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It is important to study the stability of plant extracts used as active ingredients in phytotherapic medicine, as degradation of the active principles directly affects the efficacy and safety of these products. Therefore, a stability study of the hydroalcoholic extract of the species: Mikania glomerata and Mikania laevigata was conducted in order to determine the speed of degradation and shelf life of these extracts, which are incorporated in cough syrup in Brazil. Leaves of both species were dried in an oven or by lyophilization (freeze-dried). Hydroalcoholic extracts underwent both accelerated stability study of six months and long-term stability study for 12 months. Samples were stored at different temperatures and every three months were analysed by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) to monitor their chemical profile, quantifying coumarin and chlorogenic acid. For all conditions of the study, a reduction of the content of the chemical marker of this species, coumarin, greater than 5% was observed, so a shelf life of two years cannot be assigned to the hydroalcoholic extracts of these species as observed in commercial extracts.

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INTRODUCTION: Mikania laevigata Sch.Bip. ex Baker and Mikania glomerata Spreng. are medicinal plants popularly known as guaco, used for inflammatory diseases of the respiratory system, included in pharmaceutical formularies and often used without distinction. However, several studies show that the chemical composition varies between these species, as well as in plants are subjected to different environmental stresses. Few studies have been carried out with the volatile compounds of guaco, even less about the changes in volatile composition due to abiotic variation. OBJECTIVE: The aim of this work was to evaluate how volatile compounds vary according to the seasons and at different times of the day and if these compounds are influenced by the variations in the growth conditions such as: temperature, luminosity and water. METHODS: The headspace volatiles of the leaves were sampled by solid phase micro extraction and analyzed by gas chromatography-mass spectrometry. Untargeted metabolomic analysis of the resulting chromatograms and chemometrics was applied. The chemical profile of the volatiles of M. laevigata and M. glomerata were different; being clearly separated in the exploratory grouping analyzes (PCA), followed by analysis of variance of the marker compounds of both species. RESULTS: Only M. laevigata contained coumarin, considered to be the chemical marker of both species and to be responsible for the therapeutic activities. There was no significant difference between the morning and afternoon collections of either species. Coumarin, α-pinene and bicyclogermacrene were more intense in the volatiles of M. laevigata throughout the year and responsible for grouping the samples of this species. For M. glomerata, hexanal and 2-hexenal were responsible for grouping the samples and were more intense in all months. The growth conditions tested affected the intensity of specific compounds in the chromatograms. Some compounds were less intense with the increase of the temperature and in the plants subjected to full sunlight. However, certain volatile compounds-such as pinenes-were more intense in plants suffering drought. CONCLUSION: The variation in composition between species of guaco was greater than those observed in the seasonal and cultivation studies, indicating that these species cannot be used indistinctly.

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Statistical mixture design extraction and fractionation of Mikania plant samples were carried out to quantitatively study seasonal and solvent composition effects as well as their interactions on secondary metabolites. The mixture design consisted of ethanol, acetone, dichloromethane and chloroform solvents and their binary, ternary and quaternary mixtures. Yields were measured for the crude extract and its neutral, organic, basic, polar and fiber fractions obtained with each solvent composition from samples harvested during 2010. Two-way ANOVA found statistically significant seasonal and solvent effects for the crude extract and all the fractions except for the polar fraction solvent. The best solvent extractor depends the harvest season. High crude, organic and polar fraction yields in the summer are correlated with the coumarin UV-vis absorbance at 274 nm. Crude yields of mixtures containing ethanol are correlated with the coumarin absorbances in summer, winter and spring whereas mixtures without ethanol show no significant correlation.

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Abstract In Brazil, Mikania glomerata Spreng. and M. laevigata Sch. Bip. ex Baker, Asteraceae, known popularly as guaco, are widely used for colds and asthma. Although coumarin is adopted as the chemical marker of both species, it was not always detected in M. glomerata, for which chlorogenic acid was identified and quantified instead. The purpose of this study was to develop and validate a method to quantify both coumarin and chlorogenic acid and apply it to extracts of plants identified as M. glomerata, M. laevigata, or as guaco, to determine the pattern of composition of these two species and to observe differences between oven-dried and lyophilized leaves. A method using ultra-high resolution liquid chromatography–mass spectrometry (UHPLC–MS) in the full scan mode was validated for selectivity, matrix effect, linearity, limits of detection and quantification, precision and accuracy. The concentration of coumarin varied between species and samples, therefore these two species should not be used interchangeably. The concentration of chlorogenic acid was also determined for all samples. The UHPLC–MS method permitted the quantification of coumarin and chlorogenic acid in 16 samples of guaco and several commercial samples were possibly misidentified.

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Resumo O Estado do Paraná destaca-se pela tradição no cultivo de plantas medicinais. O aumento em sua demanda através da busca de culturas alternativas e rentáveis e o estímulo a uma agricultura ecologicamente sustentável fomentou os agricultores a diversificarem a produção. A pesquisa de campo nos diferentes elos da cadeia produtiva do guaco permitiu construir seu fluxograma, caracterizar o sistema e fluxo de produção, os seus custos e receitas para analisar as Margens e Markups de comercialização. Os sistemas de coleta com enriquecimento e de cultivo apresentaram resultados diferenciados em relação ao seu custo de produção, onde o produtor/coletor alcança uma rentabilidade de 48,9 %, menor margem de comercialização entre todos os elos da cadeia, enquanto o produtor que cultiva o guaco como exploração obtém uma margem de comercialização de 346,4 %. As maiores margens de comercialização (1.733,3 %) estão nos elos fora da porteira das propriedades, percentual entre diferença de preço pago pelo consumidor e o preço recebido pelo produtor/coletor. Os produtores/coletores podem agregar mais renda em vários elos da cadeia produtiva através da organização dos produtores e a melhoria no processo produtivo, possibilitando a melhoria econômica e social nas propriedades familiares, reduzindo o impacto ambiental e garantindo a sustentabilidade dos sistemas de produção.

Abstract The State of Parana stands out for its tradition in the medicinal plants cultivation. The increase of the demand due to the search of alternative and profitable crops and the impulse to an environmentally sustainable agriculture encouraged farmers to diversify production. The field research about the different links of the guaco supply chain allowed the construction of its flowchart, the characterization of the production system and flow, as well as the knowledge of its costs and revenues in order to analyze the commercialization margins and markups. The collection systems with enrichment and the cultivation ones presented different results in relation to its production cost, with the producer/collector reaching a yield of 48.9%, the lowest commercialization margin among all links of the chain, while the farmer who cultivates guaco gets a commercialization margin of 346.4%. The largest commercialization margins (1,733.3%) are in the links outside the properties" gates, as the percentage difference between the price paid by the consumer and the one received by the producer/collector. Producers/collectors can add more income in several links of the supply chain through the producers" organization and by improvements in the production process, enabling the economic and social enhancement at the family farms, thus reducing the environmental impact and ensuring the sustainability of production systems.

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Statistical design mixtures of acetone, chloroform, dichloromethane and ethanol were used to study the effects of different solvents and their mixtures on the quantities of coumarin and related metabolites extracted from Mikania laevigata samples harvested in each of the four seasons. RP-HPLC-DAD and both positive and negative modes of UPLC-MS analyses were used to determine relative quantities of coumarin, o-coumaric acid and melilotic acids in each season for all the mixture design extracts. The existence and measurement of the relative abundances of melilotic acid in Mikania laevigata have not been reported previously. Highest coumarin concentrations were encountered in the summer whereas its o-coumaric acid precursor and melilotic acid were most abundant in the spring. O-coumaric and melilotic acids concentrations were strongly correlated during the year. Also solvent effects were seen to be significant. Ethanol and 1:1 binary mixtures of ethanol and acetone extracted the largest quantities of coumarin whereas ethanolic binary and ternary mixtures with chloroform and dichloromethane provided the best yields of o-coumaric and melilotic acids. Statistical mixture models indicated that synergic binary interactions, especially those involving ethanol with acetone or chloroform, are important in the Mikania extraction process.

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Cancer is the second major cause of mortality worldwide, losing only to cardiovascular disease. Nowadays, around 50% of antineoplastic drugs were discovered and isolated by indications of plants in folk medicine. In Brazilian flora there are many species of plants which have great therapeutic importance, highlighting the Mikania laevigata (Asteraceae) that has been used for their valuable properties, especially in the respiratory tract. In the present study, the compounds of M. laevigata extracts were characterized by High Resolution Mass Spectrometry (HRMS) and Gas Chromatography with Mass analysis (GC/MS-EI). Therefore, the presence of some compounds with promising biological properties as antitumor activity was detected. Coumarin (1,2-benzopyrone) was previously reported as responsible for some biological activities of this plant species. Here, the extracts were evaluated by their cytotoxic activity against tumor (Hep-2, HeLa) and non tumor (MRC-5) cell lines, presenting significant inhibitory activity of cell growth in all extracts analyzed, chloroform, ethyl acetate, hexane, ethanol, which is related to its chemical composition. From the four different extracts here tested, two of them, hexane and ethanol, presented a clear selectivity against both tumor cells lines investigated. This can be explained by variances and increase of phenolic compounds in the ethanol fraction and an association of molecules with coumarin found in the hexane fraction.

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No Brasil as espécies de Mikania glomerata Spreng. e M. laevigata Sch. Bip. ex Baker, Asteraceae, popularmente conhecidas como guaco, são amplamente utilizadas com evidências pré-clínicas para o tratamento de doenças onde estejam envolvidas suas ações broncodilatadoras, antialérgicas, antiasmáticas, anti-inflamatórias, antiulcerogênicas e relaxantes da musculatura lisa. Em virtude das propriedades terapêuticas atribuídas as essas espécies, ambas foram oficializadas na farmacopéia brasileira, e por serem bastante semelhantes, dificilmente são diferenciadas. Com o objetivo de contribuir com novas pesquisas, a presente revisão descreve, de forma sistematizada, sobre os aspectos farmacológicos, toxicológicos, químicos, agronômicos, morfoanatômicos, genéticos e o uso nos programas de fitoterapia do Brasil destas espécies. As principais diferenças e semelhanças entre as duas espécies são evidenciadas através da comparação das informações baseadas na literatura específica.

In Brazil the species of Mikania glomerata Spreng. and M. laevigata Sch. Bip. ex Baker, Asteraceae popularly known as guaco, are widely used with preclinical evidence for the treatment of diseases whose actions involve bronchodilators, antiallergic, anti-asthmatic, anti-inflammatory, antiulcerogenic and smooth muscles relaxant. Due to the therapeutic properties attributed to these species, both were included in the official Brazilian Pharmacopoeia, and because of their similarity, they are hardly differentiated. In order to contribute to the new research, this review describes their several aspects: pharmacology, toxicology, chemical, agricultural, anatomical, genetics and the use in herbal medicine programs in Brazil, in a systematic way. The main differences and similarities between the two species are evidenced by comparing the information based on literature.

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O gênero Mikania, pertencente à família Asteraceae e à tribo Eupatoriae, tem cerca de 430 espécies distribuída principalmente na América do Sul. No Brasil, o gênero está representado por aproximadamente 171 espécies. Várias espécies do gênero Mikania de hábito trepador recebem a denominação vulgar de "guaco". Mikania laevigata Sch. Bip. ex Baker, conhecida como "guaco", "guaco-de-casa" e "guaco-do-mato", é uma espécie nativa do sul do Brasil. Suas folhas são empregadas na medicina tradicional como expectorante e como anti-reumáticas. É muito semelhante morfologicamente com M. glomerata, que é a espécie oficializada pela Farmacopéia Brasileira I. Alguns estudos de M. laevigata comprovaram atividades farmacológicas como, antiinflamatória, antimutagênica, antimicrobiana e antiulcerogênica. Objetivou-se estudar a morfologia externa e a anatomia do caule e da folha de M. laevigata, com a finalidade de fornecer dados farmacognósticos referentes à identificação e à diferenciação dessa espécie das demais Mikania, visando o controle de qualidade da matéria-prima. O material botânico foi submetido às microtécnicas fotônicas e eletrônicas de varredura usuais. As características morfoanatômicas descritas para a folha e o caule de M. laevigata auxiliam na identificação da espécie.

The genus Mikania, belonging to the family Asteraceae and tribe Eupatoriae, has about 430 species mainly distributed in South America. In Brazil, the genus is represented by approximately 171 species. Various members of Mikania are lianas and commonly called "guaco". Mikania laevigata Sch. Bip. ex Baker, known as "guaco", "guaco-de-casa" and "guaco-do-mato", is native to the South of Brazil. Its leaves are used in folk medicine as expectorant and antirheumatic. It is morphologically similar to M. glomerata, whose monograph is included in the Brazilian Pharmacopoeia 1st Ed. Some studies on M. laevigata have demonstrated pharmacological activities, such as anti-inflammatory, antimutagenic, antimicrobial and anti-ulcerogenic. This work has aimed to study the morpho-anatomy of the stem and the leaf of M. laevigata, in order to supply pharmacognostic information related to the identification of this species and distinction from other Mikania for quality control purposes. The botanical material was prepared according to standard light and scanning electronic micro techniques. The morpho-anatomical characters described for the stem and the leaf contribute to the identification of this species.

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Adulterações da matéria-prima vegetal ocorrem comumente no mercado de fitoterápicos, sob a forma de substituições e/ou falsificações. Muitas vezes, espécies distintas são comercializadas em substituição à farmacopéica devido à dificuldade de obtenção desta, ou mesmo pelo emprego intencional de espécies de menor valor econômico que apresentem características morfológicas semelhantes. Portanto, faz-se necessária a realização de estudos que busquem identificar a constituição química das espécies de amplo uso medicinal, que são alvos de adulterações. Mikania glomerata e Mikania laevigata, conhecidas popularmente como "guaco", são comercializadas indistintamente. Buscando-se avaliar a composição química das duas espécies, realizou-se uma prospecção fitoquímica por CCD e foram obtidos os perfis cromatográficos por CLAE-FR. Fez-se a quantificação da cumarina empregando-se o mesmo método. Na prospecção fitoquímica por CCD, foi detectada, nas duas espécies, a presença de cumarina, triterpenos/esteróides e heterosídeos flavônicos. Os perfis cromatográficos, obtidos por CLAE-FR, mostraram-se semelhantes, observando-se a predominância de compostos polares. Na quantificação da cumarina, obtêve-se um teor de 0,30 por cento para M. glomerata e 0,43 por cento para M. laevigata, teores de acordo com o preconizado na monografia do guaco-cheiroso (mín. 0,1 por cento). M glomerata e M. laevigata apresentaram composição química semelhante e teores de cumarina próximos, sugerindo que estas podem ser utilizadas de forma indistinta.

Adulterations of plant raw materials often occur in the marketing of herbal medicine, usually in the form of substitutions and/ or fakes. Many times, different plant species are commercialized in the place of the one recognized by the pharmacopoeia. This may occur due to the difficulties of obtaining the authentic plant material, or by an intentional use of a plant species that has less economic value but shows similar morphologic features. Therefore, it is necessary to carry out studies that can identify the chemical composition of those species of wide medicinal use, which are the targets of adulterations. Mikania glomerata and Mikania laevigata, popularly known as "guaco", are indistinctly commercialized in Brazil. A phytochemical screening by TLC was carried out to evaluate the chemical composition of both species. The RP-HPLC profiles were obtained and the quantification of coumarin was determined by the same method. A phytochemical screening has shown that coumarin, triterpenes/ steroids and flavonic heterosides are the constituents detected in both species. The RP-HPLC profiles were similar between the two species and revealed the predominance of polar compounds. In the quantification of coumarin, a content of 0.30 percent (w/w) was obtained for M. glomerata and 0.43 percent (w/w) for M. laevigata. These values are in accord with the contents (min. 0.1 percent, w/w) described by the monograph of "guaco-cheiroso" in the Brazilian Pharmacopoeia. Therefore, the results indicated similar chemical profiles for M. glomerata and M. laevigata, as well as comparable coumarin contents, thus suggesting that the species might be used indistinctly.

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Os extratos etanólicos de folhas de guaco (Mikania laevigata) cultivado tradicionalmente no solo ou por hidroponia foram avaliados quanto às atividades alelopática e antimicrobiana. Para a atividade alelopática foi utilizado o ensaio de inibição da germinação de sementes de alface (Lactuca sativa), enquanto que para a atividade antimicrobiana utilizouse a técnica de difusão em disco. Observou-se um notável efeito alelopático dos extratos de ambos os cultivos, em especial do extrato etanólico do guaco tradicional, que mesmo na menor concentração testada inibiu completamente a germinação das sementes. Nenhum dos extratos etanólicos do guaco apresentou atividade antibacteriana significativa para as linhagens de Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis e Enterococcus faecium.

The ethanolic extract of "guaco" (Mikania laevigata) traditionally cultivated in the soil or in a hydroponic system were tested for allelopathic and antimicrobial activities. Allelopathic activity was evaluated by the inhibition of germination assay using lettuce seeds, and antimicrobial activity by the disc diffusion assay. A notable allelopathic effect was observed for both extracts, although a more expressive activity of traditional "guaco" was verified, since the inhibition of seeds germination was 100% even in the lower concentration. None ethanolic extract of "guaco" presented significant antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis and Enterococcus faecium.

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The Mikania genus is widely known as "guaco" and is used to treat fever, rheumatism, flu and respiratory diseases. Our previous work evidenced the synergism among M. laevigata extract components to produce desirable effects, and included the coumarin precursor, o-coumaric acid as marker. Many Mikania species are producers of ent-kaurene diterpenes which presented antiespasmodic and relaxant activities on smooth muscle. Seeking to standardize the guaco extract, which is registered in the Brazilian Pharmacopoea, this paper deals with the determination of kaurenoic acid through LC-PDA and the isolation through LC of syringaldehyde. Kaurenoic acid was not found in the extract, and syringaldehyde is one of the major compounds of pharmacopoeal extract, together with coumarin and o-coumaric acid. Samples from the lung and liver of Balb-C isogenic allergic pneumonitis bearing mice, treated with the same extract, were analyzed through GC-FID, and the fatty acid content was determined and analyzed. The results obtained by measuring the arachidonic acid (ARA) and docosahexaenoic acid (DHA) in the liver and lung of treated animals demonstrated that the fatty acid composition is distinct in both tissues, and that in the liver, only the DHA was altered as a result of the treatments. DHA is absent in the lung and in both organs, no significant difference in ARA production was observed. The aqueous extract, coumarin and o-coumaric acid stimulated DHA synthesis in the liver (p < 0.05).

O gênero Mikania é popularmente conhecido como "guaco" e é utilizado para tratar febre, reumatismo, resfriados e afecções respiratórias. Em trabalho prévio demonstramos sinergismo entre os componentes do extrato de M. laevigata para produzir os efeitos farmacológicos esperados, incluindo a cumarina e seu precursor ácido o-cumárico como marcadores. Muitas espécies de Mikania são produtoras de diterpenos ent-caurenos que apresentam atividade antispasmódica e relaxante da musculatura lisa. Buscando a padronização do extrato medicinal de guaco (preparado segundo a farmacopéia brasileira 1ª edição), este trabalho visou determinar a presença de ácido caurenóico através de CLAE-DAD e isolou siringaldeído através de CLAE semipreparativa, sendo que o primeiro não foi encontrado no extrato e o siringaldeído é um dos seus componentes majoritários. Camundongos isogênicos Balb-C portadores de pneumonite alérgica foram tratados com este extrato, e amostras de pulmão e fígado foram analisadas por CG-DIC quanto ao seu conteúdo de ácidos graxos. A quantidade de ácido araquidônico (ARA) e de ácido docosahexaenóico (DHA) encontrada demonstrou que a composição é distinta em ambos tecidos, e apenas a concentração de DHA hepático foi alterado em função do tratamento, o qual não foi encontrado no pulmão. Não foi detectada diferença significativa na produção de ARA. Tanto o extrato aquoso, quanto a cumarina e o ácido o-cumárico, estimularam a síntese de DHA no fígado (p < 0.05).

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