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1.  Metabolic engineering of flavonoids in tomato (Solanum lycopersicum): the potential for metabolomics 
Metabolomics  2007;3:399-412.
Flavonoids comprise a large and diverse group of polyphenolic plant secondary metabolites. In plants, flavonoids play important roles in many biological processes such as pigmentation of flowers, fruits and vegetables, plant-pathogen interactions, fertility and protection against UV light. Being natural plant compounds, flavonoids are an integral part of the human diet and there is increasing evidence that dietary polyphenols are likely candidates for the observed beneficial effects of a diet rich in fruits and vegetables on the prevention of several chronic diseases. Within the plant kingdom, and even within a single plant species, there is a large variation in the levels and composition of flavonoids. This variation is often due to specific mutations in flavonoid-related genes leading to quantitative and qualitative differences in metabolic profiles. The use of such specific flavonoid mutants with easily scorable, visible phenotypes has led to the isolation and characterisation of many structural and regulatory genes involved in the flavonoid biosynthetic pathway from different plant species. These genes have been used to engineer the flavonoid biosynthetic pathway in both model and crop plant species, not only from a fundamental perspective, but also in order to alter important agronomic traits, such as flower and fruit colour, resistance, nutritional value. This review describes the advances made in engineering the flavonoid pathway in tomato (Solanum lycopersicum). Three different approaches will be described; (I) Increasing endogenous tomato flavonoids using structural or regulatory genes; (II) Blocking specific steps in the flavonoid pathway by RNA interference strategies; and (III) Production of novel tomato flavonoids by introducing novel branches of the flavonoid pathway. Metabolite profiling is an essential tool to analyse the effects of pathway engineering approaches, not only to analyse the effect on the flavonoid composition itself, but also on other related or unrelated metabolic pathways. Metabolomics will therefore play an increasingly important role in revealing a more complete picture of metabolic perturbation and will provide additional novel insights into the effect of the introduced genes and the role of flavonoids in plant physiology and development.
doi:10.1007/s11306-007-0074-2
PMCID: PMC4309898
GC/MS; LC/MS; Metabolic engineering; Metabolomics; Tomato
2.  Inhibition of estrogen signaling activates the NRF2 pathway in breast cancer 
Exposure to higher levels of estrogen produces genotoxic metabolites that can stimulate mammary tumorigenesis. Induction of NF-E2-related factor 2 (NRF2)-dependent detoxifying enzymes (e.g., NAD(P)H-quinone oxidoreductase 1 (NQO1)) is considered an important mechanism of protection against estrogen-associated carcinogenesis because they would facilitate removal of toxic estrogens. Here, we studied the impact of estrogen-receptor (ER) signaling on NRF2-dependent gene transcription. In luciferase assay experiments using the 5-flanking region of the human NQO1 gene promoter, we observe that ERα ligand-binding domain (LBD) is required for estrogen inhibition of NQO1 promoter activity in estrogen-dependent breast cancer cells. Chromatin immunoprecipitation (ChIP) assay shows that estrogen recruits ERα and a class III histone deacetylase SIRT1 at the NQO1 promoter, leading to inhibition of NQO1 transcription. Inhibition of ERα expression by the antiestrogen shikonin reverses the inhibitory effect of estrogen on NQO1 expression. As a consequence, a chemoprevention study was undertaken to monitor the impact of shikonin on DNA lesions and tumor growth. Treatment of MCF-7 breast cancer cells with shikonin inhibits estrogen-induced 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of DNA damage. NQO1 deficiency promotes estrogen-dependent tumor formation, and shikonin inhibits estrogen-dependent tumor growth in an NQO1-dependent manner in MCF-7 xenografts. These results suggest that estrogen-receptor signaling pathway has an inhibitory effect on NRF2-dependent enzymes. Moreover, shikonin reverses the inhibitory effects of estrogen on this pathway and may contribute to breast cancer prevention.
doi:10.1007/s10549-010-1023-8
PMCID: PMC3417311  PMID: 20623181
Estrogen receptor; NRF2; NQO1; Chemoprevention
3.  Apigenin Inhibits Antiestrogen-resistant Breast Cancer Cell Growth through Estrogen Receptor-α-dependent and -independent Mechanisms 
Molecular cancer therapeutics  2008;7(7):2096-2108.
Breast cancer resistance to the antiestrogens tamoxifen and fulvestrant is accompanied by alterations in both estrogen-dependent and -independent signaling pathways. Consequently, effective inhibition of both pathways may be necessary to block proliferation of antiestrogen-resistant breast cancer cells. In this study, we examined the effects of apigenin, a dietary plant flavonoid with potential anticancer properties, on estrogen-responsive, antiestrogen-sensitive MCF7 breast cancer cells and two MCF7 sublines with acquired resistance to either tamoxifen or fulvestrant. We found that apigenin can function as both an estrogen and antiestrogen, in a dose-dependent manner. At low concentrations (1μM), apigenin stimulated MCF7 cell growth but had no effect on the antiestrogen-resistant MCF7 sublines. In contrast, at high concentrations (≥10μM), the drug inhibited growth of MCF7 cells and the antiestrogen-resistant sublines, and the combination of apigenin with either tamoxifen or fulvestrant demonstrated synergistic, growth-inhibitory effects on both antiestrogen-sensitive and -resistant breast cancer cells. To further elucidate the molecular mechanism of apigenin as either an estrogen or antiestrogen, effects of the drug on estrogen receptor-α (ERα) transactivation activity, mobility, stability, and ERα-coactivator interactions were investigated. Low-dose apigenin enhanced receptor transcriptional activity by promoting interaction between ERα and its co-activator AIB1 (amplified in breast cancer-1). However, higher doses (> 10μM) of apigenin inhibited ERα mobility (as determined by FRAP assays), downregulated ERα and AIB1 expression levels, and inhibited multiple protein kinases, including p38, PKA, MAPK and AKT. Collectively, these results show that apigenin can function as both an antiestrogen and protein kinase inhibitor with activity against breast cancer cells with acquired resistance to OHT or fulvestrant. We conclude that apigenin, through its ability to target both ERα-dependent and -independent pathways, holds promise as a new therapeutic agent against antiestrogen-resistant breast cancer.
doi:10.1158/1535-7163.MCT-07-2350
PMCID: PMC2559959  PMID: 18645020
apigenin; antiestrogen; fulvestrant; tamoxifen; breast cancer
4.  Fruit-Surface Flavonoid Accumulation in Tomato Is Controlled by a SlMYB12-Regulated Transcriptional Network 
PLoS Genetics  2009;5(12):e1000777.
The cuticle covering plants' aerial surfaces is a unique structure that plays a key role in organ development and protection against diverse stress conditions. A detailed analysis of the tomato colorless-peel y mutant was carried out in the framework of studying the outer surface of reproductive organs. The y mutant peel lacks the yellow flavonoid pigment naringenin chalcone, which has been suggested to influence the characteristics and function of the cuticular layer. Large-scale metabolic and transcript profiling revealed broad effects on both primary and secondary metabolism, related mostly to the biosynthesis of phenylpropanoids, particularly flavonoids. These were not restricted to the fruit or to a specific stage of its development and indicated that the y mutant phenotype is due to a mutation in a regulatory gene. Indeed, expression analyses specified three R2R3-MYB–type transcription factors that were significantly down-regulated in the y mutant fruit peel. One of these, SlMYB12, was mapped to the genomic region on tomato chromosome 1 previously shown to harbor the y mutation. Identification of an additional mutant allele that co-segregates with the colorless-peel trait, specific down-regulation of SlMYB12 and rescue of the y phenotype by overexpression of SlMYB12 on the mutant background, confirmed that a lesion in this regulator underlies the y phenotype. Hence, this work provides novel insight to the study of fleshy fruit cuticular structure and paves the way for the elucidation of the regulatory network that controls flavonoid accumulation in tomato fruit cuticle.
Author Summary
A major step in the evolution of land plants was the formation of a cuticular layer on their outer surfaces. Despite the cuticle's key role in organ development and in protecting against a variety of stresses, very little is known about the regulation of the metabolic pathways that generate its building blocks. Flavonoids, often embedded in the cuticle, have been suggested to impact the characteristics of this structure and to provide protection against radiation and pathogens. Flavonoids are an integral part of the human diet and are likely responsible for the observed beneficial effects of a fruit-rich diet. Here, we examine in detail the tomato colorless peel y mutant, which lacks the yellow flavonoid pigment naringenin chalcone, a major constituent of the fruit cuticle. Extensive transcript and metabolite profiling of this mutant revealed SlMYB12 as a key regulator in a transcription network that controls flavonoid accumulation in tomato peel. Moreover, the change in cuticle flavonoid composition enabled us to evaluate the importance of these constituents as barriers in the cuticle layer. Finally, because most commercial cultivars in the Far East are based on the y genetic background, discovery of the y gene will contribute also to tomato breeding programs.
doi:10.1371/journal.pgen.1000777
PMCID: PMC2788616  PMID: 20019811
5.  Flavonoids activate pregnane × receptor-mediated CYP3A4 gene expression by inhibiting cyclin-dependent kinases in HepG2 liver carcinoma cells 
BMC Biochemistry  2010;11:23.
Background
The expression of the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is regulated by the pregnane × receptor (PXR), which is modulated by numerous signaling pathways, including the cyclin-dependent kinase (Cdk) pathway. Flavonoids, commonly consumed by humans as dietary constituents, have been shown to modulate various signaling pathways (e.g., inhibiting Cdks). Flavonoids have also been shown to induce CYPs expression, but the underlying mechanism of action is unknown. Here, we report the mechanism responsible for flavonoid-mediated PXR activation and CYP expression.
Results
In a cell-based screen designed to identify compounds that activate PXR-mediated CYP3A4 gene expression in HepG2 human carcinoma cells, we identified several flavonoids, such as luteolin and apigenin, as PXR activators. The flavonoids did not directly bind to PXR, suggesting that an alternative mechanism may be responsible for flavonoid-mediated PXR activation. Consistent with the Cdk5-inhibitory effect of flavonoids, Cdk5 and p35 (a non-cyclin regulatory subunit required to activate Cdk5) were expressed in HepG2. The activation of Cdk5 attenuated PXR-mediated CYP3A4 expression whereas its downregulation enhanced it. The Cdk5-mediated downregulation of CYP3A4 promoter activity was restored by flavonoids, suggesting that flavonoids activate PXR by inactivating Cdk5. In vitro kinase assays showed that Cdk5 directly phosphorylates PXR. The Cdk kinase profiling assay showed that apigenin inhibits multiple Cdks, suggesting that several Cdks may be involved in activation of PXR by flavonoids.
Conclusions
Our results for the first time link the stimulatory effect of flavonoids on CYP expression to their inhibitory effect on Cdks, through a PXR-mediated mechanism. These results may have important implications on the pharmacokinetics of drugs co-administered with herbal remedy and herbal-drug interactions.
doi:10.1186/1471-2091-11-23
PMCID: PMC2901217  PMID: 20553580
6.  Flavonoids Induce the Synthesis and Secretion of Neurotrophic Factors in Cultured Rat Astrocytes: A Signaling Response Mediated by Estrogen Receptor 
Neurotrophic factors are playing vital roles in survival, growth, and function of neurons. Regulation of neurotrophic factors in the brain has been considered as one of the targets in developing drug or therapy against neuronal disorders. Flavonoids, a family of multifunctional natural compounds, are well known for their neuronal beneficial effects. Here, the effects of flavonoids on regulating neurotrophic factors were analyzed in cultured rat astrocytes. Astrocyte is a major secreting source of neurotrophic factors in the brain. Thirty-three flavonoids were screened in the cultures, and calycosin, isorhamnetin, luteolin, and genistein were identified to be highly active in inducing the synthesis and secretion of neurotrophic factors, including nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF). The inductions were in time- and dose-dependent manners. In cultured astrocytes, the phosphorylation of estrogen receptor was triggered by application of flavonoids. The phosphorylation was blocked by an inhibitor of estrogen receptor, which in parallel reduced the flavonoid-induced expression of neurotrophic factors. The results proposed the role of flavonoids in protecting brain diseases, and therefore these flavonoids could be developed for health food supplement for patients suffering from neurodegenerative diseases.
doi:10.1155/2013/127075
PMCID: PMC3708423  PMID: 23878590
7.  Crossbreeding of transgenic flax plants overproducing flavonoids and glucosyltransferase results in progeny with improved antifungal and antioxidative properties 
Molecular Breeding  2014;34(4):1917-1932.
Flavonoids are a large group of secondary plant metabolites with many important functions; they play a role in fruit, flower and seed pigmentation and are involved in multiple protective mechanisms. They are very active natural antioxidants, acting as antimicrobial compounds in defense against pathogens, and they protect the plant against various stress factors, including excessive solar radiation and temperature. They are also an animal deterrent. Flax is already a very useful crop plant with nutritional and biomedical applications. With increased phenylpropanoid content, flax plants could be used in the production of improved dietary supplements and antimicrobial agents. The main aim of this study was to engineer a flax variety with increased flavonoid content by crossing two transgenic flax varieties that have heightened flavonoid levels. A mother plant that over expresses genes encoding the flavonoid biosynthesis pathway enzymes chalcone synthase, chalcone isomerase and dihydroflavonol reductase was crossed with plants overexpressing the glucosyltransferase (GT) gene. It was expected that the progeny would display better properties thanks to the simultaneous increases in flavonoid synthesis and stability. In comparison to the control and parental plants, plants of the selected flax lines were found to have increased contents of flavonoids and other phenylpropanoids, including phenolic acids, in their stems and seeds. A significant increase in the secoisolariciresinol diglucoside content was found in the seeds. The antioxidative properties of extracts from W92 × GT crossbreed plants were higher than the control (non-transgenic) and parental plants. These results correlated with the increase in the susceptibility of the crossbreeds to Fusarium infection. The increased flavonoid content did not cause any negative phenotypic changes or reduce the yield of seeds.
Electronic supplementary material
The online version of this article (doi:10.1007/s11032-014-0149-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s11032-014-0149-5
PMCID: PMC4257994  PMID: 25506258
Flax; Flavonoids; Fusarium; Cross breeding; Antioxidants; Antifungal activity
8.  Hydrophobic flavonoids from Scutellaria baicalensis induces colorectal cancer cell apoptosis through a mitochondrial-mediated pathway 
International journal of oncology  2013;42(3):1018-1026.
Scutellaria baicalensis extract (SbE) has chemopreventive effects on several cancers. Baicalin, a hydrophilic flavonoid in SbE, also may have opposing effects that decrease the anti-tumor potential of SbE against colorectal cancer. In this study, after removing baicalin, we prepared an aglycon-rich fraction (ARF) of SbE and evaluated its antiproliferative activity and mechanism of action. The flavonoids in ARF, baicalin fraction (BF) and SbE were determined by HPLC. The effects of ARF, BF, SbE and representative flavonoids on the proliferation of HCT-116 and HT-29 human colorectal cancer cells were determined by an MTS assay. Cell cycle, cyclins A and B1 expression and cell apoptosis were assayed using flow cytometry. Apoptosis-related gene expression was visualized by quantitative real-time polymerase chain reaction (PCR), and mitochondrial membrane potential was estimated after staining with JC-1. HPLC analysis showed that ARF contained two hydrophobic flavonoids baicalein and wogonin, and BF contained only baicalin. SbE had little antiproliferative effect on colorectal cancer cells, even increasing cancer cell growth at certain concentrations. ARF showed potent antiproliferative effects on cancer cells. In contrast, BF increased cancer cell growth. ARF arrested cells in the S and G2/M phases, increased expression of cyclins A and B1, and significantly induced cell apoptosis. Multiple genes in the mitochondrial pathway are involved in ARF-induced apoptosis, and subsequent cellular functional analysis validated this pathway. These results suggest that knocking out baicalin from SbE produces a ARF that significantly inhibits the growth of colorectal cancer cells, and the mitochondrial apoptotic pathway is part of hydrophobic flavonoids-induced apoptosis.
doi:10.3892/ijo.2013.1777
PMCID: PMC3576930  PMID: 23337959
Scutellaria baicalensis; hydrophobic flavonoids; baicalin; baicalein; wogonin; colorectal cancer; mitochondrial apoptotic pathway
9.  Flavonoids and Age Related Disease: Risk, benefits and critical windows 
Maturitas  2010;66(2):163-171.
Plant derived products are consumed by a large percentage of the population to prevent, delay and ameliorate disease burden; however, relatively little is known about the efficacy, safety and underlying mechanisms of these traditional health products, especially when taken in concert with pharmaceutical agents. The flavonoids are a group of plant metabolites that are common in the diet and appear to provide some health benefits. While flavonoids are primarily derived from soy, many are found in fruits, nuts and more exotic sources, e.g., kudzu. Perhaps the strongest evidence for the benefits of flavonoids in diseases of aging relates to their effect on components of the metabolic syndrome. Flavonoids from soy, grape seed, kudzu and other sources all lower arterial pressure in hypertensive animal models and in a limited number of tests in humans. They also decrease the plasma concentration of lipids and buffer plasma glucose. The underlying mechanisms appear to include antioxidant actions, central nervous system effects, gut transport alterations, fatty acid sequestration and processing, PPAR activation and increases in insulin sensitivity. In animal models of disease, dietary flavonoids also demonstrate a protective effect against cognitive decline, cancer and metabolic disease. However, research also indicates that the flavonoids can be detrimental in some settings and, therefore, are not universally safe. Thus, as the population ages, it is important to determine the impact of these agents on prevention/attenuation of disease, including optimal exposure (intake, timing/duration) and potential contraindications.
doi:10.1016/j.maturitas.2010.01.010
PMCID: PMC2879453  PMID: 20181448
soy; kudzu; metabolic syndrome; complimentary medicine; dietary supplements
10.  Recent advances in understanding the anti-diabetic actions of dietary flavonoids 
The Journal of nutritional biochemistry  2013;24(11):10.1016/j.jnutbio.2013.06.003.
Flavonoids are polyphenolic compounds that are abundant in fruits and vegetables and increasing evidence demonstrates a positive relationship between consumption of flavonoid-rich foods and disease prevention. Epidemiological, in vitro and animal studies support the beneficial effects of dietary flavonoids on glucose and lipid homeostasis. It is encouraging that the beneficial effects of some flavonoids are at physiological concentrations and comparable to clinically-used anti-diabetic drugs; however, clinical research in this field and studies on the anti-diabetic effects of flavonoid metabolites are limited. Flavonoids act on various molecular targets and regulate different signaling pathways in pancreatic β-cells, hepatocytes, adipocytes, and skeletal myofibers. Flavonoids may exert beneficial effects in diabetes by (i) enhancing insulin secretion and reducing apoptosis and promoting proliferation of pancreatic β-cells, (ii) improving hyperglycemia through regulation of glucose metabolism in hepatocytes, (iii) reducing insulin resistance, inflammation and oxidative stress in muscle and fat, and (iv) increasing glucose uptake in skeletal muscle and white adipose tissue. This review highlights recent findings on the anti-diabetic effects of dietary flavonoids, including flavan-3-ols, flavanones, flavonols, anthocyanidins, flavones, and isoflavones, with particular emphasis on the studies that investigated the cellular and molecular mechanisms involved in the beneficial effects of the compounds.
doi:10.1016/j.jnutbio.2013.06.003
PMCID: PMC3821977  PMID: 24029069
diabetes; flavonoids; insulin; glucose; β-cells; liver; fat; muscle; hyperglycemia; polyphenols; islets; pancreatic β-cells
11.  Evaluation of Estrogenic Potential of Flavonoids Using a Recombinant Yeast Strain and MCF7/BUS Cell Proliferation Assay 
PLoS ONE  2013;8(10):e74881.
Phytoestrogens are of interest because of their reported beneficial effects on many human maladies including cancer, neurodegeneration, cardiovascular disease and diabetes. Furthermore, there is a search for compounds with estrogenic activity that can replace estrogen in hormone replacement therapy during menopause, without the undesirable effects of estrogen, such as the elevation of breast cancer occurrence. Thus, the principal objective of this study was to assess the estrogenic activity of flavonoids with different hydroxylation patterns: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone via two different in vitro assays, the recombinant yeast assay (RYA) and the MCF-7 proliferation assay (E-screen), since the most potent phytoestrogens are members of the flavonoid family. In these assays, kaempferol was the only compound that showed ERα-dependent transcriptional activation activity by RYA, showing 6.74±1.7 nM EEQ, besides acting as a full agonist for the stimulation of proliferation of MCF-7/BUS cells. The other compounds did not show detectable levels of interaction with ER under the conditions used in the RYA. However, in the E-screen assay, compounds such as galangin, luteolin and fisetin also stimulated the proliferation of MCF-7/BUS cells, acting as partial agonists. In the evaluation of antiestrogenicity, the compounds quercetin, chrysin and 3-hydroxyflavone significantly inhibited the cell proliferation induced by 17-β-estradiol in the E-screen assay, indicating that these compounds may act as estrogen receptor antagonists. Overall, it became clear in the assay results that the estrogenic activity of flavonoids was affected by small structural differences such as the number of hydroxyl groups, especially those on the B ring of the flavonoid.
doi:10.1371/journal.pone.0074881
PMCID: PMC3788058  PMID: 24098354
12.  Transport and accumulation of flavonoids in grapevine (Vitis vinifera L.) 
Plant Signaling & Behavior  2008;3(9):626-632.
Flavonoids are a group of secondary metabolites widely distributed in plants that represent a huge portion of the soluble phenolics present in grapevine (Vitis vinifera L.). These compounds play different physiological roles and are often involved in protection against biotic and abiotic stress. Even if the flavonoid biosynthetic pathways have been largely characterized, the mechanisms of their transport and accumulation in cell wall and vacuole are still not completely understood. This review analyses the known mechanisms of flavonoid uptake and accumulation in grapevine, with reference to the transport models and membrane carrier proteins described in other plant species. The effect of different environmental factors on flavonoid biosynthesis and transporters is also discussed.
PMCID: PMC2634543  PMID: 19513253
ABC proteins; active transport; bilitranslocase; biotic and abiotic stress; flavonoid; secondary metabolites
13.  Anti-diabetic functions of soy isoflavone genistein: mechanisms underlying effects on pancreatic β-cell function 
Food & function  2013;4(2):200-212.
Type 2 diabetes is a result of chronic insulin resistance and loss of functional pancreatic β-cell mass. Strategies to preserve β-cell mass and a greater understanding of the mechanisms underlying β-cell turnover are needed to prevent and treat this devastating disease. Genistein, a naturally-occurring soy isoflavone, is reported to have numerous health benefits attributed to multiple biological functions. Over the past 10 years, numerous studies have demonstrated that genistein has anti-diabetic effects, in particular, direct effects on β-cell proliferation, glucose-stimulated insulin secretion and protection against apoptosis, independent of its functions as an estrogen receptor agonist, antioxidant, or tyrosine kinase inhibitor. Effects are structure-specific and not common to all flavonoids. While there are limited data on the effects of genistein consumption in humans with diabetes, there are a plethora of animal and cell-culture studies that demonstrate, at physiologically-relevant concentrations (<10 µM), a direct effect of genistein on β-cells. The effects appear to involve cAMP/PKA signaling and there are some studies that suggest an effect on epigenetic regulation of gene expression. This review focuses on the anti-diabetic effects of genistein in both in-vitro and in-vivo models and potential mechanisms underlying its direct effects on β-cells.
doi:10.1039/c2fo30199g
PMCID: PMC3678366  PMID: 23160185
14.  Flavonoids as Therapeutic Compounds Targeting Key Proteins Involved in Alzheimer’s Disease 
ACS Chemical Neuroscience  2013;5(2):83-92.
Alzheimer’s disease is characterized by pathological aggregation of protein tau and amyloid-β peptides, both of which are considered to be toxic to neurons. Naturally occurring dietary flavonoids have received considerable attention as alternative candidates for Alzheimer’s therapy taking into account their antiamyloidogenic, antioxidative, and anti-inflammatory properties. Experimental evidence supports the hypothesis that certain flavonoids may protect against Alzheimer’s disease in part by interfering with the generation and assembly of amyloid-β peptides into neurotoxic oligomeric aggregates and also by reducing tau aggregation. Several mechanisms have been proposed for the ability of flavonoids to prevent the onset or to slow the progression of the disease. Some mechanisms include their interaction with important signaling pathways in the brain like the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways that regulate prosurvival transcription factors and gene expression. Other processes include the disruption of amyloid-β aggregation and alterations in amyloid precursor protein processing through the inhibition of β-secretase and/or activation of α-secretase, and inhibiting cyclin-dependent kinase-5 and glycogen synthase kinase-3β activation, preventing abnormal tau phosphorylation. The interaction of flavonoids with different signaling pathways put forward their therapeutic potential to prevent the onset and progression of Alzheimer’s disease and to promote cognitive performance. Nevertheless, further studies are needed to give additional insight into the specific mechanisms by which flavonoids exert their potential neuroprotective actions in the brain of Alzheimer’s disease patients.
doi:10.1021/cn400213r
PMCID: PMC3930994  PMID: 24328060
Flavonoids; Alzheimer’s disease; amyloid precursor protein; amyloid beta; BACE-1; tau; signaling
15.  Structure and function of enzymes involved in the biosynthesis of phenylpropanoids 
As a major component of plant specialized metabolism, phenylpropanoid biosynthetic pathways provide anthocyanins for pigmentation, flavonoids such as flavones for protection against UV photodamage, various flavonoid and isoflavonoid inducers of Rhizobium nodulation genes, polymeric lignin for structural support and assorted antimicrobial phytoalexins. As constituents of plant-rich diets and an assortment of herbal medicinal agents, the phenylpropanoids exhibit measurable cancer chemopreventive, antimitotic, estrogenic, antimalarial, antioxidant and antiasthmatic activities. The health benefits of consuming red wine, which contains significant amounts of 3,4′,5-trihydroxystilbene (resveratrol) and other phenylpropanoids, highlight the increasing awareness in the medical community and the public at large as to the potential dietary importance of these plant derived compounds. As recently as a decade ago, little was known about the three-dimensional structure of the enzymes involved in these highly branched biosynthetic pathways. Ten years ago, we initiated X-ray crystallographic analyses of key enzymes of this pathway, complemented by biochemical and enzyme engineering studies. We first investigated chalcone synthase (CHS), the entry point of the flavonoid pathway, and its close relative stilbene synthase (STS). Work soon followed on the O-methyl transferases (OMTs) involved in modifications of chalcone, isoflavonoids and metabolic precursors of lignin. More recently, our groups and others have extended the range of phenylpropanoid pathway structural investigations to include the upstream enzymes responsible for the initial recruitment of phenylalanine and tyrosine, as well as a number of reductases, acyltransferases and ancillary tailoring enzymes of phenylpropanoid-derived metabolites. These structure–function studies collectively provide a comprehensive view of an important aspect of phenylpropanoid metabolism. More specifically, these atomic resolution insights into the architecture and mechanistic underpinnings of phenylpropanoid metabolizing enzymes contribute to our understanding of the emergence and on-going evolution of specialized phenylpropanoid products, and underscore the molecular basis of metabolic biodiversity at the chemical level. Finally, the detailed knowledge of the structure, function and evolution of these enzymes of specialized metabolism provide a set of experimental templates for the enzyme and metabolic engineering of production platforms for diverse novel compounds with desirable dietary and medicinal properties.
doi:10.1016/j.plaphy.2007.12.009
PMCID: PMC2860624  PMID: 18272377
Phenylpropanoid pathway; Secondary metabolism; Lignin; Flavonoids; Anthocyanins; Plant enzymes; Structure
16.  Hydrophobic flavonoids from Scutellaria baicalensis induce colorectal cancer cell apoptosis through a mitochondrial-mediated pathway 
International Journal of Oncology  2013;42(3):1018-1026.
Scutellaria baicalensis extract (SbE) has been shown to exert chemopreventive effects on several types of cancer. Baicalin, a hydrophilic flavonoid found in SbE, may have opposing effects that decrease the antitumor potential of SbE against colorectal cancer. In this study, after removing baicalin, we prepared an aglycone-rich fraction (ARF) of SbE and evaluated its anti-proliferative activity and mechanisms of action. The flavonoids found in ARF, baicalin fraction (BF) and SbE were determined by high-performance liquid chromatography (HPLC). The effects of ARF, BF, SbE and representative flavonoids on the proliferation of HCT-116 and HT-29 human colorectal cancer cells were determined by an MTS assay. The cell cycle, the expression of cyclins A and B1 and cell apoptosis were assayed using flow cytometry. Apoptosis-related gene expression was visualized by quantitative real-time polymerase chain reaction (PCR), and mitochondrial membrane potential was estimated following staining with JC-1. HPLC analysis showed that ARF contained two hydrophobic flavonoids, baicalein and wogonin, and that BF contained only baicalin. SbE had little anti-proliferative effect on the colorectal cancer cells; cancer cell growth was even observed at certain concentrations. ARF exerted potent anti-proliferative effects on the cancer cells. By contrast, BF increased cancer cell growth. ARF arrested cells in the S and G2/M phases, increased the expression of cyclins A and B1, and significantly induced cell apoptosis. Multiple genes in the mitochondrial pathway are involved in ARF-induced apoptosis, and subsequent cellular functional analysis validated the involvement of this pathway. These results suggest that removing baicalin from SbE produces an ARF that significantly inhibits the growth of colorectal cancer cells, and that the mitochondrial apoptotic pathway plays a role in hydrophobic flavonoid-induced apoptosis.
doi:10.3892/ijo.2013.1777
PMCID: PMC3576930  PMID: 23337959
Scutellaria baicalensis; hydrophobic flavonoids; baicalin; baicalein; wogonin; colorectal cancer; mitochondrial apoptotic pathway
17.  A role for hippocampal PSA-NCAM and NMDA-NR2B receptor function in flavonoid-induced spatial memory improvements in young rats 
Neuropharmacology  2014;79(100):335-344.
The increase in incidence and prevalence of neurodegenerative diseases highlights the need for a more comprehensive understanding of how food components may affect neural systems. In particular, flavonoids have been recognized as promising agents capable of influencing different aspects of synaptic plasticity resulting in improvements in memory and learning in both animals and humans. Our previous studies highlight the efficacy of flavonoids in reversing memory impairments in aged rats, yet little is known about the effects of these compounds in healthy animals, particularly with respect to the molecular mechanisms by which flavonoids might alter the underlying synaptic modifications responsible for behavioral changes. We demonstrate that a 3-week intervention with two dietary doses of flavonoids (Dose I: 8.7 mg/day and Dose II: 17.4 mg/day) facilitates spatial memory acquisition and consolidation (24 recall) (p < 0.05) in young healthy rats. We show for the first time that these behavioral improvements are linked to increased levels in the polysialylated form of the neural adhesion molecule (PSA-NCAM) in the dentate gyrus (DG) of the hippocampus, which is known to be required for the establishment of durable memories. We observed parallel increases in hippocampal NMDA receptors containing the NR2B subunit for both 8.7 mg/day (p < 0.05) and 17.4 mg/day (p < 0.001) doses, suggesting an enhancement of glutamate signaling following flavonoid intervention. This is further strengthened by the simultaneous modulation of hippocampal ERK/CREB/BDNF signaling and the activation of the Akt/mTOR/Arc pathway, which are crucial in inducing changes in the strength of hippocampal synaptic connections that underlie learning. Collectively, the present data supports a new role for PSA-NCAM and NMDA-NR2B receptor on flavonoid-induced improvements in learning and memory, contributing further to the growing body of evidence suggesting beneficial effects of flavonoids in cognition and brain health.
Highlights
•We have identified novel targets for the flavonoid actions in the brain.•Flavonoid ingestion facilitates hippocampal-dependent learning in healthy animals.•Flavonoids regulate hippocampal PSA-NCAM in the Dentate Gyrus of the hippocampus.•Flavonoids modulate the levels of hippocampal glutamate receptors (NMDA-NR2B).
doi:10.1016/j.neuropharm.2013.12.003
PMCID: PMC4062943  PMID: 24333331
Memory; Learning; Flavonoid; Hippocampus; BDNF; PSA-NCAM; NMDR2B
18.  Antiestrogenic Activity of Flavonoid Phytochemicals Mediated via the c-Jun N-terminal Protein Kinase Pathway 
Flavonoid phytochemicals act as both agonists and antagonists of the human estrogen receptors (ERs). While a number of these compounds act by directly binding to the ER, certain phytochemicals, such as the flavonoid compounds chalcone and flavone, elicit antagonistic effects on estrogen signaling independent of direct receptor binding. Here we demonstrate both chalcone and flavone function as cell type-specific selective ER modulators. In MCF-7 breast carcinoma cells chalcone and flavone suppress ERα activity through stimulation of the stress-activated members of the mitogen-activated protein kinase (MAPK) family: c-Jun N-terminal kinase (JNK)1 and JNK2. The use of dominant-negative mutants of JNK1 or JNK2 in stable transfected cells established that the antiestrogenic effects of chalcone and flavone required intact JNK signaling. We further show that constitutive activation of the JNK pathway partially suppresses estrogen (E2)-mediated gene expression in breast, but not endometrial carcinoma cells. Our results demonstrate a role for stress-activated MAPKs in the cell type-specific regulation of ERα function.
doi:10.1016/j.jsbmb.2012.05.004
PMCID: PMC4083692  PMID: 22634477
flavonoids; phytoestrogens; estrogen receptor; mitogen-activated protein kinase; antiestrogens; c-Jun N-terminal kinase (JNK)
19.  Mutual interactions between flavonoids and enzymatic and transporter elements responsible for flavonoid disposition via phase II metabolic pathways 
RSC advances  2012;2(21):7948-7963.
Flavonoids, existing mainly as glycosides in nature, have multiple “claimed” beneficial effects in humans. Flavonoids are extensively metabolized in enterocytes and hepatocytes by phase II enzymes such as UGTs and SULTs to form glucuronides and sulfates, respectively. These glucuronides and sulfates are subsequently excreted via ABC transporters (e.g., MRP2 or BCRP). Therefore, it is the interplay between phase II enzymes and efflux transporters that affects the disposition of flavonoids and leads to the low bioavailability of flavonoid aglycones. Flavonoids can also serve as chemical regulators that affect the activity or expression levels of phase II enzymes including UGTs, SULTs and GSTs, and transporters including P-gp, MRP2, BCRP, OATP and OAT. In general, flavonoids may exert the inhibitory or inductive effects on the phase II enzymes and transporters via multiple mechanisms that may involve different nuclear receptors. Since flavonoids may affect the metabolic pathways shared by many important clinical drugs, drug-flavonoid interaction is becoming an increasingly important concern. This review article focused on the disposition of flavonoids and effects of flavonoids on relevant enzymes (e.g. UGTs and SULTs) and transporters (e.g. MRP2 and BCRP) involved in the interplay between phase II enzymes and efflux transporters. The effects of flavonoids on other metabolic enzymes (e.g. GSTs) or transporters (e.g. P-gp, OATP and OAT) are also addressed but that is not the emphasis of this review.
doi:10.1039/C2RA01369J
PMCID: PMC4228968  PMID: 25400909
flavonoids; ABC transporter; UGT; SULT; MRP2; BCRP; P-gp; OATP; OAT; disposition; glucuronidation; sulfonation; interplay
20.  When plants produce not enough or at all: metabolic engineering of flavonoids in microbial hosts 
As a result of the discovery that flavonoids are directly or indirectly connected to health, flavonoid metabolism and its fascinating molecules that are natural products in plants, have attracted the attention of both the industry and researchers involved in plant science, nutrition, bio/chemistry, chemical bioengineering, pharmacy, medicine, etc. Subsequently, in the past few years, flavonoids became a top story in the pharmaceutical industry, which is continually seeking novel ways to produce safe and efficient drugs. Microbial cell cultures can act as workhorse bio-factories by offering their metabolic machinery for the purpose of optimizing the conditions and increasing the productivity of a selective flavonoid. Furthermore, metabolic engineering methodology is used to reinforce what nature does best by correcting the inadequacies and dead-ends of a metabolic pathway. Combinatorial biosynthesis techniques led to the discovery of novel ways of producing natural and even unnatural plant flavonoids, while, in addition, metabolic engineering provided the industry with the opportunity to invest in synthetic biology in order to overcome the currently existing restricted diversification and productivity issues in synthetic chemistry protocols. In this review, is presented an update on the rationalized approaches to the production of natural or unnatural flavonoids through biotechnology, analyzing the significance of combinatorial biosynthesis of agricultural/pharmaceutical compounds produced in heterologous organisms. Also mentioned are strategies and achievements that have so far thrived in the area of synthetic biology, with an emphasis on metabolic engineering targeting the cellular optimization of microorganisms and plants that produce flavonoids, while stressing the advances in flux dynamic control and optimization. Finally, the involvement of the rapidly increasing numbers of assembled genomes that contribute to the gene- or pathway-mining in order to identify the gene(s) responsible for producing species-specific secondary metabolites is also considered herein.
doi:10.3389/fpls.2015.00007
PMCID: PMC4310283
flavonoid biosynthesis; unnatural flavonoids; metabolic engineering; dynamic regulation; metabolic control; secondary metabolites; combinatorial biosynthesis
21.  Phytoestrogens: food or drug? 
Within the past several years, the relation between diet and health has been accepted by the mainstream nutrition community and in this connection interest in the physiological role of bioactive compounds present in plants has dramatically increased over the last decade.
The phytoestrogens are bioactive molecules present as nutritional constituents of widely consumed vegetables. Their name derives from the fact that they are able to bind to estrogen receptors and to induce an estrogenic/antiestrogenic response in target tissues. Natural estrogens are involved in a multiplicity of programmed events in target tissues as uterus, breast, pituitary gland and hormone responsive tumors. Phytoestrogens are present in many human foodstuffs including fruits (plum, pear, apple grape berries, …), vegetables (beans, sprouts, cabbage, spinach, soybeans, grains, hops, garlic, onion,…), wine, tea, and they have been identified in a number of botanical dietary supplements. They include a wide variety of structurally different compounds such as isoflavones, mainly found in soy, lignans found in grains, stilbenes found in the skin of grapes. Other less investigated compounds include flavones, flavans, isoflavanes and coumestans. The estrogenic or antiestrogenic activity of any chemicals depends on the ability of the compound to interact with the ERs (ERα , ERβ ).
This article reported the knowledge about the activity of phytoestrogens from a pharmacological point of view for their estrogenicity or antiestrogenicity.
PMCID: PMC2781234  PMID: 22461212
phytoestrogens; nutrition; bioactive compounds; soy; bone health
22.  Synergistic Action of Flavonoids, Baicalein, and Daidzein in Estrogenic and Neuroprotective Effects: A Development of Potential Health Products and Therapeutic Drugs against Alzheimer's Disease 
Despite the classical hormonal effect, estrogen has been reported to mediate neuroprotection in the brain, which leads to the searching of estrogen-like substances for treating neurodegenerative diseases. Flavonoids, a group of natural compounds, are well known to possess estrogenic effects and used to substitute estrogen, that is, phytoestrogen. Flavonoid serves as one of the potential targets for the development of natural supplements and therapeutic drugs against different diseases. The neuroprotection activity of flavonoids was chosen for a possible development of anti-Alzheimer's drugs or food supplements. The estrogenic activity of two flavonoids, baicalein and daidzein, were demonstrated by their strong abilities in stimulating estrogen receptor phosphorylation and transcriptional activation of estrogen responsive element in MCF-7 breast cells. The neuroprotection effects of flavonoids against β-amyloid (Aβ) were revealed by their inhibition effects on in vitro Aβ aggregation and Aβ-induced cytotoxicity in PC12 neuronal cells. More importantly, the estrogenic and neuroprotective activities of individual flavonoid could be further enhanced by the cotreatment in the cultures. Taken together, this synergistic effect of baicalein and daidzein might serve as a method to improve the therapeutic efficacy of different flavonoids against Aβ, which might be crucial in developing those flavonoidsin treating Alzheimer's disease in the future.
doi:10.1155/2013/635694
PMCID: PMC3766606  PMID: 24058373
23.  Environmental and dietary estrogens and human health: is there a problem? 
Environmental Health Perspectives  1995;103(4):346-351.
It has been hypothesized that organochlorine pesticides and other environmental and dietary estrogens may be associated with the increased incidence of breast cancer in women and decreased sperm concentrations and reproductive problems in men. However, elevation of organochlorine compounds such as dichlorodipehenyldichloroethylene (DDE) and polychlorinated biphenyls (PCBs) in breast cancer patients is not consistently observed. Reanalysis of the data showing that male sperm counts decreased by over 40% during 1940 to 1990 indicated that inadequate statistical methods were used and that the data did not support a significant decline in sperm count. Humans are exposed to both natural and industrial chemicals which exhibit estrogenic and antiestrogenic activities. For example, bioflavonoids, which are widely distributed in foods, and several industrial compounds, including organochlorine pesticides and various phenolic chemicals, exhibit estrogenic activity. Humans are also exposed to chemicals which inhibit estrogen-induced responses such as the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin and related chlorinated aromatics, polynuclear aromatic hydrocarbon combustion products, and indole-3-carbinol, which is found in cruciferous vegetables. Many of the weak estrogenic compounds, including bioflavonoids, are also antiestrogenic at some concentrations. A mass balance of dietary levels of industrial and natural estrogens, coupled with their estimated estrogenic potencies, indicates that the dietary contribution of estrogenic industrial compounds is 0.0000025% of the daily intake of estrogenic flavonoids in the diet.(ABSTRACT TRUNCATED AT 250 WORDS)
Images
PMCID: PMC1519094  PMID: 7607134
24.  Lasofoxifene (CP-336,156), a novel selective estrogen receptor modulator, in preclinical studies 
Estrogen replacement therapy is reported to reduce the incidence of vertebral fractures in postmenopausal women, however, its compliance is limited because of side effects and safety concerns. Estrogen’s side effects on breast and uterine tissues leading to the potential increased risk of uterine and breast cancer limit widespread estrogen usage. Thus, there is a significant medical need for a therapy that protects against postmenopausal bone loss but is free of estrogen’s negative effects on reproductive tissues. Selective estrogen receptor modulators (SERMs) have been investigated as an alternative to hormone replacement therapy. One such compound, raloxifene, has been approved for the prevention and treatment of osteoporosis.
Lasofoxifene (LAS), a new, nonsteroidal, and potent SERM, is an estrogen antagonist or agonist depending on the target tissue. LAS selectively binds with high affinity to human estrogen receptors. In ovariectomized (OVX) rat studies, LAS prevented the decrease in femoral bone mineral density, tibial and lumbar vertebral trabecular bone mass at an ED100 of about 60 μg/kg/day. LAS inhibited the activation of trabecular and endocortical bone resorption and bone turnover in tibial metaphyses and diaphyses, and lumbar vertebral body in OVX rats. In addition, LAS decreased total serum cholesterol, inhibited body weight gain and increased soleus muscle weight in OVX rats. Similarly, LAS prevented bone loss induced by orchidectomy or aging in male rats by decreasing bone resorption and bone turnover while it had no effect in the prostate. Further, LAS decreased total serum cholesterol in intact aged male rats or in orchidectomized male rats. Synergestic skeletal effects were found with LAS in combination with bone anabolic agents such as prostaglandin E2 (PGE2), parathyroid hormone (PTH) or a growth hormone secretagoue (GHS) in OVX rats. In combination with estrogen, LAS inhibited the uterine stimulating effects of estrogen but did not block the bone protective effects of estrogen. In immature and aged female rats, LAS did not affect the uterine weight and uterine histology. In OVX adult female rats, LAS slightly but significantly increased uterine weight. These results demonstrated that LAS produced effects on the skeleton indistinguishable from estrogen in female and male rats. However, unlike estrogen, LAS had little effect on uterine weight and cellular proliferation of uterus in female rats. In preclinical anti-tumor studies, LAS inhibited human breast cancer growth in mice bearing MCF7 tumors, prevented NMU-induced mammary carcinomas and possessed chemotherapeutic effects in NMU-induced carcinomas in rats.
Therefore, we conclude that LAS possesses the antiestrogenic effects in breast tissue and estrogenic effects in bone and serum cholesterol, but lacks estrogen’s side effects on uterine tissue. These data support the therapeutic potential of LAS for the prevention and treatment of postmenopausal bone loss and mammary carcinomas in humans.
doi:10.1007/s11357-002-0007-y
PMCID: PMC3455753  PMID: 23604900
25.  INTERACTIONS OF FLAVONES AND OTHER PHYTOCHEMICALS WITH ADENOSINE RECEPTORS 
Dietary flavonoids have varied effects on animal cells, such as inhibition of platelet binding and aggregation, inhibition of inflammation, and anticancer properties, but the mechanisms of these effects remain largely unexplained. Adenosine receptors are involved in the homeostasis of the immune, cardiovascular, and central nervous systems, and adenosine agonists/antagonists exert many similar effects. The affinity of flavonoids and other phytochemicals to adenosine receptors suggests that a wide range of natural substances in the diet may potentially block the effects of endogenous adenosine. We used competitive radioligand binding assays to screen flavonoid libraries for affinity and a computational CoMFA analysis of flavonoids to compare steric and electrostatic requirements for ligand recognition at three subtypes of adenosine receptors. Flavone derivatives, such as galangin, were found to bind to three subtypes of adenosine receptors in the μM range. Pentamethylmorin (Ki 2.65 μM) was 14- to 17-fold selective for human A3 receptors than for A1 and A2A receptors. An isoflavone, genistein, was found to bind to A1 receptors. Aurones, such as hispidol (Ki 350 nM) are selective A1 receptor antagonists, and, like genistein, are present in soy. The flavones, chemically optimized for receptor binding, have led to the antagonist, MRS 1067 (3,6-dichloro-2′-(isopropoxy)-4′-methylflavone), which is 200-fold more selective for human A3 than A1 receptors. Adenosine receptor antagonism, therefore, may be important In the spectrum of biological activities reported for the flavonoids.
PMCID: PMC3429336  PMID: 12083460

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