Validation of reference genes for quantitative gene expression in the Lippia alba polyploid complex (Verbenaceae).

Lippia alba (Verbenaceae) is one of the most studied species of the genus Lippia, mainly due to its medicinal properties. The species was described as a polyploid complex with five cytotypes. The comparison of gene expression in species with several ploidal levels needs to be conducted carefully due to possible changes in gene regulation. Quantitative reverse transcription PCR (qRT-PCR) is a widely used method for transcript abundance analyses in plants. Besides being an extremely powerful technique, relative quantification by Real-Time quantitative PCR (RT-qPCR) needs the normalization with a stable reference gene. We evaluated the stability of nine candidate reference genes in Lippia alba with different ploidal levels using NormFinder, geNorm, and RefFinder software. The product of each primer showed a single peak in the melting curve. The R2 value ranged from 0.998 to 1000 and primers efficiency ranged from 98.95% to 129%. The CIT gene came up as a stable housekeeping gene, being appropriate for studies in polyploid accessions of Lippia alba. Considering that polyploidy is widely documented in Angiosperms, the results can be used not only for further gene expression studies in L. alba but also as a possible reference gene for other polyploid complexes. Differential stability among different genes highlights the importance of the validation of reference genes used for RT-qPCR approach in polyploid studies.

Genetic diversity and chemical variability of Lippia spp. (Verbenaceae).

BACKGROUND: The genus Lippia comprises 150 species, most of which have interesting medicinal properties. Lippia sidoides (syn. L. origanoides) exhibits strong antimicrobial activity and is included in the phytotherapy program implemented by the Brazilian Ministry of Health. Since species of Lippia are morphologically very similar, conventional taxonomic methods are sometimes insufficient for the unambiguous identification of plant material that is required for the production of certified phytomedicines. Therefore, genetic and chemical analysis with chemotype identification will contribute to a better characterization of Lippia species. METHODS: Amplified Length Polymorphism and Internal Transcribed Spacer molecular markers were applied to determine the plants' genetic variability, and the chemical variability of Lippia spp. was determined by essential oil composition. RESULTS: Amplified Length Polymorphism markers were efficient in demonstrating the intra and inter-specific genetic variability of the genus and in separating the species L. alba, L. lupulina and L. origanoides into distinct groups. Phylogenetic analysis using Amplified Length Polymorphism and markers produced similar results and confirmed that L. alba and L. lupulina shared a common ancestor that differ from L. origanoides. Carvacrol, endo-fenchol and thymol were the most relevant chemical descriptors. CONCLUSION: Based on the phylogenetic analysis it is proposed that L. grata should be grouped within L. origanoides due to its significant genetic similarity. Although Amplified Length Polymorphism and Internal Transcribed Spacer markers enabled the differentiation of individuals, the genotype selection for the production of certified phytomedicines must also consider the chemotype classification that reflects their real medicinal properties.

Antigenotoxic Effect Against Ultraviolet Radiation-induced DNA Damage of the Essential Oils from Lippia Species.

The antigenotoxicity against ultraviolet radiation (UV)-induced DNA damage of essential oils (EO) from Lippia species was studied using SOS Chromotest. Based on the minimum concentration that significantly inhibits genotoxicity, the genoprotective potential of EO from highest to lowest was Lippia graveolens, thymol-RC ≈ Lippia origanoides, carvacrol-RC ≈ L. origanoides, thymol-RC > Lippia alba, citral-RC ≈ Lippia citriodora, citral-RC ≈ Lippia micromera, thymol-RC > L. alba, myrcenone-RC. EO from L. alba, carvone/limonene-RC, L. origanoides, α-phellandrene-RC and L. dulcis, trans-ß-caryophyllene-RC did not reduce the UV genotoxicity at any of the doses tested. A gas chromatography with flame ionization detection analysis (GC-FID) was conducted to evaluate the solubility of the major EO constituents under our experimental conditions. GC-FID analysis showed that, at least partially, major EO constituents were water-soluble and therefore, they were related with the antigenotoxicity detected for EO. Constituents such as p-cymene, geraniol, carvacrol, thymol, citral and 1,8-cineole showed antigenotoxicity. The antioxidant activity of EO constituents was also determined using the oxygen radical antioxidant capacity (ORAC) assay. The results showed that the antigenotoxicity of the EO constituents was unconnected with their antioxidant activity. The antigenotoxicity to different constituent binary mixtures suggests that synergistic effects can occur in some of the studied EO.

Quimiotipos, Extracción, Composición y Aplicaciones del Aceite Esencial de Lippia alba / Quimiotipos, extração, composição e uso do óleo essencial de Lippia alba / Chemotypes, extraction, chemical composition and use of lippia alba essential oil

RESUMO Lippia alba é uma planta amplamente distribuída nas zonas tropicais, subtropicais e temperadas das Américas, África e Ásia. O óleo essencial de L. alba tem sido amplamente estudado, entretanto apresenta variações de produção. Portanto este estudo teve como objetivo realizar uma revisão dos principais quimiotipos, métodos de extração, composição e aplicação do óleo essencial de L. alba. Neste estudo são discutidos os principais quimiotipos e sua relação com fatores genéticos e características morfológicas. Também são discutidos os fatores que afetam o rendimento de produção, composição química, métodos de extração e do uso e da atividade biológica do óleo essencial de L. alba. Apesar da vasta literatura sobre os óleos essenciais de L. alba, ainda desenvolvimento de aplicações para a produção de cosméticos, fármacos e alimentos, bem como faltam definições agronomicas sobre o cultivo e melhoramento desta planta.

ABSTRACT Lippia alba is a plant widely distributed in tropical, subtropical and temperate zones of the Americas, Africa and Asia. The essential oil of L. alba has been widely studied and there are many variations in the production process. Therefore, this study is aimed at conducting a review of the main chemotypes, extraction methods, composition and application of the essential oil of L. alba. In this study, the main chemotypes and its relation to genetic and morphological characteristics are discussed. It also discusses the factors that affect the yield, chemical composition, extraction methods and the use and the biological activity of the essential oil of L. alba. Despite the vast literature on the essential oils of L. alba, there is still a lack of development in its application for the production of cosmetics, pharmaceuticals and food, as well as a lack of agronomic definitions for its cultivation and genetic improvement.

Potencial do óleo essencial de erva-luísa (Aloysia citriodora Palau) no controle de Fusarium sp. in vitro / Essential oil potential herb-luisa (Aloysia citriodora Palau) in the control of Fusarium sp. in vitro

RESUMO O presente trabalho teve como objetivo principal avaliar o potencial do uso do óleo essencial de Aloysia citriodora no controle in vitro de Fusarium sp., isolado de plântulas de beterraba infectadas com o mesmo. O trabalho foi realizado por meio de dois experimentos: um sobre o efeito do óleo essencial no crescimento micelial, e outro sobre o efeito na germinação de conídios do fungo. No primeiro trabalho, avaliou-se em delineamento inteiramente ao acaso, o efeito das concentrações 0,0155%; 0,0315%; 0,0625%; 0,125%; 0,250% e 0,500% do óleo essencial de A. citriodora em placas de Petri® com meio de cultivo BDA, mais a testemunha, com meio BDA puro. Cada placa foi considerada uma repetição, as quais foram incubadas a 24ºC±1ºC e submetidas a fotoperíodo de doze horas. Avaliou-se o crescimento radial do patógeno em função do crescimento micelial do patógeno quando este atingia as bordas da primeira placa. No segundo experimento, as mesmas concentrações foram testadas, nas mesmas condições de incubação, no entanto, em lâminas de microscopia com meio BD. Utilizou-se delineamento inteiramente ao acaso, sendo considerada como unidade experimental cada lâmina utilizada. Avaliou-se 24 horas após a incubação, em microscópio óptico, a germinação dos primeiros vinte conídios visualizados a partir do canto esquerdo superior para o direito. Os resultados do segundo experimento foram expressos em porcentagem de germinação de conídios. Os resultados obtidos permitiram concluir que este óleo essencial possui efeito fungistático e fungicida sobre o crescimento micelial e na germinação de conídios de Fusarium sp.. Além disso este efeito é maior em função do aumento da concentração do óleo essencial.

ABSTRACT This study aimed to evaluate the potential use of Aloysia citriodora essential oilisolated from beet seedlings infected with it. The study was performed by conducting two experiments evaluating the effect of the essential oil on mycelial growth and fungus conidia germination. In the first study, in a completely randomized design, the effect concentrations (0.0155%; 0.0315%; 0.0625%; 0.125%; 0.250% and 0.500%) of essential oil of A. citriodorawas evaluated on Petri® dish with a PDA cultivation medium, plus the control, with half pure PDA. Each plate was taken as a repeat, and incubated at 24°C±1°C and a photoperiod of twelve hours. The radial growth of the pathogen, when the first plate was hit by the mycelial growth of the pathogen on its edges was evaluated. In the second experiment, the same concentrations were tested under the same incubationconditions, however, on microscope slides with half PD medium. The design was used completely randomized, each microscope slide used was considered as one experimental unit. Germination of the first 2 conidia strains, viewed from the upper left to the right was evaluated24 hours after incubation, using an optical microscope. The results of the second experiment were expressed as a percentage of conidia germination. The results obtained showed that this essential oil has fungistatic and fungicidal effect on the mycelial growth and at the conidia germination of Fusarium sp., which has a greater effect with increasing concentrations.

Análise fitoquímica e atividade antifúngica do óleo essencial de folhas de Lippia sidoides Cham. e do Timol contra cepas de Candida spp / Phytochemical analysis and antifungal activity of the essential oil of Lippia sidoides Cham. and of the Thymol against Candida strains

RESUMO A resistência de fungos do gênero Candida aos fármacos químicos tem lançado o desafio de se identificar novas substâncias que possuam atividade antibiótica ou venham a modular o efeito de produtos atualmente usados contra candidíase. O presente estudo avaliou a atividade antifúngica do óleo essencial de Lippia sidoides Cham. e do timol, sobre cepas de Candida. Inicialmente os produtos foram testados frente a 16 cepas fúngicas pela técnica de difusão em meio sólido, o que permitiu selecionar linhagens para continuidade da pesquisa. Com as linhagens de Candida krusei (CK LMBM 01, CK LMBM 02), Candida albicans (CA LM 62) e Candida tropicalis (CT LM 20), procedeu-se, por microdiluição em caldo, a determinação da Concentração Inibitória Mínima (CIM) e em meio sólido, a Concentração Fungicida Mínima (CFM) dos produtos foi identificada. O microcultivo das leveduras em meio empobrecido foi realizado para verificação de alterações morfológicas e, além disso, uma análise da composição química do óleo foi realizada por Cromatografia Gasosa acoplada à espectrometria de massas (CG-EM). Nesta análise, o constituinte majoritário foi o timol (84,95%), seguido de compostos como p-cimeno e Éter metil carvacrol, entre outros. A CIM do óleo essencial de Lippia sidoides Cham. frente às cepas variou entre 64 e 256 μg/mL, enquanto a CIM do timol foi estabelecida entre 32 e 64 μg/mL. A CFM do óleo essencial foi determinada entre 128 e 512 μg/mL e para o timol foram encontrados valores entre 64 e 128 μg/mL. Em relação à análise micromorfológica, verificada nas concentrações de CIM e CIM x 2, o óleo essencial inibiu o dimorfismo das cepas CK 01 e CT 20 na CIM e quando foi ensaiado o timol, este, na CIM, impediu a transição morfológica das cepas CK 01 e CA 62. Uma redução da morfogênese também foi obsevada na cepa CT 20, porém apenas em CIM x 2 e de forma mais discreta. Os resultados enaltecem o potencial antifúngico de L. sidoides e de seu composto majoritário timol tanto no combate à Candida quanto na neutralização de um dos fatores de virulência, a capacidade invasiva por formação de hifas e pseudohifas verificado na condição patogênica da candidíase. Estes dados são promissores e poderão incentivar futuras pesquisas sobre os aspectos fitoquímicos, toxicológicos e farmacológicos tanto do óleo essencial de Lippia sidoides como também de seus componentes químicos.

ABSTRACT The resistance of the Candida against drugs has been a challenge to the discovery of new substances with antimicrobial or modulatory effects that could be used against the cadidiasis. This work evaluated the antifungal activity of the essential oil of Lippia sidoides Cham. and of the Thymol against Candida strains. The products were tested towards 16 strains of Candida using the diffusion method, which allowed to select the strains in order to proceed with the research. The strains of Candida krusei (CK LMBM 01, CK LMBM 02), Candida albicans (CA LM 62) and Candida tropicalis (CT LM 20) were assayed by the microdilution method so that the Minimal Inhibitory Concentration (MIC) and the Minimal Fungicide Concentration (MFC) could be determined. The morphogenesis of the Candida was evaluated using poor environment in order to observe morphological changes. The composition of the essential oil was determined by GC-MS. The main compound observed was the thymol (84.95%). The MIC of the essential oil of L. sidoides and Thymol ranged between 64 to 256 μg/mL, and between 32 to 64 μg/mL respectively. The MFC of the essential oil and the thymol varied between 128 to 512 μg/mL and 64 to 128 μg/mL respectively. The morphogenesis of different Candida strains was inhibited in the MIC and MICx2 to the essential oil and thymol. The results indicated the antifungal potential of the L. sidoides and of the Thymol due to the inhibition of the invasive capacity, one of the most important virulence factors for the candidiasis` development. These results are promising to new researches about the phytochemical, toxicological and pharmacological aspects of the essential oil of L. sidoides and its phytochemical compounds.

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.

Avaliação da atividade antibacteriana do óleo essencial de Lippia origanoides frente à Staphylococcus sp. isolados de alimentos de origem animal / Assessment of the antibacterial activity of the essential oil of Lippia origanoides in relation to Staphylococcus sp. isolated from food of animal origin

Objetivou-se no trabalho avaliar a ação antibacteriana do óleo essencial de Lippia origanoides frente à isolados de Staphylococcus sp. de alimentos de origem animal. Para tanto, realizou-se análise química da composição do óleo, teste de sensibilidade das bactérias frente a dez antibióticos de uso terapêutico e ao óleo essencial de Lippia origanoides, além da determinação da concentração inibitória mínima (CIM) e da concentração bactericida mínima (CBM). A análise cromatográfica do óleo apresentou o timol como composto majoritário (48,70%), além de pequena quantidade de carvacrol (1,14%). No teste de sensibilidade frente aos antibióticos, 75% dos isolados apresentaram resistência a, no mínimo, três antibióticos. Em relação ao óleo essencial, os isolados de Staphylococcus sp oriundos de leite bovino mostraram-se mais resistentes e os isolados de carne ovina apresentaram-se mais sensíveis. A CIM foi maior para os Staphylococcus sp. isolados de leite bovino e leite ovino (60µL/mL). Enquanto que para os isolados de carcaça de ovinos e queijo, a CIM foi de 15µL/mL e 30µL/mL respectivamente. A CBM, consequentemente, foi maior para os isolados de leite bovino e leite ovino, sendo de 240µL/mL e 120µL/mL respectivamente. O óleo essencial de Lippia origanoides apresenta atividade antimicrobiana sobre Staphylococcus sp. isolados de alimentos.

The aim of this study was to evaluate the antibacterial activity of the essential oil of Lippia origanoides in relation to Staphylococcus sp. isolated from food of animal origin. For this purpose, we performed the chemical analysis to determine the oil composition, evaluated the bacteria sensibility to ten antibiotics of therapeutic use and determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The chromatographic analysis showed that tymol was the main compound (48.70%) and that carvacrol was present in a small amount (1.14%). In the test of antibiotic susceptibility, 75% of the microorganism isolates were resistant to at least three of the ten antibiotics tested. The Staphylococcus sp isolated from bovine milk was more resistant to the essential oil of Lippia origanoides, and the microorganism isolated from sheep meat was more susceptible to this oil. The Staphylococcus sp. isolated from bovine or sheep milk showed a MIC of 60µL/mL and for those isolated from sheep carcass and cheese the MIC was 15µL/mL and 30µL/mL, respectively. Consequently, the MBC was higher for isolates from cow's (240µL/mL) and sheep's milk (120µL/mL) when compared with other food sources. These results suggest that the essential oil of Lippia origanoides has antimicrobial activity against staphylococcus sp. isolated from food.

Relationship between pollen morphology and chromosome numbers in Brazilian species of Lippia L. (Verbenaceae).

The pollen morphology and exine structure of 17 species of Lippia L. were investigated in this work using light and scanning electron microscopy. Among the species studied, 14 showed tricolporate pollen grains, two had tri- and tetracolporate pollen grains and a single species exhibited, only tetracolporate pollen. The amb ranged from triangular to square, and the shape varied from oblate-spheroidal to prolate-spheroidal. Three different types of exine ornamentation were observed: psilate, scabrate and perforate. In addition to morphological data, we found positive association between the chromosome numbers and size of pollen grains, and also between the length and width of the colpi. The results indicate that the characteristics of pollen grains in Lippia may be used as an additional taxonomic character of the genus.

Acaricidal activity of Lippia gracilis essential oil and its major constituents on the tick Rhipicephalus (Boophilus) microplus.

The present study aimed to evaluate the activity of Lippia gracilis Schauer essential oil obtained from different L. gracilis genotypes and their major components, carvacrol and thymol against Rhipicephalus (Boophilus) microplus (cattle tick) larvae and engorged females. The larval test was performed parallel to the adult immersion test for engorged females for four L. gracilis genotypes. Similar tests were further performed for their major compounds carvacrol and thymol. Carvacrol (LC50 of 0.22 and 4.46 mg/mL, to larvae and engorged females, respectively) was more efficient than thymol (LC50 of 3.86 and 5.50 mg/mL, to larvae and engorged females, respectively). The lethal concentrations obtained for the isolated essential oil from genotypes LGRA-201 against larvae (1.31 mg/mL) and LGRA-106 against engorged females (4.66 mg/mL) confirmed the acaricidal activity of L. gracilis essential oil and its effectiveness in controlling the southern cattle tick.

Karyological studies in Brazilian species of Lippia L. (Verbenaceae).

The genus Lippia (Verbenaceae) comprise around 160 species spread out mainly in South and Central Americas with few African species, some of them with potential medicinal use. Brazil is one the most important centers of diversity with approximately 75% of the species described so far. Innumerous species are endemic and poorly studied especially at a cytological level. Here, chromosomal length, karyomorphology and chromosome asymmetry of twelve Brazilian species of Lippia were evaluated [L. alba (Miller) N.E.Brown, L. diamantinensis Glaz., L. florida Cham., L. hermanioides Cham., L. lacunosa Mart. & Schauer, L. lupulina Cham., L. pohliana Schauer, L. pseudothea (St. Hil) Schauer, L. rosella Moldenke, L. rotundifolia Cham., L. rubella Moldenke and L. sidoides Cham.]. The analysis suggested that the genus has a variable chromosome number (from 2n = 20 to 2n = 56) originated by dysploidy and polyploidy. This is the first description of chromosome morphology for 11 of the 12 Lippia species studied.

Isolation and characteristics of eight novel polymorphic microsatellite loci in Lippia alba (Verbenaceae).

PREMISE OF THE STUDY: A set of eight microsatellite (simple sequence repeat [SSR]) markers for Lippia alba, an important medicinal and cosmetic plant, was developed to aid studies of genetic diversity and to define efficient strategies for breeding programs. METHODS AND RESULTS: Using a (CT)(8)- and (GT)(8)-enriched library, a total of 11 SSR loci were developed and optimized in L. alba. Of the 11 loci, eight were found to be polymorphic after screening 61 accessions from two populations. The parameters used to characterize loci were expected heterozygosity (H(e)) and number of alleles. A total of 44 alleles were identified, with an average of 5.5 alleles per loci, which were moderately to highly informative according to H(e). CONCLUSIONS: These new SSR markers have potential for informing genetic diversity, allele mining, and mapping studies and will be used to generate information for breeding programs of L. alba.

Karyotype analysis, DNA content and molecular screening in Lippia alba (Verbenaceae)

Cytogenetic analyses, of pollen viability, nuclear DNA content and RAPD markers were employed to study three chemotypes of Lippia alba (Mill.) (Verbenaceae) in order to understand the genetic variation among them. Different ploidy levels and mixoploid individuals were observed. This work comprises the first report of different chromosome numbers (cytotypes) in L. alba. The chromosome numbers of La2-carvone and La3-linalool chemotypes suggested that they are polyploids. Flow cytometric analysis showed an increase of nuclear DNA content that was not directly proportional to ploidy level variation. A cluster analysis based on RAPD markers revealed that La3-linalool shares genetic markers with La1-citral and La2-carvone. The analysis showed that the majority of genetic variation of La3-linalool could be a consequence of ixoploidy. ur data indicates that sexual reproduction aong those three chemotypes is unlikely and suggests the beginning of reproductive isolation. The results demonstrated that chromosome analysis, nuclear DNA content estimation and RAPD markers constitute excellent tools for detecting genetic variation among L. alba chemotypes.

Análises citogenéticas, de viabilidade do pólen, do conteúdo de DNA nuclear e marcadores RAPD foram empregadas no estudo de três quimiotipos de Lippia alba (Mill.) (Verbenaceae) visando contribuir para o entendimento da variação genética entre os mesmos. Diferentes níveis de ploidia e indivíduos mixoploides foram observados. Este trabalho compreende o primeiro relato de diferentes números cromossômicos (citótipos) em L. alba. Os números cromossômicos dos quimiotipos La2-carvona e La3-linalol sugere que eles seja poliploides. A análise da citometria de fluxo mostrou um aumento do conteúdo de DNA nuclear que não foi diretamente proporcional à variação no nível de ploidia. A análise de agrupamento baseada nos marcadores RAPD demonstrou que La3-linalol compartilha marcadores genéticos com La1-citral e La2-carvona. A análise mostrou que a maior parte da variação genética de La3-linalol pode ser consequência da mixoploidia. Nossos dados indicam que a reprodução sexual entre os três quimiotipos parece improvável, sugerindo o início de isolamento reprodutivo. Os resultados demonstraram que a análise cromossômica, a quantificação do DNA nuclear estimado e os marcadores RAPD constituem excelentes ferramentas para detecção de variação genética entre quimiotipos de L. alba.

Karyotype analysis, DNA content and molecular screening in Lippia alba (Verbenaceae).

Cytogenetic analyses, of pollen viability, nuclear DNA content and RAPD markers were employed to study three chemotypes of Lippia alba (Mill.) (Verbenaceae) in order to understand the genetic variation among them. Different ploidy levels and mixoploid individuals were observed. This work comprises the first report of different chromosome numbers (cytotypes) in L. alba. The chromosome numbers of La2-carvone and La3-linalool chemotypes suggested that they are polyploids. Flow cytometric analysis showed an increase of nuclear DNA content that was not directly proportional to ploidy level variation. A cluster analysis based on RAPD markers revealed that La3-linalool shares genetic markers with La1-citral and La2-carvone. The analysis showed that the majority of genetic variation of La3-linalool could be a consequence of ixoploidy. ur data indicates that sexual reproduction aong those three chemotypes is unlikely and suggests the beginning of reproductive isolation. The results demonstrated that chromosome analysis, nuclear DNA content estimation and RAPD markers constitute excellent tools for detecting genetic variation among L. alba chemotypes.

Lippia javanica (Burm F) Spreng: its general constituents and bioactivity on mosquitoes.

Mosquito repellent plants are used in the rural areas of Zimbabwe despite the fact that very few of them have been biologically evaluated. Leaves of the plant Lippia javanica, were collected from Mumurwi village, Zimbabwe and evaluated for repellency against laboratory reared Aedes aegypti mosquitoes. Major plant compounds were identified using Thin Layer Chromatography (TLC) and Mass Spectrometry (MS). Fraction 'A' contained coumarins, flavonoids and essential oils and offered a protection time of 8 and 5.5hrs in choice and non-choice experiments respectively. Fraction 'B1' contained flavonoids with a protection time of 1 and 0.5hrs in choice and non choice experiments respectively. Fraction 'B2' contained coumarins and essential oils and offered a protection time of 2hrs in either test. No major compounds were identified from the following fractions: 'C1', 'C2', 'D1', 'E1', 'E2', 'F1' and 'F2' and all of them failed to give 100% repellence. The 'C1' supernatant fraction contained coumarins and provided protection from mosquito bites for 1 and 0.5 hrs in choice and non-choice experiments respectively. Fraction 'C2' did not have the major compounds but gave a protection time of 1 hr in either experiment. Fraction 'D2' contained essential oils only and it provided a protection time of 2.5hrs in choice experiments. Analysis by MS showed the presence of alpha pinene, 1,3-5 cycloheptatriene, beta phellandrene, (+)-2-carene, 3-carene, eucalyptol and caryophyllene oxide. L. javanica offered protection from mosquito bites for 8hrs (choice) and 5.5hrs (non choice experiments). The combined presence of coumarins, flavonoids and essential oils have an additive effect compared with individual plant fractions.

Genetic diversity and spatial genetic structure within a population of an aromatic shrub, Lippia origanoides (Verbenaceae), in the Chicamocha Canyon, northeastern Colombia.

The geographical scale of genetic structure in a continuous population is highly dependent on its breeding system and dispersion capabilities, and this knowledge is important for the study of population dynamics as well as for conservation purposes. In the present study, spatial autocorrelation statistics and intersimple sequence repeat (ISSR) markers were used to describe the genetic structure of a natural population of a prominent aromatic plant, Lippia origanoides, native to the Chicamocha Canyon in northeastern Colombia. For this purpose, individuals were sampled from two localities within the Chicamocha Canyon, where the species is abundant and continuously distributed. Cluster (principal coordinate analysis (PCO) and unweighted pair group method using arithmetic averages (UPGMA)), analysis of molecular variance (AMOVA) and Bayesian analyses revealed a low level of genetic differentiation among the two localities, suggesting that they belong to a single population. Genetic diversity levels in this population, described as the percentage of polymorphic loci (P=86.21%) and quantified using Shannon's diversity index (I=0.453) and the average panmictic heterozygosity (HB=0.484), were shown to be comparable to or higher than that in other plant species with allogamous breeding systems and to other related Verbenaceae species. Fine-scale autocorrelation analyses showed a pattern consistent with the classical model of isolation by distance with moderate but significant levels of local spatial structure. Our results suggest that sampling individuals at distances greater than approximately 1.2 km may result in the collection of different genotypes, which could help preserve the levels of genetic diversity in a propagation programme. The causes of this spatial pattern are currently unknown and could be influenced by many contemporary factors such as restricted seed dispersal and/or short-distance pollen movement, among others.

[Oregano: properties, composition and biological activity]. / El orégano: propiedades, composición y actividad biológica de sus componentes.

The oregano spice includes various plant species. The most common are the genus Origanum, native of Europe, and the Lippia, native of Mexico. Among the species of Origanum. their most important components are the limonene, gamma-cariofilene, rho-cymenene, canfor, linalol, alpha-pinene, carvacrol and thymol. In the genus Lippia, the same compounds can be found. The oregano composition depends on the specie, climate, altitude, time of recollection and the stage of growth. Some of the properties of this plant's extracts are being currently studied due to the growing interest for substituting synthetic additives commonly found in foods. Oregano has a good antioxidant capacity and also presents antimicrobial activity against pathogenic microorganisms like Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, among others. These are all characteristics of interest for the food industry because they may enhance the safety and stability of foods. There are also some reports regarding the antimutagenic and anticarcinogenic effect of oregano; representing an alternative for the potential treatment and/or prevention of certain chronic ailments, like cancer.