Kinetic study of flavonoid reactions with stable radicals

The antiradical activities of some flavonols (kaempferol, quercetin, robinetin, quercetagetin, and myricetin), flavones (apigenin, baicalein, and luteolin), flavanones (naringenin and dihydroquercetin), and flavanols [(+)-catechin and (-)-epicatechin] were determined by measuring the reaction kinetics with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and alpha,gamma-bisdiphenylene-beta-phenylallyl (BDPA) radicals. The reactions, which follow the mixed second-order rate law, were investigated under pseudo-first-order conditions by use of a large excess of flavonoids, and their stoichiometry was determined by spectrophotometric titration. The results confirm stoichiometric factors of 1, 2, and 3 for flavonoids with one, two, and three hydroxyl groups in the B-ring, respectively, excluding kaempferol, which, despite a single OH group in the B-ring, has a factor of 2, which is explained by the 3-OH group supporting the reaction with free radicals. Structure-activity considerations indicate for the present series of flavonoids the importance of multiple OH substitutions and conjugation. The logarithms of reaction rate constants with the OH, DPPH, and BDPA radicals correlate well with the reduction potential of the flavonoids.     

Flavonol profiles of Vitis vinifera red grapes and their single-cultivar wines

The main flavonols found in seven widespread Vitis vinifera red grape cultivars include the 3-glucosides and 3-glucuronides of myricetin and quercetin and the 3-glucosides of kaempferol and isorhamnetin. In addition, the methoxylated trisubstituted flavonols, laricitrin and syringetin, were predominantly found as 3-glucosides. As minority flavonols, the results suggest the detection of the 3-galactosides of kaempferol and laricitrin, the 3-glucuronide of kaempferol, and the 3-(6' '-acetyl)glucosides of quercetin and syringetin. The flavonol profiles based on the eight above-mentioned flavonols allowed the cultivar differentiation of the grape samples. With regard to flavonol biosynthesis in the berry skin, quercetin 3-glucuronide predominated at véraison, followed by quercetin 3-glucoside, and only trace amounts of trisubstituted flavonols were detected. The proportion of quercetin 3-glucoside remained almost constant during berry ripening, whereas the proportion of quercetin 3-glucuronide decreased and the other flavonols, especially myricetin 3-glucoside, increased their importance. In wines, flavonol 3-glycosides coexisted with their corresponding free aglycones released by hydrolysis. The presence of laricitrin, syringetin, and laricitrin 3-glucoside in red wines is reported here for the first time. The extent of hydrolysis was widely variable among wines made from the same grape cultivar, and the results suggest the influence of the type of aglycone and glycoside on the rate of hydrolysis. Due to hydrolysis, the differentiation of single-cultivar wines gave acceptable results only when aglycone-type flavonol profiles were used.     

Influence of domestic processing and storage on flavonol contents in berries

Effects of domestic processing and storage on the flavonols quercetin, myricetin, and kaempferol in five berries were studied using an optimized RP-HPLC method with UV and diode array detection after an acid hydrolysis of the corresponding glycosides. In fresh berries, the total content of flavonols was highest in lingonberry (169 mg/kg) and black currant (157 mg/kg), intermediate in bilberry (41 mg/kg) and strawberry (17 mg/kg), and lowest in red raspberry (9.5 mg/kg). Cooking strawberries with sugar to make jam resulted in minor losses (quercetin 15%, kaempferol 18%). During cooking of bilberries with water and sugar to make soup, 40% of quercetin was lost. Traditional preservation of crushed lingonberries in their own juice caused a considerable (40%) loss of quercetin. Only 15% of quercetin and 30% of myricetin present in unprocessed berries were retained in juices made by common domestic methods (steam-extracted black currant juice, unpasteurized lingonberry juice). Cold-pressing was superior to steam-extraction in extracting flavonols from black currants. During 9 months of storage at 20 C, quercetin content decreased markedly (40%) in bilberries and lingonberries, but not in black currants or red raspberries. Myricetin and kaempferol were more susceptible than quercetin to losses during storage.     

Content of the flavonols myricetin, quercetin, and kaempferol in finnish berry wines

The amounts of myricetin, quercetin, and kaempferol were analyzed in 16 red and 2 white berry and grape wines after acid hydrolysis using an RP-HPLC method with diode array detection. The red berry wines analyzed were made mainly from black currant, crowberry, and bog whortleberry, i.e., berries rich in flavonols. The red grape wines were made mainly from Cabernet Sauvignon or Merlot grapes in several countries. The white wines studied were gooseberry and white currant wines and Chardonnay and Riesling wines. The amount of myricetin ranged from 3.8 to 22.6 mg L(-1) in red berry wines and from 0 to 14.6 mg L(-1) in red grape wines. The amount of quercetin was from 2.2 to 24.3 mg L(-1) red berry wines and from <1.2 to 19.4 mg L(-1) in red grape wines. Low levels of kaempferol were found in all red berry wines and in 9 red grape wines. The total concentration of these flavonols was from 6 to 46 mg L(-1) (mean 20 mg L(-1)) in red berry wines and from 4 to 31 mg L(-1) (mean 15 mg L(-1)) in red grape wines. Small amounts of quercetin were found in white currant and gooseberry wines, whereas no flavonols were detected in white grape wines. These results demonstrate that the contents of flavonols in red     

Flavonols from saffron flower: tyrosinase inhibitory activity and inhibition mechanism.

A common flavonol, kaempferol, isolated from the fresh flower petals of Crocus sativus L. (Iridaceae) was found to inhibit the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase with an ID(50) of 67 microgram/mL (0.23 mM). Interestingly, its 3-O-glycoside derivatives did not inhibit this oxidation. The inhibition kinetics analyzed by a Lineweaver-Burk plot found kaempferol to be a competitive inhibitor, and this inhibitory activity presumably comes from its ability to chelate copper in the enzyme. This copper chelation mechanism can be applicable for all of the flavonols as long as their 3-hydroxyl group is free. However, quercetin, kaempferol, and galangin each affect the oxidation of L-tyrosine in somewhat different ways.     

Metabolite profiling of grape: Flavonols and anthocyanins

Flavonols are products of the flavonoid biosynthetic pathway, which also give rise to anthocyanins and condensed tannins in grapes. We investigated their presence in the berry skins of 91 grape varieties (Vitis vinifera L.), in order to produce a classification based on the flavonol profile. The presence of laricitrin 3-O-galactoside and syringetin 3-O-galactoside in red grapes is reported here for the first time. In red grapes, the main flavonol was quercetin (mean = 43.99%), followed by myricetin (36.81%), kaempferol (6.43%), laricitrin (5.65%), isorhamnetin (3.89%), and syringetin (3.22%). In white grapes, the main flavonol was quercetin (mean = 81.35%), followed by kaempferol (16.91%) and isorhamnetin (1.74%). The delphinidin-like flavonols myricetin, laricitrin, and syringetin were missing in all white varieties, indicating that the enzyme flavonoid 3',5'-hydroxylase is not expressed in white grape varieties. The pattern of expression of flavonols and anthocyanins in red grapes was compared, in order to gain information on the substrate specificity of enzymes involved in flavonoid biosynthesis.     

The UVA and aqueous stability of flavonoids is dependent on B-ring substitution

Flavonols such as kaempferol and quercetin are believed to provide protection against ultraviolet (UV)-induced damage to plants. Recent in vitro studies have examined the ability of flavonols to protect against UV-induced damage to mammalian cells. Stability of flavonols in cell culture media, however, has been problematic, especially for quercetin, one of the most widely studied flavonols. As part of our investigations into the potential for flavonols to protect skin against UV-induced damage, we have determined the stability of a series of flavonols that differ only in the number of substituents on the B-ring. We measured the stability of these flavonols over time to UVA radiation, Dulbecco's modified Eagle's medium (DMEM), and Dulbecco's phosphate-buffered saline (DPBS) using high performance liquid chromatography with UV detection (HPLC-UV). The identification of the breakdown products of flavonols was accomplished by using a hybrid quadrupole linear ion trap mass spectrometer coupled with liquid chromatography. Tandem mass spectrometric analysis (MS/MS) of flavonol photoproducts was confirmed by comparing with the known standard samples. We have determined that flavonol stability decreases with increasing B-ring substitution, suggesting that future investigation of potential photoprotective flavonols will need to be cognizant of this trend.     

Characterization of flavonol conjugates in immature leaves of pak choi [Brassica rapa L. Ssp. chinensis L. (Hanelt.)] by HPLC-DAD and LC-MS/MS

The flavonoid composition of immature leaves of pak choi [Brassica rapa L. ssp. chinensis L. (Hanelt.)] was investigated. Flavonol aglycone content was measured in 11 pak choi varieties, indicating significant differences (P < 0.05) in content between varieties and relatively high contents of kaempferol and isorhamnetin. Levels of quercetin ranged from 3.2 to 6.1 mg/100 g of dry weight (DW), whereas levels of isorhamnetin and kaempferol were significantly higher (8.1-35.1 and 36.0-102.6 mg/100 g of DW, respectively). A large number of glycoside and hydroxycinnamic acid derivatives of quercetin, kaempferol, and isorhamnetin were identified in cv. 'Shanghai' by LC/UV-DAD/ESI-MS/MS. The UV-DAD data allowed identification of hydroxycinnamic acid derivatives, but detailed MS/MS fragmentations were required for the structure elucidation. Pak choi could be a potentially important source of dietary flavonols, in particular, kaempferol and isorhamnetin.     

Investigation of flavonoids bearing different substituents on ring C and their cu2+ complex binding with bovine serum albumin: structure-affinity relationship aspects

The effects of 1:1 flavonoid-Cu(2+) complexes of four flavonoids with different C-ring substituents, quercetin (QU), luteolin (LU), taxifolin (TA), and (+)-catechin (CA), on bovine serum albumin (BSA) were investigated and compared with corresponding free flavonoids by spectroscopic analysis in an attempt to characterize the chemical association taking place. The results indicated that all of the quenching mechanisms were based on static quenching combined with nonradiative energy transfer. Cu(2+) chelation changed the binding constants for BSA depending on the structures of flavonoids and the detected concentrations. The reduced hydroxyl groups, increased steric hindrance, and hydrophilicity of Cu(2+) chelation may be the main reasons for the reduced binding constants, whereas the formation of stable flavonoid-Cu(2+) complexes and synergistic action could increase the binding constants. The changed trends of critical energy transfer distance (R(0)) for Cu(2+) chelation were contrary to those of binding constants.     

Polymorphism for novel tetraglycosylated flavonols in an Eco-model crucifer, Barbarea vulgaris

Nineteen apparent flavonoids were determined by HPLC-DAD in foliage of a chemotype (G-type) of Barbarea vulgaris , and four were isolated. Two were novel tetraglycosylated flavonols with identical glycosylation patterns, kaempferol 3-O-(2,6-di-O-β-d-glucopyranosyl)-β-d-glucopyranoside-7-O-α-l-rhamnopyranoside (1) and quercetin 3-O-(2,6-di-O-β-d-glucopyranosyl)-β-d-glucopyranoside-7-O-α-l-rhamnopyranoside (2). The identification of d/l configuration was tentatively based on susceptibility to α-l-rhamnosidase and β-d-glucosidases. A characteristic feature of 1 and 2 was appreciable water solubility, an expected consequence of the extensive glycosylation. A less complex pair of flavonols comprised 3-O-β-d-glucopyranoside-7-O-α-l-rhamnopyranosides of kaempferol and quercetin. Two natural chemotypes of B. vulgaris differed in levels of 1 and 2, with the P-type deficient in 1 and 2 and the insect-resistant G-type rich in 1 (ca. 3-4 μmol/g dry wt) and with moderate levels of 2 (ca. 0.3-0.8 μmol/g dry wt). However, there was only modest seasonal variation in flavonols 1 and 2, in contrast to a strong seasonal variation in insect resistance.