To elucidate the bioavailability of luteolin and its glycosides in Chrysanthemum morifolium flowers, the absorption and metabolism of luteolin from them was investigated in rats and Caco-2 cells using HPLC and LC-MS. After oral administration of C. morifolium extract (1.7 g/kg body weight (bw), equivalent to 22.8 and 58.3 μmol/kg bw of luteolin and luteolin-7-O-glucoside, respectively) to rats, luteolin and its glycosides were quickly absorbed and luteolin, luteolin monoglucoside, and luteolin monoglucuronide were detected in the plasma. Their levels were highest at 1 h after administration (0.76 ± 0.27 μM). These compounds were also detected in media on the basolateral side from Caco-2 cells treated with the C. morifolium extract. These results suggest that luteolin and luteolin monoglucoside are rapidly absorbed after administration of C. morifolium flower extract and that luteolin, luteolin monoglucoside, and luteolin monoglucuronide may circulate in humans.

Cytochrome P450 (CYP) 1B1 catalyzes 17β-estradiol (E2) to predominantly carcinogenic 4-hydroxy-E2, whereas CYP1A1 and 1A2 convert E2 to non-carcinogenic 2-hydroxy-E2. Hence, selective inhibition of CYP1B1 is recognized to be beneficial for the prevention of E2 related breast cancer. In this study, we first evaluated the structure–property relationship of 18 major flavonoids on inhibiting enzymatic activity of CYP1A1, 1A2 and 1B1 by using an ethoxyresorufin O-deethylation assay. Flavones and flavonols indicated relatively strong inhibitory effects on CYP1s compared with flavanone that does not have the double bond between C-positions 2 and 3 on the C-ring. Flavonoids used in this study selectively inhibited CYP1B1 activity. In particular, methoxy types of flavones and flavonols such as chrysoeriol and isorhamnetin showed strong and selective inhibition against CYP1B1. To understand why selective inhibition was observed, we carried out a molecular docking analysis of these methoxyflavonoids with the 2–3 double bond and CYP1s. The results suggested that chrysoeriol and isorhamnetin fit well into the active site of CYP1B1, but do not fit into the active site of CYP1A2 and 1A1 because of steric collisions between the methoxy substituent of these methoxyflavonoids and Ser-122 in CYP1A1 and Thr-124 in CYP1A2. In conclusion, our results demonstrate: (1) strong inhibitory effects of flavonoids on CYP1 activities require the 2–3 double bond on the C-ring; (2) methoxyflavonoids with the 2–3 double bond had strong and selective inhibition against CYP1B1, suggesting chemopreventive flavonoids for E2 related breast cancer; and (3) binding specificity of these methoxyflavonoids is based on the interactions between the methoxy groups and specific CYP1s residues.Molecular docking study suggested selective inhibition of human CYP1B1 by methoxyflavonoids.

The physiology and tissue distribution of bilberry anthocyanins were studied in mice. After oral administration of bilberry extract (100 mg/kg body weight), both unmodified and methylated anthocyanins appeared in the plasma. The plasma concentration of total anthocyanins reached a maximum of 1.18 ± 0.3 μM after 15 min and then sharply decreased. Their urinary excretion was highest between 0 and 6 h after administration and had ceased by 24 h. The total quantities of bilberry anthocyanins excreted into urine represented 1.88% (range, 0.62% to 2.45%) of consumed anthocyanins. Thirteen anthocyanins were identified in bilberry extracts. Of these, malvidin-3-glucoside and -3-galactoside were the principal anthocyanins in the plasma 60 min after administration. When mice were maintained for 2 weeks on a diet containing 0.5% of bilberry extracts, the plasma concentration of anthocyanins reached a maximum of 0.26 μM. Anthocyanins were detected only in the liver, kidney, testes, and lung, with maximum tissue concentrations of 605, 207, 149, and 116 pmol/g, respectively. In these organs, malvidin-3-glucoside and -3-galactoside were the predominant anthocyanins. Anthocyanins were not detectable in the spleen, thymus, heart, muscle, brain, white fat, or eyes. We conclude that bilberry anthocyanins were absorbed into the body and distributed in specific organs, particularly the liver, kidney, and testis. The most common anthocyanins in tissues were malvidin glycosides.

Cytochrome P450 (CYP) 1 families including CYP1A1, 1A2 and 1B1 are well known to be deeply involved in the initiation of several cancers, due to the fact that they activate environmental pro-carcinogens to form ultimate carcinogens. Benzo[a]pyrene (BaP) is one of the major classes of prototypical pro-carcinogen. It is activated by the CYP1 family to its ultimate carcinogenic forms, mainly BaP-7,8-diol-9,10-epoxide (BPDE), and it forms adducts with DNA. This has been recognized to be a major initiation pathway for cancer. Our previous study demonstrated that chrysoeriol, which is a dietary methoxyflavonoid, selectively inhibited CYP1B1 enzymatic activity and might protect the CYP1B1 related-diseases such as breast cancer. In the present study, we further examined the effects of chrysoeriol on the other initiation pathway of cancer relating to the CYP1 family with BaP in human breast cancer MCF-7 cells. The effects of chrysoeriol on the formation of BPDE-DNA adducts were analyzed specifically using the liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. When MCF-7 cells were incubated with 2 μM BaP for 24 h, three types of BPDE-dG adducts, especially (+)-trans-BPDE-dG as the dominant adduct, were detected. Co-treatment of MCF-7 cells with 10 μM chrysoeriol and BaP remarkably reduced (+)-trans-BPDE-dG formation. Chrysoeriol (1–10 μM) dose-dependently inhibited both EROD activity and the gene expressions of CYP1A1, 1B1 and 1A2 stimulated by treatment with BaP. In addition, the same amounts of chrysoeriol significantly inhibited the binding of BaP to the aryl hydrocarbon receptor (AhR), which is the key factor concerning the induction of the CYP1 families. In conclusion, our results clearly indicate that chrysoeriol inhibited the formation of BPDE-DNA adducts via regulation of the AhR pathway stimulated by BaP. As a consequence chrysoeriol may be involved in the chemoprevention of environmental pro-carcinogens such as BaP.

A 17β-estradiol (E2) is hydrolyzed to 2-hydroxy-E2 (2-OHE2) and 4-hydroxy-E2 (4-OHE2) via cytochrome P450 (CYP) 1A1 and 1B1, respectively. In estrogen target tissues including the mammary gland, ovaries, and uterus, CYP1B1 is highly expressed, and 4-OHE2 is predominantly formed in cancerous tissues. In this study, we investigated the inhibitory effects of chrysoeriol (luteorin-3′-methoxy ether), which is a natural methoxyflavonoid, against activity of CYP1A1 and 1B1 using in vitro and cultured cell techniques. Chrysoeriol selectively inhibited human recombinant CYP1B1-mediated 7-ethoxyresorufin-O-deethylation (EROD) activity 5-fold more than that of CYP1A1-mediated activity in a competitive manner. Additionally, chrysoeriol inhibited E2 hydroxylation was catalyzed by CYP1B1, but not by CYP1A1. Methylation of 4-OHE2, which is thought to be a detoxification process, was not affected by the presence of chrysoeriol. In human breast cancer MCF-7 cells, chrysoeriol did not affect the gene expression of CYP1A1 and 1B1, but significantly inhibited the formation of 4-methoxy E2 without any effects on the formation of 2-methoxy E2. In conclusion, we present the first report to show that chrysoeriol is a chemopreventive natural ingredient that can selectively inhibit CYP1B1 activity and prevent the formation of carcinogenic 4-OHE2 from E2.