Optimization of Flavonoid Production in Plant Cell Culture of Thevetia peruviana Elicited with Methyl Jasmonate and Salicylic Acid

Abstract Thevetia peruviana is a medicinal plant that has valuable secondary terpenoid-type metabolites and phenolic compounds. Some flavonoid compounds of pharmaceutical interest stand out in the latter group. The concentration of these bioactive compounds in natural conditions is limited by environmental; therefore, it has been considered necessary to make in vitro plant cell suspension cultures that admit the use of elicitors to increase the content of active principles. Accordingly, in this study, for the optimization of flavonoid production in cell suspension culture of T. peruviana, different parameters related to elicitation with methyl jasmonate (MeJa), and salicylic acid (SA) were evaluated, at stirred flask scale. Firstly, 3 μM MeJa and 300 μM SA were added separately in cell cultures of T. peruviana, to assess their potential effects. Secondly, several experimental conditions were evaluated, for optimization purpose. In the first part, MeJa and SA increased the total flavonoid content, in 1.07 and 1.3 times, respectively, compared to the control culture; in the second part, total flavonoid content produced in MeJa mediated cell suspension cultures were 4.14 mg QE/g DW (milligrams of quercetin equivalent per gram of dry biomass) with: concentration 0.3 μM, addition time day 5 and harvest time 90 h. On the other hand, total flavonoid content produced in SA mediated cell suspension cultures were 3.75 mg QE/g DW with: concentration 100 μM, addition time day 0 and harvest time 96 h. Elicitation of cell suspension cultures of T. peruviana with MeJa and SA under their ideal parameter values increased flavonoid content.

Phytochemical Screening of Callus and Cell Suspensions Cultures of Thevetia peruviana

Abstract Thevetia peruviana is an ornamental shrub grown-up in many tropical region of the world. This plant produces secondary metabolites with biological properties of interest for the pharmaceutical industry. The objective was to determine the secondary metabolites profile of callus and cell suspension cultures of T. peruviana and compare them with those from explant (fruit pulp). Extracts in 50% aqueous ethanol and ethyl acetate were prepared. The phytochemical analysis was performed using standard chemical tests and thin layer chromatography. In addition, total phenolic and flavonoids compounds (TPC and TFC), total cardiac glycosides (TCG) and total antioxidant activity (TAA) was determined during the cell suspension growth. Phenolic chemical profile was also analyzed by high performance liquid chromatography (HPLC). Common metabolites (alkaloids, amino acids, antioxidants, cardiac glycosides, leucoanthocyanidins, flavonoids, phenols, sugars and triterpenes) were detected in all samples. The maximum production of extracellular TCG, TPC, TFC and TAA in cells suspensions were at 6-12 days; in contrast, intracellular content was relatively constant during the exponential grown phase (0 to 12-days). HPLC analysis detected one compound with retention time at 11.6 min; this compound was tentatively identified as dihydroquercetin, a flavonoid with anti-cancer properties. These results provide evidence on the utility of the in vitro cell cultures of T. peruviana for valuable pharmaceutical compounds production.

CARDIAC GLYCOSIDES, PHENOLIC COMPOUNDS AND ANTIOXIDANT ACTIVITY FROM PLANT CELL SUSPENSION CULTURES OF Thevetia peruviana / PRODUCCIÓN DE GLÍCOSIDOS CARDIOTÓNICOS, COMPUESTOS FENÓLICOS Y ACTIVIDAD ANTIOXIDANTE EN CULTIVOS DE CÉLULAS VEGETALES EN SUSPENSIÓN DE Thevetia peruviana

Cell suspension cultures of Thevetia peruviana were established under dark for 19 days to investigate kinetic behavior related to biomass, substrate, cardiac glycoside, polyphenols, reactive oxygen species and anti-oxidant activity. The results showed high biomass production (18.80gDW/L) as well as sucrose consumption in 7 days. Preferential glucose over fructose consumption was observed. Intracellular production of cardiac glycosides reached 2.58mg DE/gDW at day 19. Highest extracellular production was reached between day 2 and 7 (6.19mg DE/L). Highest extracellular phenolic content was 80.61 ± 5.16mg GAE/L at day 7. Intracellular phenolic content increased to 2.76 ± 0.14mg GAE/gFW at day 7 and remained constant until day 19. ROS production at day 7 could be related to sucrose and glucose total consumption. Pearson Product-Moment Correlation Coefficient (ρ) showed that the phenolic compounds in cell suspension cultures of T. peruviana were responsible for the observed anti-oxidant activity. All together, these results give the first steps in metabolic behavior in cell suspension cultures of T. peruviana.

Se establecieron cultivos en suspensión de la especie vegetal Thevetia peruviana en oscuridad, durante 19 días, para estudiar el comportamiento cinético de producción de biomasa, consumo de sustrato, producción de glicósidos cardiotónicos, polifenoles, especies reactivas de oxígeno y actividad antioxidante. Los resultados mostraron una alta producción de biomasa (18,80g PS/L), al igual que consumo total de sacarosa, a los 7 días de cultivo. Se observó un consumo preferencial de glucosa sobre fructosa durante todo el cultivo. La producción de glicósidos cardiotónicos intracelulares alcanzó valores de 2,58mg ED/g PS, al día 19. La mayor producción extracelular (6,19mg ED/L), se alcanzó entre los días 2 y 7. El mayor contenido de compuestos fenólicos extracelular fue de 80,61 ± 5,16mg GAE/L, en el día 7. El contenido de compuestos fenólicos intracelulares incrementó a 2,76 ± 0,14mg AGE/gPF, al día 7 y se mantuvo constante, hasta el día 19. La producción de EROs, al día 7, puede estar relacionada con el consumo total de sacarosa y glucosa. El coeficiente de correlación producto-momento de Pearson (ρ) indicó que los compuestos fenólicos en cultivos celulares en suspensión de T. peruviana eran los responsables de la actividad antioxidante observada. En conjunto, estos resultados brindan las primeras bases relacionadas al comportamiento metabólico de cultivos celulares en suspensión, de T. peruviana.

Molecular identification of species of the Genus Euglossa Latreille (Hymenoptera: Apidae, Euglossini)

Euglossine bees interact with more than 60 plant families of the Neotropical region. The richness and abundance of these bees have been intensively studied in different ecosystems using the methodology of capturing males with chemical baits. Females are poorly known for most of the species and morphological characters for their taxonomic classification have not yet been described. The purpose of this study was to use allozymes and restriction patterns of the mitochondrial regions 16S and Cyt b to identify species of Euglossa Latreille. Bees were collected while visiting Thevetia peruviana (Apocynaceae) flowers in five cities of the state of São Paulo, Brazil. Three Euglossa species were identified among the 305 individuals collected. Euglossa cordata (L.) was the only species found in all cities, while E. securigera Dressler and E. townsendi Cockerell were restricted to two and one cities respectively. EST-3 was a diagnostic marker, whereas ICD, MDH, ME and PGM were informative for species identification when used in combination. Restriction by VspI of the amplified 16S fragment differentiated the three species and showed intraspecific polymorphism for E. cordata and E. securigera. The Cyt b region showed distinctive patterns for E. townsendi but it was not possible to differentiate the other two species. Our results describe potentially useful genetic markers for the identification of Euglossa spp. at the species and group level.

As abelhas euglossíneas interagem com mais de 60 famílias de plantas da Região Neotropical. Sua riqueza e abundância têm sido intensamente estudadas em diferentes ecossistemas utilizando-se a metodologia de captura de machos em iscas-armadilhas. As fêmeas, entretanto, são pouco conhecidas para a maioria das espécies, e caracteres morfológicos que permitam sua identificação taxonômica não têm sido descritos. O propósito deste trabalho foi utilizar alozimas e padrões de restrição das regiões mitocondriais 16S e Cit b para identificar espécies do gênero Euglossa Latreille. As abelhas foram coletadas enquanto visitavam flores de Thevetia peruviana (Apocynaceae) em cinco cidades do estado de São Paulo. Foram identificadas três espécies de Euglossa entre os 305 indivíduos coletados. Euglossa cordata (L.) foi a única espécie presente em todas as cidades, enquanto E. securigera Dressler e E. townsendi Cockerell foram encontradas em duas e uma cidade, respectivamente. EST-3 mostrou-se ser marcador diagnóstico, enquanto ICD, MDH, ME e PGM foram locos informativos para a identificação de espécies quando considerados conjuntamente. A restrição com VspI da região 16S amplificada, além de diferenciar as três espécies, apresentou polimorfismo intraespecífico para E. cordata e E. securigera. A região Cit b apresentou padrões característicos para E. townsendi, mas não permitiu diferenciar as outras duas espécies. Os resultados descrevem marcadores genéticos potencialmente úteis para a identificação de Euglossa spp. ao nível de espécie e grupo de espécies.