A report on identification of sequence polymorphism in barcode region of six commercially important Cymbopogon species.

CYMBOPOGON: is an important member of grass family Poaceae, cultivated for essential oils which have greater medicinal and industrial value. Taxonomic identification of Cymbopogon species is determined mainly by morphological markers, odour of essential oils and concentration of bioactive compounds present in the oil matrices which are highly influenced by environment. Authenticated molecular marker based taxonomical identification is also lacking in the genus; hence effort was made to evaluate potential DNA barcode loci in six commercially important Cymbopogon species for their individual discrimination and authentication at the species level. Four widely used DNA barcoding regions viz., ITS 1 & ITS 2 spacers, matK, psbA-trnH and rbcL were taken for the study. Gene sequences of the same or related genera of the concerned loci were mined from NCBI domain and primers were designed and validated for barcode loci amplification. Out of the four loci studied, sequences from matK and ITS spacer loci revealed 0.46% and 5.64% nucleotide sequence diversity, respectively whereas the other two loci i.e., psbA-trnH and rbcL showed 100% sequence homology. The newly developed primers can be used for barcode loci amplification in the genus Cymbopogon. The identified Single Nucleotide Polymorphisms from the studied sequences may be used as barcodes for the six Cymbopogon species. The information generated can also be utilized for barcode development of the genus by including more number of Cymbopgon species in future.

Efeito moluscicida do óleo essencial de Cymbopogon winterianus Jowitt (Poaceae) sobre Lymnaea columella (Say, 1817) e Biomphalaria tenagophila (D'Orbigny, 1835) / Molluscicidal effect of essential oil of Cymbopogon winterianus Jowitt (Poaceae) on Lymnaea columella (Say, 1817) and Biomphalaria tenagophila (D'Orbigny, 1835)

RESUMO O uso das substâncias moluscicidas convencionais no controle de planorbídeos vetores constitui-se uma importante ferramenta no combate da fasciolose hepática e esquistossomose. Sendo, portanto, de extrema relevância para a pecuária e para os serviços de Vigilância Epidemiológica. Por outro lado, a seleção de caramujos resistentes a tais substâncias e sua baixa seletividade estimulam a busca por novas substâncias. Neste sentido, o presente trabalho foi desenvolvido para avaliar o efeito do óleo essencial de Cymbopogon winterianusJowitt sobre Lymnaea columella e Biomphalaria tenagophila, hospedeiros intermediários de Fasciola hepatica e esquistossomose mansônica, respectivamente. O óleo essencial foi extraído a partir de folhas frescas utilizando o sistema Clevenger. A análise qualitativa foi realizada por meio de cromatografia gasosa acoplada a espectrometria de massas (CG/EM) e a quantificação dos constituintes presentes no óleo foi determinada por cromatografia gasosa acoplada ao detector de ionização de chama (CG/DIC). O efeito moluscicida foi avaliado utilizando seis moluscos de cada espécie e o óleo essencial de C. winterianus nas concentrações finais de 10, 20, 30, 40, 60, 80 e 100 ppm. A análise por cromatografia gasosa do óleo essencial possibilitou a identificação dos componentes majoritários geraniol (28,62%), citronelal (23,62%) e citronelol (17,10%). Os valores de DL100 e DL50para os moluscos das espécies L. columella e B. tenagophila foram, respectivamente, 60 e 40 ppm; 80 ppm e 60 ppm. O óleo de Cymbopogon winterianus demonstrou-se uma alternativa promissora para o controle dos moluscos, sendo a espécie L. columella mais sensível ao mesmo.

ABSTRACT Conventional molluscicides have been employed to control of planorbids vectors and are an important tool in order to control the hepatic fascioliasis and schistosomiasis. Thus, these substances have been showinggreat relevance for both Veterinary and Livestock Services as well as for Epidemiology and Disease Surveillance. On the other hand, the process of drug pressure for the selection of resistant snails to such components and their low selectivity have stimulated the search for new substances. Since researches on new drugs are the starting point to assist on themolluscs control, this work was developed in order to evaluate the effect of Cymbopogon winterianus Jowitt essential oil on L. columella and B. tenagophila, intermediate hosts of Fasciola hepatica and Schistosoma mansoni, respectively. The essential oil was obtained from fresh leaves by hydrodistillation using a Clevenger apparatus. A qualitative analysis was performed by gas chromatography together with a mass spectrometry one (GC/MS) and the chemical constituent content was determined by gas chromatography with a flame ionization detector (GC/FID). The molluscicidal effect was evaluated through the use of six snails of each species and C. winterianus essential oil at 10, 20, 30, 40, 60, 80 and 100 ppm. The result of the gas chromatographic analysis for the essential oil showed geraniol (28.62%), citronellal (23.62%) and citronellol (17.10%) as the major chemical components . The DL100 and DL50 values for L. columella and B. tenagophila species were, respectively, 60 and 40 ppm; 80 ppm and 60 ppm. L. columella had demonstrated more sensitivity to this essential oil than theB. tenagophila species. The C. winterianus essential oil proved to be a promising alternative for the control of these molluscs being the L. columella species the most sensitive of them.

Atividade fungitóxica in vitro dos óleos essenciais de Lippia sidoides Cham., Cymbopogon citratus(D. C. ) Stapf. e de seus constituintes majoritários no controle de Rhizoctonia solani e Sclerotium rolfsii / Fungitoxicity in vitro of essential oils from Lippia sidoides Cham., Cymbopogon citratus (D. C. ) Stapf. and their major constituents in the control of Rhizoctonia solani and Sclerotium rolfsii

RESUMO O objetivo deste estudo foi avaliar o potencial fungitóxicos dos óleos essenciais de Cymbopogon citratus, Lippia sidoides, e de seus constituintes majoritários, sobre o crescimento micelial dos fitopatógenos Rhizoctonia solani e Sclerotium rolfsii. A caracterização química do óleo de L. sidoides demonstrou a presença do carvacrol (33,27%) e o 1,8-cineol (24,41%) como seus componentes majoritários. Enquanto que o citral (77,6%) foi o constituinte majoritário do óleo essencial de C. citratus. A avaliação do potencial fungitóxico dos óleos essenciais e de seus constituintes majoritários foi realizada por meio de ensaios in vitro, avaliando a inibição do crescimento micelial dos microrganismos. Ambos os óleos essenciais inibiram totalmente o crescimento micelial de R. solani na concentração de 400 µg mL-1. O crescimento micelial de S. rolfsii foi inibido pelo óleo essencial de C. citratus na concentração de 300 µg mL-1 e pelo óleo essencial de L. sidoides na concentração de 400 µg mL-1. Em relação aos constituintes majoritários, o 1,8-cineol não apresentou efeito fungitóxico nas concentrações avaliadas. No entanto, o carvacrol e o citral foram mais efetivos que os óleos essenciais havendo ausência de crescimento micelial de R. solani e de S. rolfsii nas concentrações de 200 µg mL-1 e 225 µg mL-1, respectivamente.

ABSTRACT The objective of this study was to evaluate the fungitoxic potentials of the essential oils of Cymbopogon citratus, Lippia sidoides, and of its major constituents, on the mycelial growth of phytopathogens Rhizoctonia solani and Sclerotium rolfsii. The chemical characterization of L. sidoides oil showed the presence of carvacrol (33.27%) and of 1,8-cineole (24.41%) as its major components, whereas citral (77.6%) was the major constituent of C. citratus essential oil. The evaluation of the fungitoxic potential of the essential oils and of its major constituents was performed through in vitro assays, the microorganisms mycelial growth inhibition. Both essential oils totally inhibited the mycelial growth of R. solani at 400 µg mL-1. Regarding the major constituents, the 1,8-cineole did not show fungitoxic effect at the concentrations evaluated. However, the carvacrol and the citral were more effective than the essential oils and there was no mycelial growth of R. solani and of S. rolfsii at the concentrations of 200 µg mL-1 and 225 µg mL-1, respectively.

Chemical composition, cytotoxicity and in vitro antitrypanosomal and antiplasmodial activity of the essential oils of four Cymbopogon species from Benin.

ETHNOPHARMACOLOGICAL RELEVANCE: Cymbopogon species are largely used in folk medicine for the treatment of many diseases some of which related to parasitical diseases as fevers and headaches. As part of our research on antiparasitic essential oils from Beninese plants, we decided to evaluate the in vitro antiplasmodial and antitrypanosomal activities of essential oils of four Cymbopogon species used in traditional medicine as well as their cytotoxicity. MATERIALS AND METHODS: The essential oils of four Cymbopogon species Cymbopogon citratus (I), Cymbopogon giganteus (II), Cymbopogon nardus (III) and Cymbopogon schoenantus (IV) from Benin obtained by hydrodistillation were analysed by GC/MS and GC/FID and were tested in vitro against Trypanosoma brucei brucei and Plasmodium falciparum respectively for antitrypanosomal and antiplasmodial activities. Cytotoxicity was evaluated in vitro against Chinese Hamster Ovary (CHO) cells and the human non cancer fibroblast cell line (WI38) through MTT assay to evaluate the selectivity. RESULTS: All tested oils showed a strong antitrypanosomal activity with a good selectivity. Sample II was the most active against Trypanosoma brucei brucei and could be considered as a good candidate. It was less active against Plasmodium falciparum. Samples II, III and IV had low or no cytotoxicity, but the essential oil of Cymbopogon citratus (I), was toxic against CHO cells and moderately toxic against WI38 cells and needs further toxicological studies. Sample I (29 compounds) was characterised by the presence as main constituents of geranial, neral, ß-pinene and cis-geraniol; sample II (53 compounds) by trans-p-mentha-1(7),8-dien-2-ol, trans-carveol, trans-p-mentha-2,8-dienol, cis-p-mentha-2,8-dienol, cis-p-mentha-1(7),8-dien-2-ol, limonene, cis-carveol and cis-carvone; sample III (28 compounds) by ß-citronellal, nerol, ß-citronellol, elemol and limonene and sample IV (41 compounds) by piperitone, (+)-2-carene, limonene, elemol and ß-eudesmol. CONCLUSIONS: Our study shows that essential oils of Cymbopogon genus can be a good source of antitrypanosomal agents. This is the first report on the activity of these essential oils against Trypanosoma brucei brucei, Plasmodium falciparum and analysis of their cytotoxicity.

Efeito do óleo de capim limão (Cymbopogon flexuosus Stapf) no controle do carrapato dos bovinos / Effect of lemongrass (Cymbopogon flexuosus Stapf) oil on the control of cattle ticks

Esta pesquisa foi conduzida com o objetivo de avaliar o efeito in vitro e in vivo do óleo essencial de capim limão (Cymbopogon flexuosus Stapf) sobre o carrapato dos bovinos [Rhipicephalus (Boophilus) microplus]. Na experimentação in vitro foi utilizado o grupo controle negativo e oito concentrações do óleo de capim-limão (0,5; 1; 2; 5; 10; 20; 50; 100%), em fêmeas ingurgitadas do carrapato. A eficácia de controle foi de 0; 20; 39; 99,5; 100; 100; 100; 100 e 100%, respectivamente. Para a experimentação in vivo foram constituídos três grupos (controle negativo, óleo de capim limão a 2,0% - nível estimado mediante análise de regressão, correspondendo a 95% de eficácia de controle do carrapato da pesquisa in vitro e amitraz a 0,025%), com dezoito vacas da raça Holandesa. Antes (média dos dias -3, -2, -1) e após a aplicação do produto (1, 2, 3, 5, 7, 10, 14 e 21 dias), foram contadas fêmeas ingurgitadas do carrapato. A eficácia de controle foi de 0; 54 e 74,5%, respectivamente, 21dias após o tratamento. Após a aplicação dos tratamentos (controle negativo x tratamento fitoterápico), foram avaliadas as variáveis fisiológicas: frequência cardíaca, respiratória, temperatura do globo ocular e temperatura da pele; os resultados foram similares entre os tratamentos.

This research was aimed at evaluating in vitro and in vivo effects of lemongrass (Cymbopogon flexuosus Stapf) oil on cattle ticks (Rhipicephalus (Boophilus) microplus). Negative control group and eight concentrations of lemongrass oil (0.5; 1; 2; 5; 10; 20; 50; 100%), were used on in vitro trials with engorged female ticks. The efficacy of control ticks was 0; 20; 39; 99.5; 100; 100; 100; 100 and 100%, respectively. For the in vivo trial, eighteen Holstein cows were allocated to three groups (negative control, lemongrass oil at 2.0% - level estimated by regression analysis, accounting for 95% efficacy of control ticks on in vitro trial, and amitraz at 0.025%). Engorged female ticks were counted before (mean of days -3, -2, -1) and after treatment (1, 2, 3, 5, 7, 10, 14 and 21 days). The efficacy of control ticks was 0; 54 and 74.5%, respectively, at 21 days after treatment. Physiologic variables were evaluated after treatments (negative control x phytotherapic treatment). Similar results were found between the treatments for physiological variables.

Bioatividade de extratos hidroalcoólicos de Cymbopogon citratus (DC. ) Stapf. sobre picão-preto (Bidens pilosa L.) e alface (Lactuca sativa L.) / Bioactivity of hydroalcoholic extracts of Cymbopogon citratus (DC. ) Stapf. on germination and early growth of beggartick (Bidens pilosa L.) and lettuce (Lactuca sativa L.)

Este trabalho visou avaliar a bioatividade de extratos hidroalcoólicos de capim-limão sobre germinação e crescimento inicial da planta daninha picão-preto (Bidens pilosa L.) e da planta teste alface (Lactuca sativa L.). A partir de maceração hidroalcoólica de folhas secas e rasuradas (127,46 g) de capim-limão em 1L de álcool de cereais (70%), foram preparados os tratamentos como extratos hidroalcoólicos (HA) pela diluição (v/v) do macerado filtrado em água deionizada na proporção 1:1 (HA1:1) e 1:2 (HA1:2); dos quais foram obtidos os respectivos extratos secos (ES), a partir da evaporação da fase líquida de duas alíquotas de 50 mL de cada extrato HA, que após re-suspendidas em igual volume de água, sendo uma autoclavada (1 atm por 15 minutos), resultando nos extratos secos de HA1:1 e HA1:2 autoclavados (ES1:1A e ES1:2A) e nos extratos secos não autoclavados (ES1:1 e ES1:2); e água (AG) como controle. No primeiro dia dos bioensaios, foram aplicados 2 mL dos tratamentos, em DIC, com cinco repetições. Avaliou-se a porcentagem de germinação (G%), Índice de Velocidade de Germinação (IVG), altura da parte aérea (AA) e comprimento de radícula (CR) de aquênios (25) de picão-preto e de alface distribuídos em placas de Petri e mantidos em câmara de germinação (B.O.D.) por duas semanas. Os extratos HA1:1 e HA1:2 inibiram a G%, AA e CR das duas espécies. Todos os extratos secos reduziram a G%, IVG e o CR da alface. Para o picão preto, apenas o extrato ES1:1 reduziu a G% e o IVG foi reduzido por todos os extratos, a exceção do ES1:1A, mas nenhum extrato influenciou o crescimento inicial desta espécie.

This study aimed to evaluate the bioactivity of hydroalcoholic extract of lemongrass on the germination and early growth of the weed plant beggartick (Bidens pilosa L.) and the test plant lettuce (Lactuca sativa L.). From the hydroalcoholic maceration of dried and cut leaves of lemon grass (127.46 g) in 1 L grain alcohol (70%), the following treatments were prepared, hydro-alcoholic (HA) extracts by diluting (v/v) the macerate in deionized water at 1:1 (HA1:1) and 1:2 (HA1:2) proportions, obtaining the respective dried extracts (DE) from the evaporation of the liquid phase of two 50-mL aliquots of each HA extract; after resuspension in the same water volume, one aliquot was autoclaved (1 atm for 15 minutes) resulting in HA1:1 and HA1:2 dried autoclaved extracts (DE1:1A and DE1:2A) and in dried non-autoclaved extracts (ES1:1 and ES1:2), and water as control. On the first day of bioassays, 2mL of treatments were applied, in completely randomized design, with 5 replicates. Germination percentage (G%), Germination Speed Index (GSI), shoot height (SH) and radicle length (RL) of achenes (25) of beggartick and lettuce were evaluated after having been distributed in Petri dishes and kept in a germination chamber (B.O.D.) for two weeks. HA1:1 and HA1:2 extracts inhibited G%, SH and RL of both species. All dried extracts reduced G%, GSI and CR of lettuce. For beggartick, only ES1:1 extract reduced G% while GSI was reduced in all extracts, except ES1:1A, but no extract influenced the early growth of this species.

Controle da ferrugem das folhas do capim-limão [Cymbopogon citratus (DC. ) Stapf] com produtos naturais / Rust control in leaves of lemongrass [Cymbopogon citratus (DC. ) Stapf] by using natural products

Cymbopogon citratus (DC.) Stapf é uma planta medicinal pertencente a família Poaceae, perene, importante para a indústria farmacêutica e alimentícia devido ao óleo essencial que produz. Um problema limitante para a produção comercial é a ferrugem, doença cujo agente etiológico é o fungo Puccinia nakanishikii Dietel. O presente trabalho objetivou avaliar a ação de produtos naturais no controle da doença in vitro e in vivo. Para os testes in vitro, empregaram-se os seguintes produtos sobre a germinação de esporos do patógeno: óleos essenciais de eucalipto, palma-rosa, cravo-da-índia, menta, limão, citronela e mil-folhas; extrato pirolenhoso de eucalipto e mentol cristalizado, em diferentes concentrações. Para os testes de campo foram selecionados quatro produtos, o óleo de citronela e de eucalipto, o extrato pirolenhoso, e o mentol. In vitro, todos os agentes mostraram-se promissores no controle do patógeno. Quando em condições in vivo, sob alta severidade da doença no ato da avaliação, os produtos testados foram capazes de reduzir a severidade da doença em comparação com a testemunha. O controle da doença com os agentes naturais variou de 38% (extrato pirolenhoso) a 61%(óleo essencial de eucalipto).

Cymbopogon citratus (DC.) Stapf is a medicinal plant that belongs to the Poaceae family; it is perennial and important for the pharmaceutical and food industry due to the essential oil it produces. A limiting problem for commercial production is rust, a disease that has as etiological agent the fungus Puccinia nakanishikii Dietel. The present study aimed to evaluate the action of natural products on the disease control, in vitro and in vivo. For the in vitro tests, the following products were employed on the germination of the pathogen spores: essential oils from eucalyptus, palmarosa, clove, peppermint, lemon, citronella and yarrow, pyroligneous extract from eucalyptus, and crystallized menthol, at different concentrations. Four products were selected for the tests in the field: citronella and eucalyptus oil, pyroligneous extract and menthol. In vitro, all agents showed promising in controlling the pathogen. Under in vivo conditions and great severity of the disease at evaluation, the tested products were capable of reducing the disease severity when compared to the control. The disease control by the natural products varied from 38% (pyroligneous extract) to 61% (eucalyptus essential oil).

Development of simple sequence repeat markers in cymbopogon species.

The genus Cymbopogon comprises about 140 species, which produce characteristic aromatic essential oils. However, the phenotypic identification of species of Cymbopogon has been difficult as a result of widespread occurrence of natural variants, which differ in ploidy levels and chemotaxonomic complexities. Therefore, we have developed a set of simple sequence repeat markers from a genomic library of Cymbopogon jwarancusa to help in the precise identification of the species (including accessions) of Cymbopogon. For this purpose, we isolated 16 simple sequence repeat containing genomic deoxyribonucleic acid clones of C. jwarancusa, which contained a total of 32 simple sequence repeats with a range of 1 to 3 simple sequence repeats per clone. The majority (68.8%) of the 32 simple sequence repeats comprised dinucleotide repeat motifs followed by simple sequence repeats with trinucleotide (21.8%) and other higher order repeat motifs. Eighteen (81.8%) of the 22 designed primers for the above simple sequence repeats amplified products of expected sizes, when tried with genomic DNA of C. jwarancusa, the source species. Thirteen (72.2%) of the 18 functional primers detected polymorphism among the three species of Cymbopogon (C. flexuosus, C. pendulus and C. jwarancusa) and amplified a total of 95 alleles (range 1-18 alleles) with a PIC value of 0.44 to 0.96 per simple sequence repeat. Thus, the higher allelic range and high level of polymorphism demonstrated by the newly developed simple sequence repeat markers are likely to have many applications such as in improvement of essential oil quality by authentication of Cymbopogon species and varieties and mapping or tagging the genes controlling agronomically important traits of essential oils, which can further be utilized in marker assisted breeding.