High-performance liquid chromatographic assay of glycosyltransferases using flavonoids as substrate

Pace, Mario; Agnellini, Dario; Gardana, Claudio; Mauri, Pierluigi; Pietta, Piergiorgio

doi:10.1016/0021-9673(94)00943-4

Cited by 8 publications

(3 citation statements)

References 10 publications

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“…Many different roles have been ascribed to the UGTs, and stabilization, detoxification, and solubilization of different cellular metabolites are among them. The UGTs convert reactive or toxic aglycones into the stable and nonreactive storage forms (6) that are easily conveyed through physiological fluids (28) to the vacuole (27). Glucosylation increases the polarity of compounds (1) and affects the action of plant hormones (27).…”

Section: Resultsmentioning

confidence: 99%

Ectopic Expression of Anthocyanin 5-O-Glucosyltransferase in Potato Tuber Causes Increased Resistance to Bacteria

Lorenc-Kukuła

Jafra

Oszmiański

et al. 2004

J. Agric. Food Chem.

119

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The principal goal of this paper was to investigate the significance of anthocyanin 5-O-glucosyltransferase (5-UGT) for potato tuber metabolism. The ectopic expression of a 5-UGT cDNA in the tuber improved the plant's defense against pathogen infection. The resistance of transgenic lines against Erwinia carotovora subsp. carotovora was about 2-fold higher than for nontransformed plants. In most cases the pathogen resistance was accompanied by a significant increase in tuber yield. To investigate the molecular basis of transgenic potato resistance, metabolic profiling of the plant was performed. In tuber extracts, the anthocyanin 3,5-O-substituted level was significantly increased when compared to that of the control plant. Of six anthocyanin compounds identified, the highest quantity for pelargonidin 3-rutinoside-5-glucoside acylated with p-coumaric acid and peonidin 3-rutinoside-5-glucoside acylated with p-coumaric acid was detected. A significant increase in starch and a decrease in sucrose level in transgenic tubers have been detected. The level of all other metabolites (amino acids, organic acids, polyamines, and fatty acids) was quite the same as in nontransformants. The plant resistance to bacterial infection correlates with anthocyanin content and sucrose level. The properties of recombinant glucosyltransferase were analyzed in in vitro experiments. The enzyme kinetics and its biochemical properties were similar to those from other sources.

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Section: Resultsmentioning

confidence: 99%

Ectopic Expression of Anthocyanin 5-O-Glucosyltransferase in Potato Tuber Causes Increased Resistance to Bacteria

Lorenc-Kukuła

Jafra

Oszmiański

et al. 2004

J. Agric. Food Chem.

119

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“…However, it was possible to differentiate kaempferol glycosides from those of quercetin by the presence of the shoulder on band II of the spectra of the latter. This fact, rarely found in the literature for identification purposes [19] could avoid the hydrolysis step and it could constitute a valid tool for provisional identification of these structures in complex samples such as green beans. Also, one daidzein glycoside was tentavely identified (peak XI).…”

Section: Chromatographic Separation and Identification Of Phenolics Imentioning

confidence: 97%

Identification and quantitation of phenolics from green beans by high-performance liquid chromatography

Escarpa

González

2000

Chromatographia

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KeyWordsColumn liquid chromatography Green beans Phenolics Identification and quantitation SummaryHigh-performance liquid chromatography with diode-array detection has been used for the separation and quantitation of phenohcs from fresh and processed green beans. Whole beans, pods and seeds of green beans were studied separately Chromatographic profiles from pods were more complex than those found in seeds. Flavonol glycosides were confined to quercetin and kaempferol classes and they were typical of external parts of the fruit. Flavan-3-ols monomers ((+) catechin and (-) epicatechin) as well as procyanidins structures were identified in pods and seeds. Chromatographic profiles from processed beans revealed a polyphenolic composition similar to those found in pod fresh green beans. The chromatographic method was carefully validated in regard to precision and accuracy High reproducibility of peak area (RSD < 3%) and calibration slopes (RSD < 4%) was obtained. Recoveries between 94-104% revealed good accuracy for the overall method. Application to quantitative determination in a representative number of samples allowed good knowledge of the phenolic composition of green beans (Phaseolus vulgaris v. vulgaris).

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“…Although glycoside hydrolysis has been performed to obtain a simpler chromatogram, hydrolysis never yields the exact number of glycosides involved and time-consuming sample treatment is required prior to analysis. Since the importance of the glycosides has been already confirmed [9], their investigation requires the development of suitable chromatographic methods to carry out the separation of polyphenolic compounds from vegetables and avoid both the hydrolysis step and the ensuing lengthy and difficult sample treatments.…”

Section: Introductionmentioning

confidence: 99%

Optimization and Validation of a Fast Liquid Gradient for Determination of Prominent Flavan-3-ols and Flavonols in Fresh Vegetables

Escarpa

Pérez-Cabrera

González

2000

J. High Resol. Chromatogr.

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A fast high‐performance liquid chromatography method was used for analysis of prominent flavan‐3‐ols and flavonols in vegetables. Gradient elution with phosphoric acid‐acetonitrile mixtures and phosphoric acid‐methanol mixtures allowed fast and complete separation of the studied phenolic compounds within analysis times less than 10 min. The development of two elution gradients using methanol and acetonitrile as modifiers proved to be an excellent approach for the verification of the real polyphenolic composition in vegetables samples because the two optimized methods allowed the separation of the same number of compounds in the same elution order. Diode‐array detection was employed for the provisional identification of phenolic compounds that were not available as standards. We preferred methanol as a modifier because it was less toxic and cheaper than acetonitrile. Detection limits ranged between 0.12 and 0.59 μg mL–1. High recoveries of phenolics from fresh vegetables were measured in all studied cases, independent of the phenolic structure, matrix, and vegetable in question. High levels of procyanidins between 150 and 450 mg kg–1 were found in all studied vegetables. Quantification of quercetin and kaempferol glycosides was only possible in marrow and onion, respectively.

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High-performance liquid chromatographic assay of glycosyltransferases using flavonoids as substrate

Cited by 8 publications

References 10 publications

Ectopic Expression of Anthocyanin 5-O-Glucosyltransferase in Potato Tuber Causes Increased Resistance to Bacteria

Ectopic Expression of Anthocyanin 5-O-Glucosyltransferase in Potato Tuber Causes Increased Resistance to Bacteria

Identification and quantitation of phenolics from green beans by high-performance liquid chromatography

Optimization and Validation of a Fast Liquid Gradient for Determination of Prominent Flavan-3-ols and Flavonols in Fresh Vegetables

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