The LC-MS/MS technique was applied to the stability study of several flavonoids and phenolic acids in honey samples during the ultrasonic extraction (USE) and microwave-assisted extraction (MAE). Phenolic compounds from the standard mixture were stable under ultrasounds action with the mean recovery of (90.4%±7.1%), but during microwave-assisted extraction the benzoic acid derivatives and aglycones of flavonoids showed lower recovery (70-80%). In honey matrix, the phenolic acids and the glycosides exhibited the high stability for MAE and USE treatments. However, the recoveries of tested aglycones were below 10%. In the presence of an artificial sugar matrix, flavonols were almost completely degraded after successive treatment under MAE and USE conditions. The obtained results indicated that standard addition method for flavonoids quantification in honey samples should not be recommended. Application of the USE conditions provided higher and/or similar extraction yields for phenolic acids than usually applied shaking with solvent. It also allowed shortening the time required for the whole sample preparation procedure. Phenolic acids and glycosides such as quercetrin, rutin and hesperidin appeared to be stable under such conditions.
The phenolic compounds were extracted from green and yellow leaves, stalks, and seeds of garlic ( Allium ursinum L.). The extracts were analyzed by liquid chromatography-photodiode array detector-electrospray ionization-tandem mass spectrometry (LC-PDA-ESI-MS/MS). In total, 21 compounds were detected. The flavonol derivatives were identified on the basis of their ultraviolet (UV) spectra and fragmentation patterns in collision-induced dissociation experiments. On the basis of accurate MS and MS/MS data, six compounds were newly identified in bear’s garlic, mainly the kaempferol derivatives. As far as the investigated parts of garlic are concerned, the kaempferol derivatives were found to be predominant in yellow leaves [2362.96 mg/100 g of dry matter (dm)], followed by green leaves (1856.31 mg/100 g of dm). Seeds contained the minimal phenolic compounds, less than stalks. The yellow leaves of A. ursinum possessed a much larger content of compounds acylated with p-coumaric acid than green leaves (1299.97 versus 855.67 mg/100 g of dm, respectively). The stalks and seeds contained much more non-acetylated than acetylated flavonoid glycosides with p-coumaric acid compounds (162.4 versus 62.82 mg/100 g of dm and 105.49 versus 24.18 mg/100 g of dm, respectively).
Flavonols kaempferol, quercetin, myricetin and gossypetin, and flavones apigenin, acacetin, luteolin, orientin and tricin, are subjected to two AlCl(3) spectrophotometric methods used for determination of total flavonoid content. The method developed by Woisky and Salatino involves addition of AlCl(3) solution to the flavonoid solution and recording of optical density at 420nm. All flavonols except kaempferol have absorption maxima above 440nm and so readings at 420nm are erroneous. Among flavones, all except for luteolin and orientin, have absorption maxima below 400nm. Further, addition of CH(3)COOK and recording the absorbance at 415nm, as modified by Chang et al., works well for flavonols kaempferol, quercetin and myricetin, but not for gossypetin. The flavones luteolin and orientin absorbed above 400nm, whereas all others absorbed below 400nm. Examination of the results of both methods indicates they are inadequate, and should not to be considered as universal and standard methods for total flavonoid determination.
INTRODUCTION: Propolis is a chemically complex resinous substance collected by honeybees (Apis mellifera) from tree buds, comprising plant exudates, secreted substances from bee metabolism, pollen and waxes. Its chemical composition depends strongly on the plant sources available around the beehive, which have a direct impact in the quality and bioactivity of the propolis. Being as Portugal is a country of botanical diversity, the phenolic characterisation of propolis from the different regions is a priority. OBJECTIVE: Extensive characterisation of the phenolic composition of Portuguese propolis from different continental regions and islands. METHOD: Forty propolis ethanolic extracts were analysed extensively by liquid chromatography with diode-array detection coupled to electrospray ionisation tandem mass spectrometry (LC-DAD-ESI-MS(n) ). RESULTS: Seventy-six polyphenols were detected in the samples and two groups of propolis were established: the common temperate propolis, which contained the typical poplar phenolic compounds such as flavonoids and their methylated/esterified forms, phenylpropanoid acids and their esters, and an uncommon propolis type with an unusual composition in quercetin and kaempferol glycosides - some of them never described in propolis. CONCLUSION: The method allowed the establishment of the phenolic profile of Portuguese propolis from different geographical locations, and the possibility to use some phenolic compounds, such as kaempferol-dimethylether, as geographical markers. Data suggest that other botanical species in addition to poplar trees can be important sources of resins for Portuguese propolis. Copyright © 2012 John Wiley & Sons, Ltd.
A high-performance liquid chromatographic method with gradient elution and diode-array detection was developed to quantify free phenolic acids (gallic, vanillic, chlorogenic, caffeic, syringic, p-coumaric, ferulic, sinapic, salycilic, elagic and trans-cinnamic), flavonoids (catechin, epicatechin, rutin, myricetin and quercetin) and juglone in walnut leaves. Chromatographic separation was performed on a Hypersil Gold C18 column (5 µm particle size, 250 × 4.6 mm) and detection was conducted at three different wavelengths (254, 278 and 300 nm) according to the absorption maxima of the analyzed compounds. Validation procedures were conducted and the method was proven to be precise, accurate and sensitive. The developed method has been applied to analyze walnut leaves samples from nine different cultivars, with the same agricultural, geographical and climatic conditions. The experimental results revealed high concentrations of myricetin, catechin hydrate and rutin, and low concentrations of quercetin and epicatechin aglycones. Ellagic acid was established as the dominating phenolic acid of walnut leaves, followed by trans-cinnamic, chlorogenic and caffeic acids. Juglone content varied between 44.55 and 205.12 mg/100 g fresh weight. Significant differences were detected among cultivars for the concentration levels of phenolics.
A novel homoflavanol derivative (1) with an unprecedented skeleton, as well as two known flavonoids kaempferol (2) and quercetin (3), was isolated from the plant Pteridium aquilinum (L.) Kuhn, and the structure and relative stereochemistry of the new compound (1) were elucidated on the basis of spectroscopic data.
Staphylococcus aureus is an important etiological agent responsible for healthcare-associated infections. In this study, the effect of flavonoids on the inhibition of S. aureus PriA (SaPriA), an essential helicase for DNA replication restart, which is critical for bacterial survival, was investigated. Using vanadate-sensitive colorimetric assay, the concentration of phosphate, from ATP hydrolysis by SaPriA, was decreased to 37 and 69 %, respectively, in the presence of 35 μM kaempferol and myricetin. The effect of quercetin, galangin, dihydromyricetin, and myricitrin was insignificant. From titration curve, IC50 of kaempferol for SaPriA was determined to be 22 ± 2 μM. Using fluorescence quenching, we identified that kaempferol can bind to SaPriA with K d of 9.1 ± 3.2 μM. To our knowledge, these preliminary results constituted the first study regarding that naturally occurring product such as flavonols kaempferol and myricetin can be potent inhibitors targeting PriA.
Flavonoids are a group of phenolic secondary plant metabolites that are ubiquitous in plant-based diets. Data from anthropological, observational and intervention studies have shown that many flavonoids are bioactive. For this reason, there is an increasing interest in investigating the potential health effects of these compounds. The translation of these findings into the context of the health of the general public requires detailed information on habitual dietary intake. However, only limited data are currently available for European populations.
Metabolite profile, antioxidant and antinociceptive activities of Syringa vulgaris bark and leaf methanolic extracts were investigated. By means of HPLC-DAD-ESI-TOF and HPLC-DAD-ESI-MS/MS, a total of 33 phenolics were identified, including 15 secoiridoids, 6 phenylpropanoids, 3 flavonoids, 3 lignans and 6 low molecular weight phenols. Validated quantitative analysis show that syringin (2.52%) and rutin (1.13%) are the main phenolic compounds in bark and leaf, respectively. Notable radical scavenging and antinociceptive activities of the bark and leaf extracts were confirmed by in vitro DPPH● and ABTS●+ assays and by in vivo hot-plate method in mice, respectively. Our results could lay the scientific basic of future clinical perspectives of lilac bark and leaf.
Herein, the polyphenolic content in extracts of Ruppia cirrhosa (Petagna) Grande and Ruppia maritima L.was fully characterized for the first time. High amounts of the main compound chicoric acid (CA) (≤30.2 ± 4.3 mg/g) were found in both Ruppia species. In addition, eight flavonoids, namely the 3-O-glucopyranosides and 3-O-galactopyranosides, as well as malonylated 3-O-glycosides of quercetin and isorhamnetin, were isolated and identified. The antioxidant activity of Ruppia cirrhosa extracts and isolated compounds was investigated spectrophotometrically by a 1,1-diphenyl-2-picrylhydrazyl (DPPH·) radical scavenging assay. IC50 values were 31.8-175.7 μg/mL for Ruppia cirrhosa extracts and 12.1-88.4 μg/mL for isolated flavonoids. Both individual and total phenolic and flavonoid content were quantified in crude extracts using analytical HPLC. The relative high amount of total flavonoids ranged from 5.9 to 14.7 mg/g in both species, with concentrations of individual flavonoids ranging from 0.4 to 2.9 mg/g dry weight. The content of chicoric acid was twofold more in Ruppia maritima than in Ruppia cirrhosa. Seasonal variation of the quantitative content in Ruppia cirrhosa was examined. Total flavonoid content ranged from 8.4 mg/g in October to 14.7 mg/g in August, whereas the highest concentration of chicoric acid was observed in March (29.2 mg/g).