable of reducing the mechanical hypersensitivity presented in all persistent models of hypersensitivity (inflammatory pain, chronic nerve constriction and cancer pain), with emphasis for ANCh. These results suggest that both chalcones could represent good strategies for the control of acute and chronic pain, without important side effects. ANCh seems to involve cell migration and cytokines production as the main mechanism, together with interference in PGE2 neuronal sensitization pathway. In vivo and in silico analyses reinforce the potential characteristics of the compounds to become future drugs.

Antiproliferative and toxicological properties of methanolic extract obtained from Solanum capsicoides All. seeds and carpesterol.

Natural products are considered important sources of potential chemotherapeutic agents. Here, we evaluated the antiproliferative activity and the toxicological effects of the methanolic extract and a pure compound obtained from Solanum capsicoides seeds. The phytochemical profile was analyzed by chromatographic and spectroscopy methods. The acute toxicity was assessed in mice orally treated with the extract (2000 mg/kg), in vitro hemolytic activity and micronucleus test. The mutagenicity, developmental toxicity, and lethal dose (LD50) of carpesterol were estimated by the Toxicity Estimation Software Tool (TEST) software. A sulforhodamine B assay was employed to evaluate the antiproliferative activity. The toxicological assays did not observe signs of toxicity, either during the behavioral observations or in the autopsies, as well as no mutagenicity and hemolytic activity. The carpesterol did not present mutagenic effect and hemolytic activity but presents developmental toxicology and LD50 of 410 mg/kg in toxicity estimations by the TEST software. The S. capsicoides extract exhibited antiproliferative activity mainly in leukemia (K562) cell lineage. However, carpesterol presented antiproliferative activity in glioma (U251), breast (MCF-7), kidney (786-0), ovary (OVCAR-03), and K562 cell lineages. The data obtained show that S. capsicoides extract presents antiproliferative and does not present toxicological effects. In addition, it was shown for the first time the antiproliferative and toxicological parameters of carpesterol.

Rubus imperialis (Rosaceae) extract and pure compound niga-ichigoside F1: wound healing and anti-inflammatory effects.

Here, we evaluate the anti-inflammatory and wound-healing effects of methanolic crude extract obtained from aerial parts (leaves and branches) of Rubus imperialis Chum. Schl. (Rosaceae) and the pure compound niga-ichigoside F1. Anti-inflammatory activity was determined in vivo and in vitro, and the healing effect was evaluated in surgical lesions in mice skin. The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) assay and H2O2-induced oxidative stress were used to determine antioxidant activity. The efferocytosis activity was also determined. The data obtained show that the extract of R. imperialis promote reduction in the inflammatory process induced by lipopolysaccharide (LPS) or carrageenan in the air pouch model; the effects could be reinforced by nitric oxide reduction in LPS-stimulated neutrophils, and an increase in the efferocytosis. The extract showed wound healing property in vitro and in vivo, scavenging activity for DPPH, and cytoprotection in the H2O2-induced oxidative stress in L929 cells. In addition, the compound niga-ichigoside F1 was able to reduce the NO secretion; however, it did not present wound-healing activity in vitro. Together, the data obtained point out the modulatory actions of R. imperialis extract on leukocyte migration to the inflamed tissue, the antioxidant, and the pro-resolutive activity. However, the R. imperialis anti-inflammatory activity may be mediated in parts by niga-ichigoside F1, and on wound healing do not correlated with niga-ichigoside F1.