Quantitative NMR analysis of L-Dopa in seeds from two varieties of Mucuna pruriens.

INTRODUCTION: L-Dopa, a key neurotransmitter used to treat neural disorders such as Parkinson's disease, is found in the seeds of the genus Mucuna at a sufficient concentration for possible commercial use. OBJECTIVE: To develop a simple and reliable method to extract L-Dopa from M. pruriens seeds in an aqueous medium and then quantitate this compound using a 1 H qNMR method (internal standard); and also to evaluate the accuracy and reproducibility of this method with an NMR calibration curve. METHODOLOGY: The extraction method of L-Dopa from M. pruriens was optimized. The quantitation with single point quantitative NMR (qNMR) and NMR calibration curve was based on the resonance properties of the main functional groups of the L-Dopa molecule, in particular the signals of the three aromatic protons, which were compared with the signal of an internal standard such as syringic acid. The accuracy (precision and trueness) and reproducibility of both NMR techniques were evaluated. RESULTS: The methods of single point qNMR and NMR calibration curve, applied to the seeds of two M. pruriens varieties, gave very similar L-Dopa contents: 3.0-3.2% and 3.0-3.1%, respectively. CONCLUSION: The statistical analysis confirmed the accuracy and reproducibility of this single point qNMR method (internal standard) for determining L-Dopa, as well as other commercial preparations of this species, without performing an NMR calibration curve.

Produtividade de vinagreira, pimenta rosa e carobinha cultivadas em sucessão a mucuna preta e feijão de porco / Productivity of roselle, Brazilian peppertree, and carobinha grown in succession to black velvet bean and jack bean

RESUMO O uso de adubos verdes e/ou de plantas de cobertura é uma técnica que vem sendo difundida para o cultivo de espécies medicinais, por garantir a sustentabilidade do solo agrícola, ser flexível em seu uso e por melhorar a atividade biológica do solo. O objetivo do trabalho foi avaliar o uso de culturas de cobertura para incrementar a produção de massa de plantas de vinagreira, pimenta rosa e carobinha. Foram avaliadas duas espécies de leguminosas tropicais (mucuna preta e feijão de porco) como cobertura do solo e uma testemunha (sem cultura de cobertura) e três espécies medicinais (vinagreira, pimenta rosa e carobinha). O experimento foi arranjado em esquema de parcelas subdivididas, tendo nas parcelas as coberturas vegetais e nas subparcelas, as plantas medicinais. As plantas para a cobertura vegetal foram propagadas por semeadura direta no local de cultivo, enquanto as medicinais foram transplantadas diretamente entre a palhada da cobertura vegetal. A produção da mucuna preta foi de 8,37 t ha-1 de massa seca e do feijão de porco, de 14,37 t ha-1 de massa seca. O uso das culturas de cobertura contribuiu para uma maior biomassa microbiana do solo. As produções de massas frescas de folhas, caules e frutos de vinagreira e de frutos da pimenta rosa foram maiores quando cultivadas em sucessão às coberturas vegetais do solo, independente da espécie. As produções da carobinha foram semelhantes quando cultivadas em áreas com ou sem a cobertura. Visando à maior produtividade da vinagreira e pimenta rosa, são indicadas as culturas antecessoras feijão de porco e mucuna preta.

ABSTRACT The use of green manure and/or cover crops is a technique that has been widespread for the cultivation of medicinal species, since it ensures the sustainability of agricultural soil, is flexible in its use, and improves biological activity in the soil. The purpose of this study was to evaluate the use of cover crops to increase biomass production of roselle, Brazilian peppertree, and carobinha plants. Two species of tropical legumes (black velvet bean and jack bean) were evaluated as ground covers, and also assessed were one control plot (without cover crops) and three medicinal species (roselle, Brazilian peppertree, and carobinha). The experiment was arranged in a split-plot design, where the plots had cover crops and the subplots contained the medicinal plants. The plants for the cover crop were propagated by direct sowing in the growing site, while the medicinal ones were transplanted directly between the cover crop. The dry mass production of the velvet beans and jack beans were 8.37 t ha-1 and 14.37 t, respectively. The use of cover crops contributed to the increased microbial biomass of the soil. The production of dry mass of leaves, stems, and fruits of roselle and fruits of Brazilian peppertree were higher when grown in succession to the crop cover for the soil, regardless of the species. The production of carobinha was similar when grown in areas with or without the cover. To increase the yield of roselle and Brazilian peppertree, preceding crops of jack bean and black velvet bean are recommended.

The use of cycleave PCR for the differentiation of the rejuvenating herb species Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea), and Mucuna macrocarpa (Black Kwao Khruea), and the simultaneous detection of multiple DNA targets in a DNA admixture.

Kwao Khruea, the tuberous roots of Pueraria candollei Graham ex Benth. (White Kwao Khruea), Butea superba Roxb. (Red Kwao Khruea), and Mucuna macrocarpa Wall. (Black Kwao Khruea), are used as rejuvenating herbs in traditional medicine in many tropical countries. Although Kwao Khruea has attracted strong interest because of its rejuvenation properties, each species is used for specific purposes and effects. P. candollei shows estrogenic effects in females. In contrast, B. superba and M. macrocarpa show androgenic effects in males. The potential misidentification of dried tuberous roots of various Kwao Khruea species might cause problems in the drug market, especially when they are reduced into powders. A cycleave PCR, which is based on the sequence of chloroplast matK gene, was developed to differentiate P. candollei, B. superba, and M. macrocarpa. The results showed that cycleave PCR is able to identify specific Kwao Khruea species. A multiplex cycleave PCR was optimized for the simultaneous detection of two different DNA targets in a DNA admixture. The specificity of this technique was confirmed by its ability to distinguish M. macrocarpa from five related Mucuna species. Cycleave PCR can be a specific, sensitive, and rapid method for the identification of medicinal plants and crude plant samples.

Discrimination of the Thai rejuvenating herbs Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea), and Mucuna collettii (Black Kwao Khruea) using PCR-RFLP.

The tuberous roots of Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea) and Mucuna collettii (Black Kwao Khruea), which belong to the family Leguminosae, are used as rejuvenating herbs in traditional Thai medicine. Although all of these species have an indication for rejuvenation, each differs in its medicinal properties. Two varieties of P. candollei, var. mirifica and var. candollei, affect females, whereas B. superba and M. collettii exhibit effects on males. However, the identification of these roots according to the name "Kwao Khruea" is confusing due to the similarity in their features. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was utilised to identify plant origin. The partial matK gene was amplified and subjected to restriction enzyme digestion with DdeI and TaqI. The restriction fragments generated differed in number and size. To test the reliability of the method, an admixture of the different Kwao Khruea species containing equal amounts of DNA was tested. The results showed combined restriction patterns, and each species could be detected in the background of the others. The method was also used to authenticate eight different crude drugs sold as various types of Kwao Khruea in Thai markets. The results showed that the misidentification of commercial drugs remains a problem in crude drug markets. The PCR-RFLP analysis developed here provides a simple and accurate discrimination of these rejuvenating "Kwao Khruea" species.