Naturally occurring flavonoid

Eupatorin is a naturally occurring flavone that inhibits cell proliferation in human tumor cells. Here we demonstrate that eupatorin arrests cells at the G2-M phase of the cell cycle and induces apoptotic cell death involving activation of multiple caspases, mitochondrial release of cytochrome c and poly(ADP-ribose) polymerase cleavage in human leukemia cells. This flavonoid induced the phosphorylation of members of the mitogen-activated protein kinases and cell death was attenuated by inhibition of c-jun N-terminal kinases/stress activated protein kinases. Eupatorin-induced cell death is mediated by both the extrinsic and the intrinsic apoptotic pathways and through a mechanism dependent on reactive oxygen species generation [1].

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Eupatorin Caused a Cytotoxic Effect in 4T1 Cells Proliferation [2]
Eupatorin caused a time (24, 48, and 72 hours) and dosage (0.16-20 µg/mL) dependent inhibition of cell proliferation toward 4T1 cells (Table 2; Figure 2). At 24 hours, the IC50 value of eupatorin was higher than 20 µg/mL. When the incubation time was extended for 48 hours, the 4T1 cells exhibited an IC50 value of 6.00 µg/mL. At 72 hours, the IC50 of 4T1 cells was 5 µg/mL. Cytotoxicity of eupatorin was lower than the positive control doxorubicin with the IC50—1.50, 0.90, and 0.60 µg/mL at 24, 48, and 72 hours, respectively (Figure 2).

Eupatorin is a polymethoxy flavone extracted from Orthosiphon stamineus and was reported to exhibit cytotoxic effects on several cancer cell lines. However, its effect as an anti–breast cancer agent in vivo has yet to be determined. This study aims to elucidate the potential of eupatorin as an anti–breast cancer agent in vivo using 4T1 challenged BALB/c mice model. In this article, BALB/c mice (20-22 g) challenged with 4T1 cells were treated with 5 mg/kg or 20 mg/kg eupatorin, while the untreated and healthy mice were fed with olive oil (vehicle) via oral gavage. After 28 days of experiment, the mice were sacrificed and blood was collected for serum cytokine assay, while tumors were harvested to extract RNA and protein for gene expression assay and hematoxylin-eosin staining. Organs such as spleen and lung were harvested for immune suppression and clonogenic assay, respectively. Eupatorin (20 mg/kg) was effective in delaying the tumor development and reducing metastasis to the lung compared with the untreated mice. Eupatorin (20 mg/kg) also enhanced the immunity as the population of NK1.1+ and CD8+ in the splenocytes and the serum interferon-γ were increased. Concurrently, eupatorin treatment also has downregulated the expression of pro-inflammatory and metastatic related genes (IL-1β. MMP9, TNF-α, and NF-κB). Thus, this study demonstrated that eupatorin at the highest dosage of 20 mg/kg body weight was effective in delaying the 4T1-induced breast tumor growth in the animal model [2].

Cell culture and Cytotoxicity Assays [1]
HL-60, U937 and Molt-3 cells grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin. The cytotoxicity of Eupatorin was analyzed by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described and the concentration required to reduce cell viability by 50% (IC50) was determined graphically using the curve fitting algorithm of the computer software Prism 4.0 (GraphPad). Values are means ± SEs from three independent experiments, each performed in triplicate.

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Intracellular Reactive Oxygen Species (ROS) Determination [1]
The generation of peroxides and superoxide was monitored by flow cytometry with the probes 2′,7′-dichlorodihydrofluorescein diacetate (H2-DCF-DA) and dihydroethidium (DHE), respectively. H2-DCF-DA is incorporated into cells and deacetylated by intracellular esterases to yield the nonfluorescent 2′,7′-dichlorodihydrofluorescein (H2-DCF). H2-DCF and DHE are oxidized by H2O2 (and other peroxides) and superoxide anions, respectively, to yield the highly fluorescent compounds 2′,7′-dichlorofluorescein (DCF) and ethidium. The intensity of fluorescence of DCF and ethidium is proportional to the amount of peroxide and superoxide, respectively, produced by cells. Cells were treated with or without Eupatorin and H2-DCF-DA (2 µM) or DHE (2 µM) was added to the medium 30 min before the end of incubation with Eupatorin. Cells were irradiated with an argon laser at 488 nm, and fluorescence was detected at 525 nm (DCF) and 568 nm (DHE) in the flow cytometer.

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Cell culture and Cytotoxicity Assays [2]
HL-60, U937 and Molt-3 cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin. The cytotoxicity of Eupatorin was analyzed by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described and the concentration required to reduce cell viability by 50% (IC50) was determined graphically using the curve fitting algorithm of the computer software Prism 4.0. Values are means ± SEs from three independent experiments, each performed in triplicate.

In Vivo Study of Eupatorin Compounds as an Antitumor Agent [2]
For in vivo study, female Balb/c mice (total N = 32) aged between 4 and 5 weeks were used and acclimatized in plastic cages (4 mice/cage) for 1 week (22 ± 1 °C; 12-hour dark/light cycle). All mice were given distilled water and standard pellet diet ad libitum until the ideal body weight (BW; 20-22 g) was achieved. Then, mice were randomly divided into 4 groups: (1) healthy group (n = 8); (2) untreated group (n = 8); (3) low-dose Eupatorin (dosage: 5 mg/kg BW) group (n = 8); and (4) high-dose Eupatorin (dosage: 20 mg/kg BW) group (n = 8). Each mouse from groups 2, 3, and 4 was challenged with 4T1 cells by injecting orthotopically 1 × 105 cells/mice into the mammary fat pad. After 5 days of injection, tumor volume was measured by electronic caliper and calculated using the following formula:
Tumorvolume:0.5236×length×width×height⁡(mm3)
All mice were recorded with tumor sized ~0.10 mm3. The 4T1-induced mice in groups 3 and 4 were treated with 100 µL of Eupatorin dissolved in olive oil at respective dosage via force-feeding once daily. In addition, untreated and healthy mice were fed with 100 µL of olive oil (vehicle) via force-feeding once daily. After 28 days of treatment, all mice were anaesthetized with 2% isoflurane and sacrificed by cervical dislocation. Prior to treatment, all the mice were numbered and weighed and the initial tumor volumes were recorded. Overview of the experimental design is summarized in Figure 1.

[1]. Eupatorin-induced cell death in human leukemia cells is dependent on caspases and activates the mitogen-activated protein kinase pathway. PLoS One. 2014 Nov 12;9(11):e112536.

[2]. Eupatorin Suppressed Tumor Progression and Enhanced Immunity in a 4T1 Murine Breast Cancer Model. Integr Cancer Ther. 2020 Aug 24;19:1534735420935625.

Eupatorin is a trimethoxyflavone that is 6-hydroxyluteolin in which the phenolic hydogens at positions 4', 6 and 7 have been replaced by methyl groups. It has a role as a Brassica napus metabolite, an apoptosis inducer, a vasodilator agent, a calcium channel blocker, an anti-inflammatory agent, a P450 inhibitor and an antineoplastic agent. It is a dihydroxyflavone, a trimethoxyflavone and a polyphenol. It is functionally related to a 6-hydroxyluteolin.
Eupatorin has been reported in Salvia plebeia, Achillea setacea, and other organisms with data available.

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In summary, the findings of the present study indicate that Eupatorin induces cell death in human leukemia cell lines at concentrations which might be achieved in vivo. Eupatorin induces cytotoxicity via G2-M phase cell-cycle arrest and apoptosis through a caspase-dependent mechanism, is associated with cytochrome c release, and is dependent on ROS generation. Eupatorin induces the activation of the MAPK pathway and activation of JNK/SAPK is essential for cell death. These results provide a basis to further evaluate the potential applications of eupatorin and/or structurally similar compounds in the fight against cancer.[1]

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The present study has demonstrated that the Eupatorin at the highest dosage of 20 mg/kg BW is effective for delaying the 4T1-induced breast tumor growth in the animal model. This study signifies the in vivo efficacy and the potential of eupatorin for breast cancer therapeutic purposes.[2]

C18H16O7

344.3154

344.089

C, 62.79; H, 4.68; O, 32.53

855-96-9

97214

Light yellow to yellow solid powder

1.4±0.1 g/cm3

587.0±50.0 °C at 760 mmHg

194-196°C

216.0±23.6 °C

0.0±1.7 mmHg at 25°C

1.627

2.96

2

7

4

25

520

0

COC1=C(C=C(C=C1)C2=CC(=O)C3=C(C(=C(C=C3O2)OC)OC)O)O

KLAOKWJLUQKWIF-UHFFFAOYSA-N

InChI=1S/C18H16O7/c1-22-12-5-4-9(6-10(12)19)13-7-11(20)16-14(25-13)8-15(23-2)18(24-3)17(16)21/h4-8,19,21H,1-3H3

5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-6,7-dimethoxychromen-4-one

Eupatorin; NSC 106402; 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-6,7-dimethoxychromen-4-one; 5,3'-Dihydroxy-6,7,4'-trimethoxyflavone; UNII-3J474AV6MY; 5-Hydroxy-2-(3-hydroxy-4-methoxyphenyl)-6,7-dimethoxy-4H-1-benzopyran-4-one; Flavone, 3',5-dihydroxy-4',6,7-trimethoxy-; ...; 855-96-9;

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 : ~250 mg/mL (~726.07 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.04 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 20.8 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: ≥ 2.08 mg/mL (6.04 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 20.8 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.)