Andean berry (Vaccinium meridionale Swartz) juice in combination with Aspirin modulated anti-inflammatory markers on LPS-stimulated RAW 264.7 macrophages.

V. meridionale Swartz is an underutilized Andean Berry that has been linked to several health benefits potentially derived from its anti-inflammatory effects. This research aimed to evaluate the impact of Andean Berry Juice (ABJ) combined with Aspirin in the modulation of anti-inflammatory markers from LPS-stimulated RAW 264.7 macrophages. The chemical characterization of ABJ showed a high content of polyphenols, mainly gallic acid (659-75 µg/g) and cyanidin chloride (418.61 µg/mL). Compared to LPS-stimulated macrophages, ABJ, Aspirin, and its combination reduced NO and ROS production (3.26-42.55 and 17.59-65.68%, respectively). In comparison, the half inhibitory concentration of NO reduction (IC50) was found at 7.69% v/v (ABJ) and 24.48 mM (Aspirin). Compared to the pro-inflammatory control (LPS), ABJ reduced IL-1ß, MCP-1, and GCSF; Aspirin decreased IL1R1, MCP-1, GMCSF, GCSF, and TNF-α; and the ABJ + Aspirin treatment reduced IL1R, GMCSF, and CXCL10. The in silico interaction of cytokines and the prediction of potential binding interactions suggested CCR1, CCR5, and NF-kB modulation. These results showed the anti-inflammatory potential of underutilized South American berries and their co-adjuvant effect with known drugs such as Aspirin in the resolution of inflammatory-derived conditions. This is the first report of the anti-inflammatory effects of V. meridionale Swartz juice in combination with Aspirin on LPS-challenged RAW 264.7 macrophages.

Dichotomous response of Malassezia-infected macrophages to Malassezia pachydermatis and Malassezia furfur.

Malassezia pachydermatis and Malassezia furfur are lipophilic yeasts of the cutaneous microbiome, although these organisms are occasionally responsible for serious invasive infections in neonates. Since phagocytosis is an important mechanism mediating the adaptive immune response, here we evaluated the phagocytosis capacity and production of nitric oxide and cytokine by macrophages after challenged with M. furfur CBS-1878 and M. pachydermatis CBS-1696. The phagocytic indexes was determined using RAW 264.7 cultivated or not with M. furfur or M. pachydermatis in the concentrations of 5:1 or 2:1 (yeasts:macrophages ratio) for 6 h, 24 h, and 48 h following the challenges. Evaluation of nitric oxide and pro- and anti-inflammatory cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17A, interferon (IFN)-γ and tumor necrosis factor [TNF]-α) by Griess method and flow cytometry, respectively, were performed in the different intervals by collecting the cell culture supernatant. Results showed a higher phagocytic index in the 5:1 ratio in 24 h for both species. Malassezia pachydermatis-infected macrophages had superior phagocytic indexes than M. furfur-infected macrophages. Phagocytosis evaluation at 48 h showed significant microorganisms proliferation and macrophages death, particularly in macrophages infected with M. pachydermatis, suggesting yeast evasion mechanism. Significant variations in the nitric oxide production were observed in macrophages infected with both species. Levels of TNF-α and IL-4 cytokines have increased in M. furfur and M. pachydermatis macrophage-infected cultures, respectively. The low microbicidal activity and the presence of pro- and anti-inflammatory cytokines reinforce the dichotomous character of the relation of these yeasts with the host, acting as a commensal in the cutaneous microbiome or causing infection.

Evaluation of Strontium-Containing PCL-PDIPF Scaffolds for Bone Tissue Engineering: In Vitro and In Vivo Studies.

Bone tissue engineering (BTE) has the general objective of restoring and improving damaged bone. A very interesting strategy for BTE is to combine an adequate polymeric scaffold with an osteoinductive compound. Strontium is a divalent cation that can substitute calcium in hydroxyapatite and induce both anabolic and anti-catabolic effects in bone. On the other hand, systemic increases in Sr2+ levels can provoke adverse cardiovascular effects. In the present study we have developed a compatibilized blend of poly-ε-caprolactone (PCL) and polydiisopropyl fumarate (PDIPF) enriched with 1% or 5% Sr2+ and evaluated the applicability of these biomaterials for BTE, both in vitro and in vivo. In vitro, whereas Blend + 5% Sr2+ was pro-inflammatory and anti-osteogenic, Blend + 1% Sr2+ released very low quantities of the cation; was not cytotoxic for cultured macrophages; and showed improved osteocompatibility when used as a substratum for primary cultures of bone marrow stromal cells. In vivo, implants with Blend + 1% Sr2+ significantly increased bone tissue regeneration and improved fibrous bridging (vs. Blend alone), while neither inducing a local inflammatory response nor increased serum levels of Sr2+. These results indicate that our compatibilized blend of PCL-PDIPF enriched with 1% Sr2+ could be useful for BTE.

Dichotomous response of Malassezia-infected macrophages to Malassezia pachydermatis and Malassezia furfur

Malassezia pachydermatis and Malassezia furfur are lipophilic yeasts of the cutaneous microbiome, although these organisms are occasionally responsible for serious invasive infections in neonates. Since phagocytosis is an important mechanism mediating the adaptive immune response, here we evaluated the phagocytosis capacity and production of nitric oxide and cytokine by macrophages after challenged with M. furfur CBS-1878 and M. pachydermatis CBS-1696. The phagocytic indexes was determined using RAW 264.7 cultivated or not with M. furfur or M. pachydermatis in the concentrations of 5:1 or 2:1 (yeasts:macrophages ratio) for 6 h, 24 h, and 48 h following the challenges. Evaluation of nitric oxide and pro- and anti-inflammatory cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17A, interferon (IFN)-gama and tumor necrosis factor [TNF]-a) by Griess method and flow cytometry, respectively, were performed in the different intervals by collecting the cell culture supernatant. Results showed a higher phagocytic index in the 5:1 ratio in 24 h for both species. Malassezia pachydermatis-infected macrophages had superior phagocytic indexes than M. furfur-infected macrophages. Phagocytosis evaluation at 48 h showed significant microorganisms proliferation and macrophages death, particularly in macrophages infected with M. pachydermatis, suggesting yeast evasion mechanism. Significant variations in the nitric oxide production were observed in macrophages infected with both species. Levels of TNF-a and IL-4 cytokines have increased in M. furfur and M. pachydermatis macrophage-infected cultures, respectively. The low microbicidal activity and the presence of pro- and anti-inflammatory cytokines reinforce the dichotomous character of the relation of these yeasts with the host, acting as a commensal in the cutaneous microbiome or causing infection.

Anti-inflammatory effect of geranium nanoemulsion macrophages induced with soluble protein of Candida albicans.

Pelargonium graveolens is a member of the Geraniaceae family and has been used in folk medicine in many countries because of its anti-inflammatory activity. No studies have yet been reported to evaluate the anti-inflammatory activity of a nanoemulsion containing geranium oil (GO) model in macrophages. In this study the anti-inflammatory effect of Geranium nanoemulsion (NEG) macrophages induced with soluble proteins of Candida albicans was investigated. GO presented citronellol (17.74%) and geraniol (14.43%) as main constituents. The characterization in NEG was demonstrated, showing the particle size of 164 ± 3.5 nm, PDI of 0.12 ± 0.006 and zeta potential -10 mV ± 1.7. The MIC obtained for NEG and GO were 3.64 µg ml-1 and 1.82 µg ml-1, respectively. The viability of the macrophages treated with NEG and GO concentrations (1/2 x, 1x and 2x MIC) was evaluated. There was a significant reduction of viability and the MTT assay was not confirmed after the LDH assay. Anti-inflammatory activity was evaluated by determining nitric oxide (NO), cytokines (interleukin IL-1, IL-6 and IL-10), tumor necrosis factor-α (TNF) and the expression levels gene of interleukin (IL-2), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). The apoptosis inhibition capacity was assessed by determination of INFγ, caspase 3 and caspase 8. The results indicated that there was a significant increase of NO in the levels after treatment with NEG and significantly reduced levels after treatment with GO. The cytokines (IL-1, IL-6, IL-10, and TNF) were evaluated and NEG (½ x, 1x MIC) decreased IL-1 levels by 1.25-1.37 times, respectively. The NEG did not decrease IL-6 levels and a significant increase was observed for IL-10. GO significantly decreased IL-6 and IL-10 levels. There was a significant decrease in IL-2 and COX-2 levels and increased levels of iNOs. The levels of IFNγ and caspase-3 after treatment with NEG decreased indicating an anti-inflammatory effect and can inhibit apoptosis. Finally, the levels of caspase-8 do not change. Thus, pretreatment with NEG induced an anti-inflammatory effect against soluble proteins of C. albicans model macrophages.

Toxicological evaluation of the natural products and some semisynthetic derivatives of Heterotheca inuloides Cass (Asteraceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Heterotheca ineuloides Cass (Asteraceae), popularly known as árnica mexicana, is widely used in Mexican traditional medicine to treat bruises, dermatological problems, rheumatic pains, and other disorders as cancer. The major constituents in H. inuloides are cadinane type sesquiterpenes, flavonoids and phytosterols. Compounds with a cadinane skeleton have been proved to possess cytotoxic activity against human-tumor cell lines and brine shrimp, and display toxic effects in different animal species. Although this plant has been widely used, there is little available information on the safety and toxicity especially of pure compounds. AIM OF THIS STUDY: Evaluate the potential toxicity of the natural products isolated from H. inuloides and some semisynthetic derivatives. MATERIALS AND METHODS: The toxic aspects of the following natural products isolated from dried flowers of H. inuloides: 7-hydroxy-3,4-dihydrocadalene (1), 7-hydroxycadalene (2), 3,7-dihydroxy-3(4H)-isocadalen-4-one (3), (1R,4R)-1-hydroxy-4H-1,2,3,4- tetrahydrocadalen-15-oic acid (4), D-chiro-inositol (5), quercetin (6), quercetin-3,7,3'-trimethyl ether (7), quercetin-3,7,3',4'-tetramethyl ether (8), eriodictyol-7,4'-dimethyl ether (9), α-spinasterol (10), caryolan-1,9ß-diol (11) and 7-(3,3-dimethylallyloxy)-coumarin (12) as well as the toxic aspects of the semisynthetic compounds 7-acetoxy-3,4-dihydrocadalene (13), 7-benzoxy-3,4-dihydrocadalene (14), 7-acetoxycadalene (15), 7-benzoxycadalene (16), quercetin pentaacetate (17), 7-hydroxycalamenene (18), 3,8-dimethyl-5-(1-methylethyl)-1,2-naphthoquinone (19), and 4-isopropyl-1,6-dimethylbenzo[c]oxepine-7,9-dione (20). Toxic activities of compounds were determined by sulforhodamine B (SRB) assay, Artemia salina assay, RAW264.7 macrophage cells. Additionally, the acute toxicity in mouse of compound 1, the major natural sesquiterpene isolated from the acetone extract, was evaluated. RESULTS: The best cytotoxicity activity was observed for mansonone C (19) on K562 cell line with IC50 1.45 ± 0.14 µM, for 7-hydroxycadalene (2) on HCT-15 cell line with IC50 18.89 ± 1.2 µM, and for quercetin pentaacetate (17) on MCF-7 cell line with IC50 22.57 ± 2.4 µM. Sesquiterpenes mansonone C (19) and 7-hydroxy-3,4-dihydrocadalene (1) caused the strongest deleterious effects against A. salina with IC50 39.4 ± 1.07, and 45.47 ± 1.74 µM, respectively. The number of viable RAW 264.7 cells was reduced with sesquiterpenes 1 and 2 by more than 90%. In addition, the acute study of 1 revealed no lethal effects at 300 mg/kg body weight, however, a reduction in the body weight of mice, morphological changes in the tissues of the liver and kidney and toxic signs were observed at very high doses (2000 mg/kg). CONCLUSION: The results provided evidence for the cytotoxicity of Mexican arnica (H. inuloides) metabolites and may be correlated with one of the popular uses of this plant, in traditional Mexican medicine, as anticancer remedy. Among the active compounds contained in the acetone extract, the cytotoxic activity is mainly ascribable to cadinene type sesquiterpenes. In addition, evidence of acute toxicity suggests that 7-hydroxy-3,4-dihydrocadalene (1) may lead to toxicity at very high doses.

In vivo and in vitro anti-inflammatory activity of Cryptostegia grandiflora Roxb. ex R. Br. leaves

BACKGROUND: Despite Cryptostegia grandiflora Roxb. ex R. Br. (Apocynaceae) leaves are widely used in folk Caribbean Colombian medicine for their anti-inflammatory effects, there are no studies that support this traditional use. Therefore, this work aimed to evaluate the effect of the total extract and primary fractions obtained from Cryptostegia grandiflora leaves, using in vivo and in vitromodels of inflammation, and further get new insights on the mechanisms involved in this activity. RESULTS: Ethanolic extract of Cryptostegia grandiflora leaves, and its corresponding ether and dichloromethane fractions, significantly reduced inflammation and myeloperoxidase activity (MPO) in ear tissue of mice treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Histological analysis revealed a reduction of edema and leukocyte infiltration. Complementarily, we demonstrated that extract and fractions reduced nitric oxide (NO•) and prostaglandin E2 (PGE2) production in LPS-stimulated RAW 264.7 macrophages, as well as scavenging activity on DPPH and ABTS radicals. CONCLUSIONS: Our results demonstrated for the first time the anti-inflammatory activity of Cryptostegia grandiflora leaves, supporting its traditional use. This activity was related to inhibition of MPO activity, and PGE2 and NO• production. These mechanisms and its antioxidant activity could contribute, at least in part, to the anti-inflammatory effect showed by this plant.

Efecto de Rhizophora mangle L sobre la producción de anión superóxido en macrófagos murinos RAW 264, 7 / Effect of Rhizophora mangle L on the superoxide anion production in RAW 264, 7 murine macrophages

Introducción: el extracto acuoso de la corteza de Rhizophora mangle L. (mangle rojo) posee varias propiedades farmacológicas: en el tratamiento de la mastitis bovina, la curación de heridas,las infecciones uterinas y las úlceras gastroduodenales; debido a sus propiedades antiséptica, cicatrizante, antiinflamatoria y antioxidante. Sin embargo, no se han completado los estudios de la actividad antioxidante a todos los niveles de complejidad para dilucidar los mecanismos de acción involucrados en este efecto farmacológico. Objetivo: determinar el efecto del extracto acuoso de Rhizophora mangle y su fracción polifenólica sobre la producción de anión superóxido en una línea celular de macrófagos murinos RAW 264,7, estimulados con forbol 12-myristato 13-acetato o lipopolisacárido. Métodos: la evaluación de la actividad antioxidante del extracto de Rhizophora mangle y su fracción polifenólica sobre la producción de anión superóxido en macrófagos RAW 264.7, activados con lipopolisacárido o forbol 12-myristato 13-acetato, se determinó mediante el método de reducción del ferricitocromo c. Resultados: el extracto de Rhizophora mangle y su fracción polifenólica, inhibieron la producción de anión superóxido en macrófagos RAW 264,7, activados con ambos tipos de agentes de forma dependiente de la concentración de taninos. La comparación de las líneas de regresión reveló diferencias significativas (p< 0,05), resultando este efecto superior en la fracción para ambos tipos de agentes, y en presencia de lipopolisacárido para ambas muestras. Conclusiones: el extracto acuoso de Rhizophora mangle mostró actividad antioxidante a nivel celular, evidenciada por la reducción del estrés oxidativo en macrófagos mediante la inhibición de la producción de anión superóxido. A su vez se demostró que los compuestos polifenólicos presentes en el extracto fueron los principales responsables de los efectos antioxidantes observados en este estudio(AU)

Introduction: the aqueous extract of Rhizophora mangle L (red mangrove) bark has some pharmacological properties, that is, the treatment of bovine mastitis, wound healing, uterine infections and stomach ulcers, due to its antiseptic, healing, antiinflammatory and antioxidant properties. However, the studies of antioxidant activity at all complexity levels have not been completed in order to elucidate the mechanisms of action involved in this pharmacological effect. Objective: to determine the effect of Rhizophora mangle aqueous extract and its polyphenolic fraction on superoxide anion production in a cellular line of RAW 264,7 murine macrophages, stimulated with phorbol 12-myristate 13-acetate or lipopolysaccharide. Methods: the evaluation of the antioxidant activity of Rhizophora mangle extract and its polyphenolic fraction on superoxide anion production in RAW 264.7 macrophages activated with lipopolysaccharide or phorbol 12-myristate 13-acetate was made using the C ferricytochrome reduction method. Results: Rhizophora mangle extract and its polyphenolic fraction inhibit the superoxide anion production in RAW 264.7 macrophages activated with both types of agents depending on tannin concentration. The comparison of the regression lines showed significant differences (p< 0.05), resulting this effect higher in the fraction for both types of agents, and the presence of lipopolysaccharide for both samples. Conclusions: Rhizophora mangle aqueous extract showed antioxidant activity at cellular level, evidenced by reduction of oxidative stress in macrophages, through the inhibition of superoxide anion production. At the same time, it was shown that polyphenolic compounds, present in the extract, were the main responsible for the antioxidant effects observed in this study(AU)

Efecto de Rhizophora mangle L sobre la producción de anión superóxido en macrófagos murinos RAW 264, 7 / Effect of Rhizophora mangle L on the superoxide anion production in RAW 264, 7 murine macrophages

Introducción: el extracto acuoso de la corteza de Rhizophora mangle L. (mangle rojo) posee varias propiedades farmacológicas: en el tratamiento de la mastitis bovina, la curación de heridas,las infecciones uterinas y las úlceras gastroduodenales; debido a sus propiedades antiséptica, cicatrizante, antiinflamatoria y antioxidante. Sin embargo, no se han completado los estudios de la actividad antioxidante a todos los niveles de complejidad para dilucidar los mecanismos de acción involucrados en este efecto farmacológico. Objetivo: determinar el efecto del extracto acuoso de Rhizophora mangle y su fracción polifenólica sobre la producción de anión superóxido en una línea celular de macrófagos murinos RAW 264,7, estimulados con forbol 12-myristato 13-acetato o lipopolisacárido. Métodos: la evaluación de la actividad antioxidante del extracto de Rhizophora mangle y su fracción polifenólica sobre la producción de anión superóxido en macrófagos RAW 264.7, activados con lipopolisacárido o forbol 12-myristato 13-acetato, se determinó mediante el método de reducción del ferricitocromo c. Resultados: el extracto de Rhizophora mangle y su fracción polifenólica, inhibieron la producción de anión superóxido en macrófagos RAW 264,7, activados con ambos tipos de agentes de forma dependiente de la concentración de taninos. La comparación de las líneas de regresión reveló diferencias significativas (p< 0,05), resultando este efecto superior en la fracción para ambos tipos de agentes, y en presencia de lipopolisacárido para ambas muestras. Conclusiones: el extracto acuoso de Rhizophora mangle mostró actividad antioxidante a nivel celular, evidenciada por la reducción del estrés oxidativo en macrófagos mediante la inhibición de la producción de anión superóxido. A su vez se demostró que los compuestos polifenólicos presentes en el extracto fueron los principales responsables de los efectos antioxidantes observados en este estudio

Introduction: the aqueous extract of Rhizophora mangle L (red mangrove) bark has some pharmacological properties, that is, the treatment of bovine mastitis, wound healing, uterine infections and stomach ulcers, due to its antiseptic, healing, antiinflammatory and antioxidant properties. However, the studies of antioxidant activity at all complexity levels have not been completed in order to elucidate the mechanisms of action involved in this pharmacological effect. Objective: to determine the effect of Rhizophora mangle aqueous extract and its polyphenolic fraction on superoxide anion production in a cellular line of RAW 264,7 murine macrophages, stimulated with phorbol 12-myristate 13-acetate or lipopolysaccharide. Methods: the evaluation of the antioxidant activity of Rhizophora mangle extract and its polyphenolic fraction on superoxide anion production in RAW 264.7 macrophages activated with lipopolysaccharide or phorbol 12-myristate 13-acetate was made using the C ferricytochrome reduction method. Results: Rhizophora mangle extract and its polyphenolic fraction inhibit the superoxide anion production in RAW 264.7 macrophages activated with both types of agents depending on tannin concentration. The comparison of the regression lines showed significant differences (p< 0.05), resulting this effect higher in the fraction for both types of agents, and the presence of lipopolysaccharide for both samples. Conclusions: Rhizophora mangle aqueous extract showed antioxidant activity at cellular level, evidenced by reduction of oxidative stress in macrophages, through the inhibition of superoxide anion production. At the same time, it was shown that polyphenolic compounds, present in the extract, were the main responsible for the antioxidant effects observed in this study