Anti-inflammatory, anti-osteoclastogenic and antioxidant effects of Malva sylvestris extract and fractions: in vitro and in vivo studies

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOGiven their medical importance, natural products represent a tremendous source of drug discovery. The aim of this study was to investigate Malva sylvestris L. extract and fractions and their pharmacological activities followed by chemical identification. The aqueous fraction (AF) was identified as the bioactive fraction in the in vitro and in vivo assays. The AF controlled the neutrophil migration to the peritoneal cavity by 66%, inhibited the antiedematogenic activity by 58.8%, and controlled IL-1 beta cytokine expression by 54%. The in vitro viability tests showed a concentration-dependent effect, where the MSE and fractions at concentrations under 10 mu g/ mL were non-toxic to cells. Transcriptional factors of carbonic anhydrase II (CAII), cathepsin K (Ctsk) and tartrate-resistant acid phosphatase (TRAP) were analyzed by qPCR in RAW 264.7 cell lines. The gene expression analysis showed that the AF was the only treatment that could downregulate all the study genes: CAII, Ctsk and TRAP (p<0.05). TRAP staining was used to evaluate osteoclast formation. AF treatments reduced the number of osteoclastogenesis 2.6-fold compared to the vehicle control group. Matrix metalloproteinase 9 (MMP-9) activity decreased 75% with the AF treatment. Moreover, the bioactive fraction had the ability to regulate the oxidation pathway in the ABTS (2,2-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) assay with an activity equivalent to 1.30 mu mol Trolox/g and DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals 1.01 g/L. Positive ion ESI-mass spectrometry for molecular ions at m/z 611 and 633 confirmed rutin as the major compound in the AF. The AF of M. sylvestris presented anti-inflammatory, controlled osteoclastogenic mechanisms and antioxidant abilities in different in vitro and in vivo methods. In addition, we suggest that given its multi-target activity the bioactive fraction may be a good candidate in the therapy of chronic inflammatory diseases.Given their medical importance, natural products represent a tremendous source of drug discovery. The aim of this study was to investigate Malva sylvestris L. extract and fractions and their pharmacological activities followed by chemical identification. The aqueous fraction (AF) was identified as the bioactive fraction in the in vitro and in vivo assays. The AF controlled the neutrophil migration to the peritoneal cavity by 66%, inhibited the antiedematogenic activity by 58.8%, and controlled IL-1 beta cytokine expression by 54%. The in vitro viability tests showed a concentration-dependent effect, where the MSE and fractions at concentrations under 10 mu g/ mL were non-toxic to cells. Transcriptional factors of carbonic anhydrase II (CAII), cathepsin K (Ctsk) and tartrate-resistant acid phosphatase (TRAP) were analyzed by qPCR in RAW 264.7 cell lines. The gene expression analysis showed that the AF was the only treatment that could downregulate all the study genes: CAII, Ctsk and TRAP (p<0.05). TRAP staining was used to evaluate osteoclast formation. AF treatments reduced the number of osteoclastogenesis 2.6-fold compared to the vehicle control group. Matrix metalloproteinase 9 (MMP-9) activity decreased 75% with the AF treatment. Moreover, the bioactive fraction had the ability to regulate the oxidation pathway in the ABTS (2,2-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) assay with an activity equivalent to 1.30 mu mol Trolox/g and DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals 1.01 g/L. Positive ion ESI-mass spectrometry for molecular ions at m/z 611 and 633 confirmed rutin as the major compound in the AF. The AF of M. sylvestris presented anti-inflammatory, controlled osteoclastogenic mechanisms and antioxidant abilities in different in vitro and in vivo methods. In addition, we suggest that given its multi-target activity the bioactive fraction may be a good candidate in the therapy of chronic inflammatory diseases119FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2011/23980-

Phenolic composition and antioxidant activity of agroindustrial residues

Atualmente, são produzidas milhões de toneladas de resíduos provenientes do processamento agroindustrial. Muitos deles são ricos em compostos bioativos sendo potenciais fontes naturais dessas substâncias. Assim, este trabalho teve como objetivo avaliar o teor de compostos fenólicos totais, a atividade antioxidante e a composição fenólica de três resíduos gerados por agroindústrias brasileiras: bagaço de uva Isabel (BI) (Vitis labrusca), bagaço de uva Verdejo (BV) (Vitis vinifera) e bagaço de goiaba (BG) (Psidium guajava). Os resultados do teor de compostos fenólicos totais (mg GAE g-1) encontrados nos extratos etanólicos e aquosos dos resíduos foram, respectivamente: BV (20,94&plusmn;0,46; 8,03&plusmn;0,43)> BI (16,57&plusmn;0,19; 4,41&plusmn;0,01)> BG (3,41&plusmn;0,09; 1,88&plusmn;0,06). Alta atividade antioxidante, principalmente em BV e BI, foi verificada nos ensaios realizados (ABTS &#9679;, DPPH &#9679; e auto-oxidação do sistema beta-caroteno/ácido linoléico). Uma forte correlação positiva entre atividade antioxidante e o teor de compostos fenólicos totais foi encontrada. Os compostos fenólicos encontrados, por cromatografia gasosa com espectrometria de massas (CG-EM), foram: ácido gálico, epicatequina, quercetina (BV, BI e BG); ácido isovanílico (BI, BG); ácido p-cumárico (BI); ácido caféico e resveratrol (BV, BI). Esses resultados mostram que os resíduos agroindustriais analisados, particularmente os vinícolas, são ricos em substâncias bioativas e podem ser explorados pela indústria de alimentos e farmacêutica.Nowadays, the agro-industrial processing produces millions of tons of wastes. Many of them are rich in bioactive compounds, being a potential natural source of these substances. This study aimed to evaluate the content of total phenolics, antioxidant activity and phenolic composition of residues generated by three Brazilian agribusiness: Isabel grape pomace (PI) (Vitis labrusca), Verdejo grape pomace (PV) (Vitis vinifera) and guava pomace (PG) (Psidium guajava). The results of total phenolics content (mg GAE g-1) found in the ethanol and aqueous extracts of residues were: PV (20.94&plusmn;0.46; 8.03&plusmn;0.43)>PI (16.57&plusmn;0.19; 4.41&plusmn;0.01)>PG (3.41&plusmn;0.09; 1.88&plusmn;0.06). High antioxidant activity of these extracts, particularly PV and PI, was found by the methods DPPH &#9679;, ABTS &#9679; and beta-carotene bleaching method. A strong positive correlation between antioxidant activity and content of total phenolic compounds was found. The following phenolic compounds were found by gas chromatography with mass spectrometry (GC-MS): gallic acid, epicatechin, quercetin (PV, PI and PG); isovanilic acid (PI, PG), p-coumaric acid (PI), caffeic acid and resveratrol (PV, PI). The results show that these residues, particularly the wineries, are rich in bioactive substances and should be exploited by the food industry and pharmaceuticals

Extraction yield, antioxidant activity andphenolics from grape, mango and peanut agro-industrial by-products Rendimento de extração, atividade antioxidante e compostos fenólicos dos subprodutos agro industriais de uva, manga e amendoim

ABSTRACT The objective of this study was to determine and correlate the extraction yields, antioxidant activity, total phenolics and total flavonoids from grape, mango and peanut agro-industrial by-products. The β-carotene/linoleic acid autoxidatio

Composição fenólica e atividade antioxidante de resíduos agroindustriais

Atualmente, são produzidas milhões de toneladas de resíduos provenientes do processamento agroindustrial. Muitos deles são ricos em compostos bioativos sendo potenciais fontes naturais dessas substâncias. Assim, este trabalho teve como objetivo avaliar o teor de compostos fenólicos totais, a atividade antioxidante e a composição fenólica de três resíduos gerados por agroindústrias brasileiras: bagaço de uva Isabel (BI) (Vitis labrusca), bagaço de uva Verdejo (BV) (Vitis vinifera) e bagaço de goiaba (BG) (Psidium guajava). Os resultados do teor de compostos fenólicos totais (mg GAE g-1) encontrados nos extratos etanólicos e aquosos dos resíduos foram, respectivamente: BV (20,94±0,46; 8,03±0,43)> BI (16,57±0,19; 4,41±0,01)> BG (3,41±0,09; 1,88±0,06). Alta atividade antioxidante, principalmente em BV e BI, foi verificada nos ensaios realizados (ABTS , DPPH e autooxidação do sistema beta-caroteno/ácido linoléico). Uma forte correlação positiva entre atividade antioxidante e o teor de compostos fenólicos totais foi encontrada. Os compostos fenólicos encontrados, por cromatografia gasosa com espectrometria de massas (CG-EM), foram: ácido gálico, epicatequina, quercetina (BV, BI e BG); ácido isovanílico (BI, BG); ácido p-cumárico (BI); ácido caféico e resveratrol (BV, BI). Esses resultados mostram que os resíduos agroindustriais analisados, particularmente os vinícolas, são ricos em substâncias bioativas e podem ser explorados pela indústria de alimentos e farmacêutica

Functional properties of peanut genotypes adapted to the Brazilian semiarid region: reactive oxygen species scavenging activity and accessibility of polyphenols

O amendoim é um alimento conhecido pelo seu alto conteúdo proteico e lipídico, também, destaca-se com alto teor de compostos fenólicos, conhecidos por suas propriedades antioxidantes. A cultura do amendoim possui uma ampla adaptabilidade às condições tropicais e o seu cultivo no nordeste brasileiro possui um papel importante para a renda dos pequenos agricultores da região. Em atendimento a esses agricultores, a Empresa Brasileira de Pesquisa Agropecuária (Embrapa) detém o programa de melhoramento genético do amendoim para ambientes semiáridos, no desenvolvimento de cultivares tolerantes ao estresse hídrico. No presente estudo, um total de 14 genótipos pertencentes Banco Ativo de Germoplasma da Embrapa (sendo 6 genótipos tolerantes, 1 médio tolerante e 7 não tolerantes à seca) foram analisados quanto à composição fenólica e o potencial no sequestro de espécies reativas do oxigênio, além da análise da bioacessibilidade desses compostos após a digestão simulada in vitro, nunca antes determinados para tais genótipos. Este trabalho está organizado em quatro capítulos, divididos na seguinte forma: Capítulo I destina-se à revisão da literatura; Capítulo II descreve o estudo com os genótipos BR1 (tolerante à seca) e LViPE-06 (não tolerante à seca) para determinar as melhores condições de obtenção do extrato rico em compostos fenólicos com atividade antioxidante e para determinar o perfil fenólico dos cotilédones e películas pela técnica LC-ESI-QTOF-MS; Capítulo III, com base nas condições estabelecidas para a extração, foram produzidos os extratos (cotilédones e películas) dos 14 genótipos de amendoins e analisados quanto ao sequestro de cinco diferentes espécies reativas de oxigênio; Capítulo IV, a acessibilidade dos compostos fenólicos majoritários do amendoim, inclusive o ácido p-cumárico, do genótipo BR1 foi determinada por meio da digestão simulada in vitro. Os valores estabelecidos de temperatura e grau de hidratação do etanol para a obtenção dos extratos dos cotilédones foram de 60°C e 35% e para os extratos das películas os valores foram de 40°C e 60%, que permitiram obter extratos com os maiores teores de compostos fenólicos e atividade antioxidante. Por meio da técnica LC-ESI-QTOF-MS os principais compostos fenólicos presentes nos extratos dos cotilédones foram os derivados do ácido p-cumárico e do p-cumaroil e nos extratos das películas, as procianidinas oligoméricas do tipo A. Quanto ao sequestro de espécies reativas, os extratos de películas se mostraram excelentes, especialmente no sequestro do radical hidroxila cujos valores do IC50 foram inferiores à concentração de 0,1 &mu;g/mL e, concentrações maiores dos extratos (IC50= 29,07 - 42,84 &mu;g/mL) foram necessárias para o sequestro do peróxido de hidrogênio. Entre os extratos dos cotilédones, se destacou o extrato do amendoim do genótipo BR1, no sequestro do radical superóxido (inibição de 28,85% na concentração de 50 &mu;g/mL), peróxido de hidrogênio (IC25= 304,61 &mu;g/mL) e radical peroxila (738,97 &mu;mol Trolox/g). A acessibilidade de sete compostos fenólicos majoritários de amendoim variou de 7 a 100% sendo que o ácido p-cumárico se apresentou em maior concentração após a digestão simulada in vitro (252,86 &mu;g/g) quando comparada à do extrato da amostra não digerida (68,55 &mu;g/g).Peanut is a food known for its high protein and lipid content as well as for its high content of phenolic compounds, which are described to have antioxidant properties. Peanut cultivation has a wide adaptability to tropical conditions and plays an important role in income generation among small local farmers in northeastern Brazil. To meet these farmers\' needs, the Brazilian Agricultural Research Corporation (Embrapa) holds a peanut genetic improvement program for semi-arid environments through which they develop cultivars tolerant to water stress. In this study, a total of fourteen genotypes from the Embrapa\'s Germplasm Active Collection (six drought-tolerant genotypes, one mild tolerant and seven non-drought tolerant) were analyzed for their phenolic composition, potential for scavenging reactive oxygen species, and bio-accessibility after in vitro simulated digestion, which have never been investigated for such genotypes. This study is organized into four chapters, as follows: Chapter I corresponds to the literature review; Chapter II describes the study with BR1 (drought tolerant) and LViPE-06 (non-drought tolerant) genotypes to determine the optimal conditions to obtain a phenolics-rich extract with antioxidant activity, as well as to determine the phenolic profile of peanut cotyledon and skin by the LC-ESI-QTOF-MS technique; Chapter III, based on the conditions established for extraction, cotyledon and skin extracts of the fourteen peanut genotypes were produced, which were analyzed for their scavenging capacity with five different reactive oxygen species; Chapter IV, the accessibility of the major phenolic compounds present in BR1 peanut genotype, including p-coumaric acid, was determined by in vitro simulated digestion. The optimal temperature and ethanol hydration to obtain the peanut cotyledon and skin extracts were 60°C and 35% and 40°C and 60%, respectively, which yielded extracts with the highest contents of phenolic compounds and strong antioxidant activity. The analysis by LC-ESI-QTOF-MS identified the main phenolic compounds present in the extracts, namely: derivatives of p-coumaric acid and p-cumaroil in cotyledon extracts, and type A oligomeric procyanidins in skin extracts. Skin extracts showed excellent reactive species scavenging activity, especially of hydroxyl radical, as IC50 values were lower than 0.1 &mu;g/mL; higher concentrations of the extracts (IC50 = 29.07-42.84 &mu;g / mL) were required for scavenging hydrogen peroxide. Among the cotyledon extracts, the extract from peanut genotype BR1 showed promising capacity to scavenge superoxide radical (inhibition of 28.85% at 50 &mu;g/mL), hydrogen peroxide (IC25 = 304.61 &mu;g/mL) and peroxyl radical (738.97 &mu;mol Trolox/g). The bio-accessibility of seven major phenolic compounds ranged from 7% to 100%, with a higher concentration of p-coumaric acid found after in vitro simulated digestion (252.86 &mu;g/g) as compared to the undigested extract (68.55 &mu;g/g)

Extraction yield, antioxidant activity andphenolics from grape, mango and peanut agro-industrial by-products

ABSTRACT: The objective of this study was to determine and correlate the extraction yields, antioxidant activity, total phenolics and total flavonoids from grape, mango and peanut agro-industrial by-products. The β-carotene/linoleic acid autoxidation system and scavenging capacity for DPPH and ABTS free radicals assays were used. The results were expressed in terms of lyophilized sample or dry extract. Mango bagasse exhibited the highest extraction yield (37.07%) followed by peanut skin (15.17%) and grape marc (7.92%). In terms of lyophilized sample, total phenolics did not vary significantly among the residues evaluated (average of 60.33mg EAG g-1); however, when they were expressed as dry extract grape marc exhibited the highest total phenolic (768.56±116.35mg GAE g-1), followed by peanut skin (404.40±13.22mg GAE g-1) and mango bagasse (160.25±4.52mg GAE g-1), Peanut skin exhibited the highest content of total flavonoids (2.44mg QE g-1), while grape marc (1.76mg QE g-1) and mango bagasse (1.70 mg QE g-1) showed no significant differences. The extraction yield showed strong negative linear correlation with total phenolic and total flavonoid. This study showed that peanut skin was the sample with the highest antioxidant activity and it was strongly influenced by total flavonoids. All extracts of byproducts showed antioxidant activity comparable to α-tocopherol, and they can be a source of natural compounds with potential to replace synthetic antioxidants such as BHT

Active Antioxidant Phenolics from Brazilian Red Propolis: An Optimization Study for Their Recovery and Identification by LC-ESI-QTOF-MS/MS

Brazilian red propolis (BRP) is a natural product widely known for its phenolic composition and strong antioxidant properties. In this study, we used the Box–Behnken Design (BBD) with Surface Response Methodology to optimize the extraction conditions for total phenolic content (TPC) and Trolox equivalent antioxidant capacity(TEAC) of bioactive phenolics from BRP. The extraction time, ethanol/water concentration and temperature, were tested. All variables had significant effects (p  ≤ 0.05), with a desirability coefficient of 0.88. Under optimized conditions (90% ethanol at 80 °C for 30 min), the BRP extract showed a TPC of 129.00 ± 2.16 mg GAE/g and a TEAC of 3471.76 ± 53.86 µmol TE/g. Moreover, FRAP and ORAC assays revealed that the optimized BRP extract had 1472.86 ± 72.37 µmol Fe2+/g and 4339.61 ± 114.65 µmol TE/gof dry weight, respectively. Thirty-two phenolic compounds were tentatively identified by LC-QTOF-ESI-MS/MS, of which thirteen were found for the first time in BRP, including four flavones, one flavanol, two flavanones, two chalcones, and four isoflavonoids. Thus, our results highlight the importance of BRP as a source of a wide variety of phenolic compounds with significant antioxidant properties

Anti-inflammatory and antioxidant potential, in vivo toxicity, and polyphenolic composition of Eugenia selloi B.D.Jacks. (pitangatuba), a Brazilian native fruit.

Brazilian native fruits are a rich source of polyphenolic compounds that can act as anti-inflammatory and antioxidant agents. Here, we determined the polyphenolic composition, anti-inflammatory mechanism of action, antioxidant activity and systemic toxicity in Galleria mellonella larvae of Eugenia selloi B.D.Jacks. (synonym Eugenia neonitida Sobral) extract (Ese) and its polyphenol-rich fraction (F3) obtained through bioassay-guided fractionation. Phenolic compounds present in Ese and F3 were identified by LC-ESI-QTOF-MS. The anti-inflammatory activity of Ese and F3 was tested in vitro and in vivo through NF-κB activation, cytokine release and neutrophil migration assays. The samples were tested for their effects against reactive species (ROO•, O2•-, HOCl and NO•) and for their toxicity in Galleria mellonella larvae model. The presence of hydroxybenzoic acid, ellagitannins and flavonoids was identified. Ese and F3 reduced NF-κB activation, cytokine release and neutrophil migration, with F3 being three-fold more potent. Overall, F3 exhibited strong antioxidant effects against biologically relevant radicals, and neither Ese nor F3 were toxic to G. mellonella larvae. In conclusion, Ese and F3 revealed the presence of phenolic compounds that decreased the inflammatory parameters evaluated and inhibited reactive oxygen/nitrogen species. E. selloi is a novel source of bioactive compounds that may provide benefits for human health

Comprehensive characterization of bioactive phenols from new brazilian superfruits by LC-ESI-QTOF-MS, and their ROS and RNS scavenging effects and anti-inflammatory activity

Brazilian native fruits (BNF) remain unexplored and underutilized resources with a high potential to improve human health and wellness. In our study, five new BNF (Eugenia stipitata, Sageretia elegans, Byrsonima arthropoda, Spondias mombin and Rubus rosaefolius) were evaluated for their phenolic composition by LC-ESI-QTOF-MS; and for their ROS and RNS scavenging effects (ROO center dot, O-2(center dot-), NO center dot, HOCl); in vivo anti-inflammatory activity (neutrophil migration); and in vivo acute toxicity in Galleria mellonella. Eighty-six phenolic compounds were identified, including hydroxybenzoic acids, hydroxycinnamic acids, flavonoids, anthocyanins and ellagitannins, several of which had never been reported in BNF. The BNF exhibited high antioxidant effects against biologically relevant radicals, and treated animals showed decreased neutrophil influx and NF-kB activation. Thus, these BNF are good sources of antioxidant and anti-inflammatory molecules that can be beneficial for human health as functional foods. Based on their bioactivity, they can be considered as new Brazilian superfruits281178188CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP307893/2016-2não tem2013/13190-2; 2014/12606-3; 2016/02926; 2017/0989

Exploration of avocado by-products as natural sources of bioactive compounds.

This study aimed to evaluate the antioxidant, anti-inflammatory, and cytotoxic properties and phenolic composition of peel and seed of avocado varieties Hass and Fuerte using green solvents. Ethanol soluble compounds were identified in peel and seed of both varieties using HPLC-MS/MS and quantified using HPLC-DAD. Agro-industrial by-products of both varieties exhibited high radical scavenging activity against synthetic free radicals (DPPH and ABTS) and reactive oxygen species (peroxyl, superoxide, and hypochlorous acid) and high ability to reduce Fe3+ to Fe2+. The main compounds with significant contribution to the antioxidant activity determined by online HPLC-ABTS●+ analyses were procyanidin B2 and epicatechin in the peel and trans-5-O-caffeoyl-D-quinic acid, procyanidin B1, catechin, and epicatechin in the seed. Peel of Fuerte significantly suppressed TNF-α and nitric oxide (NO) release (459.3 pg/mL and 8.5 μM, respectively), possibly because of the high phenolic content and antioxidant activity detected. Avocado agro-industrial by-products can be used for food and pharmaceutical purposes due to their antioxidant and anti-inflammatory properties