Natural flavonoid; CYP1A enzyme

HepG2 cell growth is inhibited by diosmetin in a concentration-dependent manner. HepG2 cells treated with diosmetin are distorted and some take on a round, floating appearance, whereas untreated cells develop normally and have a normal skeleton [1].

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Diosmetin (Dio) is a major active component of flavonoid compounds. A previous study demonstrated that Dio exhibited anticancer activity and induced apoptosis in HepG2 human hepatoma cells via cytochrome P450, family 1-catalyzed metabolism. The present study observed that cell proliferation of HepG2 cells was inhibited by Dio treatment and tumor protein p53 was significantly increased following Dio treatment. Following addition of recombinant transforming growth factor‑β (TGF‑β) protein to Dio‑treated HepG2 cells, cell growth inhibition and cell apoptosis was partially reversed. These findings suggest a novel function for the TGF‑β/TGF‑β receptor signaling pathway and that it may be a key target of Dio‑induced cell apoptosis in HepG2 cells.

In cerulein-induced acute pancreatitis, pretreatment with diosmetin significantly decreased serum levels of lipase and amylase, tissue damage, secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, myeloperoxidase (MPO) activity, trypsinogen-activating peptide (TAP) levels, expression of inducible nitric oxide synthase (iNOS), and nuclear factor (NF)-κB [2].

HepG-2 cells were maintained in a humidified atmosphere of 5% CO2 at 37°C, and cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin. HepG2 cells were grown in standard media, and when 60–70% confluent, the cells were treated with different concentrations of Diosmetin (5, 10 and 15 µg/ml) or TGF-β protein/ Diosmetin (10 µg/ml) for 24 h. Images were captured by microscopy (magnification, ×100).[1]

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HepG2 cell density was adjusted to 2×104 cells/100 µl, and the cells were seeded into 96-well plates and placed in an incubator overnight (37°C in 5% CO2) to allow for attachment and recovery. MTT and CCK8 analyses were performed separately. Briefly, cells were pretreated with 5, 10, 15 and 20 µg/ml Diosmetin for 24 h. A total of 20 µl MTT solution (5 mg/ml in PBS) solution was transferred to each well to yield a final 120 µl/well and to separate wells a total of 10 µl CCK8 (5 mg/ml in PBS) was transferred. The plates were incubated for 4 h at 37°C in 5% CO2 and the absorbance was recorded using the EnSpire™ 2300 Multilabel Plate Reader at wavelengths of 595 nm and 450 nm, respectively. The half maximal inhibitory concentration (IC50) of Diosmetin was calculated using software.[2]

Diosmetin was dissolved in vehicle (2% DMSO). Then three doses (25 mg/kg, 50 mg/kg, 100 mg/kg) were used to pretreat cerulein-induced AP. AP was induced by seven injections of cerulein (50 ug/kg, i.p. at intervals of 1 h) as described previously. The normal control mice were given saline (0.9% NaCl) solution intraperitoneally instead of cerulein (n=8 for each group). Vehicle or diosmetin (p.o.) was administered 2 h before the first cerulein injection. All animals were sacrificed at 12 h after the first injection of cerulein, a time point at which pancreatic damage had already peaked. The effect of diosmetin was evaluated by the level of serum amylase, an indicator which was usually considered to be closely related to pancreatic damage, to get an optimal dose. The optimal dose of diosmetin (100 mg/kg) was used for the next series of experiment. Then 72 mice were divided into three groups randomly: group 1, normal control; group 2, cerulein + vehicle-treated; group 3, cerulein + diosmetin-treated. The induction of AP and administration of diosmetin or vehicle were performed the same as the preliminary study. Mice were sacrificed at 6 h, 9 h and 12 h after the first cerulein injection, 8 mice at every time point in each group. Blood samples were taken to determine the serum amylase, lipase and cytokine levels. A portion of the tail of the pancreas was fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 12 h, embedded in paraffin, and cut into 5-μm thick sections which were stained with hematoxylin and eosin to observe the morphological changes under a light microscope by standard procedures. The rest portion of each pancreas was stored at -80°C for further investigation.[2]

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100 mg/kg

Mice: Experimental acute pancreatitis is induced in mice by seven intraperitoneal injection of cerulein (50 μg/kg) at hourly intervals. Diosmetin (100 mg/kg) or vehicle is pretreated 2 h before the first cerulein injection. After 6 h, 9 h, 12 h of the first cerulein injection, the severity of acute pancreatitis is evaluated biochemically and morphologically

Absorption, Distribution and Excretion
Diosmin is hydrolyzed to its aglycone diosmetin by intestinal microflora enzymes before its absorption into the body.

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Metabolism / Metabolites
... Diosmetin was metabolised to the structurally similar flavone luteolin in MDA-MB 468 cells, whereas no metabolism was seen in MCF-10A cells...
Various types of tumors are known to overexpress enzymes belonging to the CYP1 family of cytochromes P450. The present study aimed to characterize the metabolism and further antiproliferative activity of the natural flavonoid diosmetin in the CYP1-expressing human hepatoma cell line HepG2. Diosmetin was converted to luteolin in HepG2 cells after 12 and 30 hr of incubation. In the presence of the CYP1A inhibitor alpha-naphthoflavone, the conversion of diosmetin to luteolin was attenuated. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays revealed luteolin to be more cytotoxic than diosmetin. The antiproliferative effect of diosmetin in HepG2 cells was attributed to blockage at the G2/M phase as determined by flow cytometry. Induction of G2/M arrest was accompanied by up-regulation of phospho-extracellular-signal-regulated kinase (p-ERK), phospho-c-jun N-terminal kinase, p53 and p21 proteins. More importantly, induction of G2/M arrest and p53 and p-ERK up-regulation were reversed by the application of the CYP1 inhibitor alpha-naphthoflavone. Taken together, the data provide new evidence on the tumor-suppressing role of cytochrome P450 CYP1A enzymes and extend the hypothesis that the anticancer activity of dietary flavonoids is enhanced by P450-activation.
CYP1A1 and CYP1B1 are two extrahepatic enzymes that have been implicated in carcinogenesis and cancer progression. Selective inhibition of CYP1A1 and CYP1B1 by dietary constituents, notably the class of flavonoids, is a widely accepted paradigm that supports the concept of dietary chemoprevention. In parallel, recent studies have documented the ability of CYP1 enzymes to selectively metabolize dietary flavonoids to conversion products that inhibit cancer cell proliferation. In the present study /the authors/ have examined the inhibition of CYP1A1 and CYP1B1-catalyzed EROD activity by 14 different flavonoids containing methoxy- and hydroxyl-group substitutions as well as the metabolism of the monomethoxylated CYP1-flavonoid inhibitor acacetin and the poly-methoxylated flavone eupatorin-5-methyl ether by recombinant CYP1A1 and CYP1B1. The most potent inhibitors of CYP1-EROD activity were the methoxylated flavones acacetin, diosmetin, eupatorin and the di-hydroxylated flavone chrysin, indicating that the 4'-OCH(3) group at the B ring and the 5,7-dihydroxy motif at the A ring play a prominent role in EROD inhibition. Potent inhibition of CYP1B1 EROD activity was also obtained for the poly-hydroxylated flavonols quercetin and myricetin. HPLC metabolism of acacetin by CYP1A1 and CYP1B1 revealed the formation of the structurally similar flavone apigenin by demethylation at the 4'-position of the B ring, whereas the flavone eupatorin-5-methyl ether was metabolized to an as yet unidentified metabolite assigned E(5)M1. Eupatorin-5-methyl ether demonstrated a submicromolar IC50 in the CYP1-expressing cancer cell line MDA-MB 468, while it was considerably inactive in the normal cell line MCF-10A. Homology modeling in conjunction with molecular docking calculations were employed in an effort to rationalize the activity of these flavonoids based on their CYP1-binding mode. Taken together the data suggest that dietary flavonoids exhibit three distinct modes of action with regard to cancer prevention, based on their hydroxyl and methoxy decoration: (1) inhibitors of CYP1 enzymatic activity, (2) CYP1 substrates and (3) substrates and inhibitors of CYP1 enzymes.
Flos Chrysanthemi (the flower of Chrysanthemum morifolium Ramat.) is widely used in China as a food and traditional Chinese medicine for many diseases. Luteolin and apigenin are two main bioactive components in Flos Chrysanthemi, and chrysoeriol and diosmetin are two methylated metabolites of luteolin in vivo by cathechol-O-methyltransferase (COMT). However, there was /a/ lack of pharmacokinetic information of chrysoeriol and diosmetin after oral administration of Flos Chrysanthemi extract (FCE). The present study aimed to develop an HPLC-UV method for simultaneous determination of rat plasma concentration of luteolin, apigenin, chrysoeriol and diosmetin and utilize it in pharmacokinetic study of the four compounds after orally giving FCE to rats. The method was successfully validated and applied to the pharmacokinetic study when oral administration of FCE to rats with or without co-giving a COMT inhibitor, entacapone. Chrysoeriol and diosmetin were detected in rat plasma after oral administration of FCE and their concentrations were significantly decreased after co-giving entacapone... In conclusion, a sensitive, accurate and reproducible HPLC-UV method for simultaneous determination of luteolin, apigenin, chrysoeriol and diosmetin in rat plasma were developed, pharmacokinetics of chrysoeriol and diosmetin combined with luteolin and apigenin were characterized after oral administration of FCE to rats, which gave us more information on pharmacokinetics and potential pharmacological effects of FCE in vivo.
Diosmetin has known human metabolites that include (2S,3S,4S,5R)-3,4,5-Trihydroxy-6-[5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxochromen-7-yl]oxyoxane-2-carboxylic acid.

[1]. Diosmetin induces apoptosis by upregulating p53 via the TGF-β signal pathway in HepG2 hepatoma cells. Mol Med Rep. 2016 Jul;14(1):159-64.

[2]. Diosmetin ameliorates the severity of cerulein-induced acute pancreatitis in mice by inhibiting the activation of the nuclear factor-κB. Int J Clin Exp Pathol. 2014 Apr 15;7(5):2133-42.

Diosmetin is a monomethoxyflavone that is the 4'-methyl ether derivative of luteolin. It is a natural product isolated from citrus fruits which exhibits a range of pharmacological activities. It has a role as an antioxidant, an antineoplastic agent, a plant metabolite, a tropomyosin-related kinase B receptor agonist, an apoptosis inducer, an angiogenesis inhibitor, a cardioprotective agent, a bone density conservation agent, an anti-inflammatory agent and a vasodilator agent. It is a monomethoxyflavone, a trihydroxyflavone and a 3'-hydroxyflavonoid. It is functionally related to a luteolin. It is a conjugate acid of a diosmetin-7-olate.
Diosmetin is an O-methylated flavone and the aglycone part of the flavonoid glycosides diosmin that occurs naturally in citrus fruits. Pharmacologically, diosmetin is reported to exhibit anticancer, antimicrobial, antioxidant, oestrogenic and anti-inflamatory activities. It also acts as a weak TrkB receptor agonist.
Diosmetin has been reported in Lepisorus ussuriensis, Taraxacum sinicum, and other organisms with data available.
See also: Agathosma betulina leaf (part of).

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Mechanism of Action
Various types of tumors are known to overexpress enzymes belonging to the CYP1 family of cytochromes P450. The present study aimed to characterize the metabolism and further antiproliferative activity of the natural flavonoid diosmetin in the CYP1-expressing human hepatoma cell line HepG2. Diosmetin was converted to luteolin in HepG2 cells after 12 and 30 hr of incubation. In the presence of the CYP1A inhibitor alpha-naphthoflavone, the conversion of diosmetin to luteolin was attenuated. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays revealed luteolin to be more cytotoxic than diosmetin. The antiproliferative effect of diosmetin in HepG2 cells was attributed to blockage at the G2/M phase as determined by flow cytometry. Induction of G2/M arrest was accompanied by up-regulation of phospho-extracellular-signal-regulated kinase (p-ERK), phospho-c-jun N-terminal kinase, p53 and p21 proteins. More importantly, induction of G2/M arrest and p53 and p-ERK up-regulation were reversed by the application of the CYP1 inhibitor alpha-naphthoflavone. Taken together, the data provide new evidence on the tumor-suppressing role of cytochrome P450 CYP1A enzymes and extend the hypothesis that the anticancer activity of dietary flavonoids is enhanced by P450-activation.
CYP1A1 and CYP1B1 are two extrahepatic enzymes that have been implicated in carcinogenesis and cancer progression. Selective inhibition of CYP1A1 and CYP1B1 by dietary constituents, notably the class of flavonoids, is a widely accepted paradigm that supports the concept of dietary chemoprevention. In parallel, recent studies have documented the ability of CYP1 enzymes to selectively metabolize dietary flavonoids to conversion products that inhibit cancer cell proliferation. In the present study /the authors/ have examined the inhibition of CYP1A1 and CYP1B1-catalyzed EROD activity by 14 different flavonoids containing methoxy- and hydroxyl-group substitutions as well as the metabolism of the monomethoxylated CYP1-flavonoid inhibitor acacetin and the poly-methoxylated flavone eupatorin-5-methyl ether by recombinant CYP1A1 and CYP1B1. The most potent inhibitors of CYP1-EROD activity were the methoxylated flavones acacetin, diosmetin, eupatorin and the di-hydroxylated flavone chrysin, indicating that the 4'-OCH(3) group at the B ring and the 5,7-dihydroxy motif at the A ring play a prominent role in EROD inhibition. Potent inhibition of CYP1B1 EROD activity was also obtained for the poly-hydroxylated flavonols quercetin and myricetin. HPLC metabolism of acacetin by CYP1A1 and CYP1B1 revealed the formation of the structurally similar flavone apigenin by demethylation at the 4'-position of the B ring, whereas the flavone eupatorin-5-methyl ether was metabolized to an as yet unidentified metabolite assigned E(5)M1. Eupatorin-5-methyl ether demonstrated a submicromolar IC50 in the CYP1-expressing cancer cell line MDA-MB 468, while it was considerably inactive in the normal cell line MCF-10A. Homology modeling in conjunction with molecular docking calculations were employed in an effort to rationalize the activity of these flavonoids based on their CYP1-binding mode. Taken together the data suggest that dietary flavonoids exhibit three distinct modes of action with regard to cancer prevention, based on their hydroxyl and methoxy decoration: (1) inhibitors of CYP1 enzymatic activity, (2) CYP1 substrates and (3) substrates and inhibitors of CYP1 enzymes.
The binding mechanism of molecular interaction between diosmetin and human serum albumin (HSA) in a pH 7.4 phosphate buffer was studied using atomic force microscopy (AFM) and various spectroscopic techniques including fluorescence, resonance light scattering (RLS), UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by diosmetin was a static quenching procedure. The binding constants and number of binding sites were evaluated at different temperatures. The RLS spectra and AFM images showed that the dimension of the individual HSA molecules were larger after interaction with diosmetin. The thermodynamic parameters, /changes in enthalpy and entropy/ were calculated to be -24.56 kJ/mol and 14.67 J/mol/K, respectively, suggesting that the binding of diosmtin to HSA was driven mainly by hydrophobic interactions and hydrogen bonds. The displacement studies and denaturation experiments in the presence of urea indicated site I as the main binding site for diosmetin on HSA. The binding distance between diosmetin and HSA was determined to be 3.54 nm based on the Forster theory. Analysis of CD and FT-IR spectra demonstrated that HSA conformation was slightly altered in the presence of diosmetin.
The survival of osteoblasts is one of the determinants of the development of osteoporosis. This study /investigates/ the osteoblastic differentiation induced by diosmetin, a flavonoid derivative, in osteoblastic cell lines MG-63, hFOB, and MC3T3-E1 and bone marrow stroma cell line M2-10B4. Osteoblastic differentiation was determined by assaying alkaline phosphatase (ALP) activity and mineralization degree and measuring various osteoblast-related markers using ELISA. Expression and phosphorylation of Runt-related transcription factor 2 (Runx2), protein kinase Cdelta (PKCdelta), extracellular signal-regulated kinase (ERK), p38, and c-jun-N-terminal kinase (JNK) was assessed by immunoblot. Rac1 activity was determined by immunoprecipitation, and Runx2 activity was assessed by EMSA. Genetic inhibition was performed by small hairpin RNA plasmids or small interfering RNA (siRNA) transfection. Diosmetin exhibited an effect on osteoblastic maturation and differentiation by means of ALP activity, osteocalcin, osteopontin, and type I collagen production, as well as Runx2 upregulation. Induction of differentiation by diosmetin was associated with increased PKCdelta phosphorylation and the activations of Rac1 and p38 and ERK1/2 kinases. Blocking PKCdelta by siRNA inhibition significantly decreased osteoblastic differentiation by inhibiting Rac1 activation and subsequently attenuating the phosphorylation of p38 and ERK1/2. In addition, blocking p38 and ERK1/2 by siRNA transfection also suppressed diosmetin-induced cell differentiation. /This shows/ that diosmetin induced osteoblastic differentiation through the PKCdelta-Rac1-MEK3/6-p38 and PKCdelta-Rac1-MEK1/2- ERK1/2-Runx2 pathways and that it is a promising agent for treating osteoporosis.

C16H12O6

300.2629

300.063

C, 64.00; H, 4.03; O, 31.97

520-34-3

Diosmetin-d3;1189728-54-8

5281612

Light yellow to yellow solid powder

1.5±0.1 g/cm3

576.7±50.0 °C at 760 mmHg

256-258ºC

220.3±23.6 °C

0.0±1.7 mmHg at 25°C

1.697

3.1

3

6

2

22

462

0

O1C2=C([H])C(=C([H])C(=C2C(C([H])=C1C1C([H])=C([H])C(=C(C=1[H])O[H])OC([H])([H])[H])=O)O[H])O[H]

MBNGWHIJMBWFHU-UHFFFAOYSA-N

InChI=1S/C16H12O6/c1-21-13-3-2-8(4-10(13)18)14-7-12(20)16-11(19)5-9(17)6-15(16)22-14/h2-7,17-19H,1H3

5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.33 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 10 mg/mL (33.30 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)