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The establishment of a reliable and sensitive method for the detection of flavonoids, such as kaempferol (Kae) and quercetin (Que), is important and challenging in food chemistry and pharmacology because numerous structural analogues may interfere with the detection. Until now, designing an efficient switch-on fluorescence sensing strategy for Kae and Que was still in the unachievable stage. In this work, a switch-on near-infrared (NIR) luminescence sensing assay for Kae and Que was fabricated based on a metal-organic framework (MOF) called IQBA-Yb for the first time. The fluorescence enhancing mechanism was that analytes served as additional "antenna" of Yb 3+ , leading to the efficient switch-on NIR emission under excitation at 467 nm. Meanwhile, the combination results of experiment and theoretical calculation revealed that there existed hydrogen bonds between Kae, Que, and the MOF skeleton, further promoting the energy transfer between the analyte and Yb 3+ and facilitating fluorescence enhancement response. The developed probe possessed excellent sensing capability for Kae and Que, accompanied by a wide linear range (0.04-70, 0.06-90 μM), low detection limit (0.01, 0.06 μM), and short response time (20 min, 6 min), which was used to determine the Kae and Que contents in Green Lake and eatable Que samples with satisfactory results.

Because of the rapid increase in bacterial resistance, there is an urgent need for developing new antimicrobial agents to combat multidrug-resistant pathogens. In this study, we designed and synthesized a series of kaempferol derivatives as antimicrobial agents biomimicking the structural properties and biological functions of host defense peptides. After fine-tuning of hydrophobic and cationic hydrophilic moieties linked to the flavone scaffold of kaempferol, we obtained a lead compound ( 52 ) that displayed high membrane selectivity (>128), poor hemolytic activity, low cytotoxicity to mammalian cells, and excellent activity against Gram-positive bacteria (minimum inhibitory concentrations = 1.56 μg/mL), including methicillin-resistant Staphylococcus aureus . Compound 52 can kill bacteria quickly by destroying the bacterial membranes and avoid developing bacterial resistance. Moreover, compound 52 exhibited potent in vivo antibacterial activity against S. aureus in a murine corneal infection model. These results indicated that compound 52 had the therapeutic potential as a novel membrane-active antimicrobial to combat Gram-positive bacterial infections.

Oilseed rape ( Brassica napus L.) is the second highest yielding oil crop worldwide. In addition to being used as an edible oil and a feed for livestock, rapeseed has high ornamental value. In this study, we identified and characterized the main floral major constituents, including phenolic acids and flavonoids components, in rapeseed accessions with different-colored petals. A total of 144 constituents were identified using ultrahigh-performance liquid chromatography-HESI-mass spectrometry (UPLC-HESI-MS/MS), 57 of which were confirmed and quantified using known standards and mainly contained phenolic acids, flavonoids, and glucosinolates compounds. Most of the epicatechin, quercetin, and isorhamnetin derivates were found in red and pink petals of B. napus , while kaempferol derivates were in yellow and pale white petals. Moreover, petal-specific compounds, including a putative hydroxycinnamic acid derivative, sinapoyl malate, 1- O -sinapoyl-β-d-glucose, feruloyl glucose, naringenin-7- O -glucoside, cyanidin-3-glucoside, cyanidin-3,5-di- O -glucoside, petunidin-3- O -β-glucopyranoside, isorhamnetin-3- O -glucoside, kaempferol-3- O -glucoside-7- O -glucoside, quercetin-3,4'- O -di-β-glucopyranoside, quercetin-3- O -glucoside, and delphinidin-3- O -glucoside, might contribute to a variety of petal colors in B. napus . In addition, bound phenolics were tentatively identified and contained three abundant compounds ( p -coumaric acid, ferulic acid, and 8- O -4'-diferulic acid). These results provide insight into the molecular mechanisms underlying petal color and suggest strategies for breeding rapeseed with a specific petal color in the future.

By means of activity-guided fractionation using taste dilution analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography-time-of-flight mass spectrometry, one-/two-dimensional nuclear magnetic resonance spectroscopy, LC-MS/MS quantitation, dose-over-threshold considerations, and sensory spiking experiments, kaempferol 3- O-(2‴- O-sinapoyl-β-sophoroside), exhibiting a bitter taste above the low threshold concentration of 3.4 μmol/L, was found for the first time as the key molecule contributing to the unpleasant bitter taste of rapeseed (canola) protein isolates. This finding opens new avenues for a biorefinery approach targeting an off-taste removal.

Human colonic bacteria have an important impact on the biotransformation of flavonoid glycosides and their conversion can result in the formation of bioactive compounds. However, information about the microbial conversion of complex glycosylated flavonoids and the impact on the gut microbiota are still limited. In this study, in vitro fermentations with selected flavonoid O- and C-glycosides and three different fecal samples were performed. As a result, all flavonoid glycosides were metabolized via their aglycones yielding smaller substances. Main metabolites were 3-(4-hydroxyphenyl)propionic acid, 3-phenylpropionic acid, and phenylacetic acid. Differences in the metabolite formation due to different time courses between the donors were determined. Therefore, from all fermentations, the ones with a specific donor were always slower resulting in a lower number of metabolites compared to the others. For example, tiliroside was totally degraded from 0 h (105 ± 13.2 μM) within the first 24 h, while in the fermentations with fecal samples from other donors, tiliroside (107 ± 52.7 μM at 0 h) was not detected after 7 h anymore. In general, fermentation rates of C-glycosides were slower compared to the fermentation rates of O-glycosides. The O-glycoside tiliroside was degraded within 4 h while the gut microbiota converted the C-glycoside vitexin within 13 h. However, significant changes (p < 0.05) in the microbiota composition and short chain fatty acid levels as products of carbohydrate fermentation were not detected between incubations with different phenolic compounds. Therefore, microbiota diversity was not affected and a significant prebiotic effect of phenolic compounds cannot be assigned to flavonoid glycosides in food-relevant concentrations.

Kaempferol-3-O-β-d-glucopyranoside (astragalin, AS), a major flavonoid that exists in various plants, exerts antioxidant, antitumor, anti-human immunodeficiency virus (HIV), and anti-inflammatory effects. However, the low water solubility of AS limits its use. In this study, we used cyclodextrin glucanotransferase (CGTase) with maltose (G2) as a donor molecule to enzymatically modify AS to improve its water solubility and physiochemical properties. We isolated the glycosylated astragalin (G1-AS) and identified the structure of G1-AS as kaempferol-3-O-β-d-glucopyranosyl-(1→4)-O-α-d-glucopyranoside, where one glucose residue was transferred to AS. G1-AS retained the antioxidative activity of the original AS compound; however, the solubility of G1-AS was 65-fold higher than that of AS. In addition, G1-AS showed enhanced anti-inflammatory effects and aldose reductase inhibitory activity compared to AS when applied to rat lenses.

To study the effect of metabolic conjugation of flavonoids on the potential to inhibit protein kinase activity, the inhibitory effects of the dietary flavonol kaempferol and its major plasma conjugate kaempferol-3-O-glucuronide on protein kinases were studied. To this end, the inhibition of the phosphorylation activity of recombinant protein kinase A (PKA) and of cell lysate from the hepatocellular carcinoma cell line HepG2 on 141 putative serine/threonine phosphorylation sites derived from human proteins was assessed. Glucuronidation reduced the inhibitory potency of kaempferol on the phosphorylation activity of PKA and HepG2 lysate on average about 16 and 3.5 times, respectively, but did not appear to affect the target selectivity for kinases present in the lysate. The data demonstrate that, upon glucuronidation, kaempferol retains part of its intrinsic kinase inhibition potential, which implies that K3G does not necessarily need to be deconjugated to the aglycone for a potential inhibitory effect on protein kinases.

The purpose of this work was to determine the kaempferol content in three red wines of Calabria, a southern Italian region with a great number of certified food products. Considering that wine cultivar, climate, and soil influence the qualitative and quantitative composition in flavonoids of Vitis vinifera L. berries, the three analyzed samples were taken from the 2013 vintage. Moreover, the Gaglioppo samples, with assigned Controlled Origin Denomination (DOC), were also investigated in the production of years 2008, 2010, and 2011. In addition to the analysis of kaempferol, which is present in higher concentration than in other Italian wines, in vitro assays were performed to evaluate, for the first time, the inhibition of the human monoamine oxidases (hMAO-A and hMAO-B). Molecular recognition studies were also carried out to provide insight into the binding mode of kaempferol and selectivity of inhibition of the hMAO-A isoform.

Death-associated protein kinase 1 (DAPK1) is a 160 kDa serine/threonine protein kinase that belongs to the Ca(2+)/calmodulin-dependent protein kinase subfamily. DAPK1 is a possible target for the treatment of acute ischemic stroke and endometrial adenocarcinomas. In the present study, we investigated the binding characteristics of 17 natural flavonoids to DAPK1 using a 1-anilinonaphthalene-8-sulfonic acid competitive binding assay and revealed that morin was the strongest binder among the selected compounds. The crystallographic analysis of DAPK1 and 7 selected flavonoid complexes revealed the structure-binding affinity relationship in atomic-level detail. It was suggested that the high affinity of morin could be accounted for by the ionic interaction between 2'-OH and K42 and that such an interaction would not take place with either cyclin-dependent protein kinases or PIM kinases because of their broader entrance regions. Thus, morin would be a more selective inhibitor of DAPK1 than either of these other types of kinases. In addition, we found that the binding of kaempferol to DAPK1 was associated with a chloride ion. The present study provides a better understanding of the molecular properties of the ATP site of DAPK1 and may be useful for the design of specific DAPK1 inhibitors.

Xanthine oxidase (XO), a key enzyme in purine catabolism, is widely distributed in human tissues. It can catalyze xanthine to generate uric acid and cause hyperuricemia and gout. Inhibition kinetics assay showed that kaempferol inhibited XO activity reversibly in a competitive manner. Strong fluorescence quenching and conformational changes of XO were found due to the formation of a kaempferol-XO complex, which was driven mainly by hydrophobic forces. The molecular docking further revealed that kaempferol inserted into the hydrophobic cavity of XO to interact with some amino acid residues. The main inhibition mechanism of kaempferol on XO activity may be due to the insertion of kaempferol into the active site of XO occupying the catalytic center of the enzyme to avoid the entrance of the substrate and inducing conformational changes of XO. In addition, luteolin exhibited a stronger synergistic effect with kaempferol than did morin at the lower concentration.

Flavonoids are polyphenolic compounds required in the fertilization process in many, if not all, plants. However, the exact biological mechanism(s) and the interacting proteins are unknown. To determine the characteristics important in activating or inhibiting the pollination sequence, a structure-activity relationship analysis of natural and synthetic flavonols was conducted. Flavonol analogues were synthesized through a modified "one-pot" procedure that utilized a Baker-Venkataraman type rearrangement and a Suzuki-Miyaura cross-coupling of a halo-flavonol with an organotrifluoroborate. Of the flavonols tested, kaempferol was the only compound to act as a full agonist. The other smaller, less sterically hindered flavonols (galangin, kaempferide, and 4'-methyl flavonol) acted as partial agonists. Larger more hydrophobic flavonol analogues (3'- and 4'-benzoyl, 3'- and 4'-phenyl, and 3'- and 4'-iodo flavonols) had minimal or no agonist activity. Competition assays between kaempferol and these minimally activating flavonols showed that these analogues inhibited the action of kaempferol in a manner consistent with noncompetitive antagonism. The results suggest that steric hindrance is the most important factor in determining a good agonist. Hydrogen bonding also had a positive effect as long as the substituent did not cause any steric hindrance.

The utilization of kaempferol and its glycosides in food and pharmaceutical industries could be improved by the formation of inclusion complexes with cyclodextrins at different pH. This study explores the complexation of kaempferol-4'-glucoside with sulfobutyl ether-β-cyclodextrin (SBE-β-CD), hydroxypropyl-β-cyclodextrin (HP-β-CD), and methylated-β-cyclodextrin (M-β-CD) in phosphate buffer solutions of different pH using isothermal titration calorimetry, UV-vis absorption and proton nuclear magnetic resonance spectroscopy at 298.2 K. Experimental results showed that kaempferol-4'-glucoside binds with the three β- cyclodextrins in the same 1:1 stoichiometry. The rank order of stability constants is SBE-β-CD > HP-β-CD > M-β-CD at the same pH level and pH 6.0 > pH 7.4 > pH 9.0 for the same cyclodextrin. The binding of kaempferol-4'-glucoside with the three β-cyclodextrin derivatives is synergistically driven by enthalpy and entropy at pH 6.0 and enthalpy-driven at pH 7.4 and 9.0. The possible inclusion mode was that in the cavity of β-CD is included the planar benzopyranic-4-one part of the kaempferol-4'-glucoside.

Flavonoids are polyphenolic compounds with various claimed health benefits, but the extensive metabolism by uridine-5'-diphospho-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) in liver and intestine led to poor oral bioavailabilities. The effects of structural changes on the sulfonation of flavonoids have not been systemically determined, although relevant effects of structural changes on the glucuronidation of flavonoids had. We performed the regiospecific sulfonation of sixteen flavonoids from five different subclasses of flavonoids, which are represented by apigenin (flavone), genistein (isoflavone), naringenin (flavanone), kaempherol (flavonol), and phloretin (chalcone). Additional studies were performed using 4 monohydroxyl flavonoids with a -OH group at the 3, 4', 5 or 7 position, followed by 5 dihydroxyl flavonoids, and 2 trihydroxyl flavonoids by using expressed human SULT1A3 and Caco-2 cell lysates. We found that these compounds were exclusively sulfated at the 7-OH position by SULT1A3 and primarily sulfated at the 7-OH position in Caco-2 cell lysates with minor amounts of 4'-O-sulfates formed as well. Sulfonation rates measured using SULT1A3 and Caco-2 cell lysates were highly correlated at substrate concentrations of 2.5 and 10 μM. Molecular docking studies provided structural explanations as to why sulfonation only occurred at the 7-OH position of flavones, flavonols and flavanones. In conclusion, molecular docking studies explain why SULT1A3 exclusively mediates sulfonation at the 7-OH position of flavones/flavonols, and correlation studies indicate that SULT1A3 is the main isoform responsible for flavonoid sulfonation in the Caco-2 cells.

Kaempferol (KAE) is a strong antioxidant flavonoid compound, but its clinical application is limited by quantity and poor dissolution property. However, the dissolution mechanism of a kaempferol nanoparticle formulation (KAEN) has not yet been elucidated. The aim of the present study was therefore to use a nanoparticle engineering process to resolve the dissolution problem. Our data indicated that KAEN effectively increased the dissolution percentage by particle size reduction, high encapsulation efficiency, amorphous transformation, and hydrogen-bond formation with excipients. In addition, we used several different antioxidant activity assays to evaluate KAE and KAEN. The data indicated that KAEN retained potent antioxidant activity after the nanoparticle engineering process and showed better antioxidant activity than KAE dissolved in water (P < 0.05). According to these findings, we concluded that KAEN could be a low-dose alternative to KAE in health food and future clinical research.

A mechanochemical-assisted extraction (MCAE) method was proposed and investigated for the fast extraction of two kaempferol glycosides (kaempferol-3-O-[2-O-β-D-galactopyranosyl-6-O-α-L-rhamnopyranosyl]-β-D-glucopyranoside and kaempferol-3-O-[2-O-β-D-xylopyranosyl-6-O-α-L-rhamnopyranosyl]-β-D-glucopyranoside) from Camellia oleifera Abel. meal. The effects of operating parameters in terms of NaOH content, grinding time, extraction time, and ratio of solution to solid were evaluated by means of response surface methodology (RSM). Under the optimal conditions with a ratio of material to NaOH of 20:1 (g/g), a milling time of 15 min, and a ratio of solution to solid of 20:1 (mL/g) for 60 min, the maximum extraction yields of the two kaempferol glycosides reached 13.34 and 13.83%, respectively. The antioxidant activity of kaempferol glycosides extract was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay and ferric thiocyanate (FTC) assay. Compared with the heat reflux extraction (HRE) method, the yield and the antioxidant activities of the extracts from MCAE with water as solvent were higher and stronger.

Kaempferol 3-O-(3'',6''-di-O-E-p-coumaroyl)-β-D-glucopyranoside (1), an optimal metabolite of Scots pine seedlings for protection of deep-lying tissue against damaging UV-B, represents a typical acylated flavonol 3-O-glycoside. This compound was synthesized for the first time via two approaches. The first approach, starting with kaempferol, featured formation of the flavonol 3-O-glycosidic linkage with a glycosyl bromide under conventional PTC conditions. In the second approach, 5,7,4'-tri-O-benzyl-kaempferol was readily prepared from 2',4',6'-trihydroxyacetophenone and p-hydroxybenzoic acid, which was coupled with a glucopyranosyl o-hexynylbenzoate under the catalysis of a gold(I) complex to provide the desired 3-O-glycoside in excellent yield. A variety of the glycosyl o-hexanylbenzoates equipped with the 2-O-benzoyl group were also proven to be highly efficient donors for construction of the flavonol 3-O-glycosidic linkages.

The complexation of kaempferol with cyclodextrins (CDs) (beta-, G(2)-beta-, and HP-beta-CDs) in basic medium was studied, and the complexation constants (K(c)) were calculated by using enzymatic, solubility, and fluorometric methods. This is the first time that a decrease in fluorescence has been observed as result of the analyte complexation by CDs. The highest K(c) value for kaempferol complexation was obtained for HP-beta-CDs. To establish the validity of the fluorometric method for determining the K(c) between kaempferol and CDs, the same parameters were also determined by enzymatic and solubility methods. The enzymatic method was carried out by using horseradish peroxidase as oxidative enzyme, and the K(c) values obtained were similar to those obtained by using the solubility method. However, the fluorometric method underestimated the K(c) value by about 1.2-fold with respect to the other methods used. In all cases HP-beta-CDs showed the highest K(c) value, indicating that they are more efficient in the formation of inclusion complexes with kaempferol.

A strategy to assemble cells on a solid support surface, here the surface of a gold electrode, is developed by transfecting cells with thiolated DNA molecules which have been immobilized on the gold electrode surface in advance. This strategy to assemble cells can present a general and convenient method for cell assembly on a solid support surface. What is more interesting, since efficient electric communication via the thiolated DNA between the electroactive species inside the cells and the substrate electrode can be achieved, an approach to "look" into the inner part of the cells is proposed. For the test in this work, one drug, kaempferol, and one dye molecule, methylene blue, inside the cells have been detected by the commonly used electrochemical method linear scan voltammetry, and satisfactory results have been obtained.

Atherosclerosis is a chronic inflammatory disease of the arterial wall. Kaempferol and rhamnocitrin (kaempferol 7-O-methyl ether) are two anti-inflammatory flavonoids commonly found in plants. The aim of this study is to investigate the function of kaempferol and rhamnocitrin on prevention of atherosclerosis. Chemical analyses demonstrated that kaempferol and rhamnocitrin were scavengers of DPPH (1,1-diphenyl-2-picrylhydrazyl) with IC50 of 26.10 +/- 1.33 and 28.38 +/- 3.07 microM, respectively. Copper-induced low-density lipoprotein (LDL) oxidation was inhibited by kaempferol and rhamnocitrin, with similar potency, as measured by decreased formation of malondialdehyde and relative electrophoretic mobility (REM) on agarose gel, while rhamnocitrin reduced delayed formation of conjugated dienes better than kaempferol. Cholesterol-laden macrophages are the hallmark of atherogenesis. The class B scavenger receptor, CD36, binds oxidized low-density lipoprotein (oxLDL), is found in atherosclerotic lesions, and is up-regulated by oxLDL. Addition of kaempferol and rhamnocitrin (20 microM) caused significant reductions in cell surface CD36 protein expression in THP-1-derived macrophages (p < 0.05). Reverse transcription quantitative PCR (RT-Q-PCR) showed that kaempferol and rhamnocitrin (20 microM) decreased oxLDL-induced CD36 mRNA expression (p < 0.01 and p < 0.05, respectively). Kaempferol- and rhamnocitrin-treated macrophages also showed reduction in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide perchlorate (DiI)-labeled oxLDL uptake. Current evidences indicate that kaempferol and rhamnocitrin not only protect LDL from oxidation but also prevent atherogenesis through suppressing macrophage uptake of oxLDL.

A short synthesis of Kaempferitrin (1), a 3,7-diglycosylflavone, is reported. Key features include the synthesis of a protected form of kaempferol in which all four hydroxy groups are differentiated and the first bis-glycosylation of a dihydroxyflavone. This synthesis will allow the preparation of derivatives for further explorations into the origins of this compound's biological activity.

Four different colored beans (white, red, pinto, and black beans) were investigated for factors affecting iron bioavailability using an in vitro digestion/human Caco-2 cell model. Iron bioavailability from whole beans, dehulled beans, and their hulls was determined. The results show that white beans contained higher levels of bioavailable iron compared to red, pinto, and black beans. These differences in bioavailable iron were not due to bean-iron and bean-phytate concentrations. Flavonoids in the colored bean hulls were found to be contributing to the low bioavailability of iron in the non-white colored beans. White bean hulls contained no detectable flavonoids but did contain an unknown factor that may promote iron bioavailability. The flavonoids, kaempferol and astragalin (kaempferol-3-O-glucoside), were identified in red and pinto bean hulls via HPLC and MS. Some unidentified anthocyanins were also detected in the black bean hulls but not in the other colored bean hulls. Kaempferol, but not astragalin, was shown to inhibit iron bioavailability. Treating in vitro bean digests with 40, 100, 200, 300, 400, 500, and 1000 microM kaempferol significantly inhibited iron bioavailability (e.g., 15.5% at 40 microM and 62.8% at 1000 microM) in a concentration-dependent fashion. Thus, seed coat kaempferol was identified as a potent inhibitory factor affecting iron bioavailability in the red and pinto beans studied. Results comparing the inhibitory effects of kaempferol, quercitrin, and astragalin on iron bioavailability suggest that the 3',4'-dihydroxy group on the B-ring in flavonoids contributes to the lower iron bioavailability.

The work investigates the possibility of the protective action of kaempferol on phosphatidylcholine liposome membranes exposed to the pro-oxidative action of diphenyltin dichloride (DPhT) and triphenyltin chloride (TPhT) induced by UV radiation (lambda = 253.7 nm). The concentrations of kaempferol and its equimolar mixtures with DPhT and TPhT were determined so that they induce 50% inhibition in oxidation of liposomes irradiated with UV. They are 11.6, 10.0, and 4.5 microM/L, which constitute the following sequence of antioxidative activity: kaempferol/triphenyltin > kaempferol/diphenyltin > kaempferol. This relationship is confirmed by the results on the antiradical ability of kaempferol and its mixtures with DPhT and TPhT toward the free radical 2,2-diphenyl-1-picrylhydrazyl. Similar sequences obtained in both studies suggest a possible mechanism of the antiradical action of the mixtures as free radical scavengers. Kaempferol's ability, then documented, to form complexes with phenyltins indicates (a) a possible way to liquidate the peroxidation caused by the free radical forms of phenyltins and (b) the stabilizing role of chelating in the antioxidative action of the kaempferol/phenyltins. The differentiation in the action of the compounds studied may, among others, result from different localizations in the liposome membrane, which is indicated by the results of the fluorometric studies.