Natural flavone; anti-inflammatory, anti-tumor, anti-oxidant, neuroprotective, anti-fungal activities; estrogen

The compound chyrsin, which is mostly present in gum, honey, and passionflower, has the potential to be used as a preventative and therapeutic tool since it reduces the growth of many types of malignancies in humans. Chrysin exhibits anticancer properties in a range of cancer types; nevertheless, its exact function in ovarian cancer remains unclear, despite the fact that it accumulates poorly and functions as an adjuvant to chemotherapy in conjunction with conventional treatment drugs. In ES2 and OV90 cells, chrysin increases reactive oxygen species (ROS), produces cytoplasmic Ca2+ levels, and induces mitochondrial membrane potential (MMP) to activate MAPK and PI3K/AKT dyes. Chrysin alters the nuclear factor NF-κB signal amplifier cascade dyes and canonical Wnt signal. When compared to untreated control cells, chyrosin induced the phosphorylation of AKT and P70S6K proteins in ES2 and OV90 cells. Furthermore, JNK, p38, and ERK1/2 proteins are phosphorylated by Chrysin epidermal activation medications [1].

Determination of cellular reactive oxygen species (ROS)[1]
Intracellular reactive oxygen species (RO)S production was estimated using 2′,7′-dichlorofluorescein diacetate which is oxidized to fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of peroxides. Cells were detached with trypsin-EDTA, collected by centrifugation, and washed with PBS. The cells were treated with 10 µM DCFH-DA for 30 min at 37°C, washed with PBS twice, and then treated with increasing concentrations of chrysin (0, 5, 10, 20, and 50 µM) or hydrogen peroxide (H2O2) as a positive control for 1 hr at 37°C in a CO2 incubator. The treated cells were washed again with PBS, and then the fluorescent DCF intensity was analyzed using a flow cytometer.

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Mitochondrial membrane potential (MMP) assay[1]
Changes in mitochondrial membrane potential (MMP) were analyzed using a mitochondrial staining kit. The ovarian cancer cells (5 × 105 cells) were seeded in six-well plates and treated with increasing concentrations of chrysin (0, 5, 10, 20, and 50 µM) for 48 hr at 37°C in a CO2 incubator. The supernatants were removed from the culture dishes, and the adherent cells were detached with trypsin-EDTA. The cells were collected by centrifugation, resuspended in staining solution containing 200× JC-1 at 1× staining buffer, and incubated at 37°C in a CO2 incubator for 20 min. The stained cells were collected by centrifugation, washed once with 1× JC-1 staining buffer, and the cell suspension was centrifuged once more, followed by resuspension of the cells in 1 ml staining buffer. The fluorescence intensity was determined using a FACS Calibur instrument.

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Measurement of intracellular free Ca2+ concentration[1]
ES2 and OV90 (5 × 105) cells were seeded in six-well plates and incubated for 24 hr in serum-free medium when the cells reached 70–80 % confluency. Then, the cells were treated with chrysin, 2-aminoethoxydiphenyl borate (2-APB), and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM) for 48 hr at 37°C in a CO2 incubator. The supernatants were removed from the culture dishes, and adherent cells were detached with trypsin-EDTA, collected by centrifugation, resuspended in 3 µM fluo-4, and incubated at 37°C in a CO2 incubator for 20 min. The stained cells were subsequently washed with PBS, and the fluorescence intensity was analyzed using a flow cytometer.

Proliferation assay[1]
The proliferation assays were conducted using a cell proliferation enzyme-linked immunosorbent assay (ELISA) 5-bromo-2′-deoxyuridine (BrdU) kit according to the manufacturer's instructions. Briefly, ES2 and OV90 cells were seeded in a 96-well plate, and then treated with chrysin (0, 5, 10, 20, 50, and 100 μM) with or without inhibitors (20 µM LY294002, PI3K/AKT; 10 µM U0126, ERK1/2; 10 µM SP600125, JNK; and 20 µM SB203580, p38) in a final volume of 100 µl/well. After a 48-hr incubation, 10 µM BrdU was added to the cell culture, followed by an additional 2-hr incubation at 37°C. After labeling the cells with BrdU, they were fixed and then incubated with the anti-BrdU-peroxidase (POD) working solution for 90 min. The anti-BrdU-POD binds to the BrdU incorporated into newly synthesized cellular DNA and these immune complexes were detected by analyzing their reaction with the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. The absorbance values of the reaction product were measured at 370 and 492 nm using an ELISA reader.

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Immunofluorescence analysis The effects of chrysin on the expression of proliferating cell nuclear antigen (PCNA) were determined using immunofluorescence microscopy. Cells were incubated with or without chrysin (20 μM) for 48 hr at 37°C in a CO2 incubator and probed with mouse anti-human monoclonal PCNA at a final dilution of 1:100 (2 μg/ml). The cells were then incubated with goat anti-mouse IgG Alexa 488 at a 1:200 dilution for 1 hr at room temperature. Cells were then washed with 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and stained with 4′,6-diamidino-2-phenylindole (DAPI). For the primary antibodies, the images were captured using an LSM710 confocal microscope.

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Annexin V and propidium iodide (PI) staining The naringenin-induced apoptosis of endometriosis cells was analyzed using a fluorescein isothiocyanate (FITC) Annexin V apoptosis detection kit I. The cells (5 × 105 cells) were seeded in six-well plates and treated with increasing concentrations of chrysin (0, 5, 10, 20, 50, and 100 µM) for 48 hr at 37°C in a CO2 incubator. The supernatants were removed from culture dishes, and the adherent cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA). The cells were collected by centrifugation, washed with PBS, and resuspended in 1× binding buffer at 1 × 106 cells/ml. Then, 100 µl of the cell suspension (1 × 10~6 cells) was transferred to a 5-ml culture tube and incubated with 5 µl each of FITC Annexin V and 5 propidium iodide for 15 min at room temperature in the dark. Then, 400 µl of 1× binding buffer was added in a 5-ml culture tube. The fluorescence intensity was determined using a fluorescence-activated cell sorting (FACS) Calibur instrument.

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Cell cycle analysis Cells (5 × 105 cells) were seeded in six-well plates and treated with increasing concentrations of chrysin (0, 5, 10, 20, and 50 µM) for 48 hr at 37°C in a CO2 incubator. After treatment, the cells were centrifuged, washed twice with cold 0.1% BSA in PBS, and fixed in 70% ethanol at 4°C for 24 hr. The ES2 and OV90 cells were then centrifuged, and the supernatant was discarded. The pellets were washed twice with 0.1% BSA in PBS and stained with PI in 100 µg/ml RNase A for 30 min in the dark. The fluorescence intensity was analyzed using a flow cytometer.

Metabolism / Metabolites
Chrysin has known human metabolites that include (2S,3S,4S,5R)-3,4,5-trihydroxy-6-(5-hydroxy-4-oxo-2-phenylchromen-7-yl)oxyoxane-2-carboxylic acid.

[1]. Chrysin Attenuates Progression of Ovarian Cancer Cells by Regulating Signaling Cascades and Mitochondrial Dysfunction. J Cell Physiol. 2017 Aug 17.

Chrysin is a dihydroxyflavone in which the two hydroxy groups are located at positions 5 and 7. It has a role as an anti-inflammatory agent, an antineoplastic agent, an antioxidant, a hepatoprotective agent, an EC 2.7.11.18 (myosin-light-chain kinase) inhibitor and a plant metabolite. It is a dihydroxyflavone and a 7-hydroxyflavonol.
Chrysin has been reported in Apis, Populus yunnanensis, and other organisms with data available.
5,7-Dihydroxyflavone is found in carrot. Chrysin is a naturally occurring flavone chemically extracted from the blue passion flower (Passiflora caerulea). Honeycomb also contains small amounts. It is also reported in Oroxylum indicum or Indian trumpetflower. (Wikipedia).

C15H10O4

254.24

254.057

C, 70.86; H, 3.96; O, 25.17

480-40-0

480-40-0

5281607

Light yellow to yellow solid powder

1.4±0.1 g/cm3

491.9±45.0 °C at 760 mmHg

284-286 °C(lit.)

192.5±22.2 °C

0.0±1.3 mmHg at 25°C

1.699

2.88

2

4

1

19

384

0

O1C(=C([H])C(C2=C(C([H])=C(C([H])=C12)O[H])O[H])=O)C1C([H])=C([H])C([H])=C([H])C=1[H]

RTIXKCRFFJGDFG-UHFFFAOYSA-N

InChI=1S/C15H10O4/c16-10-6-11(17)15-12(18)8-13(19-14(15)7-10)9-4-2-1-3-5-9/h1-8,16-17H

5,7-dihydroxy-2-phenylchromen-4-one

Galangin flavanone; 57Dihydroxyflavone; NP005901; 5,7-Dihydroxyflavone; Chrysine; 5,7-Dihydroxy-2-phenyl-4H-chromen-4-one; 5,7-dihydroxy-2-phenylchromen-4-one; Crysin; 4H-1-Benzopyran-4-one, 5,7-dihydroxy-2-phenyl-; NP-005901; NP 005901

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)

DMSO: ~51 mg/mL (~200.6 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.83 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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: ≥ 2.5 mg/mL (9.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly.

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