Structure-activity relationships of flavonoids for vascular relaxation in porcine coronary artery

Flavonoids are polyphenolic compounds that are widespread in the plant kingdom, and structure-activity relationships (SAR) for vascular relaxation effects were examined for 17 of them using porcine coronary arteries. Density functional theory was employed to calculate the chemical parameters of these compounds. The order of potency for vascular relaxation was as follows: flavones (apigenin and luteolin) >or= flavonols (kaempferol and quercetin)>isoflavones (genistein and daidzein)>flavanon(ol)es (naringenin)>chalcones (phloretin)>anthocyanidins (pelargonidin)>flavan(ol)es ((+)-catechin and (-)-epicatechin). SAR analysis revealed that for good relaxation activity, the 5-OH, 7-OH, 4'-OH, C2=C3 and C4=O functionalities were essential. Comparison of rutin with quercetin, genistin with genistein, and puerarin with daidzein demonstrated that the presence of a glycosylation group greatly reduced relaxation effect. Total energy and molecular volume were also predictive of their relaxation activities. Our findings indicated that the most effective relaxing agents are apigenin, luteolin, kaempferol and genistein. These flavonoids possess the key chemical structures demonstrated in our SAR analysis.     

Chemical structure of flavonols in relation to modulation of angiogenesis and immune-endothelial cell adhesion

The antioxidant activity of flavonoids has been suggested to contribute to several health benefits associated with the consumption of fruits and vegetables. Four flavonols - myricetin (M), quercetin (Q), kaempferol (K) and galangin (G), all with different numbers of hydroxyl moieties (-OH) - were examined for their antioxidant activity and cytotoxicity on human umbilical vein endothelial cells (HUVECs) and for their potential antiangiogenic and cell adhesion effects. The relative antioxidant capacity of these flavonols in cell culture medium (cell-free system) and their intracellular antioxidant activity were M = Q > K = G, which correlated respectively with the presence of 3, 2, 1 and 0 moieties of -OH on their B-ring. The higher the numbers of -OH moieties on the B-ring the less toxic the flavonol was to HUVEC, and the LD50 was determined as: M (100 microM) > Q (50 microM) > K (20 microM) > G (10 microM). These flavonols at approximately 0.5 LD50 doses suppressed the vascular endothelial growth factor (VEGF)-stimulated HUVEC tubular structure formation by: M (47%) > Q (37%) > K (15%) > G (14%), which was not linearly associated with their numbers of -OH moieties. However, the magnitude of flavonols' suppression of activated U937 monocytic cells adhesion to HUVEC was associated with the number of -OH moieties on the B-ring. This was prominent when U937 cells were pretreated with these flavonols. In contrast, the numbers of -OH moiety had no apparent influence on the adhesion or expression of adhesion molecules when activated HUVECs were pretreated with these flavonols. The presence of different numbers of -OH moieties on the B-ring of the flavonols may contribute to their antioxidant activity as well as their toxicity and may play an important role in their potency for biological action such as angiogenesis and immune-endothelial cell adhesion, which, respectively, are important processes in the development of cancer and atherosclerosis.     

Dietary flavonoids attenuate tumor necrosis factor alpha-induced adhesion molecule expression in human aortic endothelial cells. Structure-function relationships and activity after first pass metabolism

Flavonoids have been suggested to exert human health benefits by anti-oxidant and anti-inflammatory mechanisms. In this study, we investigated whether and by what mechanisms dietary flavonoids inhibit expression of cellular adhesion molecules, which is relevant to inflammation and atherosclerosis. We found that the capacity of flavonoids to inhibit tumor necrosis factor alpha-induced adhesion molecule expression in human aortic endothelial cells was dependent on specific structural features of the flavonoids. The 5,7-dihydroxyl substitution of a flavonoid A-ring and 2,3-double bond and 4-keto group of the C-ring were the main structural requirements for inhibition of adhesion molecule expression. In striking contrast, hydroxyl substitutions of the B- and C-rings but not the A-ring were essential for antioxidant activity. Hence, only hydroxyl flavones, such as apigenin and chrysin, and flavonols, such as galangin, kaempferol, and quercetin, were able to inhibit endothelial adhesion molecule expression, whereas flavone, chromone, the flavanone, naringenin, and the flavanol, (-)-epicatechin, were ineffectual. At low concentrations, the active flavonoids significantly attenuated expression of E-selectin and intercellular adhesion molecule 1 but not vascular cell adhesion molecule 1. In addition, exposure of apigenin and kaempferol to cultured hepatocytes, mimicking first pass metabolism, greatly diminished the inhibitory effect of flavonoids on endothelial intercellular adhesion molecule 1 expression. We conclude that the effect of dietary flavonoids on endothelial adhesion molecule expression depends on their molecular structure, concentration, and metabolic transformation but not their antioxidant activity.     

Antiangiogenic peptides and proteins: from experimental tools to clinical drugs

The formation of a 'tumor-associated vasculature', a process referred to as tumor angiogenesis, is a stromal reaction essential for tumor progression. Inhibition of tumor angiogenesis suppresses tumor growth in many experimental models, thereby indicating that tumor-associated vasculature may be a relevant target to inhibit tumor progression. Among the antiangiogenic molecules reported to date many are peptides and proteins. They include cytokines, chemokines, antibodies to vascular growth factors and growth factor receptors, soluble receptors, fragments derived from extracellular matrix proteins and small synthetic peptides. The polypeptide tumor necrosis factor (TNF, Beromun) was the first drug registered for the regional treatment of human cancer, whose mechanisms of action involved selective disruption of the tumor vasculature. More recently, bevacizumab (Avastin), an antibody against vascular endothelial growth factor (VEGF)-A, was approved as the first systemic antiangiogenic drug that had a significant impact on the survival of patients with advanced colorectal cancer, in combination with chemotherapy. Several additional peptides and antibodies with antiangiogenic activity are currently tested in clinical trials for their therapeutic efficacy. Thus, peptides, polypeptides and antibodies are emerging as leading molecules among the plethora of compounds with antiangiogenic activity. In this article, we will review some of these molecules and discuss their mechanism of action and their potential therapeutic use as anticancer agents in humans.     

血管内皮生长因子和抗肿瘤血管新生药物研究进展

肿瘤的生长与迁移离不开新血管的形成,这使得抗血管新生成为肿瘤治疗的重要途径之一。血管内皮生长因子（VEGF）是针对内皮细胞作用最强、特异性最高的血管新生促进因子,因而VEGF是抗肿瘤治疗的重要靶点。我们简要介绍了VEGF的一些生物学特点及肿瘤血管新生,着重介绍了一些抗血管新生药物的最新研究成果及其临床应用。     

Four glucosyltransferases from rice: cDNA cloning, expression, and characterization

Four UDP-dependent glucosyltransferase (UGT) genes, UGT706C1, UGT706D1, UGT707A3, and UGT709A4 were cloned from rice, expressed in Escherichia coli, and purified to homogeneity. In order to find out whether these enzymes could use flavonoids as glucose acceptors, apigenin, daidzein, genistein, kaempferol, luteolin, naringenin, and quercetin were used as potential glucose acceptors. UGT706C1 and UGT707A3 could use kaempferol and quercetin as glucose acceptors and the major glycosylation position was the hydroxyl group of carbon 3 based on the comparison of HPLC retention times, UV spectra, and NMR spectra with those of corresponding authentic flavonoid 3-O-glucosides. On the other hand, UGT709A4 only used the isoflavonoids genistein and daidzein and transferred glucose onto 7-hydroxyl group. In addition, UGT706D1 used a broad range of flavonoids including flavone, flavanone, flavonol, and isoflavone, and produced at least two products with glycosylation at different hydroxyl groups. Based on their substrate preferences and the flavonoids present in rice, the in vivo function of UGT706C1, UGT706D1, and UGT707A3 is most likely the biosynthesis of kaempferol and quercetin glucosides.     

Regioselective synthesis of plant (iso)flavone glycosides in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The flavonoids genistein, biochanin A, luteolin, quercetin, and kaempferol are plant natural products with potentially useful pharmacological and nutraceutical activities. These natural products usually exist in plants as glycosides, and their glycosylation has a remarkable influence on their pharmacokinetic properties. The glycosyltransferases UGT71G1 and UGT73C8 from Medicago truncatula are excellent reagents for the regioselective glycosylation of (iso)flavonoids in Escherichia coli grown in Terrific broth. Ten to 20 mg/L of either genistein or biochanin A 7-O-glucoside was produced after feeding genistein or biochanin A to E. coli expressing UGT71G1, and similar levels of luteolin 4'-O- and 7-O-glucosides were produced after feeding luteolin to cultures expressing UGT73C8. For the production of kaempferol 3-O-glucoside or quercetin 3-O-glucoside, the Phe148Val or Tyr202Ala mutants of UGT71G1 were employed. Ten to 16 mg/L of either kaempferol 3-O- or quercetin 3-O-glucosides were produced on feeding kaempferol or quercetin to E. coli expressing these enzymes. More than 90% of the glucoside products were released to the medium, facilitating their isolation.     

Dietary flavonoids interact with trace metals and affect metallothionein level in human intestinal cells

Flavonoids are natural compounds found in food items of plant origin. The study examined systematically the interaction of structurally diverse dietary flavonoids with trace metal ions and the potential impact of dietary flavonoids on the function of intestinal cells. Spectrum analysis was first performed to determine flavonoid-metal interaction in the buffer. Among the flavonoids tested, genistein, biochanin-A, naringin, and naringenin did not interact with any metal ions tested. Members of the flavonol family, quercetin, rutin, kaempferol, flavanol, and catechin, were found to interact with Cu(II) and Fe(III). On prolonged exposure, quercetin also interacted with Mn(II). Quercetin at 1:1 ratio to Cu(II) completely blocked the Cu-dependent color formation from hematoxylin. When quercetin was added to the growth medium of cultured human intestinal cells, Caco-2, the level of metal binding antioxidant protein, metallothionein, decreased. The effect of quercetin on metallothionein was dose and time-dependent. Genistein and biochanin A, on the contrary, increased the level of metallothionein. The interaction between dietary flavonoids and trace minerals and the effect of flavonoids on metallothionein level imply that flavonoids may affect metal homeostasis and cellular oxidative status in a structure-specific fashion.     

Catechin conjugated with fatty acid inhibits DNA polymerase and angiogenesis

Catechins in green tea have anticancer and antiangiogenesis activities, with epigallocatechin-3-gallate (EGCG) being the most potent antiangiogenic tea catechin. This study examined whether chemical modification of catechin enhanced anticancer and antiangiogenic effects. Catechin, conjugated with fatty acid (acyl-catechin), strongly inhibited DNA polymerase, HL-60 cancer cell growth, and angiogenesis. Catechin conjugated with stearic acid [(2R,3S)-3',4',5,7-tetrahydroxyflavan-3-yl octadecanoate; catechin-C18] was the strongest inhibitor in DNA polymerase alpha and beta and angiogenesis assays. Catechin-C18 also suppressed human endothelial cell (HUVEC) tube formation on the reconstituted basement membrane, suggesting that it affected not only DNA polymerases but also signal transduction pathways in HUVECs. These data indicate that acyl-catechins target both DNA polymerases and angiogenesis as anticancer agents. These results suggest that acylation of catechin is an effective chemical modification to improve the anticancer activity of catechin.     

Stable free radical scavenging and antiperoxidative properties of resveratrol compared in vitro with some other bioflavonoids

Stable free radical scavenging and antiperoxidative activities of resveratrol, a component of grapes and red wine, were evaluated and compared with some other known bioflavonoids (quercetin, catechin, kaempferol, myricetin, fisetin, ellagic acid and naringenin) widely present in the plant kingdom. Free radical scavenging activity was measured in an in vitro chemical system (DPPH assay), while for antiperoxidative activity, biological system comprising of hepatic and pulmonary homogenates was employed. Antiradical activity assay showed quercetin and myricetin to be stronger antiradical agents than resveratrol. Structure-activity study revealed that O-dihydroxy group on ring B of flavonoid plays a crucial role. A double bond at 2-3 position conjugated with a 4-oxo function and hydroxy groups at positions 3 and 5 also contribute towards antiradical activity of flavonoids. Resveratrol exhibited stronger antiradical activity than kaempferol and naringenin and was also more efficient than alpha-tocopherol, a known strong endogenous non-flavonoid antioxidant, used for comparison. In vitro antiperoxidative assay showed fisetin as the strongest and kaempferol as the weakest antioxidant. Resveratrol was found to be stronger antioxidant than catechin, myricetin, kaempferol and naringenin, but was weaker than quercetin, fisetin and alpha-tocopherol. Antiradical and antiperoxidative activities of resveratrol may explain its beneficial effects in disease states. Assays exhibited no direct correlation between antiradical and antiperoxidative activities of the phenolics.     

8-prenylnaringenin, a novel phytoestrogen, inhibits angiogenesis in vitro and in vivo

8-Prenylnaringenin is a recently discovered phytoestrogen. Using an in vitro model of angiogenesis in which endothelial cells can be induced to invade a three-dimensional collagen gel within which they form capillary-like tubes, we demonstrate that 8-prenylnaringenin inhibits angiogenesis induced by basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or the synergistic effect of the two cytokines in combination, with an IC(50) of between 3 and 10 microM. This effect was seen with bovine microvascular endothelial cells derived from the adrenal cortex (BME cells) and with endothelial cells from the bovine thoracic aorta (BAE cells). The inhibitory effects of 8-prenylnaringenin were found to be roughly equipotent to those of genistein that has previously been shown to inhibit angiogenesis in vitro. Early chorioallantoic membrane (CAM) assay results showed reductions in both vessel lengths and vein diameters, with similar potency in the 8-prenylnaringenin and genistein groups. Similar effects on the CAM vessels were seen when the two substances were co-added. These findings suggest that 8-prenylnaringenin has potential therapeutic applications for diseases in which angiogenesis is an important component.     

An Overview of DNA and RNA Bindings to Antioxidant Flavonoids

In this report we are examining how the antioxidant flavonoids can prevent DNA damage and what mechanism of action is involved in the process. Flavonoids are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. We study the interactions of quercetin (que), kaempferol (kae), and delphinidin (del) with DNA and transfer RNA in aqueous solution at physiological conditions, using constant DNA or RNA concentration 6.25 mmol (phosphate) and various pigment/polynucleotide(phosphate) ratios of 1/65 to 1 (DNA) and 1/48 to 1/8 (tRNA). The structural analysis showed quercetin, kaempferol, and delphinidin intercalate DNA and RNA duplexes with minor external binding to the major or minor groove and the backbone phosphate group with overall binding constants for DNA adducts K _que = 7.25 (±0.65) × 10⁴ M⁻¹, K _kae = 3.60 (±0.33) × 10⁴ M^−1, and K _del = 1.66 (±0.25) × 10⁴ M⁻¹ and for tRNA adducts K _que = 4.80 (±0.50) × 10⁴ M⁻¹, K _kae = 4.65 (±0.45) × 10⁴ M^−1, and K _del = 9.47 (±0.70) × 10⁴ M⁻¹. The stability of adduct formation is in the order of del>que>kae for tRNA and que>kae>del for DNA. Low flavonoid concentration induces helical stabilization, whereas high pigment content causes helix opening. A partial B to A-DNA transition occurs at high drug concentration, while tRNA remains in A-family structure. The antioxidant activity of flavonoids changes in order delphinidin>quercetin>kaempferol. The results show intercalated flavonoids can make them strong antioxidants to protect DNA from harmful free radical reactions.     

Isolation and HPLC quantitative analysis of antioxidant flavonoids from Alternanthera tenella Colla

Phytochemical analysis of the antioxidant ethanolic extract of Alternanthera tenella Colla led to the isolation of six flavonoids, acacetin 8-C-[alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranoside] (1), 2"-O-alpha-L-rhamnopyranosyl-vitexin (2), 2"-O-beta-D-glucopyranosyl-vitexin (3), vitexin (4), quercetin (5) and kaempferol (6). All the structures were established by ESI-MS and NMR spectroscopic methods. Antioxidant capacity of extract, fractions and isolated compounds was determined using the oxygen radical absorbance capacity (ORAC) assay and extract, fractions and flavonoids isolated showed antioxidant activity in vitro. Moreover, the total soluble phenolic contents of the extract and fractions were measured using the Folin-Ciocalteau reagent and the quantitative analysis of flavone C-glycosides major constituents was performed by HPLC.     

Antiangiogenic and Toxic Effects of Genistein,Usnic Acid,and Their Copper Complexes in Zebrafish Embryos at Different Developmental Stages

Angiogenesis plays a major role in the normal embryonic development and in diseases such as cancer. Drugs that control angiogenesis are an alternative way to tackle this disease. The polyphenols usnic acid ( 3 ), genistein ( 5 ), and daidzein ( 6 ) were tested for antiangiogenic and unwanted effects in zebrafish embryos whose blood vessel system resembles that of mammals. The established tyrosine kinase inhibitors axitinib ( 1 ) and tyrphostin AG490 ( 2 ) were included for comparison. All compounds except 6 caused distinct antiangiogenic effects such as a concentration‐dependent reduction of intersegmental vessels, dorsal longitudinal anastomotic vessels, subintestinal veins and secondary sprouts. As side effects, pericardial oedema and the impairment of blood flow were observed. Usnic acid ( 3 ), genistein ( 5 ) and Cu(II)‐genisteinate ( 7 ) gave rise to a curvature of the spine. Compounds 5 and 7 also induced cell death in the head of the embryos at higher doses. All effects were more pronounced when the compounds had been applied at an early stage (24 hpf) rather than at 48 hpf. The copper complexes 4 and 7 showed a stronger antiangiogenic effect than the free ligands 3 and 5 . The genistein complex 7 was antiangiogenic at doses so low that side effects were tolerable, and thus it may be a potential anticancer drug candidate.     

The effects of UV-B radiation on genetic and biochemical changes of Pelargonium graveolens L′Her

Ultraviolet radiation induces biochemical and genetic changes in plants. The aim of this study was to investigate the effects of UV-B radiation on genetic stability, phenolic compounds and antioxidant activity of Pelargonium graveolens L′Her. Plant cuttings were exposed to 0, 0.12. 0.26 and 0.38 W/m2 of UV-B radiation. Results indicated that by increasing the UV-B radiation intensity, total phenols, flavonoids and anthocyanin contents, Phenylalanine ammonia lyase activity and antioxidant capacity were increased. Analysis of four flavonols (quercetin, myricetin, kaempferol and rutin) contents of leaves extract by HPLC indicated that these four flavonols were enhanced in all treated plants and also the ratio of quercetin to kaempferol (Q/K) showed a significant increase (P ≤ 0.05) in UV-B treated plants in compare to control. To evaluate the genetic variation in treated plants, 10 ISSR primers were used. The highest level of percentage of polymorphism (P%), Shannon index (I), number of effective allele (Ne) and Nei’ genetic diversity (He), were observed at the highest UV-B radiation (0.38 W/m2). The AMOVA analysis also showed a significant genetic differentiation (P ≥ 0.001) among the studied groups, and confirmed the differentiation of groups obtained by the cluster analysis of molecular data. Overall, these results showed that biochemical changes in different intensities of UV-B were in line with genetic variations, so that the highest biochemical and genetic variations were observed in 0.38 W/m2 treatment.     

The splanchnic metabolism of flavonoids highly differed according to the nature of the compound

The absorption and splanchnic metabolism of different flavonoids (namely quercetin, kaempferol, luteolin, eriodictyol, genistein, and catechin) were investigated in rats after an in situ perfusion of jejunum plus ileum (14 nmol/min). Net transfer across the brush border ranged widely according to the perfused compound (from 78% for kaempferol to 35% for catechin). This variation seems linked to the lipophilicity of a given flavonoid rather than to its three-dimensional structure. Except for catechin, conjugated forms of perfused flavonoids were also detected in the intestinal lumen, but the extent of this secretion depended on the nature of the perfused compounds (52% for quercetin to 11% for genistein). For some of the perfused aglycones, biliary secretion was an important excretion route: 30% of the perfused dose for genistein but only 1% for catechin. Thus the splanchnic metabolism of flavonoid is controlled by several factors: 1) the efficiency of their transfer through the brush border, 2) the intensity of the intestinal secretion of conjugates toward the mucosal and serosal sides, respectively, and 3) the biliary secretion of conjugates. These data suggested that the splanchnic metabolism of perfused flavonoids depends on the nature of the compound considered, which in turn influences their availability for peripheral tissues.     

Intrauterine exposure to flavonoids modifies antioxidant status at adulthood and decreases oxidative stress-induced DNA damage

Maternal intake of flavonoids, known for their antioxidant properties, may affect the offspring's susceptibility to developing chronic diseases at adult age, especially those related to oxidative stress, via developmental programming. Therefore, we supplemented female mice with the flavonoids genistein and quercetin during gestation, to study their effect on the antioxidant capacity of lung and liver of adult offspring. Maternal intake of quercetin increased the expression of Nrf2 and Sod2 in fetal liver at gestational day 14.5. At adult age, in utero exposure to both flavonoids resulted in the increased expression of several enzymatic antioxidant genes, which was more pronounced in the liver than in the adult lung. Moreover, prenatal genistein exposure induced the nonenzymatic antioxidant capacity in the adult lung, partly by increasing glutathione levels. Prenatal exposure to both flavonoids resulted in significantly lower levels of oxidative stress-induced DNA damage in liver only. Our observations lead to the hypothesis that a preemptive trigger of the antioxidant defense system in utero had a persistent effect on antioxidant capacity and as a result decreased oxidative stress-induced DNA damage in the liver.     

BJ-1108, a 6-Amino-2,4,5-Trimethylpyridin-3-ol Analog,Inhibits Serotonin-Induced Angiogenesis and Tumor Growth through PI3K/NOX Pathway

5-Hydroxytryptamine (5-HT) induces proliferation of cancer cells and vascular cells. In addition to 5-HT production by several cancer cells including gastrointestinal and breast cancer, a significant level of 5-HT is released from activated platelets in the thrombotic environment of tumors, suggesting that inhibition of 5-HT signaling may constitute a new target for antiangiogenic anticancer drug discovery. In the current study we clearly demonstrate that 5-HT-induced angiogenesis was mediated through the 5-HT₁ receptor-linked Gβγ/Src/PI3K pathway, but not through the MAPK/ERK/p38 pathway. In addition, 5-HT induced production of NADPH oxidase (NOX)-derived reactive oxygen species (ROS). In an effort to develop new molecularly targeted anticancer agents against 5-HT action in tumor growth, we demonstrate that BJ-1108, a derivative of 6-amino-2,4,5-trimethylpyridin-3-ol, significantly inhibited 5-HT-induced angiogenesis. In addition, BJ-1108 induced a significant reduction in the size and weight of excised tumors in breast cancer cell-inoculated CAM assay, showing proportionate suppression of tumor growth along with inhibition of angiogenesis. In human umbilical vein endothelial cells (HUVECs), BJ-1108 significantly suppressed 5-HT-induced ROS generation and phosphorylation of PI3K/Akt but not of Src. Unlike NOX inhibitors, BJ-1108, which showed better antioxidant activity than vitamin C, barely suppressed superoxide anion induced by mevalonate or geranylgeranyl pyrophosphate which directly activates NOX without help from other signaling molecules in HUVECs, implying that the anti-angiogenic action of BJ-1108 was not mediated through direct action on NOX activation, or free radical scavenging activity. In conclusion, BJ-1108 inhibited 5-HT-induced angiogenesis through PI3K/NOX signaling but not through Src, ERK, or p38.