Carqueja (Baccharis trimera) Protects against Oxidative Stress and -Amyloid-Induced Toxicity in Caenorhabditis elegans

Carqueja (Baccharis trimera) is a native plant found throughout South America. Several studies have shown that Carqueja has antioxidant activity in vitro, as well as anti-inflammatory, antidiabetic, analgesic, antihepatotoxic, and antimutagenic properties. However, studies regarding its antioxidant potential in vivo are limited. In this study, we used Caenorhabditis elegans as a model to examine the antioxidant effects of a Carqueja hydroalcoholic extract (CHE) on stress resistance and lifespan and to investigate whether CHE has a protective effect in a C. elegans model for Alzheimer's disease. Here, we show for the first time, using in vivo assays, that CHE treatment improved oxidative stress resistance by increasing survival rate and by reducing ROS levels under oxidative stress conditions independently of the stress-related signaling pathways (p38, JNK, and ERK) and transcription factors (SKN-1/Nrf and DAF-16/Foxo) tested here. CHE treatment also increased the defenses against -amyloid toxicity in C. elegans, in part by increasing proteasome activity and the expression of two heat shock protein genes. Our findings suggest a potential neuroprotective use for Carqueja, supporting the idea that dietary antioxidants are a promising approach to boost the defensive systems against stress and neurodegeneration

O extrato hidroalcoólico de guaraná (Paullinia cupana) atenua fenótipos patológicos associados ao mal de Alzheimer e à doença de Huntington no organismo modelo Caenorhabditis elegans.

Programa de Pós-Graduação em Biotecnologia. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto.Submitted by Oliveira Flávia ([email protected]) on 2015-05-11T17:46:22Z No. of bitstreams: 2 license_rdf: 22190 bytes, checksum: 19e8a2b57ef43c09f4d7071d2153c97d (MD5) DISSERTAÇÃO_ExtratoHidroalcoólicoGuaraná.pdf: 1613708 bytes, checksum: 9552d2234067a5d074db03b5670bb5ed (MD5)Approved for entry into archive by Gracilene Carvalho ([email protected]) on 2015-05-12T13:29:37Z (GMT) No. of bitstreams: 2 license_rdf: 22190 bytes, checksum: 19e8a2b57ef43c09f4d7071d2153c97d (MD5) DISSERTAÇÃO_ExtratoHidroalcoólicoGuaraná.pdf: 1613708 bytes, checksum: 9552d2234067a5d074db03b5670bb5ed (MD5)Made available in DSpace on 2015-05-12T13:29:37Z (GMT). No. of bitstreams: 2 license_rdf: 22190 bytes, checksum: 19e8a2b57ef43c09f4d7071d2153c97d (MD5) DISSERTAÇÃO_ExtratoHidroalcoólicoGuaraná.pdf: 1613708 bytes, checksum: 9552d2234067a5d074db03b5670bb5ed (MD5) Previous issue date: 2015O Guaranazeiro (Paullinia cupana) é uma planta nativa das regiões amazônicas rica em fitoquímicos, como polifenóis e metilxantinas. O extrato de guaraná, obtido das sementes do Guaranazeiro, é largamente consumido no Brasil na forma de bebidas, como refrigerantes e energéticos, e como suplemento alimentar. Estudos anteriores demonstraram que o extrato de guaraná possui propriedades antioxidantes, antimicrobianas, anti-obesogênicas, antimutagênicas e anticarcinogênicas. Quanto ao Sistema Nervoso Central (SNC), foram relatadas propriedades estimulantes, bem como efeitos protetores em modelo in vitro para a Doença de Parkinson e capacidade de inibir a agregação do peptídeo β-amilóide (βA), proteína associada ao Mal de Alzheimer. Entretanto, estudos sobre as propriedades biológicas do guaraná e seus efeitos sobre o SNC em modelos in vivo são limitados e não foram bem explorados. Neste estudo, o nematóide Caenorhabditis elegans foi utilizado para testar as atividades antioxidantes e os efeitos protetores do Extrato Hidroalcoólico de Guaraná (EHG) em modelos transgênicos para o Mal de Alzheimer e a Doença de Huntington. Os resultados demonstram que o EHG produzido possui propriedades antioxidantes in vitro, evidenciadas por sua capacidade de neutralizar o radical DPPH em 54,37%, 49,36% e 48,30% para as concentrações de 5, 10 e 50 mg/mL, respectivamente. Ainda, o EHG também demonstrou capacidade antioxidante in vivo, uma vez que reduziu os níveis de ERO em animais tipo selvagem sob condições normais e de estresse oxidativo. O EHG nas concentrações encolhidas para os ensaios in vivo (10 mg/mL e 50 mg/mL) não foi tóxico para os vermes, uma vez que não interferiu no seu desenvolvimento, avaliado pela medida do tamanho corporal. Ao contrário, o tratamento com EHG diminui o acúmulo de pigmentos associados ao envelhecimento observados através do corante vermelho Nilo. O tratamento com o EHG foi capaz de atenuar o fenótipo de paralisia induzido pela toxicidade do peptídeo βA em dois modelos C. elegans para o Mal de Alzheimer. Quanto aos modelos C. elegans para a Doença de Huntington, o EHG foi capaz de reduzir a agregação de proteínas poliglutamínicas (PoliQ40) expressas no tecido muscular dos vermes, bem como reduzir a neurotoxicidade da proteína Huntingtina (Q150). Para testar se os efeitos benéficos do EHG estavam associados a uma diminuição da patogenicidade da bactéria Escherichia coli utilizada como alimento para os vermes, os efeitos do EHG sobre o crescimento bacteriano foram testados, sendo que nenhum efeito bactericida ou bacteriostático foi observado. Os resultados obtidos sugerem que o EHG age através de efeitos antioxidantes, uma vez que foi capaz de reduzir os níveis de espécies reativas de oxigênio (ERO) em animais expressando o peptídeo βA, além de ativar a expressão da enzima superóxido dismutase (SOD-3), uma enzima antioxidante. O EHG não foi capaz de diminuir a expressão do mRNA para βA, porém foi capaz de ativar a expressão da chaperonina HSP-16, aumentar a atividade do proteassoma e proteger o modelo contra o estresse térmico, um conhecido indutor de dano proteico, sugerindo uma atividade sobre a homeostase proteica. Os resultados obtidos até o momento demonstram que o EHG possui propriedades antioxidantes in vitro e in vivo, modula a homeostase proteica e que seu consumo pode ser benéfico no tratamento de doenças neurodegenerativas como o Mal de Alzheimer e a Doença de Huntington.Paullinia cupana, or guaranázeiro, is a native plant from the Amazon region rich in phytochemicals such as polyphenols and methylxanthines. The guaraná extract, obtained from guaranazeiro’s seeds, is widely consumed as beverages, such as soft and energy drinks, and as food supplement. Previous investigations have described properties such as antioxidant, antimicrobial, anti-obesogenic, antimutagenic and anticarcinogenic for guaraná. Regarding the central nervous system (CNS), stimulant properties have been described, as well as protective effects in an in vitro model for Parkinson’s Disease and a capability to inhibit amyloid-β (Aβ) peptide aggregation in vitro. However, studies about the biological properties of guaraná are limited and the effects of guaraná over the CNS are not well explored. In this study, we used Caenorhabditis elegans as a model to test both the antioxidant and the protective properties of guaraná hydro‐alcoholic extract (GHE) in wild-type and transgenic animals for Alzheimer (AD) and Huntington (HD) diseases. Our results demonstrate that GHE has in vitro antioxidant activity, shown by its capacity in neutralize the DPPH radical by 54,37%, 49,36% e 48,30% for the concentrations of 5, 10 e 50 mg/mL, respectively. Also, GHE demonstrated an in vivo antioxidant property, since it reduced ROS levels in wild type animals under standart and stress conditions. GHE treatment is not toxic since it did not interfere with the animals’ development assessed by the body length. GHE treatment also reduced the level of fluorescent pigments associated to aging indicated by the red nile staining. GHE attenuated the pathological phenotypes of paralysis induced by Aβ in two C. elegans AD models. Concerning C. elegans models for Huntington Disease, GHE reduced the aggregation of polyglutamine proteins (PoliQ40) in the muscle of the animals, as well as reduced Huntingtin protein’s neurotoxicity. Our findings suggest that GHE’s beneficial effects might be due its antioxidant properties, since it was able to reduce reactive oxygen species’ (ROS) production in animals expressing Aβ and activate the expression of superoxide dismutase (SOD-3), an antioxidant enzyme. GHE treatment did not reduced the expression of Aβ mRNA; however it activated the expression of the heat shock protein HSP-16, a chaperonine, increased the proteasome activity and boosted animals’ thermal stress resistance, which suggests that GHE can interfere with protein homeostasis . Our results so far show guaraná has antioxidant properties in vitro and in vivo, modulates protein homeostasis and suggests that GHE consumption might be advantageous in the treatment of Alzheimer and Huntington diseases

O extrato hidroalcoólico de guaraná (Paullinia cupana) atenua fenótipos patológicos associados ao mal de Alzheimer e à doença de Huntington no organismo modelo Caenorhabditis elegans.

Programa de Pós-Graduação em Biotecnologia. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto.O Guaranazeiro (Paullinia cupana) é uma planta nativa das regiões amazônicas rica em fitoquímicos, como polifenóis e metilxantinas. O extrato de guaraná, obtido das sementes do Guaranazeiro, é largamente consumido no Brasil na forma de bebidas, como refrigerantes e energéticos, e como suplemento alimentar. Estudos anteriores demonstraram que o extrato de guaraná possui propriedades antioxidantes, antimicrobianas, anti-obesogênicas, antimutagênicas e anticarcinogênicas. Quanto ao Sistema Nervoso Central (SNC), foram relatadas propriedades estimulantes, bem como efeitos protetores em modelo in vitro para a Doença de Parkinson e capacidade de inibir a agregação do peptídeo β-amilóide (βA), proteína associada ao Mal de Alzheimer. Entretanto, estudos sobre as propriedades biológicas do guaraná e seus efeitos sobre o SNC em modelos in vivo são limitados e não foram bem explorados. Neste estudo, o nematóide Caenorhabditis elegans foi utilizado para testar as atividades antioxidantes e os efeitos protetores do Extrato Hidroalcoólico de Guaraná (EHG) em modelos transgênicos para o Mal de Alzheimer e a Doença de Huntington. Os resultados demonstram que o EHG produzido possui propriedades antioxidantes in vitro, evidenciadas por sua capacidade de neutralizar o radical DPPH em 54,37%, 49,36% e 48,30% para as concentrações de 5, 10 e 50 mg/mL, respectivamente. Ainda, o EHG também demonstrou capacidade antioxidante in vivo, uma vez que reduziu os níveis de ERO em animais tipo selvagem sob condições normais e de estresse oxidativo. O EHG nas concentrações encolhidas para os ensaios in vivo (10 mg/mL e 50 mg/mL) não foi tóxico para os vermes, uma vez que não interferiu no seu desenvolvimento, avaliado pela medida do tamanho corporal. Ao contrário, o tratamento com EHG diminui o acúmulo de pigmentos associados ao envelhecimento observados através do corante vermelho Nilo. O tratamento com o EHG foi capaz de atenuar o fenótipo de paralisia induzido pela toxicidade do peptídeo βA em dois modelos C. elegans para o Mal de Alzheimer. Quanto aos modelos C. elegans para a Doença de Huntington, o EHG foi capaz de reduzir a agregação de proteínas poliglutamínicas (PoliQ40) expressas no tecido muscular dos vermes, bem como reduzir a neurotoxicidade da proteína Huntingtina (Q150). Para testar se os efeitos benéficos do EHG estavam associados a uma diminuição da patogenicidade da bactéria Escherichia coli utilizada como alimento para os vermes, os efeitos do EHG sobre o crescimento bacteriano foram testados, sendo que nenhum efeito bactericida ou bacteriostático foi observado. Os resultados obtidos sugerem que o EHG age através de efeitos antioxidantes, uma vez que foi capaz de reduzir os níveis de espécies reativas de oxigênio (ERO) em animais expressando o peptídeo βA, além de ativar a expressão da enzima superóxido dismutase (SOD-3), uma enzima antioxidante. O EHG não foi capaz de diminuir a expressão do mRNA para βA, porém foi capaz de ativar a expressão da chaperonina HSP-16, aumentar a atividade do proteassoma e proteger o modelo contra o estresse térmico, um conhecido indutor de dano proteico, sugerindo uma atividade sobre a homeostase proteica. Os resultados obtidos até o momento demonstram que o EHG possui propriedades antioxidantes in vitro e in vivo, modula a homeostase proteica e que seu consumo pode ser benéfico no tratamento de doenças neurodegenerativas como o Mal de Alzheimer e a Doença de Huntington.Paullinia cupana, or guaranázeiro, is a native plant from the Amazon region rich in phytochemicals such as polyphenols and methylxanthines. The guaraná extract, obtained from guaranazeiro’s seeds, is widely consumed as beverages, such as soft and energy drinks, and as food supplement. Previous investigations have described properties such as antioxidant, antimicrobial, anti-obesogenic, antimutagenic and anticarcinogenic for guaraná. Regarding the central nervous system (CNS), stimulant properties have been described, as well as protective effects in an in vitro model for Parkinson’s Disease and a capability to inhibit amyloid-β (Aβ) peptide aggregation in vitro. However, studies about the biological properties of guaraná are limited and the effects of guaraná over the CNS are not well explored. In this study, we used Caenorhabditis elegans as a model to test both the antioxidant and the protective properties of guaraná hydro‐alcoholic extract (GHE) in wild-type and transgenic animals for Alzheimer (AD) and Huntington (HD) diseases. Our results demonstrate that GHE has in vitro antioxidant activity, shown by its capacity in neutralize the DPPH radical by 54,37%, 49,36% e 48,30% for the concentrations of 5, 10 e 50 mg/mL, respectively. Also, GHE demonstrated an in vivo antioxidant property, since it reduced ROS levels in wild type animals under standart and stress conditions. GHE treatment is not toxic since it did not interfere with the animals’ development assessed by the body length. GHE treatment also reduced the level of fluorescent pigments associated to aging indicated by the red nile staining. GHE attenuated the pathological phenotypes of paralysis induced by Aβ in two C. elegans AD models. Concerning C. elegans models for Huntington Disease, GHE reduced the aggregation of polyglutamine proteins (PoliQ40) in the muscle of the animals, as well as reduced Huntingtin protein’s neurotoxicity. Our findings suggest that GHE’s beneficial effects might be due its antioxidant properties, since it was able to reduce reactive oxygen species’ (ROS) production in animals expressing Aβ and activate the expression of superoxide dismutase (SOD-3), an antioxidant enzyme. GHE treatment did not reduced the expression of Aβ mRNA; however it activated the expression of the heat shock protein HSP-16, a chaperonine, increased the proteasome activity and boosted animals’ thermal stress resistance, which suggests that GHE can interfere with protein homeostasis . Our results so far show guaraná has antioxidant properties in vitro and in vivo, modulates protein homeostasis and suggests that GHE consumption might be advantageous in the treatment of Alzheimer and Huntington diseases

Carqueja (Baccharis trimera) Protects against Oxidative Stress and β-Amyloid-Induced Toxicity in Caenorhabditis elegans

Carqueja (Baccharis trimera) is a native plant found throughout South America. Several studies have shown that Carqueja has antioxidant activity in vitro, as well as anti-inflammatory, antidiabetic, analgesic, antihepatotoxic, and antimutagenic properties. However, studies regarding its antioxidant potential in vivo are limited. In this study, we used Caenorhabditis elegans as a model to examine the antioxidant effects of a Carqueja hydroalcoholic extract (CHE) on stress resistance and lifespan and to investigate whether CHE has a protective effect in a C. elegans model for Alzheimer's disease. Here, we show for the first time, using in vivo assays, that CHE treatment improved oxidative stress resistance by increasing survival rate and by reducing ROS levels under oxidative stress conditions independently of the stress-related signaling pathways (p38, JNK, and ERK) and transcription factors (SKN-1/Nrf and DAF-16/Foxo) tested here. CHE treatment also increased the defenses against β-amyloid toxicity in C. elegans, in part by increasing proteasome activity and the expression of two heat shock protein genes. Our findings suggest a potential neuroprotective use for Carqueja, supporting the idea that dietary antioxidants are a promising approach to boost the defensive systems against stress and neurodegeneration

Contribution of genetic background to oxidative stress resistance induced by açaí aqueous extract (AAE) treatment.

<p>Animals were treated with 100/mL AAE or control solution (S basal) from L1 until L4 and then submitted to 7.5 mM t-BOOH in M9. Survival was measured at 3, 6, 9 and 12 h. <b>A</b>) Survival curves for transcription factor <i>daf-16 (mu86)</i> and JNK MAPK pathway <i>jnk-(gk7)</i> mutants. <b>B</b>) Survival curves for <i>skn-1(zu67)</i> and p38 MAPK pathway, <i>nsy-1(ag3)</i>, <i>sek-1(km4)</i> mutants. <b>C</b>) Survival curves for osmotic stress resistance pathway, <i>unc-43(n498n1186)</i> and <i>osr-1(rm1)</i> mutants. *p<0.05 and ***p<0.001 by the Log-rank (Mantel-Cox) test (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089933#pone-0089933-t003" target="_blank">Table 3</a> for more details).</p

Effect of açaí aqueous extract (AAE) on redox status in wild-type <i>C. elegans</i> and HUVECs.

<p><b>A</b>) <i>C. elegans</i> was treated with control solution (S basal) or 100 mg/mL AAE for 48 h and then submitted to the presence or absence of 1 mM H₂O₂ for 2 h. The results are expressed as H₂DCFDA fluorescence relative to the untreated control. <b>B</b>) HUVECs were treated with or without 2.5 mg/mL AAE for 16 h and then incubated in 0.25 mM H₂O₂ for 1 h. The fluorescence was measured by flow cytometry. The results are expressed as H₂DCFDA fluorescence relative to the untreated control. Different letters indicate significant differences by one-way ANOVA followed by Tukey's post-test. <b>C</b>) To measure the levels of total SH groups, animals were treated with control solution (S basal) or 100 mg/mL AAE from L1 until L4 and then incubated with or without 5 mM t-BOOH for 1 h. *p values were determined by a two-tailed Student's <i>t-</i>test, and groups were significantly different when p<0.05 in <i>C. elegans</i>. Transgenic worms containing reporter genes were treated with control solution (S basal) or 100 mg/mL AAE for 48 h beginning at L1 and then with or without the oxidative stress condition. After a 1-h hour recovery period, photographs were taken on a fluorescence microscope. For (<b>D</b>) <i>gcs-1::GFP</i> and (<b>E</b>) <i>gst-4::GFP</i> animals, GFP fluorescence signals were measured using NIH Image J software. Different letters correspond to significant differences by the Kruskal-Wallis test followed by Dunn's post-test.</p

Effect of açaí aqueous extract (AAE) on <i>C. elegans</i> grown under normal and stress conditions.

<p><b>A</b>) <i>fem-1(hc17)</i> mutants were treated at 25°C with control solution (S basal) or 100 mg/mL AAE beginning at L1. Surviving and dead animals were counted daily until all nematodes had died. Log-rank (Mantel-Cox) analysis showed no significant difference between the curves. <b>B</b>) Animals were treated with control solution (S basal) or 100 mg/mL AAE from L1 until L4 and then submitted to 7.5 mM t-BOOH in M9. The survival was measured at 6, 9 and 12 h. The survival curves show that AAE treatment increased <i>C. elegans</i> oxidative stress resistance. ***p<0.001 by the Log-rank (Mantel-Cox) test. <b>C</b>) Animals were treated with control solution (S basal) or 100 mg/mL AAE beginning at L1. After five days at 20°C, the animals were incubated at 35°C and survival was monitored at 6, 9 and 12 h. There was no significant difference between curves by the Log-rank (Mantel-Cox) test. <b>D</b>) Animals were treated with control solution (S basal) or 100 mg/mL AAE for 68 h beginning at L1 and then transferred to new plates containing 500 mM NaCl. The percentage of worms that moved outside a 7-mm circle was monitored at 15, 30 and 60 min. *p<0.05 by a two-tailed Student's <i>t-</i>test.</p

<i>In vitro</i> antioxidant activity of açaí aqueous extract (AAE) measured by DPPH assay.

<p>DPPH, 2,2-diphenyl-1-picrylhydrazyl; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; OP50, <i>E. coli</i> strain; KAN, Kanamycin.</p><p>Different subscript letters indicate significant differences by one-way ANOVA followed by Tukey's post-test.</p

Effect of açaí aqueous extract (AAE) on wild-type <i>C. elegans</i> body length and progeny.

<p><b>A</b>). L1 animals were treated with control solution (S basal) or 100 mg/mL AAE until L3 and then transferred onto NGM plates with OP50 until the next day. Images were captured of one-day-old animals, and body length was measured along the animal axis using NIH Image J software. There was no significant difference between groups, as determined by two-tailed Student's <i>t-</i>test. <b>B, C</b>) Progeny profiles were measured in animals treated with control solution (S basal) or 100 mg/mL AAE. Animals were transferred individually to NGM plates and moved daily until the end of the reproductive period. The results were plotted as the mean ± SEM for each day (B) and total final progeny (C). There was no significant difference between groups by a two-tailed Student's <i>t-</i>test.</p

Hypothetical model of the mode of action of açaí aqueous extract (AAE) on <i>C. elegans</i>.

<p>Text marked within a rectangle represents modulators or data observed experimentally in this manuscript. AAE modulates oxidative stress resistance by direct and indirect mechanisms. AAE removes ROS directly and prevents <i>gcs-1</i> activation and SH level reduction which in turn increases oxidative stress resistance. AAE also promotes oxidative stress resistance indirectly through DAF-16/OSR-1/UNC-43/SEK-1. In addition, AAE increases osmotic stress resistance, possibly as a result of impaired protein homeostasis and/or increased ionic strength.</p