Chrysin, Abundant in Morinda citrifolia Fruit WaterâEtOAc Extracts

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Chrysin, abundant in Morinda Citrifolia fruit water-EtOAc extracts, combined with apigenin synergistically induced apoptosis and inhibited migration in human breast and liver cancer cells Hsiu-Chen Huang, Cheng Huang, Yu Xuan Wei, Man Ching Shen, Yu-Hsuan Tu, and chia chi Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00766 • Publication Date (Web): 03 May 2016 Downloaded from http://pubs.acs.org on May 5, 2016

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Journal of Agricultural and Food Chemistry

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Chrysin, abundant in Morinda Citrifolia fruit water-EtOAc extracts, combined

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with apigenin synergistically induced apoptosis and inhibited migration in

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human breast and liver cancer cells Cheng Huang†, Yu Xuan Wei# and Man Ching Shen#, Yu-Hsuan Tu #, Chia-Chi Wang #, Hsiu Chen Huang#* † National Research Institute of Chinese Medicine, Taipei 11221, Taiwan, ROC # Department of Applied Science, National Hsinchu University of Education, Hsinchu

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30014, Taiwan, ROC

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Running title: Chrysin combined with apigenin synergistically induced apoptosis and inhibited migration

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*

Corresponding authors: Hsiu-Chen Huang Dr. Hsiu-Chen Huang Department of Applied Science National Hsinchu University of Education Address: No.521, Nanda Rd., Hsinchu City 30014, Taiwan Tel: +886-3-5213132 ext.2756 Fax: +886-3- 5257178 E-mail: [email protected]

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Abstract

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The composition of Morinda citrifolia were determined using High-Performance

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Liquid Chromatography (HPLC), and evaluated the anticancer effects of Morinda

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citrifolia extract in HepG2, Huh7, and MDA-MB-231 cancer cells. Morinda citrifolia

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fruit extracts were obtained by using five different organic solvents, including hexane

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(Hex), methanol (MeOH), ethyl acetate (EtOAc), chloroform (CHCl3), and ethanol

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(EtOH). The water-ethyl acetate extract from Morinda citrifolia fruits was found to

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have the highest anticancer activity. HPLC data revealed the predominance of chrysin

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in Water-EtOAc extracts of Morinda Citrifolia fruit. Furthermore, the combined

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effects of co-treatment with apigenin and chrysin on liver and breast cancer were

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investigated. Treatment with apigenin plus chrysin for 72-96 h reduced HepG2 and

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MDA-MB-231 cell viability, and induced apoptosis through downregulation of

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S-phase

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receptor-related protein 6 (LRP6) expression. However, the combination treatment for

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36 h synergistically decreased MDA-MB-231 cell motility but not cell viability

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through downregulation of MMP2, MMP9, fibronectin, and snail in MDA-MB-231

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cells. Additionally, chrysin combined with apigenin also suppressed tumor growth in

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human MDA-MB-231 breast cancer cells xenograft through downregulation of ki-67

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and Skp2 protein. The experimental results showed that chrysin combined with

kinase-associated

protein-2

(Skp2)

and

2


low-density

lipoprotein

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apigenin can reduce HepG2 and MDA-MB-231 proliferation, cell motility and induce

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apoptosis. It also offers opportunities for exploring new drug targets, and further

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investigations are underway in this regard.

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Keywords : Chrysin; apigenin; epithelial-mesenchymal transition; apoptosis;

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xenografts

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Introduction

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Herbal preparations from various medicinal plants have often been used for

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treatment and symptom relief. Despite the lack of solid evidence regarding their

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therapeutic benefits, around 41% of breast cancer patients are utilizing

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complementary and alternative medicines (CAM) according to data of the World

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Health Organization.1 Flavonoids as complementary medicine have been reported to

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possess various pharmacological properties including antioxidation, anti-inflammation,

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hepatoprotective, anti-atherosclerosis, anticancer, hypolipidemia, and hypoglycemia

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effects, antiviral, antibacterial, antiallergy, and so on. Flavonoids exist in plant and

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contains mamy polyphenolic compounds, such as o flavonols, flavones, flavanones

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and isoflavonoids.2 Over 4,000 varieties of biologically active flavonoids have been

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isolated and played an important role in human health. Chrysin and apigenin are

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flavones that differ in number of hydroxyl groups on their B ring. Apigenin has one

3



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hydroxyl group on its B ring on position 4' while chrysin did not have any hydroxyl

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group on its B ring. Previous studies have shown that hydroxyl group numbers on the

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B ring affected cytochrome P450 (CYP) 1 A enzyme activity.3 Although chrysin and

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apigenin have similar chemical structures and both compounds exert anticancer

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effects, they affect cancer cell growth through different mechanisms. Previous

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findings revealed enhancement in biological activities of chrysin when combined with

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apigenin.

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Both chrysin and apigenin are abundantly present in several varieties of fruits,

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vegetables, and herbs. Chrysin has prominent effects in preventing and treating cancer,

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including breast cancer and liver cancer.4 In addition, chrysin has also been reported

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to decrease chronic diseases such as diabetes5, cardiovascular diseases6, and

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inflammation. Apigenin exhibited a wide variety of anticarcinogenic effects in skin,

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prostate, colon, cervical, and breast cancer cells.7 The detailed mechanisms of chrysin

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and apigenin in breast and liver cancer prevention is still a lot unknown, and the

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synergistic anticancer effects between the two compounds also need to examine.

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Morinda Citrifolia (also called Noni) have been used to treat many diseases, including

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bacterial infections, parasitic infection, inflammation, cancer and many others.8 All

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parts of Morinda Citrifolia have been used as food and medicine including its fruit.

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Previous studies have shown that Methanol (MeOH) extracts of Morinda Citrifolia

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leaves contain many flavonoids, such as kaempferol, apigenin and luteolin. Ethyl

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acetate (EtOAc) extracts contain different flavonoids including quercetin.9 Morinda

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Citrifolia is a rich source of phytochemical constituents, namely flavonoids,

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triterpenoids, iridoids, and anthraquinones which have potent anticancer activity. Thus,

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Morinda Citrifolia has been used as complementary medicine. However, the chemical

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components of Morinda Citrifolia have not been extensively examined and their

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bioactivities merit further studies.

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This study focused on evaluating the composition and biological potential of

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Morinda Citrifolia and determining whether Morinda Citrifolia fruits contain chrysin

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and

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apigenin –induced cell growth inhibition in cancer cell and nude mice were also

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examined.

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Materials and methods

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Preparation of Morinda Citrifolia fruit extracts

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Morinda Citrifolia fruits were collected from Taiwan. For preparation of five different

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organic solvents extracts, 100 g of dried fruit of Morinda Citrifolia were immersed in

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1000 mL of Hex, CHCl3, EtOAc, EtOH or MeOH for 24 h at 4°C and then filtered

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with a filter paper. The eluates were dried after evaporation under vacuum at 55°C.

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For preparation of water-ethyl acetate (Water-EtOAc) extracts, 100 g of dried fruit of

apigenin.

The

mechanisms

responsible

for

chrysin

5


combined

with


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Morinda Citrifolia were immersed in 1000 mL of deionized water and boiled for more

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than thirty minutes. The cooling water solvent pass through a filter paper. Water

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extracts of Morinda Citrifolia fruit were evaporated to dryness using vacuum rotary

115

evaporator (80°C), followed by reconstitution with distilled water and sequential

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partitioning with equal volumes of EtOAc. Similarly, the eluates were dried by

117

evaporation under vacuum at 55°C. The concentration used in the experiment was

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determined according to the dry weight of the extract.

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Cell viability test by

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tetrazolium bromide] assay

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Cells were treated with Morinda Citrifolia extracts, chrysin, apigenin, or chrysin plus

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apigenin for 72-96 h. Cell viability was then examined using MTT assay. The MTT

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formazan dye crystals were formed by metabolically viable cells and were dissolved

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in DMSO. The DMSO solution was then collected and detected using ELISA

125

microplate readers at wavelength of 550 nm.

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Flow Cytometry

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The cells were washed with cold 1X phosphate-buffered saline (PBS). Next, the cells

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were fixed in ice-cold 80% ethanol overnight at -20℃. After fixation, cells were

129

removed ethanol and washed with cold PBS. The fixed cells were incubated in 0.5%

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Ttriton X-100/PBS/ 1mg/ml RNase for 30min at 37℃. In order to stain DNA, the

MTT [3-(4,

5-dimethylthiazol-2-yl) -2,5-diphenyl

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fixed and permeabilized cells were added 1mg/ml propidium iodide (PI) for 30min.

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Finally, fluorescence dye emitted by the PI-DNA complex was quantified by

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FACScan flow cytometry.

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High performance liquid chromatography (HPLC) analysis of Morinda Citrifolia

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extracts

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The concentrations of polyphenolic compound in Morinda Citrifolia extracts were

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determined by HPLC with C18-MS packed column. In gradient elution, the mobile

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phase consists mobile phase A (15% Acetonitrile, 4% Ethyl acetate, 0.1% Formic

139

Acid and 80.9% ddH2O) and mobile phase B (45% Acetonitrile, 4% Ethyl acetate,

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0.1% Formic Acid and 50.9% ddH2O). The polyphenols were detected by UV at 280

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nm.

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Morphological assay

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MDA-MB-231 and HepG2 cells were treated with DMSO, 10μM chrysin, 10μM

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apigenin, or 10μM chrysin plus 10μM apigenin for 96h. Cells morphology were

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examined using light microscopy.

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Trypan blue dye exclusion assay

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MDA-MB-231 and HepG2 cells were seeded into 6-well plates at 5 x 104 cell/well in

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Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum

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(FBS). After 24 hours of incubation, the medium was then changed and cells were

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treated with DMSO, 10µM chrysin, 10µM apigenin, or 10µM chrysin plus 10µM

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apigenin for 72-96 h in DMEM containing 2% FBS. After treatment with drugs for

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72-96 h, cells were harvested using treatment with 1X trypsin/EDTA solution and then

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stained with trypan blue. The dead cells stained with trypan blue in a hemocytometer

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were counted.

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Analysis of nuclear morphology

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MDA-MB-231 and HepG2 cells were plated on the coverslips in 6-well plates at 5 x

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104 cell/well and were treated with DMSO 10µM chrysin, 10µM apigenin, or 10µM

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chrysin plus apigenin for 96 h. After treatment, cells were fixed with 4%

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formaldehyde in PBS for 30 min at room temperature, and then stained the coverslips

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with cells using 4 mg/mL Hoechst 33258 for 30 min. The coverslips with Hoechst

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33258 staining were inverted onto glass slides and mounted with Vectashield. Finally,

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nuclear morphological changes was viewed under a microscope.

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Western blot analysis

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The assay were performed as we previously method10. After drugs treatment, cells

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were lysed with Gold lysis buffer and then centrifuged for 30 min at 12,000 rpm at 4

166

℃. The supernatants were collected and were quantified by the Bio-Rad assay dye.

167

Proteins

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polyvinylidene difluoride (PVDF) membrane. The membrane was then incubated with

were

separated

using

SDS-PAGE

and electrotransferred

8


onto a

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a different primary antibody, followed with horseradish peroxidase -conjugated

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secondary antibody. The blots were enhanced by chemiluminescence reagent.

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Immunofluorescence assay

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MDA-MB-231 cells were plated on coverslips in 6-well plates at 5 x 104 cell/well and

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were treated with DMSO 10µM chrysin, 10µM apigenin, or 10µM chrysin plus

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apigenin for 96 h. After treatment, the coverslips with cells were fixed with 4%

175

formaldehyde in PBS for 30 min at room temperature, and blocked at 4℃ for 1hr.

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Then, the coverslips were stained with apoptosis-inducing factor (AIF) primary

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antibodies at 4℃ overnight, follow with the fluorescein isothiocyanate-conjugated

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secondary antibodies and were viewed under a microscope.

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Soft agar assay

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MDA-MB-231 cells were treated with DMSO, 10µM chrysin, 10µM apigenin, or

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10µM chrysin plus 10µM apigenin in 20% FBS/2XDMEM medium and mixed with

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0.7% agarose. Then, the mixture was plated in 0.35% agarose. Colonies were

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observed and counted under a microscope.

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Wound-healing assay

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MDA-MB-231 cells were grown to full confluency in 6-well plates in 10%

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FBS/DMEM medium. After incubated overnight, the cells were washed with 1XPBS

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and added with drugs in 2% FBS/DMEM medium for the indicated time. Wound gap

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in MDA-MB-231 cells were produced by scratching using yellow plastic tip. The

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migration of individual cells across the wound gap was observed.

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Animals assay

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2 x 106 cancer cells were implanted subcutaneously into female BALB/c nude mice.

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which purchased from the National Laboratory Animal Breeding and Research Center

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(Taipei, Taiwan). After tumors size about 200 mm3, the mice were then randomly

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divided into 4 groups (4 mice/group) for drugs treatment by intraperitoneal (IP)

195

administration. The tumor tissues were fixed in 4% paraformaldehyde and embedded

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in paraffin for hematoxylin and eosin (H&E) and immunohistochemical (IHC)

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stainings.

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Statistical Analysis

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All values were expressed as Standard Error of the Mean (SEM). Student’s t-test was

200

used for statistical comparison. Symbol (*,#) indicates that the values are significantly

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different from the control (*,p < 0.05; **, p < 0.01; ***, p < 0.001; ＃,p < 0.05;

202

< 0.01;

203

multiple groups. Symbol (§) indicates that the values are significantly more effective

204

than either agent alone (§, p < 0.05; §§, p < 0.01; §§§, p < 0.001).

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Results

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Anticancer activities of various extracts against liver and breast cancer cells

＃＃

,p

＃＃＃

, p < 0.001). The analysis of variance (ANOVA) is used to compare

10


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Plant extracts rich in polyphenolic compounds have been safely employed as

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traditional Chinese medicine. The cell viability effect of Hex, CHCl3, EtOH, MeOH,

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EtOAc or Water-EtOAc extracts of Morinda citrifolia fruit on liver (HepG2 and Huh7)

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and breast (MDA-MB-231) cancer cells were evaluated by MTT assay. Figure 1A

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shows the growth response of three cell lines to six different extracts of Morinda

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Citrifolia fruit at various concentrations. As can be seen, the growth of HepG2 and

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Huh7 cell lines was inhibited by EtOH, EtOAc or Water-EtOAc extracts of Morinda

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Citrifolia fruit. Water-EtOAc extracts also showed the highest inhibition followed by

215

EtOAc and EtOH extracts in MDA-MB-231 cells. These data suggested that

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Water-EtOAc extracts have the highest anticancer activity. Thus, Water-EtOAc

217

extracts were selected for further analysis to evaluate the impact of anticancer activity

218

in different cancer types. To identify the mechanisms behind the antiproliferative

219

effect of Water-EtOAc extracts, MDA-MB-231, HepG2 and Huh7 cells were treated

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50-200 µg/ml Water-EtOAc extracts for 72 h and 96 h and the cell cycle distributions

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were measured using flow cytometric analysis. After Water-EtOAc extracts treatment,

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the percentage of HepG2 and MDA-MB-231 cells in the sub-G1 fraction increased

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(Figure 1B).

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Chemical composition of Water-EtOAc extracts of Morinda citrifolia fruit

225

To establish the chemical composition of Water-EtOAc

11


extracts, the


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concentrations of polyphenols were determined using HPLC. In HPLC analysis, we

227

used 21 different polyphenols standard including apigenin, chrysin, catechin, b

228

epigallocatechin gallate (EGCG), picatechin gallate (ECG), kaempferol, luteolin,

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narigenin,

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theaflavin-3-gallate (TF2), caffeic acid, chlorogenic acid, p-coumaric acid, corilagin,

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ellagic acid, gallic acid, and syringic acid to analyze the contents of these polyphenols

232

in Water-EtOAc extracts of Morinda citrifolia fruit. The contents of these polyphenols

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in WaterEtOAc extracts of Morinda citrifolia fruit were summarized in Table 1 and

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Figure 1C. The retention times of gallic acid, corilagin, and chrysin were 4.55, 10.76,

235

and 65.9 min, respectively. Three compounds in Water-EtOAc extracts have the same

236

retention time. These results indicated that Water-EtOAc extracts contained gallic acid,

237

corilagin, and chrysin at concentrations of 36.54, 28.62, and 16.35 µg/g, respectively.

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Furthermore, combinations of Water-EtOAc extracts and chrysin were injected into

239

the HPLC. As seen in Figure 1C, injection of Water-EtOAc extracts combined with

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chrysin resulted in overlapping peaks with identical HPLC retention times of 65.9 min.

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These results suggested that chrysin were predominantly present in Water-EtOAc

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extracts of Morinda citrifolia fruit.

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Anticancer activity of chrysin combined with apigenin against breast and liver

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cancer cell lines

myricetin,

quercetin,

rutin,

resveratrol,

12


theaflavin

(TF1),

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To determine the most active component of Water-EtOAc extracts of Morinda

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citrifolia fruit, the cytotoxicity of chrysin pure compounds was examined. The cell

247

viability effects of co-treatment with apigenin and chrysin on AU565, MDAMB231,

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and HepG2cells were evaluated using the MTT test. As shown in Figure 2A and 2B,

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the combination of 10 µM chrysin plus 10 µM apigenin in HepG2, MDA-MB-231,

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and AU565 cells synergistically dose- and time-dependently inhibited cancer cells

251

growth, compared with cells treated with either agent alone. The combination index

252

(CI) for chrysin in combination with apigenin consistently less than 1 represented

253

synergism (data not shown). Furthermore, the cell cycle distributions were measured

254

using flow cytometric analysis. The percentages of apoptotic HepG2 cancer cells were

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0.07, 1.57, 3.18, and 20.05% and 0.09, 3.17, 8.28, and 61.66% after treatment with

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DMSO, 10 µM chrysin, 10 µM apigenin, and 10µM chrysin plus 10µM apigenin for

257

72 h (Figure 3A) for 96 h (Figure 3B) respectively. Similar results were obtained for

258

MDA -MB-231, and AU565 cancer cells. Our results demonstrated that apigenin in

259

combination with chrysin synergistically induced HepG2, MDA-MB-231, and AU565

260

cancer cells apoptosis. After 10 µM chrysin and 10 µM apigenin cotreatment for 96 h,

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the morphology of HepG2 and MDA-MB-231 cells had more dramatic changes than

262

either agent alone (Figure 4A). The above data were assessed using the trypan blue

263

dye exclusion method and Hochest assay. Compared with either agent alone treatment,

13



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co-treatment with 10 µM chrysin plus 10 µM apigenin in HepG2 and MDA-MB-231

265

cells induced cell death (Figure 4B), DNA fragmentation and chromatin condensation

266

(Figure 4C). To identify the molecular mechanisms underlying synergistic, the

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apoptosis-related proteins such as Wnt signaling, Skp2, cyclin dependent kinase

268

(CDK), Cdc25c, and Survivin were determined by western blotting analysis. Figure

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4D shows that exposure to chrysin plus apigenin for 96 h synergistically decreased

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p-LRP6, LRP6, β-catenin, Skp2, CDK1, CDK4, Cdc25c, and Survivin in HepG2 and

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MDA-MB-231 cells. Apoptosis-inducing factor (AIF) is a critical mediator of

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caspase-independent cell death. To determine whether the combined treatment of

273

chrysin and apigenin induced caspase-independent cell death, the localization of AIF

274

in MDA-MB-231 cells was observed using immunofluorescence staining experiments.

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As shown in Figure 4E, exposure to chrysin plus apigenin for 96 h activates nuclear

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translocation of AIF.

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Antimotility activity of chrysin combined with apigenin in MDA-MB-231 cells

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Anchorage independence growth played an important role in cancer cell motility,

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invasion, and metastasis.11-13 The effects of chrysin combined with apigenin on

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anchorage independence in MDA-MB-231 cells were examined using soft agar assay.

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The colony sizes exceeding 50 µm were scored. As shown in Figure 5A, 10 µM

282

chrysin combined with 10 µM apigenin synergistically reduced the colony-forming 14


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capacity. The effects of chrysin combined with apigenin on cell motility were

284

examined using wound-healing assay. The results revealed significant cytotoxic

285

activity, of 10 µM chrysin combined with 10 µM apigenin in MDA-MB-231 cells for

286

72h. In order to decrease the interference in antimotility evaluation, the

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wound-healing assay was performed after 24-36 h exposure to 10 µM chrysin plus 10

288

µM apigenin. Figure 5B shows synergistically inhibited motility in MDA-MB-231

289

cells after exposure to 10 µM chrysin combined with 10 µM apigenin for 24-36 h.

290

However, there was no inhibition in cell growth (data not shown). Thus, these data

291

suggest that 10 µM chrysin combined with 10 µM apigenin synergistically reduced

292

MDA-MB-231 cell motility but not cell viability. Cell motility was affected by

293

change in expression of specific proteins involved in cell adhesion such as MMP2,

294

MMP9, and those related to epithelial-mesenchymal transition (EMT). To further

295

clarify whether chrysin combined with apigenin resulted from dysregulation of

296

metastasis- and EMT-related proteins such as MMP2, MMP9, fibronectin, snail, twist,

297

slug, and vimentin were examined by western blotting analysis. Exposure to chrysin

298

plus apigenin for 36 h synergistically decreased MMP2, MMP9, fibronectin, and snail

299

in MDA-MB-231 cells (Figure 5C).

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Antitumor growth of apigenin in combination with chrysin in BALB/c nude mice

301

xenograft tumor model

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To establish xenograft tumors for human breast cancer, MDA-MB-231 cells were

303

implanted subcutaneously into female BALB/c nude mice, and the tumor growth

304

inhibitory effects were observed after the intraperitoneal injection (IP) of DMSO,

305

chrysin (10 mg/kg), apigenin (10 mg/kg), or chrysin (10 mg/kg) plus apigenin (10

306

mg/kg). As shown in Figure 6A and 6B, the chrysin plus apigenin group on day 28

307

decreased tumor size, tumor weights, and the nuclear/cytoplasmic (N/C) ratio more

308

than either single agents alone treatment group. Furthermore, Cyclin A, Cyclin B,

309

Cyclin D, Cdk1/CDC2, Cdk4, Skp2, cleaved caspase 3, cleaved caspase 8 and cleaved

310

PARP expression levels in drug-treated and DMSO-treated mice were examined. The

311

chrysin plus apigenin group decreased the expression levels of Cyclin A, Skp2, and

312

Cdk1/CDC2 more than either single agents alone treatment group (Figure 6C). On the

313

other hand, the cleaved caspase 3, caspase 8 and PARP protein levels were increased

314

in the chrysin plus apigenin group. IHC data showed that, Ki-67 and Skp2 were

315

decreased in chrysin plus apigenin group more than either single agents alone

316

treatment group (Figure 6D). Our findings suggested that chrysin plus apigenin

317

co-treatment inhibited tumor growth in vitro and in vivo.

318

Discussion

319

Morinda citrifolia products have been gaining popularity because of their health

320

benefits and are sold as dietary supplements around the world. The fruit of Morinda

16


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citrifolia is widely used as traditional medicine to treat many health disorders such as

322

tumor, diabetes, arthritis, inflammation and chronic diseases.14-15 In the present study,

323

we examined the cytotoxic activity of Morinda citrifolia fruit extract with solvents of

324

different polarities. Water-EtOAc extracts of Morinda citrifolia fruit exhibited the

325

highest decrease in Huh-7, Hep-G2, and MDA-MB-231 cell viability by increasing

326

cell apoptosis. The HPLC data reported here for the first time indicated that chrysin

327

was a predominantly bioactive compound present in Water-EtOAc extracts of

328

Morinda citrifolia fruit. Combinations of phytochemicals are naturally found in plant

329

foods, and many clinical studies have also used the different phytochemicals mixtures

330

in cancer prevention. Thus, synergistic interactions between phytochemicals would

331

promote the anti-carcinogenic activity.

332

regarding synergistic interactions between phytochemicals. Thus, it's very important

333

to identify their combined effects in patients with cancer. Apigenin is also found in

334

Morinda citrifolia with no apparent toxicity reported.9 Chrysin and apigenin are both

335

abundantly present in Morinda citrifolia, but differ in their anticancer mechanism. To

336

our knowledge, this is the first report on chrysin combined with apigenin

337

synergistically inducing apoptosis in cancer cells and in nude mice xenograft tumor

338

cells through caspase-dependent and -independent pathways.

339

16

However, there was limited information

Skp2 and LRP6 have oncogenic potential and are overexpressed in breast and

17



340

liver cancer. The LRP6/Wnt/β-catenin signaling is significantly upregulated in

341

20-36% of human breast tissues 17 Skp2 is highly expressed in estrogen receptor (ER)

342

-negative tumors.18-19 Scientists recently discovered that Skp2 is a downstream target

343

gene of LRP6/Wnt/β-catenin signaling.20 Prior research has shown that the

344

combination of chrysin with 1,2,3,4,6-penta-O-galloyl-β-D-glucose (5GG) decreased

345

LRP6 and Skp2 and resulted in a synergistic inhibition of tumor cell growth.21 In this

346

current study, we found that the combination of chrysin and apigenin also inhibited

347

LRP6 and Skp2 protein expression and resulted in a synergistic inhibition of cell

348

mobility and tumor cell growth in vivo and in vitro. The overall data suggest that the

349

combination of chrysin and apigenin or 5GG could be a promising new treatments for

350

breast cancer patients.

351

EMT is the first step in the metastatic cascade. Numerous studies have indicated

352

an important role of EMT in cancer drug resistance, cancer stem cell transformation,

353

and cancer metastasis.22-26 Therefore, EMT is an important targeted therapeutic

354

strategy against invasive breast cancer. The present results demonstrated that the

355

combination of apigenin and chrysin for 36 h synergistically inhibited cancer cell

356

migration and decreased EMT-related proteins including MMP2, MMP9, fibronectin,

357

and snail in MDA-MB-231. Yang et al. (2014) reported that chrysin under serum-free

358

condition exerts anti-metastatic activities in triple-negative breast cancer (TNBC)

18


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Page 19 of 40


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cells through downregulating EMT protein expression and suppressing Akt

360

activation.27 In view of possible drug disturbance by serum, many studies preferred

361

not to use serum media in order to enhance the drug effect. However, serum is

362

required to ensure that cells are healthy and the absence of serum will cause stress to

363

the cells. Previous experience showed an optimal amount of 2% serum used during

364

drug treatment; and also indicated that chrysin combined with apigenin, in a low dose,

365

synergistically decreased EMT-related protein expression. The present findings are

366

consistent with previous results. However, whether chrysin combined with apigenin

367

could decrease EMT protein by downregulation of Akt phosphorylation needs further

368

study.

369

In summary, the abilities of Water-EtOAc extracts of Morinda citrifolia fruit to

370

induce apoptosis of breast and liver cancer cells were identified. Chrysin, abundant in

371

Morinda Citrifolia fruit water-EtOAc extracts, combined with apigenin synergistically

372

induced apoptosis in MDA-MB-231, AU565, and HepG2 cells and inhibited

373

migration in MDA-MB-231 cells and BALB/c nude mice xenografts of

374

MDA-MB-231 cells. According to the present findings, the combination of apigenin

375

and chrysin could be considered for improving effectiveness in breast or liver cancer

376

therapy.

377

19



378 379 380 381

Conflict of Interest The authors declare no conﬂict of interest. Acknowledgements This work was supported by grants from the National Science Council, Taiwan.

382

(NSC 100-2313-B-134 -001 -MY3, MOST 103-2313-B-134 -001 -MY3).

383

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Combined treatment with chrysin and 1,2,3,4,6-penta-O-galloyl-beta-D-glucose

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Figure Legends

472

Figure 1. (A) Cell viability comparison of HepG2, Huh7 and MDA-MB-231 cells

promotes

insights

into

triple-negative

breast

TNFalpha-induced

cancer

metastasis

epithelial-mesenchymal

24


by

activating

transition

in

Page 25 of 40


473

treated with Hex, CHCl3, EtOH, MeOH, EtOAc or Water-EtOAc extracts of Morinda

474

Citrifolia fruit. MTT assay showed different responses of HepG2, Huh7 and

475

MDA-MB-231 cancer cells treated with different extracts of Morinda Citrifolia fruit

476

for 72-96 h. (B) Effect of different extracts of Morinda Citrifolia fruit on cell cycle

477

distribution of HepG2 and MDA-MB-231 cancer cells. Cell cycle distribution was

478

assessed by flow cytometry. (C) Analysis of polyphenol content in Morinda Citrifolia

479

fruit. Polyphenols in Water-EtOAc extracts of Morinda Citrifolia fruit were analyzed

480

by HPLC.

481

280 nm. Peaks: 1, ellagic acid; 2, ellagic acid + gallic acid; 3, chlorogenic acid; 4,

482

catechin; 5, syringic acid + epigallocatechin gallate (EGCG) + rutin + caffeic acid; 6,

483

corilagin; 7, narigenin + p-coumaric acid; 8, picatechin gallate (ECG); 9, myricetin;

484

10, theaflavin (TF1); 11, resveratrol; 12, theaflavin-3-gallate (TF2); 13, luteolin; 14,

485

quercetin; 15, apigenin; 16, kaempferol; and 17, chrysin.

486

Figure 2. Effect of chrysin plus apigenin on cell viability of AU565, HepG2, and

487

MDA-MB-231 cancer cells. Cells were cultured in DMEM supplemented with 10%

488

fetal calf serum for 24 h, and then treated with DMSO, chrysin, apigenin, or chrysin

489

plus apigenin for 72 h (A) or 96 h (B). Cell growth inhibition was determined using

490

MTT assay. The number of viable cells after treatment is expressed as a percentage of

491

the vehicle-only control. Data are presented as mean ± SE of three independent

HPLC chromatograms of the polyphenol standard mixture recorded at

25



492

experiments. The combination of chrysin with apigenin was more effective than either

493

agent alone: *, P ＜ 0.05; **, p

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