Advances in the Biosynthesis of Pyranocoumarins: Isolation and 13C-Incorporation Analysis by High-Performance Liquid Chromatography-Ultraviolet-Solid-Phase Extraction-Nuclear Magnetic Resonance Data.

Citrus sinensis and Citrus limonia were obtained by germination from seeds, and isotopic-labeling experiments using d-[1-13C]glucose were performed with the seedlings. After 60 days, the seedlings were analyzed by high-performance liquid chromatography-ultraviolet-solid-phase extraction-nuclear magnetic resonance, data and the 13C enrichment patterns of xanthyletin and seselin indicated that the pyran ring was formed by the methylerythritol phosphate pathway and that the coumarin moiety was derived from the shikimate pathway in both compounds. This information regarding the biosynthetic pathway can be used to increase resistance against phytopathogens, because xanthyletin and seselin are reported to have antimicrobial activity on the growth of Xylella fastidiosa, which causes citrus variegated chlorosis in orange.

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.

Lippia alba (Mill.) N. E. Brown hydroethanolic extract of the leaves is effective in the treatment of migraine in women.

BACKGROUND: There is no universally accepted and effective prophylaxis of migraine headache episodes. We aimed to investigate the effects of Lippia alba (Mill.) N. E. Brown (chemotype geranial-carvenone) in the treatment of migraine patients. METHODS: Twenty-one women were enrolled in a prospective, phase 2, non-controlled cohort study to orally receive hydroethanolic extract of L. alba leaves. Symptom intensity and impact were assessed using MIDAS and HIT-6 instruments before and after 2 months of treatment. RESULTS: We observed a significant decrease on HIT-6 total score, as well as MIDAS number of missed days, number of headache days, and pain intensity. More than 70% of patients experienced a minimum 50% reduction on pain frequency. CONCLUSIONS: Treatment with L. alba leaves (geranial-carvenone) hydroethanolic extract is a cheap, safe, widely available, highly effective therapy to reduce both the intensity and the frequency of headache episodes of women with migraine. ClinicalTrials.gov Identifier: NCT01259947.

Chemical composition and therapeutic effects of Lippia alba (Mill.) N. E. Brown leaves hydro-alcoholic extract in patients with migraine.

BACKGROUND: There is no universally accepted and effective prophylaxis of migraine headache episodes. Thus we aimed to investigate the effects of Lippia alba (Mill.) N. E. Brown, an herb with many effects on central nervous system, on pain frequency and intensity of migraine patients. METHODS: Patients were enrolled in a prospective, phase 2, non-controlled cohort study to orally receive hydro-alcoholic extract of L. alba leaves. Headache intensity and frequency of episodes were recorded before and after 30-60 days of treatment. We also studied the chemical composition of its essential oil by gas chromatography-mass spectrometry. RESULTS: We described for the first time a particular L. alba chemotype with geranial and carvenone as major compounds. With treatment, both frequency and intensity of pain episodes significantly decreased from baseline to first reassessment date. More than 80% of patients experienced a minimum 50% reduction on pain intensity and frequency. No side effects were reported. CONCLUSIONS: Treatment with a geranial plus carvenone chemotype of L. alba hydro-alcoholic extract is a cheap, widely available, highly effective therapy to reduce both the intensity and the frequency of headache episodes of migraine patients with no side effects.

Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers.

Jacaranda decurrens (Bignoniaceae) is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2%) polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3% of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA) using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA) and Pearson's correlation coefficient (RAPD -0.16, p = 0.2082; AFLP 0.37, p = 0.1006) between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado.

Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers

Jacaranda decurrens (Bignoniaceae) is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2 percent) polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3 percent of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA) using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA) and Pearson's correlation coefficient (RAPD -0.16, p = 0.2082; AFLP 0.37, p = 0.1006) between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado.

Determination of lead content in medicinal plants by pre-concentration flow injection analysis-flame atomic absorption spectrometry.

INTRODUCTION: Although medicinal plants are widely used throughout the world, few studies have been carried out concerning the levels of heavy metal contaminants present. Such metals are highly toxic to living organisms even in low concentrations owing to their cumulative effect. The present paper describes the the development of a pre-concentration flow injection analysis-flame atomic absorption spectrometric system to determine the lead content in medicinal plants at the ppb level. OBJECTIVE: To develop a pre-concentration flow injection analysis-flame atomic absorption spectrometric system to determine the lead content in medicinal plants at the ppb level. METHODOLOGY: A pre-concentration flow system was coupled to a flame atomic absorption spectrometer. The plant samples were analysed after nitroperchloric digestion. The proposed system was optimised by evaluating the following parameters: nature, concentration and volume of the eluent solution, elution flow rate, elution efficiency, pre-concentration flow rate and pre-concentration time. RESULTS: The proposed system exhibited good performance with high precision and repeatability (RSD < or = 2.36%), excellent linearity (r = 0.9999), low sample consumption (10.5 mL per determination) and an analytical throughput of 55 samples/h. Lead concentrations ranged from 3.37 + or - 0.25 to 7.03 + or - 0.51 microg/g in dry material. This concentration interval is greater than that previously published in the literature. CONCLUSION: The inclusion of a pre-concentration column in the flow manifold improved the sensitivity of the spectrometer. Thus, it was possible to determine the analyte at the ng/mL level in sample solutions of medicinal plants. This is a very important accomplishment, especially when the cumulative effect of heavy metals in living organisms is considered.

The role of polar phytocomplexes on anticonvulsant effects of leaf extracts of Lippia alba (Mill.) N.E. Brown chemotypes.

OBJECTIVES: The purpose of the present work was to characterize the pharmacological profile of different L. alba chemotypes and to correlate the obtained data to the presence of chemical constituents detected by phytochemical analysis. METHODS: Essential oils from each L. alba chemotype (LP1-LP7) were characterized by gas chromatography-mass spectrometry (GC-MS) and extracted non-volatile compounds were analysed by HPLC and GC-MS. The anticonvulsant actions of the extracted compounds were studied in pentylenetetrazole-induced clonic seizures in mice and their effect on motor coordination was studied using the rota-rod test in rats. The synaptosomes and synaptic membranes of the rats were examined for the influence of LP3 chemotype extract on GABA uptake and binding experiments. KEY FINDINGS: Behavioural parameters encompassed by the pentylenetetrazole test indicated that 80% ethanolic extracts of LP1, LP3 and LP6 L. alba chemotypes were more effective as anticonvulsant agents. Neurochemical assays using synaptosomes and synaptic membranes showed that L. alba LP3 chemotype 80% ethanolic extract inhibited GABA uptake and GABA binding in a dose-dependent manner. HPLC analysis showed that LP1, LP3 and LP6 80% ethanolic extracts presented a similar profile of constituents, differing from those seen in LP2, LP4, LP5 and LP7 80% ethanolic extracts, which exhibited no anticonvulsant effect. GC-MS analysis indicated the occurrence of phenylpropanoids in methanolic fractions obtained from LP1, LP3 and LP6 80% ethanolic extracts and also the accumulation of inositol and flavonoids in hydroalcoholic fractions. CONCLUSIONS: Our results suggest that the anticonvulsant properties shown by L. alba might be correlated to the presence of a complex of non-volatile substances (phenylpropanoids, flavonoids and/or inositols), and also to the volatile terpenoids (beta-myrcene, citral, limonene and carvone), which have been previously validated as anticonvulsants.

Variabilidade sazonal de constituintes químicos (triterpenos, flavonóides e polifenóis) das folhas de Maytenus aquifolium Mart. (Celastraceae) / Seasonal variability of chemical compounds (triterpenes, flavonoids and polyphenols) from Maytenus aquifolium Mart (Celastraceae) leaves

Estudou-se a variabilidade química sazonal em Maytenus aquifolium Mart. (Celastraceae), conhecida como "espinheira santa", em exemplares cultivados em Ribeirão Preto, SP, através da análise das substâncias presentes nas folhas, ao longo das quatro estações em dois anos. O teor de fenóis totais variou de 21,96 a 45,92 mg / g (expresso em mg ácido tânico / g folhas secas), de 55,88 a 93,20 mg triterpenos totais / g folhas secas, e de 1,349 a 3,859 mg flavonóides / g folhas secas. A amostra Primavera 92 apresentou o maior teor de flavonóides e fenóis totais, e a amostra Inverno 94 apresentou o maior teor de triterpenos.

The chemical seasonal variability of Maytenus aquifolium Mart. (Celastraceae), a Brazilian medicinal plant known as "espinheira santa", was investigated on specimens cultivated at Ribeirão Preto SP, Brazil, at the four seasons of two years. The content of total phenolics, flavonoids and triterpenes varied from 21.96 to 45.92 mg total phenolics (expressed as tanic acid) / g dried leaves, 55.88 - 93.20 mg total triterpenes / g dried leaves, and 1.349 - 3.859 mg flavonoid / g dried leaves. Sample Spring 92 had the higher content of flavonoids and total phenolics, while sample Winter 94 had the highest content of triterpenes.