Separation of nordihydrocapsiate from capsiate and major capsaicinoid analogues using ultra high performance liquid chromatography.

Accurate, rapid quantitation of the capsaicinoid and capsinoid compounds produced by peppers (Capsicum spp.) is essential to assess quality. Here, we developed a rapid ultra-high performance liquid chromatography method for the simultaneous separation of five major capsaicinoids and three major capsinoids from peppers. Optimal chromatographic separation was achieved using a phenyl-hexyl stationary phase with a mobile phase of acidified water and methanol with a flow rate of 0.5 ml/min at a column temperature of 55 °C over 5 min. The method was validated by testing linearity, precision, robustness, and limits of detection and quantification. The developed method was successfully employed to profile capsaicinoids and capsinoids from different pepper cultivars. Out of the 10 pepper cultivars analysed, all three major capsinoids were detected in two cultivars. To the best of our knowledge, this is the first report of successful separation of nordihydrocapsiate from capsiate and quantification of nordihydrocapsiate.

Changes in potato phenylpropanoid metabolism during tuber development.

Phenylpropanoid metabolite and transcript expression during different developmental stages were examined in field grown potatoes. Carbohydrate and shikimic acid metabolism was assessed to determine how tuber primary metabolism influences phenylpropanoid metabolism. Phenylpropanoid concentrations were highest in immature tubers, as were some transcript levels and enzyme activities including phenylalanine ammonia lyase (PAL). Phenylpropanoid concentration differences between mature and immature tubers varied by genotype, but in some cases were approximately three-fold. The most abundant phenylpropanoid was chlorogenic acid (5CGA), which decreased during tuber maturation. Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) transcripts were highly expressed relative to other phenylpropanoid genes, but were not well correlated with 5CGA concentrations (r = -0.16), whereas HQT enzyme activity was. In contrast to 5CGA, less abundant chlorogenic isomers increased during development. Concentrations of hydroxycinnamic acid amides were higher in immature tubers, as was expression of arginine- and ornithine decarboxylases. Expression of several genes involved in carbohydrate or shikimate metabolism, including sucrose synthase and DAHP, showed similar developmental patterns to phenylpropanoid pools, as did shikimate dehydrogenase enzyme activity. Sucrose, glucose and fructose concentrations were highest in immature tubers. Exogenous treatment of potatoes with sugars stimulated phenylpropanoid biosynthesis, suggesting sugars contribute to the higher phenylpropanoid concentrations in immature tubers. These changes in phenylpropanoid expression suggest the nutritional value of potatoes varies during development.

Differential effects of environment on potato phenylpropanoid and carotenoid expression.

BACKGROUND: Plant secondary metabolites, including phenylpropanoids and carotenoids, are stress inducible, have important roles in potato physiology and influence the nutritional value of potatoes. The type and magnitude of environmental effects on tuber phytonutrients is unclear, especially under modern agricultural management that minimizes stress. Understanding factors that influence tuber secondary metabolism could facilitate production of more nutritious crops. Metabolite pools of over forty tuber phenylpropanoids and carotenoids, along with the expression of twenty structural genes, were measured in high-phenylpropanoid purple potatoes grown in environmentally diverse locations in North America (Alaska, Texas and Florida). RESULTS: Phenylpropanoids, including chlorogenic acid (CGA), were higher in samples from the northern latitudes, as was the expression of phenylpropanoid genes including phenylalanine ammonia lyase (PAL), which had over a ten-fold difference in relative abundance. Phenylpropanoid gene expression appeared coordinately regulated and was well correlated with metabolite pools, except for hydroxycinnamoyl-CoA:quinatehydroxcinnamoyl transferase (HQT; r = -0.24). In silico promoter analysis identified two cis-acting elements in the HQT promoter not found in the other phenylpropanoid genes. Anthocyanins were more abundant in Alaskan samples and correlated with flavonoid genes including DFR (r = 0.91), UFGT (r = 0.94) and F3H (r = 0.77). The most abundant anthocyanin was petunidin-3-coum-rutinoside-5-glu, which ranged from 4.7 mg g-1 in Alaska to 2.3 mg g-1 in Texas. Positive correlations between tuber sucrose and anthocyanins (r = 0.85), suggested a stimulatory effect of sucrose. Smaller variation was observed in total carotenoids, but marked differences occurred in individual carotenoids, which had over a ten-fold range. Violaxanthin, lutein or zeaxanthin were the predominant carotenoids in tubers from Alaska, Texas and Florida respectively. Unlike in the phenylpropanoid pathway, poor correlations occurred between carotenoid transcripts and metabolites. CONCLUSION: Analysis of tuber secondary metabolism showed interesting relationships among different metabolites in response to collective environmental influences, even under conditions that minimize stress. The variation in metabolites shows the considerable phenotypical plasticity possible with tuber secondary metabolism and raises questions about to what extent these pathways can be stimulated by environmental cues in a manner that optimizes tuber phytonutrient content while protecting yields. The differences in secondary metabolites may be sufficient to affect nutritional quality.

Characterization of responses of 2d array seven29 detector and its combined use with octavius phantom for the patient-specific quality assurance in rapidarc treatment delivery.

A commercial 2D array seven29 detector has been characterized and its performance has been evaluated. 2D array ionization chamber equipped with 729 ionization chambers uniformly arranged in a 27 × 27 matrix with an active area of 27 × 27 cm² was used for the study. An octagon-shaped phantom (Octavius Phantom) with a central cavity is used to insert the 2D ion chamber array. All measurements were done with a linear accelerator. The detector dose linearity, reproducibility, output factors, dose rate, source to surface distance (SSD), and directional dependency has been studied. The performance of the 2D array, when measuring clinical dose maps, was also investigated. For pretreatment quality assurance, 10 different RapidArc plans conforming to the clinical standards were selected. The 2D array demonstrates an excellent short-term output reproducibility. The long-term reproducibility was found to be within ±1% over a period of 5 months. Output factor measurements for the central chamber of the array showed no considerable deviation from ion chamber measurements. We found that the 2D array exhibits directional dependency for static fields. Measurement of beam profiles and wedge-modulated fields with the 2D array matched very well with the ion chamber measurements in the water phantom. The study shows that 2D array seven29 is a reliable and accurate dosimeter and a useful tool for quality assurance. The combination of the 2D array with the Octavius phantom proved to be a fast and reliable method for pretreatment verification of rotational treatments.

Adaptive volumetric modulated arc treatment planning for esophageal cancers using cone beam computed tomography.

PURPOSE: To assess the potential of cone beam CT (CBCT) derived adaptive RapidArc treatment for esophageal cancers in reducing the dose to organs at risk (OAR). METHODS AND MATERIALS: Ten patients with esophageal cancer were CT scanned in free breathing pattern. The PTV is generated by adding a 3D margin of 1 cm to the CTV as per ICRU 62 recommendations. The double arc RapidArc plan (Clin_RA) was generated for the PTV. Patients were setup using kV orthogonal images and kV-CBCT scan was acquired daily during first week of therapy, then weekly. These images were exported to the Eclipse TPS. The adaptive CTV which includes tumor and involved nodes was delineated in each CBCT image set for the length of the PTV. The composite CTV from first week CBCT was generated using Boolean union operator and 5 mm margin was added circumferentially to generate adaptive PTV (PTV1). Adaptive RapidArc plan (Adap_RA) was generated. NTCP and DVH of the OARs of the two plans were compared. Similarly, PTV2 was generated from weekly CBCT. PTV2 was evaluated for the coverage of 95% isodose of Adap_RA plan. RESULTS: The PTV1 and PTV2 volumes covered by 95% isodose in adaptive plans were 93.51 ± 1.17% and 94.59 ± 1.43% respectively. The lung V(10Gy,)V(20Gy) and mean dose in Adap_RA plan was reduced by 17.43% (p = 0.0012), 34.64% (p = 0.0019) and 16.50% (p = 0.0002) respectively compared to Clin_RA. The Adap_RA plan reduces the heart D(35%) and mean dose by 17.35% (p = 0.0011) and 17.16% (p = 0.0012). No significant reduction in spinal cord and liver doses were observed. NTCP for the lung (0.42% vs. 0.08%) and heart (1.39% vs. 0.090%) was reduced significantly in adaptive plans. CONCLUSION: The adaptive re-planning strategy based on the first week CBCT dataset significantly reduces the doses and NTCP to OARs.

Development, characterization and cross species amplification of polymorphic microsatellite markers from expressed sequence tags of turmeric (Curcuma longa L.).

Expressed sequence tags (ESTs) from turmeric (Curcuma longa L.) were used for the screening of type and frequency of Class I (hypervariable) simple sequence repeats (SSRs). A total of 231 microsatellite repeats were detected from 12,593 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs accounts to one SSR per 17.96 kb of EST. Mononucleotides were the most abundant class of microsatellite repeat in turmeric ESTs followed by trinucleotides. A robust set of 17 polymorphic EST-SSRs were developed and used for evaluating 20 turmeric accessions. The number of alleles detected ranged from 3 to 8 per loci. The developed markers were also evaluated in 13 related species of C. longa confirming high rate (100%) of cross species transferability. The polymorphic microsatellite markers generated from this study could be used for genetic diversity analysis and resolving the taxonomic confusion prevailing in the genus.

Isolation and amplification of genomic DNA from recalcitrant dried berries of black pepper (Piper nigrum L.)--a medicinal spice.

Black pepper is an important medicinal spice traded internationally. The extraction of high quality genomic DNA for PCR amplification from dried black pepper is challenging because of the presence of the exceptionally large amount of oxidized polyphenolic compounds, polysaccharides and other secondary metabolites. Here we report a modified hexadecyl trimethyl ammonium bromide (CTAB) protocol by incorporating potassium acetate and a final PEG precipitation step to isolate PCR amplifiable genomic DNA from dried and powdered berries of black pepper. The protocol has trade implication as it will help in the PCR characterization of traded black peppers from different countries.