Cytotoxic effects of the dietary flavones chrysin and apigenin in a normal trout liver cell line

Many flavonoids have been shown to possess prooxidant properties, capable of causing oxidative stress, especially at larger doses. Here, we examined the potential cell toxicity caused by exposure to the hydroxylated flavones chrysin, apigenin, luteolin and quercetin in comparison to the methylated flavones 5,7-dimethoxyflavone and 3',4'-dimethoxyflavone in normal Rainbow trout hepatocytes. The hydroxylated flavones, especially chrysin, demonstrated cell toxicity and inhibition of DNA synthesis at very low (2 microM) concentrations. The cytotoxicity of chrysin may partially be due to its metabolism by myeloperoxidase, which was shown to be present in these normal trout liver cells (164pmol/(min mg protein)). In contrast, methylated flavones showed no significant metabolism by myeloperoxidase and no signs of toxicity, even at much higher concentrations. These results may be useful for further investigations of cytotoxicity of dietary flavonoids.     

Apigenin induces apoptosis via downregulation of S‐phase kinase‐associated protein 2‐mediated induction of p27Kip1 in primary effusion lymphoma cells

Objective: The mechanisms that regulate mitogenic and antiapoptotic signals in primary effusion lymphoma (PEL) are not well known. In efforts to identify novel approaches to block the proliferation of PEL cells, we assessed the effect of apigenin (4′,5,7‐trihydroxyflavone), a flavonoid on a panel of PEL cell lines. Materials and methods: We studied the effect of apigenin on four PEL cell lines. Apoptosis was measured by annexin V/PI dual staining and DNA laddering. Protein expression was measured by immunoblotting. Results: Apigenin induced apoptosis in PEL cell lines in a dose dependent manner. Such effects of apigenin appeared to result from suppression of constitutively active kinase AKT resulting in down‐regulation of SKP2, hypo‐phosphorylation of Rb and accumulation of p27Kip1. Apigenin treatment of PEL cells caused dephosphorylation of p‐Bad protein leading to down regulation of the anti‐apoptotic protein, Bcl‐2 and an increase in Bax/Bcl2 ratio. Apigenin treatment also triggered Bax conformational change and subsequently translocation from cytosole to mitochondria causing loss of mitochondrial membrane potential with subsequent release of cytochrome c. Released cytochrome c onto the cytosole activated caspase‐9 and caspase‐3, followed by polyadenosin‐5′‐diphosphate‐ribose polymerase (PARP) cleavage. Finally, treatment of PEL cells with apigenin down‐regulated the expression of inhibitor of apoptosis protein (IAPs). Conclusions: Altogether, these data suggest a novel function for apigenin, acting as a suppressor of AKT/PKB pathway in PEL cells, and raise the possibility that this agent may have a future therapeutic role in PEL and possibly other malignancies with constitutive activation of the AKT/PKB pathway.     

Flavonoids from Artemisia ludoviciana var. ludoviciana

Nineteen flavonoids were isolated from Artemisia ludoviciana var. ludoviciana, including a new 2′- hydroxy- 6-methoxyflavone, 5,7,2′,4′-tetrahydroxy-6,5′-dimethoxyflavone. The known compounds include quercetagetin 3,6,3′,4′-tetramethyl ether, eupatilin, 5,7-dihydroxy-3,6,8,4′-tetramethoxyflavone, luteolin 3′,4′-dimethyl ether, jaceosidin, 5,7,4′-trihydroxy-3,6-dimethoxyflavone, tricin, hispidulin, chrysoeriol, kaempferol 3-methyl ether, apigenin, axillarin, eupafolin, selagin and luteolin together with three flavones which were previously isolated for the first time from Artemisia frigida: 5,7,4′-trihydroxy-6, 3′,5′-trimethoxyflavone, 5,7,3′-trihydroxy-6,4′,5′-trimethoxyflavone and 5,7,3′,4′-tetrahydroxy-6,5′- dimethoxyflavone.     

A distinctive flavonoid chemistry for the anomalous genus Biebersteinia

Leaf surface extracts of Biebersteinia orphanidis have yielded a complex mixture of five flavones with the unusual 5,7-dihydroxy-6,8-dimethoxy A ring substitution pattern. They are acerosin, hymenoxin, nevadensin, sudachitin and 5,7,4'-trihydroxy-6,8-dimethoxyflavone. Also present at the leaf surface are gardenin B, luteolin, apigenin, acacetin and the coumarin umbelliferone. The internal leaf flavonoids include the 7-glucosides of apigenin, luteolin and tricetin, together with the 7-rutinosides of apigenin and luteolin. This profile differs from those of B. heterostemon and B. odora. It appears that B. orphanidis is as highly distinctive in its flavonoid pattern as it is phytogeographically. The data also confirm the conclusion of other studies, including rbcL and atpB gene sequence analysis, that Biebersteinia is completely unrelated to the Geraniaceae, where it was once placed.     

Luteolin and Apigenin Attenuate 4-Hydroxy-2-Nonenal-Mediated Cell Death through Modulation of UPR,Nrf2-ARE and MAPK Pathways in PC12 Cells

Luteolin and apigenin are dietary flavones and exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) has been implicated as a causative agent in the development of neurodegenerative disorders. This study investigates the cytoprotective effects of luteolin and apigenin against 4-HNE-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Both flavones restored cell viability and repressed caspase-3 and PARP-1 activation in 4-HNE-treated cells. Luteolin also mitigated 4-HNE-mediated LC3 conversion and reactive oxygen species (ROS) production. Luteolin and apigenin up-regulated 4-HNE-mediated unfolded protein response (UPR), leading to an increase in endoplasmic reticulum chaperone GRP78 and decrease in the expression of UPR-targeted pro-apoptotic genes. They also induced the expression of Nrf2-targeted HO-1 and xCT in the absence of 4-HNE, but counteracted their expression in the presence of 4-HNE. Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin. Consistently, enhanced phosphorylation of JNK and p38 MAPK was observed in luteolin- and apigenin-treated cells. In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity. Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity.     

Temporal PTEN inactivation causes proliferation of saphenous vein smooth muscle cells of human CABG conduits

Internal mammary artery (IMA) coronary artery bypass grafts (CABG) are remarkably resistant to intimal hyperplasia (IH) as compared to saphenous vein (SV) grafts following aorto-coronary anastomosis. The reason behind this puzzling difference still remains an enigma. In this study, we examined the effects of IGF-1 stimulation on the PI3K-AKT/PKB pathway mediating proliferation of smooth muscle cells (SMCs) of IMA and SV origin and the specific contribution of phosphatase and tensin homologue (PTEN) in regulating the IGF-1-PI3K-AKT/PKB axis under these conditions. Mitogenic activation with IGF-1, time-dependently stimulated the phosphorylation of PI3K and AKT/PKB in the SV SMCs to a much greater extent than the IMA. Conversely, PTEN was found to be significantly more active in IMA SMCs. Transient overexpression of PTEN in SMCs of SV and IMA inhibited AKT/PKB activity and upstream of AKT/PKB, caused a reduction of IGF-1 receptors. Downstream, PTEN overexpression in SV SMCs induced the transactivation of tumour suppressor protein p53 by down-regulating the expression of its inhibitor MDM2. However, PTEN overexpression had no significant effect on MDM2 and p53 expression in IMA SMCs. PTEN overexpression inhibited IGF-1-induced SMC proliferation in both SV and IMA. PTEN suppression, induced by siRNA transfection of IMA SMCs diminished the negative regulation of PI3K-PKB signalling leading to greater proliferative response induced by IGF-1 stimulation. Thus, we show for the first time that early inactivation of PTEN in SV SMCs leads to temporally increased activity of the pro-hyperplasia PI3K-AKT/PKB pathway leading to IH-induced vein graft occlusion. Therefore, modulation of the PI3K-AKT/PKB pathway via PTEN might be a novel and effective strategy in combating SV graft failure following CABG.     

Investigation of Two Distinct Flavone Synthases for Plant-Specific Flavone Biosynthesis in Saccharomyces cerevisiae

Flavones are plant secondary metabolites that have wide pharmaceutical and nutraceutical applications. We previously constructed a recombinant flavanone pathway by expressing in Saccharomyces cerevisiae a four-step recombinant pathway that consists of cinnamate-4 hydroxylase, 4-coumaroyl:coenzyme A ligase, chalcone synthase, and chalcone isomerase. In the present work, the biosynthesis of flavones by two distinct flavone synthases was evaluated by introducing a soluble flavone synthase I (FSI) and a membrane-bound flavone synthase II (FSII) into the flavanone-producing recombinant yeast strain. The resulting recombinant strains were able to convert various phenylpropanoid acid precursors into the flavone molecules chrysin, apigenin, and luteolin, and the intermediate flavanones pinocembrin, naringenin, and eriodictyol accumulated in the medium. Improvement of flavone biosynthesis was achieved by overexpressing the yeast P450 reductase CPR1 in the FSII-expressing recombinant strain and by using acetate rather than glucose or raffinose as the carbon source. Overall, the FSI-expressing recombinant strain produced 50% more apigenin and six times less naringenin than the FSII-expressing recombinant strain when p-coumaric acid was used as a precursor phenylpropanoid acid. Further experiments indicated that unlike luteolin, the 5,7,4′-trihydroxyflavone apigenin inhibits flavanone biosynthesis in vivo in a nonlinear, dose-dependent manner.     

Approach to the study of C-glycosyl flavones by ion trap HPLC-PAD-ESI/MS/MS: application to seeds of quince (Cydonia oblonga)

Ion trap HPLC-PAD-ESI/MS/MS has been used to study C-glycosyl flavones in quince seeds. Comparative analysis of the ions [(M-H)-60]-, [(M-H)-90]- and [(M-H)-120]- from 6-C- and 8-C-glycosyl flavone isomers, together with their respective retention times, allowed deductions to be made about the nature of the sugar units and the positions of C-glycosylation. Vicenin-2 (6,8-di-C-glucosyl apigenin), lucenin-2 (6,8-di-C-glucosyl luteolin), stellarin-2 (6,8-di-C-glucosyl chrysoeriol), isoschaftoside (6-C-arabinosyl-8-C-glucosyl apigenin), schaftoside (6-C-glucosyl-8-C-arabinosyl apigenin), 6-C-pentosyl-8-C-glucosyl chrysoeriol and 6-C-glucosyl-8-C-pentosyl chrysoeriol were identified in quince seed.     

Analysis of flavonoids and characterization of theOsFNS gene involved in flavone biosynthesis in Rice

The major flavonoids in rice leaves were analyzed via LC-MS/MS after their total flavonoid extracts were hydrolyzed. The most abundant flavones were apigenin, luteolin, and tricetin. Of these, tricetin was methylated at its 3′ and 5′-hydroxyl group to form tricin, which was probablyO-glycosylated. Both 3′-O-methylated luteolin and luteolin were found in theC-glycosylated form while apigenin wasC-glycosylated. We also cloned and characterizedOsFNS, which catalyzes the reaction from flavanone (naringenin) to flavone (apigenin). Analysis of the reaction product with recombinant OsFNS showed that it indeed converts naringenin to apigenin.     

牛奶菜中黄酮类成分的研究

从牛奶菜(Marsdenia sinensis Hemsl)根中分离得到6个黄酮类化合物,经光谱分析确定其结构为:芹菜素(1),(2S)-柚皮素(2),木犀草素(3),异牡荆素(4),8-C-β-D-Glucopyranosyl apigeninidin(5),5,7-二羟基色原酮(6)。所有化合物均为首次从该植物中分离得到,同时对分离得到的单体化合物进行细胞毒活性测试,化合物1～5均表现出较强的细胞毒活性。     

Assessment of estrogenic activity of flavonoids from Mediterranean plants using an in vitro short-term test

Six isoflavones, daidzein (4',7,-dihydroxyisoflavone), genistein (4',5,7-trihydroxyisoflavone), genistin (genistein 7-O-beta-D-glucopyranoside), isoprunetin (4',7-dihydroxy, 5-metoxyisoflavone), isoprunetin 7-O-beta-D-glucopyranoside, isoprunetin 4',7-di-O-beta-D-glucopyranoside and four flavones, luteolin (3',4',5,7-tetrahydroxyflavone), luteolin 7-O-beta-D-glucopyranoside, luteolin 4'-O-beta-D-glucopyranoside, licoflavone C (4',5,7-trihydroxy,8-isoprenylflavone) were purified from Mediterranean plants (Genista morisii and Genista ephedroides) and their estrogenic activity was assessed by a yeast reporter gene assay (Saccharomyces cerevisiae RMY326 ER-ERE). Licoflavone C showed a powerful estrogenic activity at 10(-7) M (0.0338 microg/ml) and it was 47.45% than 10(-8) M 17beta-estradiol (0.00272 microg/ml). The estrogenicity of this flavone was found to be comparable to the activity showed by genistein at 10(-6) M (0.27 microg/ml). This study points out that a glucose substituent in flavones and isoflavones modulates the hormone-like activity in a different way. Isoflavone aglycones showed a more estrogenic activity than the corresponding glucosides. Conversely, the glucosidation made estrogenic the flavone luteolin and the position of substitution differently influenced the estrogenic activity of compounds.     

Tea prepared from Anastatica hirerochuntica seeds contains a diversity of antioxidant flavonoids, chlorogenic acids and phenolic compounds

HPLC-PDA-MS(2) was used to identify phenolic and polyphenolic compounds in an herbal tea made from seeds of Anastatica hirerochuntica, a plant found in the Sahara-Arabian deserts and used to treat a variety of ailments. Twenty compounds comprising a series of flavone C- and O-linked glycosides, phenolic acids, chlorogenic acids and flavonols were identified or partially identified on the basis of co-chromatography with reference compounds and MS(2) and MS(3) fragmentation patterns. The flavones were the principal components, occurring as luteolin, apigenin and diosmetin conjugates. The antioxidant potential of individual compounds in Anastatica was assessed using HPLC with an on-line ABTS·(+) detection system. This revealed that 14 compounds exhibited antioxidant activity. The highest contribution to the antioxidant capacity of the tea, 56%, came from 3,4-dihydroxybenzoic acid and caffeoyl- and dicaffeoylquinic acids while 6-C-glucosides of luteolin and apigenin contributed 41%.