Antiproliferative activity of flavonoids on several cancer cell lines.

Twenty-seven Citrus flavonoids were examined for their antiproliferative activities against several tumor and normal human cell lines. As a result, 7 flavonoids were judged to be active against the tumor cell lines, while they had weak antiproliferative activity against the normal human cell lines. The rank order of potency was luteolin, natsudaidain, quercetin, tangeretin, eriodictyol, nobiletin, and 3,3',4',5,6,7,8-heptamethoxyflavone. The structure-activity relationship established from comparison among these flavones and flavanones showed that the ortho-catechol moiety in ring B and a C2-C3 double bond were important for the antiproliferative activity. As to polymethoxylated flavones, C-3 hydroxyl and C-8 methoxyl groups were essential for high activity.     

Antibacterial activity of Citrus reticulata peel extracts

Citrus peels were successively extracted with hexane, chloroform and acetone using a soxhlet extractor. The hexane and chloroform extracts were fractionated into alcohol-soluble and alcohol-insoluble fractions. These fractions were tested against different gram positive and gram negative bacteria. The EtOH-soluble fraction was found to be most effective. Fractionation of EtOH-soluble fraction on silica gel column yielded three polymethoxylated flavones, namely desmethylnobiletin, nobiletin and tangeretin. Their structures were confirmed by UV, 1H, 13C NMR and mass spectral studies. The findings indicated a potential of these natural compounds as biopreservatives in food applications.     

Regulation of adipocytokine secretion and adipocyte hypertrophy by polymethoxyflavonoids, nobiletin and tangeretin

AimsThe polymethoxyflavonoids nobiletin and tangeretin possess several important biological properties such as neuroprotective, antimetastatic, anticancer, and anti-inflammatory properties. The present study was undertaken to examine whether nobiletin and tangeretin could modulate adipocytokine secretion and to evaluate the effects of these flavonoids on the hypertrophy of mature adipocytes.Main methodsAll experiments were performed on the murine preadipocyte cell line 3T3-L1. We studied the formation of intracellular lipid droplets in adipocytes and the apoptosis-inducing activity to evaluate the effects of polymethoxyflavonoids on adipocyte differentiation and hypertrophy, respectively. The secretion of adipocytokines was measured using ELISA.Key findingsWe demonstrated that the combined treatment of differentiation reagents with nobiletin or tangeretin differentiated 3T3-L1 preadipocytes into adipocytes possessing less intracellular triglyceride as compared to vehicle-treated differentiated 3T3-L1 adipocytes. Both flavonoids increased the secretion of an insulin-sensitizing factor, adiponectin, but concomitantly decreased the secretion of an insulin-resistance factor, MCP-1, in 3T3-L1 adipocytes. Furthermore, nobiletin was found to decrease the secretion of resistin, which serves as an insulin-resistance factor. In mature 3T3-L1 adipocytes, nobiletin induced apoptosis; tangeretin, in contrast, did not induce apoptosis, but suppressed further triglyceride accumulation.SignificanceOur results suggest that nobiletin and tangeretin are promising therapeutic candidates for the prevention and treatment of insulin resistance by modulating the adipocytokine secretion balance. We also demonstrated the different effects of nobiletin and tangeretin on mature adipocytes.     

台湾香檬在厦门地区的引种初报

对台湾香檬Citrus depressa植物学特性、生长性状、果实性状与营养成分、生态习性等进行适应性栽培试验与观测。结果表明,台湾香檬在厦门地区的适应性较好,植株生长快,一年梢可抽4～5次,且多批次开花结果,产量可达5460 kg·hm-2,果实川陈皮素、橘皮素等含量较高。     

Suppressive effect of nobiletin and epicatechin gallate on fructose uptake in human intestinal epithelial Caco-2 cells

Abstract

Inhibition of excessive fructose intake in the small intestine could alleviate fructose-induced diseases such as hypertension and non-alcoholic fatty liver disease. We examined the effect of phytochemicals on fructose uptake using human intestinal epithelial-like Caco-2 cells which express the fructose transporter, GLUT5. Among 35 phytochemicals tested, five, including nobiletin and epicatechin gallate (ECg), markedly inhibited fructose uptake. Nobiletin and ECg also inhibited the uptake of glucose but not of L-leucine or Gly-Sar, suggesting an inhibitory effect specific to monosaccharide transporters. Kinetic analysis further suggested that this reduction in fructose uptake was associated with a decrease in the apparent number of cell-surface GLUT5 molecules, and not with a change in the affinity of GLUT5 for fructose. Lastly, nobiletin and ECg suppressed the permeation of fructose across Caco-2 cell monolayers. These findings suggest that nobiletin and ECg are good candidates for preventing diseases caused by excessive fructose intake.     

Semisynthesis and antiproliferative evaluation of a series of 3′-aminoflavones

A series of 3′-aminoflavones 5,6,7,8-tetra- or 5,7-dioxygenated on the A-ring was synthesized from tangeretin or naringin, two natural Citrus flavonoids. These flavones were evaluated for antiproliferative activity, activation of apoptosis, and inhibition of tubulin assembly. The most antiproliferative flavones exhibit a common 5-hydroxy-6,7,8-trimethoxy substitution pattern on the A-ring.     

台湾香檬果皮川陈皮素分离制备及其抗肿瘤活性研究

以台湾香檬(Citrus depressa)果皮为原料,采用超声波提取川陈皮素并用高效液相分离鉴定,探讨台湾香檬果皮川陈皮素的抗肿瘤活性。采用MTT法检测川陈皮素对肺癌、肝癌、卵巢癌细胞株增殖的影响,通过流式细胞仪采用双染法检测川陈皮素对肝癌细胞凋亡的作用。结果表明,台湾香檬果皮川陈皮素对肿瘤细胞有一定的广谱抑制作用,并能有效促进肝癌细胞凋亡。     

A citrus polymethoxyflavonoid, nobiletin, is a novel MEK inhibitor that exhibits antitumor metastasis in human fibrosarcoma HT-1080 cells

The activation of mitogen-activated protein/extracellular signal-regulated kinase (MEK) is well known to be associated with tumor invasion and metastasis. We previously reported that a polymethoxyflavonoid, nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone), derived from Citrus depressa (Hayata), inhibits the phosphorylation of MEK and thereby suppresses matrix metalloproteinase (MMP) expression in a tumor-metastasis stimulator, 12-O-tetradecanoyl phorbol 13-acetate (TPA)-stimulated human fibrosarcoma HT-1080 cells [Mol. Cancer Ther. 3 (2004) 839-847]. In the present study, we investigated whether or not nobiletin might directly influence MEK activity to exhibit the antitumor metastatic activity in vitro. MEK kinase assay using myelin basic protein (MBP) revealed that TPA-augmented MEK activity in HT-1080 cells and that the augmented MEK activity was diminished by nobiletin treatment. In addition, the decrease in MEK activity caused by nobiletin was found to inhibit the phosphorylation of extracellular regulated kinases (ERK), a downstream signaling factor for MEK. Furthermore, when an immunoprecipitated active MEK was incubated with nobiletin under cell-free conditions, nobiletin was found to inhibit the MEK-mediated MBP phosphorylation. In contrast, other citrus polymethoxyflavonoids such as 3-hydroxy-5,6,7,8,3′,4′-hexamethoxyflavone (natsudaidain) and 3,5,6,7,8,3′,4′-heptamethoxyflavone, did not directly inhibit MEK activity. Moreover, natsudaidain and 3,5,6,7,8,3′,4′-heptamethoxyflavone exhibited no or less inhibitory effect than nobiletin on the proMMP-9/progelatinase B production in HT-1080 cells. Therefore, these results provide novel evidence that nobiletin directly inhibits MEK activity and decreases the sequential phosphorylation of ERK, exhibiting the antitumor metastatic activity by suppressing MMP expression in HT-1080 cells.     

Activation of human bitter taste receptors by polymethoxylated flavonoids

Tangeretin and nobiletin are polymethoxylated flavonoids in citrus peel. Both tangeretin and nobiletin are bitter; however, their bitterness has not been evaluated using human bitter taste receptors (hTAS2Rs). We screened 25 kinds of hTAS2Rs and found that hTAS2R14 and hTAS2R46 received both compounds.     

Nobiletin, a polymethoxylated flavonoid from citrus, shows anti-angiogenic activity in a zebrafish in vivo model and HUVEC in vitro model

Traditional Chinese medicinal herbs are a rich source of compounds with reported anti-inflammatory and anti-carcinogenic effects. Growing evidence shows the codependence of chronic inflammation and angiogenesis, and the potential benefits of targeting angiogenesis in the treatment of chronic inflammation and targeting inflammation in the treatment of diseases with impaired angiogenesis. We hypothesized that the anti-inflammatory activity of the natural compounds may owe at least some of its efficacy to their anti-angiogenic activity and hence we investigated the anti-angiogenic activity of these compounds in vivo in zebrafish embryos and in vitro in human umbilical vein endothelial cells (HUVECs). Nobiletin, a polymethoxylated flavonoid from citrus fruits, showed anti-angiogenic activity in both assays. Nobiletin inhibited the formation of intersegmental vessels (ISVs) in live transgenic zebrafish embryos expressing green fluorescent protein (GFP) in the vasculature. Cell cycle analysis of dissociated zebrafish embryo cells showed that nobiletin induced G0/G1 phase accumulation in a dose-dependent manner in GFP-positive endothelial cells. Nobiletin also dose-dependently induced VEGF-A mRNA expression. In HUVECs, nobiletin inhibited endothelial cell proliferation and, to a greater extent, tube formation in a dose-dependent manner. As in the in vivo study, nobiletin induced G0/G1 cell cycle arrest in HUVECs. However, this arrest was not accompanied by an increase in apoptosis, indicating a cytostatic effect of nobiletin. This study, for the first time, identifies nobiletin as having potent anti-angiogenic activity and suggests that nobiletin has a great potential for future research and development as a cytostatic anti-proliferative agent.     

Dietary administration of citrus nobiletin inhibits azoxymethane-induced colonic aberrant crypt foci in rats

The modifying effects of dietary feeding of a polymethoxyflavonoid nobiletin isolated from Citrus unshiu on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effects of nobiletin on cell proliferation activity of ACF using a monoclonal antibody MIB-5. Rats were given subcutaneous injections of AOM (15 mg/kg body weight) once a week for 3 weeks to induce ACF. They also received the experimental diet containing 0.01% or 0.05% nobiletin for 5 weeks, starting one week before the first dosing of AOM. AOM exposure produced 139 +/- 35 ACF/rat at the end of the study (week 5). Dietary administration of nobiletin caused significant reduction in the frequency of ACF: 70 +/- 15 (50% reduction, p<0.001) at a dose of 0.01% and 63 +/- 10 (55% reduction, p<0.001) at a dose of 0.05%. Nobiletin feeding significantly lowered MIB-5-index in ACF. Also, dietary administration of nobiletin significantly reduced prostaglandin E2 content in the colonic mucosa. These findings might suggest possible chemopreventive ability of nobiletin, through suppression of cell proliferating activity of ACF, in the development of ACF.     

Simplified Isoelectric Focusing Columns of Small Scales

ABSTRACT

Citrus plants are rich in flavonoids and beneficial for lipid metabolism. However, the mechanism has not been fully elucidated. Both citrus peel flavonoid extracts (CPFE) and a mixture of their primary flavonoid compounds, namely, nobiletin, tangeretin and hesperidin, citrus flavonoid purity mixture (CFPM), were found to have lipid-lowering effects on oleic acid-induced lipid accumulation in HepG2 cells. The carnitine palmitoyltransferase 1α (CPT1α) gene was markedly increased, while the fatty acid synthase (FAS) gene was significantly decreased by both CPFE and CFPM in oleic acid-treated HepG2 cells. Flavonoid compounds from citrus peel suppressed miR-122 and miR-33 expression, which were induced by oleic acid. Changes in miR-122 and miR-33 expression, which subsequently affect the expression of their target mRNAs FAS and CPT1α, are most likely the principal mechanisms leading to decreased lipid accumulation in HepG2 cells. Citrus flavonoids likely regulate lipid metabolism by modulating the expression levels of miR-122 and miR-33.     

Inhibitory effect of cinnamoyl compounds against human malignant cell line

In the present study, anti-proliferative effects of dietary polyphenolic compounds have been observed and demonstrated the strong anticancer efficacy of curcumin (CMN), an active constituent of dietary spice (turmeric) using human leukemia cancer cell line. CMN inhibited the proliferation of K562 leukemic cells by induction of apoptosis. The current study demonstrated synergy with combination of drug therapy, and suggested that combination of ferulic acid and cisplatin synergistically inhibited cellular proliferation. Cytotoxic synergy was observed independent of the sequence of addition of two drugs to cultured cells. The synergized growth inhibitory effect with cisplatin was probably associated with G2-M arrest in cell cycle progression. These findings suggested that among the cinnamoyl compounds, CMN was most potent and FER appeared to be a better modulating agent on human malignant cell line.     

Polymethoxylated flavones induce Ca(2+)-mediated apoptosis in breast cancer cells

Flavonoids, polyphenolic phytochemicals which include flavones and isoflavones, are present in the common human diet. It has been suggested that these compounds may exert anticancer activity; however, the mechanisms involved remain unknown. We have recently shown (Sergeev, 2004, Biochem Biophys Res Commun 321: 462-467) that isoflavones can activate the novel apoptotic pathway mediated by cellular Ca(2+). Here, we report that polymethoxyflavones (PMFs) derived from sweet orange (Citrus sinensis L.) inhibit growth of human breast cancer cells via Ca(2+)-dependent apoptotic mechanism. The treatment of MCF-7 breast cancer cells with 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (3'-OH-TtMF) induced a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) resulting from both depletion of the endoplasmic reticulum Ca(2+) stores and Ca(2+) influx from the extracellular space. This increase in [Ca(2+)](i) was associated with the activation of the Ca(2+)-dependent apoptotic proteases, mu-calpain and caspase-12, as evaluated with the calpain and caspase-12 peptide substrates and antibodies to active (cleaved) forms of the enzymes. Corresponding non-hydroxylated PMFs, 3,5,6,7,8,3',4'-heptamethoxyflavone (HpMF) and 5,6,7,3',4'-pentamethoxyflavone (PtMF), were dramatically less active in inducing Ca(2+)-mediated apoptosis. Our results strongly suggest that the cellular Ca(2+) modulating activity of flavonoids underlies their apoptotic mechanism and that hydroxylation of PMFs is critical for their ability to induce an increase in [Ca(2+)](i) and, thus, activate Ca(2+)-dependent apoptotic proteases.     

Efficient and scalable method in isolation of polymethoxyflavones from orange peel extract by supercritical fluid chromatography

Polymethoxyflavones (PMFs) from citrus genus are of particular interest because of their broad spectrum of biological activities, such as anti-inflammatory, anti-carcinogenic, and anti-atherogenic properties. Recently, the exploration into the beneficial health properties of PMFs in citrus fruits has dramatically increased. However, the supply of pure PMFs in the in vivo study is a limiting factor due to the difficulties in large-scale isolation of the interested PMFs. Therefore, the development of an efficient and a scalable separation method of PMFs is necessary and significant. In this paper, we report a newly developed method for efficient and relatively large-scale isolation of four PMFs from sweet orange (Citrus sinensis) peel by employing supercritical chromatography (SFC): nobiletin, tangeretin, 3,5,6,7,8,3',4'-heptamethoxyflavone and 5,6,7,4'-tetramethoxyflavone.     

Analyses of putative anti-cancer potential of three STAT3 signaling inhibitory compounds derived from Salvia officinalis

The extract of Salvia officinalis (Common Sage) exhibited inhibitory activity of STAT3 signal after screening of several plants extracts using the STAT3-responsive reporter system. Cirsiliol, luteolin, and carnosol were identified from the methanol extract of Silvia officinalis as inhibitors of STAT3 signaling and the effects of these three compounds on STAT3 protein or growth inhibition on cancer cells was compared. Luteolin at the dose of 90 μM clearly suppressed the phosphorylation of STAT3 induced by IL-6, while carnosol was prone to decrease total STAT3 proteins at high doses (>90 μM). Cirsiliol had almost no effect. Since the three compounds exhibited similar concentration-dependent suppression patterns in the reporter assay except for cirsiliol became plateau beyond 30 μM, these compounds appeared to function as STAT3 inhibitory factors in different ways. The direct anti-proliferative activity of three compounds was examined with or without the anti-cancer drug gefitinib using HepG2 and A549 cells. The anti-proliferative effect of the three compounds was additively enhanced by gefitinib. At the doses of 3.6 μM, statistically significant suppression of proliferation was observed in HepG2 cells only by cirsiliol among the three compounds in the absence of gefitinib but all three compounds were prone to suppress the proliferation of HepG2 cells and A549 cells dose-dependently although cirsiliol showed a modest dose-dependency and this suppression of proliferation was enhanced by the addition of gefitinib. Cirsiliol, a dimethyoxylated flavone, activated the natural killer activity of KHYG-1 cells against erythroleukemia K562 cells like a hexamethoxylated flavone, nobiletin, suggesting that it may also have an indirect anti-cancer potential through activation of NK cells. These results shed light on the putative anti-cancer potential of Salvia officinalis.     

Synthesis and biological activity of enantiomeric pairs of 5-vinylthiolactomycin congeners

Twelve enantiomeric pairs of 5-vinylthiolactomycin congeners were synthesized by employing our efficient synthetic route previously explored for the synthesis of enantiomeric pairs of thiolactomycin and its 3-demethyl derivative. From the biological activity assay carried out using the obtained congeners along with enantiomeric pairs of thiolactomycin and its 3-demethyl derivative previously prepared, it appeared evident that in vitro antibacterial and mammalian type I FAS inhibitory activity of thiolactomycin congeners can be cleanly separated by changing not only the structure but also the absolute configuration of the side chain at the C(5)-position. These studies led us to explore (S)-3-demethyl-5-(pent-1-enyl)thiolactomycin derivative [(S)-4-hydroxy-5-methyl-5-(pent-1-enyl)-5H-thiophen-2-one] which exhibits type I FAS inhibitory activity equal to that of C75, the potent inhibitor so far reported, with complete loss of in vitro antibacterial activity.     

Tangeretin inhibits streptozotocin-induced cell apoptosis via regulating NF-κB pathway in INS-1 cells

Oxidative stress is considered to play an important role in inducing the pancreatic β-cells apoptosis and promoting the development of diabetes mellitus. Tangeretin is a plant-derived flavonoid that retains antidiabetic effects. However, the role of tangeretin in streptozotocin (STZ)-induced β-cell apoptosis remains unclear. In this study, we aimed to examine the effects of tangeretin on STZ-induced cell apoptosis and the underlying mechanisms implicated in vitro. Our results showed that tangeretin improved the cell viability in STZ-induced INS-1 cells. Tangeretin reduced the increase of apoptosis ratio and revered the altered expressions of Bax and Bcl-2 caused by STZ induction. Furthermore, the impairment of insulin secretion ability as well as a reduction in messenger RNA levels of insulin 1 and 2 was significantly attenuated by tangeretin in STZ-induced INS-1 cells. Moreover, tangeretin resulted in a significant decrease in reactive oxygen species content, accompanied by an evident increase in the activities of superoxide dismutase, catalase, and glutathione peroxidase. Mechanistic studies further revealed that tangeretin inhibited the NF-κB pathway in STZ-induced INS-1 cells. These data indicated that tangeretin improved the cell apoptosis induced by STZ in INS-1 cells, which might be partly due to its antioxidant potential. Furthermore, NF-κB was found to be involved in the protective effect of tangeretin. Collectively, the results indicated that tangeretin could be used as a therapeutic approach for diabetes mellitus treatment.