Effect of Hydrogenation on Ring C of Flavonols on Their Affinity for Bovine Serum Albumin

In this paper, the effect of hydrogenation on ring C of flavonols on the affinity for bovine serum albumin was investigated. Two differently substituted B-ring hydroxylation flavonols (myricetin and quercetin) and their dihydrides (dihydromyricetin and dihydroquercetin) were used to study their affinities for BSA by quenching the intrinsic BSA fluorescence in solution. From the spectra, the bimolecular quenching constants, the binding constants, the number of binding sites and the binding distances were calculated. The hydroxylation on ring B and hydrogenation on ring C of flavonols significantly affected the binding/quenching process; in general, the hydroxylation increased the affinity and the hydrogenation decreased the affinity. For myricetin and quercetin, the binding constants (K _a) for BSA were 1.84×10⁸ L⋅mol⁻¹ and 3.83×10⁷ L⋅mol⁻¹. For dihydromyricetin, the binding constant was 1.36×10⁴ L⋅mol⁻¹, while dihydroquercetin hardly quenched the BSA intrinsic fluorescence. These results showed that hydrogen bonding and conjugative effects may play an important role in binding of flavonols to BSA. These results also showed that the properties of flavonols are related to their chemical structure.     

Inclusion complexes of modified cyclodextrins with some flavonols

The inclusion complexes of four flavonols with modified cyclodextrins (CDs) have been investigated. The effect of heptakis (2,6-di-O-methyl) β-cyclodextrin (DM-β-CD) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) on the aqueous solubility of flavonols, namely, galangin, kaempferol, quercetin, and myricetin was investigated, respectively. The increased solubility of all flavonols in the presence of CD was evidenced. The NMR experiment and molecular modeling studies showed that flavonols interact with each modified CD through different binding modes. Flavonols can complex with CDs largely by two binding modes. The first one is that B-ring of flavonols is oriented toward secondary rim of CD. The second one is that A-ring of flavonols is oriented toward secondary rim of CD. Whereas only the first mode was observed in DM-β-CD complexes, both the first and the second mode were observed in HP-β-CD complexes in this study.     

高效液相色谱法测定野葱中黄酮类化合物

建立了测定野葱中芦丁、黄酮醇类化合物的高效液相色谱方法。80%乙醇超声提取,高效液相色谱分析野葱中芦丁和黄酮醇类化合物的含量。芦丁检测条件:V(甲醇)∶V(0.2%磷酸水)=45∶55,检测波长:360nm;槲皮素、山奈酚、异鼠李素检测条件:V(甲醇)∶V(0.2%磷酸水)=40∶60,检测波长:360 nm。结果表明,野葱中芦丁含量0.22%,槲皮素、山奈酚、异鼠李素含量依次为0.42%、0、0.23%,总黄酮醇类化合物含量为1.63%。     

SUPPRESSION OF LIPID PHOTOPEROXIDATION BY QUERCETIN AND ITS GLYCOSIDES IN SPINACH CHLOROPLASTS

Abstract— Quercetin, quercitrin and rutin suppressed lipid photoperoxidation in spinach chloroplasts in the presence of 100 μ M carbonylcyanide m -chlorophenylhydrazone (CCCP) or 100 μ M methyl viologen (MV). Fifty percent inhibition of lipid peroxidation by quercetin was observed between 30 and 50 μ M . Concentrations of quercetin and rutin higher than 100 μ M were required to obtain 50% inhibition. Ouercitrin was more effective than rutin in the suppression of lipid photoperoxidation.
Photooxidation of the flavonols by chloroplasts in the presence of MV was suppressed by superoxide dismutase (SOD) more than 90%, and the rates of the oxidation decreased in order of quercetin, quer citrin and rutin suggesting that the reactivity of the flavonols with O₂-decreased in that order. The photooxidation of the flavonols by CCCP-poisoned chloroplasts was partially suppressed by SOD. Radicals generated in the course of lauroyl peroxide degradation also oxidized the flavonols and the oxidation was insensitive to SOD. In these experiments, oxidation rate of quercetin was faster than those of its glycosides. The results obtained suggest that flavonols can function as antioxidants in chloroplasts by scavenging both O₂-and the radicals formed during lipid peroxidation.     

Density‐functional theory study of the interaction mechanism and optical properties of flavonols on the boron nitride nanotubes

The flavonols are natural pigments with multiple colors. They are found ubiquitously in plants and are relevant to flower colors and the UV protection in plants. Their antioxidant, anticancer, and anti‐allergic features attract researchers much attention to explore their potential applications in biological and nanomedical areas. In this study, the interaction mechanism and optical properties of four representative flavonols, on both the surface and confined in the single‐walled boron nitride nanotubes (BNNTs), have been explored comparatively by self‐consistent density‐functional based tight‐binding method (SCC‐DFTB) and density‐functional theory (DFT). The results indicate a stronger binding when flavonols are confined inside the BNNTs. The influence of mutual interaction between flavonols and BNNTs on the excited properties and UV/vis feature of the complex structure was studied by time‐dependent DFT. Due to the interaction of flavonols with BNNTs and the weakness of the intramolecular hydrogen bond, our results indicate a red‐shift of the flavonol spectra when they are outside or inside the tube. The study concludes that the properties of flavonols can be fine‐tuned by the interaction with BNNTs.     

Antioxidative effects of flavonols and their glycosides against the free-radical-induced peroxidation of linoleic acid in solution and in micelles

The antioxidative effect of flavonols and their glycosides against the peroxidation of linoleic acid has been studied in homogeneous solution (tBuOH/H(2)O, 3:2) and in sodium dodecyl sulfate and cetyl trimethylammonium bromide micelles. The peroxidation was initiated thermally by the water-soluble initiator 2,2'-azobis(2-methylpropionamidine) dihydrochloride, and the reaction kinetics were studied by monitoring the formation of linoleic acid hydroperoxides. The synergistic antioxidant effect of the flavonols with alpha-tocopherol (vitamin E) was also studied by following the decay kinetics of alpha-tocopherol and the alpha-tocopheroxyl radical. Kinetic analysis of the antioxidative process demonstrates that the flavonols are effective antioxidants in solution and in micelles, either alone or in combination with alpha-tocopherol. The antioxidative action involves trapping the initiating radicals in solution or in the bulk-water phase of the micelles, trapping the propagating lipid peroxyl radicals on the surface of the micelles, and regenerating alpha-tocopherol by reducing the alpha-tocopheroxyl radical. It was found that the antioxidant activity of the flavonols and their glycosides depends significantly on the position and number of the hydroxy groups, the oxidation potential of the molecule, and the reaction medium. The flavonols bearing ortho-dihydroxy groups possess significantly higher antioxidative activity than those without such functionalities, and the glycosides are less active than their parent aglycones. The activity of the flavonols is higher in micelles than in solution, while the activity of alpha-tocopherol is lower in micelles than in solution. This is because the predominant factor for controlling the activity is the hydrogen-bonding interaction of the antioxidant with the micellar surface in the case of hydrophilic flavonols, while it is the inter- and intramicellar diffusion in the case of lipophilic alpha-tocopherol.     

Flavonoids inhibiting glycation of bovine serum albumin: affinity-activity relationship

Protein glycation leads to the formation of advanced glycation end-products (AGEs), which contribute to the pathogenesis of diabetic complications. The structure-activity relationship of dietary flavonoids for inhibiting the glycation of bovine serum albumin (BSA) in vitro was subjected to a detailed investigation. The structure-activity relationship revealed that: 1) the hydroxylation on ring B of the flavones enhanced the inhibition and the hydroxyl groups at the C-5 and C-7 positions of flavones favoured the inhibition; 2) the optimal number of hydroxyl groups on ring B of the flavonols was one (at the C-3 position) and the methylation of flavonols weakened the inhibition; 3) the methoxylation at the C-6 position and methylation at C-4′ position of genistein clearly enhanced the inhibition; 4) the hydroxyl groups at the C-5 and C-7 positions of flavanones were in favour of the inhibition; 5) the glycosylation of flavonoids significantly weakened the inhibition. Obvious linear affinity-activity relationships exist between the BSA-flavonoid interaction and flavonoids as BSA glycation inhibitors (R² = 0.76585). The flavonoids with a higher affinity to BSA exhibited a stronger inhibition of the glycation of BSA.     

SEPARATION OF THE MINOR FLAVONOLS FROM FLOS GOSSYPII BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

An effective high-speed countercurrent chromatography (HSCCC) method was established for further separation and purification of four minor flavonols in addition to five major flavonols which were reported by our previous study from extracts of Flos Gossypii. HSCCC was performed with three two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (7.5:15:6:7, v/v), (2.5:15:2:7, v/v) and (0:1:0:1, v/v). The separation was repeated 3 times, and 3.8 mg of 8-methoxyl-kaempferol-7-O-β-D-rhamnoside (HPLC purity 98.27%), 6.7 mg of astragalin (HPLC purity 94.18%), 3.3 mg of 4'-methoxyl-quercetin-7-O-β-D-glucoside (HPLC purity 94.30%) and 8.2 mg of hyperoside (HPLC purity 93.48%) were separated from 150 mg of the crude sample. The chemical structures of the flavonols were confirmed by MS, (1)H NMR and (13)C NMR. Meanwhile, the results indicated that the target compound with smaller K value (<0.5) can be separated by increasing column length of HSCCC. And four separation rules of flavonols according to the present study and references were summarized, which can be used as a useful guide for separation of flavonols by HSCCC.     

Profiling of In Vitro Bioaccessibility and Intestinal Uptake of Flavonoids after Consumption of Commonly Available Green Tea Types

The aim of this study was to profile the bioaccessibility and intestinal absorption of epicatechins and flavonols in different forms of green tea and its formulation: loose leaf tea, powdered tea, 35% catechins containing GTE, and GTE formulated with green tea-derived polysaccharide and flavonols (CATEPLUS™). The bioaccessibillity and intestinal absorption of epicatechins and flavonols was investigated by using an in vitro digestion model system with Caco-2 cells. The bioaccessibility of total epicatechins in loose leaf tea, powdered tea, GTE, and CATEPLUS™ was 1.27%, 2.30%, 22.05%, and 18.72%, respectively, showing that GTE and CATEPLUS™ had significantly higher bioaccessibility than powdered tea and loose leaf tea. None of the flavonols were detected in powdered tea and loose leaf tea, but the bioaccessibility of the total flavonols in GTE and CATEPLUS™ was 85.74% and 66.98%, respectively. The highest intestinal absorption of epicatechins was found in CATEPLUS™ (171.39 ± 5.39 ng/mg protein) followed by GTE (57.38 ± 9.31), powdered tea (3.60 ± 0.67), and loose leaf tea (2.94 ± 1.03). The results from the study suggest that formulating green tea extracts rich in catechins with second components obtained from green tea processing could enhance the bioavailability of epicatechins.     

Biological Activity of Extracts of Red and Yellow Fruits of Cornus mas L.—An In Vitro Evaluation of Antioxidant Activity,Inhibitory Activity against α-Glucosidase,Acetylcholinesterase, and Binding Capacity to Human Serum Albumin

Although extracts are broadly used in order to support the treatment of numerous diseases, only in a limited number of cases is the process of applying and establishing their mechanisms of action scientifically analyzed. Fruits of Cornelian cherry are an abundant source of iridoids, anthocyanins, flavonols and phenolic acids. The aim of the present study was to evaluate the in vitro bioactivity of red and yellow Cornelian cherry fruits’ extracts. The biological potential of extracts, in a broad sense, involved antioxidant activity in relation to phosphatidylcholine liposomes, inhibitory ability against α-glucosidase and acetylcholinesterase enzymes, as well as interactions with human serum albumin. Studies showed that both extracts were more effective in protecting liposome membranes against free radicals produced by AAPH in an aqueous environment due to the fact that they can be better eliminated by the hydrophilic components of the extracts than those produced by UVB radiation. Extracts exhibited inhibitory activity against acetylcholinesterase and α-glucosidase, wherein loganic acid extract showed noncompetitive inhibition of the enzyme. Moreover, extracts binded to albumin mainly through hydrogen bonds and van der Waals forces. Taken together, red and yellow cherry fruits’ extracts exhibit diverse biological properties and can be exploited as a source of natural therapeutic agents.     

HPLC-ESI-MS/MS识别蓝莓提取物中的花青素和黄酮醇

用高效液相色谱-电喷雾离子化串联质谱联用技术(HPLC-ESI-MS/MS)对蓝莓提取物中的黄酮类物质进行了分析, 可以快速地鉴定其主要成分为花青素和黄酮醇. 结构上的不同导致花青素和黄酮醇在紫外-可见光谱吸收、离子化以及裂解方式上都存在差异. 尽管花青素和黄酮醇在270 nm处都有吸收, 但两者分别在530和372 nm附近有特征吸收. 负离子模式下, 黄酮醇可以生成负离子和自由基负离子, 而正离子型的花青素则几乎不产生质谱信号, 这一显著差异可用以区别花青素和黄酮醇. 花青素与黄酮醇在串联质谱中分别拥有各自的特征产物离子更进一步证实了其结构存在差别. 该研究对于蓝莓提取物的质量分析与控制很有帮助.     

Investigation of the biotransformation of melarsoprol by electrochemistry coupled to complementary LC/ESI–MS and LC/ICP–MS analysis

Melarsoprol is the only currently available drug for treatment of the late stage of African trypanosomiasis (sleeping sickness). Unfortunately, the arsenic-containing drug causes serious side effects, for which the mechanisms have not been elucidated so far. This investigation describes the study of the melarsoprol biotransformation processes by electrochemical (EC) techniques. Based on EC, potential oxidation reactions of melarsoprol are examined. Moreover, the reactivity of melarsoprol, its metabolite melarsen oxide, and their oxidation products toward the tripeptide glutathione and the proteins hemoglobin and human serum albumin is evaluated. The combination of different analytical techniques allows the identification as well as the quantification of the biotransformation products. The hyphenation of liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI–MS) is applied for identification and structure elucidation, which implies the determination of exact masses and fragmentation patterns. For the selective detection of arsenic containing metabolites, LC coupled to inductively coupled plasma mass spectrometry is utilized. Based on the obtained data, the oxidative biotransformation of melarsoprol can be predicted, revealing novel species which have been suspected, but not been identified up to now. The results of the protein studies prove that melarsen oxide, the active derivative of melarsoprol, strongly binds to human hemoglobin and forms different adducts via the free cysteinyl groups of the hemoglobin α- and β-chain.     

Inhibition of free radical initiated peroxidation of human erythrocyte ghosts by flavonols and their glycosides

The in vitro peroxidation of human erythrocyte ghosts was used as a model to study the free radical-induced damage of biological membranes and the protective effect of flavonols and their glycosides, i.e., quercetin (Q), quercetin galactopyranoside (QG), quercetin rhamnopyranoside (QR), rutin (R), morin (MO), kaempferol (K) and kaempferol glucoside (KG). The peroxidation was initiated by a water-soluble free radical initiator 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AMPAD) at physiological temperature, and monitored by oxygen uptake. Kinetic analysis of the peroxidation process demonstrates that these flavonols and their glycosides are effective antioxidants against AMPAD-initiated oxidative damage of human erythrocyte ghosts, and that the flavonols bearing ortho-dihydroxyl groups possess significantly higher antioxidant activity than those bearing no such functionalities and the glycosides are less active than their parent aglycones.     

电喷雾质谱半定量方法研究制川乌配伍后的大鼠肠内菌生物转化

利用建立的代谢前后生物碱成分的电喷雾质谱半定量分析方法,通过分析各药对共煎液经大鼠肠内菌群代谢后主要生物碱的含量变化,对制川乌与浙贝母、法半夏、白蔹分别配伍后共煎液中双酯型、单酯型及脂型生物碱的肠内菌生物转化进行了深入研究,研究结果表明,配伍后中药浙贝母增加共煎液中双酯型生物碱含量,法半夏降低共煎液中双酯型生物碱含量,而白蔹对共煎液中双酯型生物碱含量影响不大;在代谢过程中,大鼠肠内菌群能够将复方中双酯型生物碱转化为脂型生物碱,从而达到中药配伍的减毒增效目的。本文通过化学方法和肠内菌代谢研究证明了制川乌配伍及代谢的机理。     

Synthesis of a library of glycosylated flavonols

Flavonols are an important class of natural products isolated from plants. Some glycosylated flavonols showed very interesting biological activities. A library of flavonols has been made through Algar-Flynn-Oyamada reaction from 2′-hydroxyacetophenones and benzaldehydes. Glycosylation of these flavonols with various glycosyl donors affords a library of glycosylated flavonols. These compounds are potentially useful pharmacologically active compounds and will be studied for biological activities.     

Simultaneous separation by reversed-phase high-performance liquid chromatography and mass spectral identification of anthocyanins and flavonols in Shiraz grape skin

A limitation of large-scale viticultural trials is the time and cost of comprehensive compositional analysis of the fruit by high-performance liquid chromatography (HPLC). In addition, separate methods have generally been required to identify and quantify different classes of metabolites. To address these shortcomings a reversed-phase HPLC method was developed to simultaneously separate the anthocyanins and flavonols present in grape skins. The method employs a methanol and water gradient acidified with 10% formic acid with a run-time of 48 min including re-equilibration. Identity of anthocyanins and flavonols in Shiraz (Vitis vinifera L.) skin was confirmed by mass spectral analysis.     

The In Vitro Anti-Inflammatory Activities of Galangin and Quercetin towards the LPS-Injured Rat Intestinal Epithelial (IEC-6) Cells as Affected by Heat Treatment

Flavonols possess several beneficial bioactivities in vitro and in vivo. In this study, two flavonols galangin and quercetin with or without heat treatment (100 °C for 15–30 min) were assessed for their anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated rat intestinal epithelial (IEC-6) cells and whether the heat treatment caused activity changes. The flavonol dosages of 2.5–20 μmol/L had no cytotoxicity on the cells but could enhance cell viability (especially using 5 μmol/L flavonol dosage). The flavonols could decrease the production of prostaglandin E₂ and three pro-inflammatory cytokines interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α, and simultaneously promote the production of two anti-inflammatory cytokines IL-10 and transforming growth factor-β. The Western-blot results verified that the flavonols could suppress the LPS-induced expression of TLR4 and phosphorylated IκBα and p65, while the molecular docking results also illustrated that the flavonols could bind with TLR4 and NF-κB to yield energy decreases of −(21.9–28.6) kJ/mol. Furthermore, an inhibitor BAY 11-7082 blocked the NF-κB signaling pathway by inhibiting the expression of phosphorylated IκBα/p65 and thus mediated the production of IL-6/IL-10 as the flavonols did, which confirmed the assessed anti-inflammatory effect of the flavonols. Consistently, galangin had higher anti-inflammatory activity than quercetin, while the heated flavonols (especially those with longer heat time) were less active than the unheated counterparts to exert these target anti-inflammatory effects. It is highlighted that the flavonols could antagonize the LPS-caused IEC-6 cells inflammation via suppressing TLR4/NF-κB activation, but heat treatment of the flavonols led to reduced anti-inflammatory efficacy.     

Simultaneous analysis of anthocyanins and flavonols in petals of lotus (Nelumbo) cultivars by high-performance liquid chromatography-photodiode array detection/electrospray ionization mass spectrometry

A fast and reliable HPLC method for the simultaneous separation of anthocyanins and flavonols in lotus petals was developed based on the study of four candidate solvent systems. Fifteen flavonoids were identified by high-performance liquid chromatography with photodiode array detection/mass spectrometry. Among them, two anthocyanins and nine flavonols were discovered in lotus petals for the first time. This work is valuable for both the hybrid breeding on lotus oriented to flower color and the utilization of lotus petals as functional food materials.     

Simultaneous determination of quercetin, kaempferol, and isorhamnetin in phytopharmaceuticals of Hippophae rhamnoides L. by high-performance liquid chromatography with chemiluminescence detection

A novel method based on reversed-phase high-performance liquid chromatography with chemiluminescence detection has been developed for the simultaneous determination of three flavonols including quercetin, kaempferol, and isorhamnetin. The procedure was based on the chemiluminescent enhancement by flavonols of the cerium(IV)-rhodamine 6G system in sulfuric acid medium. The effects of several parameters on the HPLC resolution and CL emission were studied systematically. Good separation was achieved with isocratic elution using a mixture of methanol and aqueous 1.0% acetic acid (37:63, v/v) within 25 min. Under optimized conditions, the linear working range covers 3 orders of magnitude with relative standard deviations below 4.5% for 11 replicate injected flavonol samples, and detection limits (S/N= 3) were 1.6 x 10(-8), 3.5 x 10(-9), and 6.5 x 10(-9) g mL(-1) for quercetin, kaempferol, and isorhamnetin, respectively. The chemiluminescence reaction was compatible with the mobile phase of high-performance liquid chromatography. The proposed method has been successfully applied to the determination of three active flavonols in phytopharmaceuticals of Hippophae rhamnoides L. After a simple extraction procedure, the repeatability and recovery were satisfactory.     

Carbon-13 NMR studies of flavonoids—I : Flavones and flavonols

¹³C-NMR spectra of hydroxylated flavones and flavonols are presented for the first time. Analyses of the spectra are derived by consideration of a series of acetophenones, cinnamic acids, flavones and flavonols of increasing oxygenation pattern. The accepted substitution additivity rules have been shown to hold for these compounds except in cases where structural modifications involve the C-3,4 and 5 positions.