Novel Fast Chromatography-Tandem Mass Spectrometric Quantitative Approach for the Determination of Plant-Extracted Phytosterols and Tocopherols.

Phytosterols and tocopherols are commonly used in food and pharmaceutical industries for their health benefits. Current analysis methods rely on conventional liquid chromatography, using an analytical column, which can be tedious and time consuming. However, simple, and fast analytical methods can facilitate their qualitative and quantitative analysis. In this study, a fast chromatography-tandem mass spectrometric (FC-MS/MS) method was developed and validated for the quantitative analysis of phytosterols and tocopherols. Omitting chromatography by employing flow injection analysis-mass spectrometry (FIA-MS) failed in the quantification of target analytes due to analyte-to-analyte interferences from phytosterols. These interferences arise from their ambiguous MS fingerprints that would lead to false identification and inaccurate quantification. Therefore, a C18 guard column with a 1.9 µm particle size was employed for FC-MS/MS under isocratic elution using acetonitrile/methanol (99:1 v/v) at a flow rate of 600 µL/min. Analyte-to-analyte interferences were identified and eliminated. The false peaks could then be easily identified due to chromatographic separation. In addition, two internal standards were evaluated, namely cholestanol and deuterated cholesterol. Both internal standards contributed to the observed analyte-to-analyte interferences; however, adequate shift in the retention time for deuterated cholesterol eliminated its interferences and allowed for an accurate quantification. The method is fast (1.3 min) compared to published methods and can distinguish false peaks observed in FIA-MS. Seven analytes were quantified simultaneously, namely brassicasterol, campesterol, stigmasterol, ß-sitosterol, α-tocopherol, δ-tocopherol, and γ-tocopherol. The method was successfully applied in the quantitative analysis of phytosterols and tocopherols present in the unsaponifiable matter of canola oil deodorizer distillate (CODD). ß-sitosterol and γ-tocopherol were the most abundant phytosterols and tocopherols, respectively.

Development and Characterization of Liposomal Formulations Containing Phytosterols Extracted from Canola Oil Deodorizer Distillate along with Tocopherols as Food Additives.

Phytosterols are plant sterols recommended as adjuvant therapy for hypercholesterolemia and tocopherols are well-established anti-oxidants. However, thermo-sensitivity, lipophilicity and formulation-dependent efficacy bring challenges in the development of functional foods, enriched with phytosterols and tocopherols. To address this, we developed liposomes containing brassicasterol, campesterol and ß-sitosterol obtained from canola oil deodorizer distillate, along with alpha, gamma and delta tocopherol. Three approaches; thin film hydration-homogenization, thin film hydration-ultrasonication and Mozafari method were used for formulation. Validated liquid chromatographic tandem mass spectrometry (LC-MS/MS) was utilized to determine the entrapment efficiency of bioactives. Stability studies of liposomal formulations were conducted before and after pasteurization using high temperature short time (HTST) technique for a month. Vesicle size after homogenization and ultrasonication (<200 nm) was significantly lower than by Mozafari method (>200 nm). However, zeta potential (-9 to -14 mV) was comparable which was adequate for colloidal stability. Entrapment efficiencies were greater than 89% for all the phytosterols and tocopherols formulated by all three methods. Liposomes with optimum particle size and zeta potential were incorporated in model orange juice, showing adequate stability after pasteurization (72 °C for 15 s) for a month. Liposomes containing phytosterols obtained from canola waste along with tocopherols were developed and successfully applied as a food additive using model orange juice.

Factors governing the regulation of Sclerotinia sclerotiorum cutinase A and polygalacturonase 1 during different stages of infection.

Sclerotinia sclerotiorum releases hydrolytic enzymes that sequentially degrade the plant cuticle, middle lamellae, and primary and secondary cell walls. The cuticle was found to be a barrier to S. sclerotiorum infection, as leaves stripped of epicuticular wax were more rapidly colonized. Consequently, the factors affecting the regulation of genes encoding polygalacturonase 1 (SsPG1) and a newly identified cutinase (SsCUTA) were examined. In vitro, SsCutA transcripts were detected within 1 h postinoculation of leaves, and expression was primarily governed by contact of mycelia with solid surfaces. Expression of SsPg1 was moderately induced by contact with solid surfaces including the leaf, and expression was restricted to the expanding margin of the lesion as the infection progressed. SsPg1 expression was induced by carbohydrate starvation but repressed by galacturonic acid. Glucose supported a basal level of SsPg1 expression but accentuated expression when provided to mycelia used to inoculate leaves. These observations were contrary to earlier reports indicating that glucose repressed SsPg1 expression while galacturonic acid induced expression. Pharmacological studies showed that disruption of calcium signalling affected SsCutA and SsPg1 expression and decreased S. sclerotiorum virulence, whereas elevated cAMP levels reduced virulence without affecting gene expression. The mechanisms involved in coordinating the expression of S. sclerotiorum hydrolytic enzymes throughout the various stages of the infection are discussed.

Expression and regulation of Sclerotinia sclerotiorum necrosis and ethylene-inducing peptides (NEPs).

Successful host colonization by necrotrophic plant pathogens requires the induction of plant cell death to provide the nutrients needed for infection establishment and progression. We have cloned two genes encoding necrosis and ethylene-inducing peptides from Sclerotinia sclerotiorum, which we named SsNep1 and SsNep2. The peptides encoded by these genes induce necrosis when expressed transiently in tobacco leaves. SsNep1 is expressed at a very low level relative to SsNep2 during infection. The expression of SsNep2 was induced by contact with solid surfaces and occurred in both the necrotic zone and at the leading margin of the infection. SsNep2 expression was dependent on calcium and cyclic adenosine monophosphate signalling, as compounds affecting these pathways reduced or abolished SsNep2 expression coincident with a partial or total loss of virulence.

Pleiotropic changes in Arabidopsis f5h and sct mutants revealed by large-scale gene expression and metabolite analysis.

Hydrocinnamic acid esters, lignin, flavonoids, glucosinolates, and salicylic acid protect plants against UV exposure, oxidative stress, diseases, and herbivores. Through the phenylpropanoid pathway, certain Brassicaceae family members, including Arabidopsis thaliana and Brassica napus, accumulate large amounts of the anti-nutritive sinapoylcholine (sinapine) in the seed. We successfully down-regulated activities of key enzymes in the pathway including F5H and SCT and achieved reduction of sinapine and lignin in B. napus seeds. Despite this success, it was unclear how multiple agronomic traits were affected in the transgenic plants. Here, we report altered large-scale gene expression of new alleles of f5h and sct mutants of A. thaliana and resultant accumulation of sinapoylglucose, disinapoylglucose, quercetin-3-O-rhamnoside, salicylic acid glucoside, and total indolyl glucosinolates in the two mutants. Expression of several flowering genes was altered in these mutants when grown under drought and NaCl treatments. Furthermore, both mutants were more susceptible to fungal infection than the wild type. Microarray experiments identified distinctive spatial and temporal expression patterns of gene clusters involved in silique/seed developmental processes and metabolite biosynthesis in these mutants. Taken together, these findings suggest that both f5h and sct mutants exhibit major differences in accumulation of diverse metabolites in the seed and profound changes in global large-scale gene expression, resulting in differential pleiotropic responses to environmental cues. Electronic supplementary material The online version of this article (doi:10.1007/s00425-009-1007-2) contains supplementary material, which is available to authorized users.