The bioconversion of the hydrophobic and volatile limonene to perillic acid, a potential anticancer agent, by the yeast Yarrowia lipolytica was studied in two steps. Firstly, experimental design was used for process optimization using high-purity limonene as substrate and secondly orange essential oil containing 89.1% limonene was used as substrate under the previously optimized conditions. Limonene concentration and pH were identified by fractional factorial design as significant factors and were optimized by central composite design.
Perillic acid can be obtained from microbial oxidation of the exocyclic methyl group of limonene. Due to the pharmacological potential of such a metabolite, the biotransformation processes leading to its synthesis have been approached in recent studies. A robust analytical method is needed to assess the performance of such studies. An analytical method was developed and validated to determine perillic acid in the supernatants of a yeast-induced bioconversion of limonene, involving gas chromatography (GC) and an acid-induced precipitation during the sample preparation. GC analysis was performed using a column with polyethylene glycol as stationary phase (HP-Innowax) which resulted in higher loads and better peak shape. The sample preparation involved the supernatant initial filtration and precipitation with 0.6 M HCl followed by centrifugation and dissolution in ethyl acetate. GC analysis conditions were oven from 50˚C to 250˚C at 20˚C·min −1 , and then held 5 min (total runtime 15 min). Injector was set at 280˚C, and detector at 300˚C. Helium was the carrier gas at 1 ml·min ; Repeatability of 2.1% RSD. Thus, a complete methodology to determine perillic acid in a bioconversion supernatant was developed and validated. This overall approach may be useful for bioconversions of monoterpenes by other microorganisms that metabolize limonene.
The specific legislation for the production and use of medicinal plants has been improved in Brazil, as far as its continuous evolution is concerned. However, there is still a failure at surveillance quality of these products. This work showed a panorama of some espinheira-santa samples (Maytenus ilicifolia) purchased from local markets, through moisture, contaminants, epicatechin and total tannin content. Contaminants were found above the maximum limits admitted in 89 % of the total samples (stems, mosses and lichens) above allowable. Regarding water content, only samples 24, 25 and 27, all from group 3, proved to be outside official limits, which must be within 8 and 12 %. For tannin a variation from 0.18 to 2.96 % could be detected (official minimum limit must be 2 %) and, for epicatechin (pharmacopoeial marker), only 37 % of total samples showed its presence, as analyzed by TLC and HPLC. Three main groups were established by Principal Component Analysis (PCA), after laboratory assays. Group 2 with the highest amounts of the characteristic metabolites epicatechin and total tannin, suggested authentic samples. Group 1 and 3 with high level of moisture and contaminants suggested false samples. These results may, in part, suggest that possible deleterious effects caused by the consumption of espinheira-santa available on the market, could be due to the unconformities observed in the analyzed samples.
Influência da técnica de extração e do tamanho de partícula do material vegetal no teor de compostos fenólicos totais da tintura das folhas de Alpinia zerumbetIgor Cardoso et al
AbstractThe specie Alpinia zerumbet (Zingiberaceae) has been use in the traditional and folk medicine as a diuretic and antihypertensive. The Herbal Medicines Formulary of the Brazilian Pharmacopoeia lists Alpinia zerumbet for the treatment of mild hypertension. This study aims to evaluate the influence of the extraction technique and particle size of the plant material in the extraction of total phenolic compounds from Alpinia zerumbet leaves. Dried leaves were extracted using a mixture of ethanol:water (7:3, v/v) combining four techniques (percolation, static maceration, ultrasound and dynamic maceration) with four ranges of particle size (not sieved, 16-32 mesh, 32-60 mesh and 60-100 mesh), totaling 16 extractions. The total phenolic compounds were quantified using the spectrophotometric method of Folin-Denis. Percolation was shown to be the most efficient method, followed by dynamic maceration, ultrasound and finally, the less efficient method, static maceration. The particle size of the plant material was not significant in the extraction of total phenolics. The percolation and the particle size ranges from 60 to 100 mesh proved to be the most efficient combination evaluated in this study.
In this report, a rapid and selective analytical method for the determination of monoterpenes in Alpinia zerumbet essential oil (AZEO) by gas chromatography (GC) flame ionization detection (FID) was developed, optimized and validated. The suitability of six different capillary columns was investigated by polarity phase constants calculated based on the methodology proposed by Rohrschneider and McReynolds. The most suitable column was then used in a central composite design applying the following GC factors to investigate responses based on peak resolutions and analysis time: initial oven temperature, heating rate and carrier gas flow rate. The optimized method has initial oven temperature of 60 o C, heating rate of 5.1 o C min -1 and flow rate of 1.0 mL min -1 using the DB-35 capillary column. The validation acceptance criteria were met in all cases. The method was successfully applied for the quantification of major monoterpenes found in AZEO in different samples of Alpinia zerumbet.
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