Proanthocyanidins, Isolated from Choerospondias axillaris Fruit Peels

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Proanthocyanidins, Isolated from Choerospondias axillaris Fruit Peels, Exhibit Potent Antioxidant Activities in Vitro and Novel Anti-angiogenic Property in Vitro and in Vivo Qian Li, Xieyi Wang, Taotao Dai, Chengmei Liu, Ti Li, David Julian McClements, Jun Chen, and Jiyan Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00236 • Publication Date (Web): 12 Apr 2016 Downloaded from http://pubs.acs.org on April 12, 2016

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Journal of Agricultural and Food Chemistry

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Proanthocyanidins, Isolated from Choerospondias axillaris Fruit

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Peels, Exhibit Potent Antioxidant Activities in Vitro and Novel Anti-

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angiogenic Property in Vitro and in Vivo

4 *

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Qian Li1, Xieyi Wang1, Taotao Dai1, Chengmei Liu1, Ti Li1 , David Julian

6

McClements , Jun Chen1, Jiyan Liu3

2*

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1

9

Nanchang 330047, Jiangxi, P.R. China

State Key Laboratory of Food Science and Technology, Nanchang University,

10

2

Department of Food Science, University of Massachusetts, Amherst, MA, 01003

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3

Jiangxi Qiyunshan Food Co., Ltd, Ganzhou 341000, Jiangxi, P.R. China

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* These authors contributed equally to this manuscript.

14

Ti Li, State Key Laboratory of Food Science and Technology, Nanchang University,

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Nanchang, 330047, Jiangxi, China Tel: +86-791-88305872. Fax: +86 791- 88334509.

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E-mail: [email protected].

17

David Julian McClements, Department of Food Science, University of Massachusetts,

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Amherst, MA 01003, USA Tel: (413) 545-1019. Fax: (413) 545-1262. E-mail:

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[email protected].

Contact information:

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Running title: Antioxidant and Anti-angiogenic Properties of Proanthocyanidins,

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from C. axillaris Fruit Peels

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ACS Paragon Plus Environment


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Abstract

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The production of new blood vessels (angiogenesis) is an important stage in the

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growth and spread of cancerous tumors. Anti-angiogenesis is one strategy for

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controlling tumor progression.

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antiangiogenic activities of a proanthocyanidins (PAs) extract from Choerospondias

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axillaris peels. HPLC-MS analysis revealed that numerous oligomeric forms of the

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PAs were detected in the PAs extract, including dimers, trimers, tetramers and flavan-

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3-ol monomers. The PAs extract possessed appreciable free radical scavenging

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activity (IC50/DPPH =164 ± 7 µg/mL, IC50/ABTS =154 ± 6 µg/mL), potent reducing power

33

(0.930 ± 0.030 g AAE/g), and strong cellular antioxidant activity (EC50 =10.2 ± 1.4

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and 38.9 ± 2.1 µg/mL without or with PBS wash, respectively). It could also retard

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various stages of angiogenesis, such as the migration of endothelial cells and the

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creation of tubes without causing toxicity to the cells. Regarding intracellular signal

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transduction, the PAs extract attenuated the phosphorylation of Akt, ERK, and

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p38MAPK dose-dependently in endothelial cells from human umbilical veins. In

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transgenic zebrafish embryo, new blood vessel formation was suppressed by PAs

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extract in a concentration-dependent manner at 72 h post fertilization. Thus, these

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results suggest that PAs from Choerospondias axillaris peels could be a good source

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of natural inhibitors to target angiogenesis.

This study evaluated the antioxidant and

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Keywords: Choerospondias axillaris; Byproduct; Proanthocyanidins; Anti-oxidant;

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Anti-angiogenesis 2


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1. Introduction The production of new capillaries (angiogenesis) is critical for tumor growth 1, 2

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and metastasis

, because of the requirement of the tumor for oxygen and nutrients

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from existing blood vessels

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angiogenesis are being investigated for the potential to prevent or treat cancer 3. In

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addition, oxidative stress plays an important role in damaging cells during the early

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stages of the development of cancer 5. Cancer cells with elevated levels of

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intracellular reactive oxygen species (ROS) up-regulate vascular endothelial growth

54

factor (VEGF) expression, which is believed to be a key element in the formation of

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new blood vessels and in tumor growth6. Thus, reducing the oxidative stress in cancer

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cells may be an effective way of inhibiting tumor angiogenesis and tumor progression.

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There is therefore considerable interest in the identification of bioactive compounds

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that can modulate angiogenesis.

3, 4

. Consequently, strategies that can inhibit tumor

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Over the last few decades, a number of different inhibitors of angiogenesis have

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been identified that can retard tumor growth by blocking the formation of blood

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vessels, including ZD6126 and SU6668. ZD6126 has tubulin-binding properties and

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the capacity to promote vascular damage in tumors 7. SU6668 is a tyrosine kinase

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inhibitor that can inhibit vascular endothelial growth factor receptor (VEGFR) and

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basic fibroblast growth factor receptor (bFGFR) tyrosine kinases 8. However, in a

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number of cases the efficacy of anti-angiogenesis treatment was only a transient effect,

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and drug resistance was developed after several months of treatments3. Besides, there

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has also been increasing evidence showing that these drugs have the potential to cause 3



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a number of adverse effects upon long-term administration. For instance, SU6668 has

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been revealed to have an unacceptable toxicity profile in Phase I studies 9, whereas

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EMD121974 has been reported to cause fatigue, rash, nausea and vomiting at doses

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up to 1200 mg/m2 10.

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The use of plants and their extracts has been widely applied for both preventive

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and curative purposes. Many natural products are relatively nontoxic and can be

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consumed as dietary supplements or food ingredients. In addition, plant extracts are

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typically compositionally complex materials that contain numerous components that

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can exhibit anti-angiogenesis by different mechanisms

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researchers have attempted to mine natural resources for anti-oxidant and anti-

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angiogenesis agents. Proanthocyanidin from cocoa tea has been shown to inhibit

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angiogenesis by attenuating the phosphorylation of ERK, Akt, and p38MAPK in a

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human microvascular endothelial cell line (HMEC) 3. Proanthocyanidins from grape

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seed inhibit angiogenesis via the down-regulation of both angiopoietin and VEGF

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signaling 12. There has already been a considerable research effort in determining the

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ability of various plants and plant extracts at exhibiting anti-angiogenic and anti-

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oxidant activities.

85

11

. Therefore, in recent years,

Choerospondias axillaris (C. axillaris) from the Anacardiaceae family is already 13

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a commercially important edible fruit that is known to have medicinal properties

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Most of the C. axillaris fruits are peeled when processed as the raw material for the

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food industry, which leads to a large amount of fruit peel being generated as a

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byproduct. Our previous studies have shown that peel extracts of C. axillaris fruit 4


.

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contain high amounts of proanthocyanidins (PAs), which are mainly composed of

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epigallocatechin, catechin, epicatechin, and their galloylated derivatives 14. In addition,

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the 60% ethanol extracts of C. axillaris peels showed potent antioxidant and anti-

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proliferative effect on HepG2 and Caco-2 cancer cell lines 15. Nevertheless, the anti-

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angiogenic and anti-oxidant activities of the PAs extracted from C. axillaris fruit

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peels have not been assessed.

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Therefore, this study aims to determine the antioxidant activity of a PAs extract

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isolated from C. axillaris fruit peels using a combination of chemical-based assays

98

and cellular-based assays. Furthermore, the antiangiogenic potential of PAs extract

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from C. axillaris fruit peels was also explored using the HUVECs for in vitro studies

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of cell proliferation, migration, and tube formation. In addition, the ability of PAs

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extract to inhibit angiogenesis was studied in vivo using a transgenic zebrafish

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embryo Tg(fli1:EGFP)y1 model.

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2. Material and methods

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2.1. Chemicals

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C. axillaris fruits were obtained from Mount Qiyun (Jiangxi Province, P.R. China) in October, 2014.

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Transgenic (Tg(fli1:EGFP)y1i) and wild-type zebrafish lines were purchased

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from Nanjing YSY Biotech Company Ltd. (Nanjing, Jiangsu, China). Both types of

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zebrafish were kept in flow-through aquaria set to a 10 h light /14 h dark photoperiod

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at a temperature of 28.5 °C. The transgenic zebrafish contained endothelial cells that

111

expressed the enhanced green fluorescent protein (EGFP) so they could be observed 5



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by confocal fluorescence microscopy.

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The acetone and methanol used in extraction and purification were obtained from

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HuShi (Shanghai, China). 2, 4, 6-tris(2-pyridyl)-s-triazine (TPTZ), L-ascorbic acid,

115

2,2'-amino-di(2-ethyl-benzothiazoline sulphonic acid-6) ammonium salt (ABTS), Z-

116

3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone; semaxanib (SU5416), 2,2'-

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diphenyl-1-picrylhydrazyl (DPPH) were obtained from Sigma-Aldrich (St. Louis, MO,

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USA). The trypsin-EDTA solution, Dulbecco’s Modified Eagle’s Medium (DMEM)

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and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Grand

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Island, NY, USA). Matrigel was purchased from Becton–Dickinson (Bedford, MA,

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USA). For Western blot assay, the primary and secondary antibodies were obtained

122

from Cell Signaling Technology (Danvers, MA, USA). Other chemicals not listed in

123

this section were obtained from Aladdin (Shanghai, China). Double distilled water

124

from a Milli-Q purification system (Bedford, MA, USA) was used for the preparation

125

of all solutions.

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2.2. Preparation of plant material and PAs extract

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Selected fresh fruits (1000 ± 7 g) were thoroughly rinsed using water, and then

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their peels were removed, freeze dried for 72 h (FreeZone Freeze Dry Systems

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LABCONCO Corp., Kansas City, MO, USA), ground into a fine powder (45.7 ± 2.5

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g), and then stored in darkness at -20 °C until used. This powder (5 g) was then

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extracted using acetone/methanol/water solution (2:2:1) (150 mL) in Erlenmeyer

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flasks for 20 min using an ultrasonic cleaner (Kunshan Ultrasonic Instrument

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Company Ltd., Shanghai, China) at ambient temperature. Extracts were then filtered

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so as to isolate the soluble compounds from the insoluble waste compounds. The 6


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extraction process was repeated twice. The solutions were then rotary-evaporated

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under vacuum (Model RE-2000A, YaRong Technology Ltd., Shanghai, China) at

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40 °C to remove the acetone. After extraction and concentration, the concentrated

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extract solution was pre-purified using a method described previously 14. After rinsing

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the HP-2MGL resin column (Mitsubishi Chemical Corp., Tokyo, Japan) (460 × 22

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mm) with double distilled water (200 mL), the fraction containing PAs was eluted

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with 60% acetone solution. The resulting eluent was concentrated by a rotary

142

evaporator to prepare a crude PAs extract followed by lyophilization. The final

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powder obtained was called crude PAs (a yield of 15% peels by dry weight). Then the

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crude PAs powder obtained above was further purified by a size exclusion column

145

(Sephadex LH-20, 500 × 22 mm, GE Health-care Bio-Sciences AB, Uppsala,

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Sweden). Briefly, 3.5 g crude PAs powder dissolved in 35% methanol was applied to

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a Sephadex LH-20 column pre-equilibrated with 35% methanol for 4 h. The column

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was then washed with 500 mL of 35% methanol to elute sugars and most of the

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interfering phenolics. Then, acetone: water (60:40, v/v; 250 mL) was used to elute the

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rest of the PAs fraction. The eluent was then rotary-evaporated under vacuum at

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40 °C to remove the organic solvents, and lyophilized to dryness. The obtained

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purified PAs powder led to a yield of 4.7% (fresh fruit peels). Finally, the purified

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PAs powder was analyzed by LC-MS and was subjected to acid catalysis to determine

154

the mean degree of polymerization (mDP).

155

2.3. Measurement of total phenolics and mDP

156

The total phenolics content of the PAs extract was measured by the Folin-

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Ciocalteau method according to the method of Ordonez, et al. with some 7



158

modifications16. Gallic acid was used to prepare the standard curve. The total

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phenolics content of PAs extract was expressed as gallic acid equivalents (GAE) i.e.,

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the mass of gallic acid (g) per unit mass of PAs powder (g).

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The mDP of PAs extract was determined by acid catalysis based on the protocol

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described by Kennedy and Jones with minor modifications 17. The reaction mixtures

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were filtered through a 0.22 µm PTFE membrane syringe Filter (Millipore, Bedford,

164

MA, USA) and immediately analyzed by liquid chromatography.

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2.4 HPLC and LC-MS analysis

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HPLC analysis was operated using a 2695 Waters HPLC system (Milford, MA,

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USA) equipped with a 2996 PAD and connected to a Micromass ZQ 4000

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electrospray mass spectrometer (Manchester, UK). Masslynx version 4.0 software

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was used for data acquisition and operating. The separation was achieved on a RP-C18

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(4.6 mm × 250 mm) column (Waters, Milford, MA, USA) and detected at 280 nm.

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The mobile phases were 0.25% (v/v) acetic acid in water (eluent A) and acetonitrile

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(eluent B) at a flow rate of 1.0 mL/min at 30 °C, using a gradient program described

173

previously with some modification 14 : from 10 to 25% B (10 min), from 25 to 35% B

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(15 min), from 35 to 40% B (5 min), from 40 to 50% B (10 min), from 50 to 100% B

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(25 min), from 100 to 10% B (10 min).

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The ESI mass spectrometer was conducted in both negative and positive ion

177

mode. Nitrogen was used as both cone gas and desolvation gas at a flow rate of 50 L/h,

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300 L/h, respectively. The desolvation temperature was set at 350 °C. The ionization

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source was conducting at 120 °C. Capillary voltage, cone voltage, extractor voltage,

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and Rf lens were set at 2.6 KV, 30 V, 2 V and 0.2 V, respectively. 8


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2.5. Antioxidant activity in vitro

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2.5.1. DPPH radical scavenging assay

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The DPPH radical scavenging activity of PAs extract was assayed according to

184

the method described by Zhou, et al. with some modifications18, as previously

185

described in our previous study15. Purified PAs powder was used at various

186

concentrations of 12.5 - 400 µg/mL. The DPPH radical scavenging activity (%) was

187

calculated by the following equation:

188

Scavenging activity (%) = [1-(Asample/ Acontrol)] ×100

(1)

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Here, Asample and Acontrol are the absorbance measurements (at 515 nm) for the sample

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and control, respectively. The IC50 value (µg/mL) refers to the effective concentration

191

of extract required to scavenge the DPPH radicals by 50%.

192

2.5.2 ABTS·+ radical scavenging assay

193

The ABTS·+ scavenging activity of PAs from C. axillaris peels was carried out

194

according to the method described by Liang, et al. with some modifications

195

percentage scavenging potency was calculated as:

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Scavenging rate (%) = [1 − (Asample / Acontrol)] × 100

19

. The

(2)

197

Here, Asample and Acontrol are the absorbance measurements (at 734 nm) for the samples

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and control, respectively. The IC50 value (µg/mL) refers to the effective concentration

199

of extract required to scavenge the ABTS·+ radicals by 50%.

200

2.5.3 Ferric-reducing antioxidant power (FRAP) assay

201

The FRAP assay was assayed according to the procedure described in the

202

literature by Benzie and Strain with some modifications20, as previously described in

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our previous study15. The results were expressed as grams of ascorbic acid equivalents 9



204

(AAE) per g PAs powder using a calibration curve.

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2.6 Cellular antioxidant activity (CAA)

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Caco-2 cells and HUVECs were obtained from Cell Bank of Institute of

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Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and

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cultured in DMEM supplemented with10% FBS and 1% penicillin/ streptomycin (v/v).

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Cells were incubated at 37°C under 5% CO2.

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2.6.1 Determination of Cytotoxicity

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Cytotoxicity toward Caco-2 cells was measured using the CCK-8 method as

212

conducted in the literature, being similar to the well-established MTT assay 21. Caco-2

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cells (1 × 104 cells/well) were seeded in 96-well plates. After 24 h of incubation at

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37 °C with 5% CO2, the old medium was removed and each tested well was washed

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with 100 µL of PBS. Then, different concentrations (0 - 300 µg/mL) of dimethyl

216

sulfoxide (DMSO)-dissolved PAs which was prepared with fresh growth medium

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were added to the tested wells, and the plates were kept at 37 °C. Control cells were

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treated only with DMSO at the maximum concentration applied in the tested extract

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concentrations, while blank wells contained 100 µL growth medium without any cells.

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After another 24 h of incubation, the culture medium was discarded, all the tested

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cells were washed twice with 100 µL of PBS. Then the WST-8 solution with growth

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medium (v/v: 1:10) was added and incubated for 3 h. The absorbance of each well

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was measured with a microplate reader (Multishan FC, Thermo Scientific, Waltham,

224

MA, USA) at 450 nm. Cytotoxicity was defined as a 10% reduction of absorbance for

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each concentration compared to the control.

10


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2.6.2. Cellular antioxidant activity of purified PAs extract

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A 20 mM DCFH-DA stock solution in DMSO was prepared and stored at -20 °C

228

until use. A stock solution of ABAP (200 mM) in water was prepared, aliquoted, and

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stored at - 80 °C until use. The cellular antioxidant activity assay protocol was based

230

on that described by Wolfe and Liu with some modifications22. Briefly, Caco-2 cells

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were cultured at a density of 1×104 /well on a 96-well microplate in 100 µL complete

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medium. After 24 hours of incubation, the old medium was removed, and the wells

233

were washed with 100 µL of PBS. Cells were then treated with 100 µL of growth

234

medium containing solvent control, control extracts, or tested extracts (0-150 µg/mL)

235

plus 50 µM DCFH-DA for 1 h. When a PBS wash was utilized, wells were washed

236

with 200 µL of PBS and then applied 800 µM ABAP in 100 µL PBS to the cells, and

237

the microplate was placed into an automated microplate reader (Varioskan Flash,

238

Thermo Scientific, Waltham, MA, USA). Emission at 538 nm was measured with

239

excitation at 485 nm every 5 min for 1 h. Each plate included triplicate control and

240

blank wells: control wells contained cells treated with DCFH-DA and oxidant; blank

241

wells contained cells treated with dye and PBS without oxidant.

242 243 244

After subtraction of the blank, the area under the curve for fluorescence vs time was integrated to calculate the CAA value at each concentration of extract as follows: CAA unit = 100 - (∫SA ⁄ ∫CA) × 100

(3)

245

Where ∫SA is the integrated area under the sample fluorescence vs time curve and ∫CA

246

is the integrated area from the control curve. The median effective dose (EC50) was

247

determined for the sample from the median effect plot of log (fa/fu) vs log (dose),

11



248

where fa is the fraction affected and fu is the fraction unaffected by the treatment. The

249

EC50 values were stated as the mean ± SD for triplicate sets of data obtained from the

250

same experiment.

251

2.7. Anti-angiogenic effects in vitro

252

2.7.1. Cell proliferation assay

253

Cytotoxicity toward HUVECs was measured using the CCK-8 assay, which was

254

similar with section 2.6.121. Absorbance values acquired for cell viability were

255

expressed as percentages of the blank control (surviving control cells) in the test.

256

2.7.2 Cell migration assay

257

The cell migration assay was conducted according to the method reported

258

previously with minor modifications 3. Briefly, cells were seeded in a 6-well plate at a

259

density of 5×105 cells/well overnight. Then the cells starved for 12 h in a serum

260

reduced medium (2%). The bottom of each well containing a HUVEC monolayer was

261

scraped (straight line) with a 200 µL sterile pipet tip. Cell debris was removed by

262

washing the plate with PBS solution. Cells were treated with different concentrations

263

(0-150 µg/mL) of PAs samples in a medium containing 2% serum and then incubated

264

at 37 °C under 5% CO2 for 18 h. The open wound area (scraped area) at 0 and 18 h

265

were photographed under 60 × magnification with a Nikon microscope (Nikon,

266

Tokyo, Japan). Data was analyzed with the TScratch software using the default

267

parameter settings 23. Percentage of the open wound area after treatment was

268

calculated and compared with the value obtained at 0 h. An increase of the percentage

269

of open wound area indicated the inhibition of cell migration. In this study, SU5416

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(1µM) was applied as a positive control. 12


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2.7.3. Tubule formation assay In vitro tests using HUVECs to evaluate the potencies of PAs samples (50, 100

273

and 150 µg/mL) on the capillary-like tubes formation was according to the method

274

described previously 3. Cells (1 ×104 cells/well) were seeded on the layer of Matrigel

275

Matrix (BD Bioscience, Franklin Lakes, NJ, USA) and exposed to various

276

concentrations (0-150 µg/mL) of PAs extract or SU5416 (10 µM) in culture medium

277

or with culture medium only (vehicle control). After incubation for 6 h at 37 °C, the

278

tubules were photographed under a microscope. Each treatment was performed in

279

triplicate. The total tubule length formation was calculated for quantification of

280

angiogenesis by the Image-Pro Plus version 6.0 (Media Cybernetics, Bethesda, MD,

281

USA). Inhibition of tubule formation was calculated as tubule length (treated) divided

282

by tubule length (control).

283

2.7.4 Western blot analysis

284

HUVECs (5×105cells/ mL) were seeded in 6-well plates and incubated for 24 h

285

to allow attachment. Then the cells were treated with various concentrations (0-200

286

µg/mL) of PAs extract and incubated for 12 h. After treatment, cells were harvested

287

and washed twice with ice-cold PBS and lysed in RIPA buffer containing protease

288

inhibitors (Beyotime Institute of Biotechnology, Nanjing, Jiangsu, China) for 30 min

289

on ice. The cell lysates were centrifuged at 12000 g for 30 min at 4 °C, and then the

290

supernatants were collected. Total protein content was determined by BCA protein

291

assay (Thermo Scientific Pierce, Rockford, IL, USA). Equal amounts (30 µg) of

292

protein samples were separated on a 12% SDS polyacrylamide gel and

293

electrophoretically transferred (100 V, 2 h) onto a NC membrane (Millipore, Billerica, 13



294

MA, USA). Afterwards, the NC membranes were blocked for 1 h using 5% BSA. The

295

following primary antibodies were used: anti-phospho-Akt, anti-phospho-ERK1/2,

296

anti-phospho-p38MAPK and anti-β-actin (Cell Signaling Technology, Beverly, MA

297

USA; 1:2000). The membranes were incubated with the above antibodies at 4°C

298

overnight. After incubation with the secondary horseradish- peroxidase-conjugated

299

antibodies (Cell Signaling Technology, Beverly, MA USA; 1:3000) for 1 h, detection

300

was performed using the ECL-Plus detection kit. (PerkinElmer Life Sciences, Inc.,

301

Boston, MA, USA). Signal intensities of p-Akt, p-ERK, and p-p38MAPK were

302

measured densitometrically (Image-Pro Plus version 6.0, Bethesda, MD, USA) and

303

expressed relative to total Akt, ERK, or p38MAPK.

304

2.8 Angiogenesis study of zebrafish embryos

305

The transgenic zebrafish line (Tg(fli1:EGFP)y1) with endothelial cells

306

expressing EGFP was cultured as described previously 3, 24. The zebrafish were fed

307

with brine shrimp and tropical fish food twice daily. Healthy embryos were chosen at

308

their 1–4 cell stage and were put into a six-well microplate with 20 embryos /well.

309

Then, the embryos were treated with various concentrations (12.5, 25, 50 µg/mL) of

310

PAs extract and SU5416 (2 µM), which were diluted using zebrafish embryos growth

311

media. Embryos receiving 0.1% DMSO served as negative controls, which were

312

shown to have no toxicity to the embryos in preliminary tests (data not shown). After

313

72 h post fertilization (hpf), the embryos were examined using an Olympus IX71S8F-

314

2 inverted microscope (Olympus, Tokyo, Japan) for the length of vessels in the sub-

315

intestinal vessel plexus (SIVs) region to assess angiogenesis. The vessel length

316

formation was calculated for quantification of angiogenesis by the Image-Pro Plus 14


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version 6.0 (Media Cybernetics, Bethesda, MD, USA)

318

2.9 Statistical analyses

319

All experiments were carried out in triplicate (n = 3). The results are expressed

320

as averages and standard deviations. An ANOVA test (SPSS 19.0, SPSS Inc.,

321

Chicago, IL, USA) was used to compare the mean values of each treatment and

322

significant differences between the means of parameters were determined using the

323

Duncan-test (p < 0.05).

324

3. Results and Discussion

325

3.1. Total phenolics content and mDP of PAs extract from C. axillaris fruits peels

326

The total phenolics content (g GAE/g purified PAs powder) was measured. The

327

amount of total phenolics in purified PAs extract from C. axillaris fruit peel was

328

0.627 ± 0.070 g GAE /g purified PAs powder, indicating that this extract contained a

329

substantial amount of phenolic compounds. The results of acid catalysis analysis by

330

HPLC-MS are shown in Table 1 and Figure 1A (Supplementary File). From Table 1,

331

we found that the extension units of PAs from C. axillaris fruits peels consisted of

332

epigallocatechin, epicatechin gallate, epicatechin and catechin, while the terminal

333

units consisted of catechin, epicatechin and epicatechin gallate. According to the

334

amount of individual compounds collected after acid catalysis, we calculated that the

335

mDP of the purified PAs extract was 3.06, indicating that oligomeric

336

proanthocyanidin was the main component in this PAs extract. Numerous oligomeric

337

forms of the molecules were also detected by HPLC-MS, including significant

338

amounts of dimers, trimers, tetramers and flavan-3-ol monomers (Supplementary File

339

Figure 1(B) and Table 1). 15



340

3.2. Chemical-based antioxidant activity assay

341

Three different in vitro (chemical-based) assays were used to evaluate the

342

antioxidant activity of the PAs extract. The free radical scavenging activities of PAs

343

extract were compared to that of ascorbic acid (Figures 1A and B). For DPPH radical

344

scavenging, the IC50 values were 164 ± 7 and 98.0 ± 5.0 µg/mL for PAs extract and

345

ascorbic acid, respectively. For ABTS radical scavenging, the IC50 values were 154 ±

346

6 and 209 ± 9 µg/mL for PAs extract and ascorbic acid, respectively. Thus, the PAs

347

extract had a lower DPPH radical scavenging activity than ascorbic acid but a higher

348

ABTS radical scavenging activity. The antioxidant activity of the PAs extract was

349

also determined using a FRAP assay, which showed that the activity increased with

350

PAs extract level (Figure 1C). The FRAP values were 0.932 ± 0.031 and 1.00 ± 0.06

351

g AAE/g for the PAs extract and ascorbic acid, respectively. There was no significant

352

difference in the ferric reducing activity between PAs extract and ascorbic acid (p >

353

0.05). The above results suggested that PAs extract had a high scavenging activity, as

354

demonstrated by the DPPH radical, ABTS radical, and potent ferric reducing

355

activities.

356

3.3 Cellular-based antioxidant activity assay

357

Unlike chemical chemical-based assays, cellular-based assays inherently account

358

for the absorption and distribution of bioactive compounds in cells, and may therefore

359

provide a more realistic model of the in vivo antioxidant activity of phytochemicals.

360

The cytotoxicity of PAs extract and ascorbic acid was determined by dividing the

361

Caco-2 cells into 96 well plates and treating them with different doses of PAs extract

362

and ascorbic acid in DMEM for 24 h to find out the safe concentration range. The 16


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results showed that all PAs extract and ascorbic acid solutions used had no significant

364

anti-proliferative activity as compared to the control when used at a concentration
8)-GCG from cocoa tea (Camellia ptilophylla) in antiangiogenesis. The Journal of nutritional biochemistry 2014, 25, 319-28. 4.

Shiozaki, T.; Fukai, M.; Hermawati, E.; Juliawaty, L. D.; Syah, Y. M.; Hakim, E. H.;

Puthongking, P.; Suzuki, T.; Kinoshita, K.; Takahashi, K.; Koyama, K., Anti-angiogenic effect of αmangostin. Journal of natural medicines 2013, 67, 202-6. 5.

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Blakey, D. C.; Westwood, F. R.; Walker, M.; Hughes, G. D.; Davis, P. D.; Ashton, S. E.; Ryan, A.

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Klohs, W. D.; Hamby, J. M., Antiangiogenic agents. Current opinion in biotechnology 1999, 10,

544-549. 9.

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Angiogenesis inhibitors in clinical development for lung cancer. Seminars in oncology 2002, 29, 66-77. 10. Holden, S.; Morrow, M.; O’Bryant, C. In Correlative biological assays used to guide dose escalation in a phase I study of the antiangiogenic αVβ3 and αVβ5 integrin antagonist EMD 121974 (EMD), Proc Am Soc Clin Oncol 2002, 28. 11. Singh, R. P.; Agarwal, R., Tumor angiogenesis: a potential target in cancer control by phytochemicals. Current cancer drug targets 2003, 3, 205-217. 12. Huang, S.; Yang, N.; Liu, Y.; Hu, L.; Zhao, J.; Gao, J.; Li, Y.; Li, C.; Zhang, X.; Huang, T., Grape seed proanthocyanidins inhibit angiogenesis via the downregulation of both vascular endothelial growth factor and angiopoietin signaling. Nutrition Research 2012, 32, 530-536. 13. Wang, H.; Gao, X. D.; Zhou, G. C.; Cai, L.; Yao, W. B., in vitro and in vivo antioxidant activity of aqueous extract from Choerospondias axillaris fruit. Food Chemistry 2008, 106, 888-895. 14. Li, Q.; Chen, J.; Li, T.; Liu, C.; Zhai, Y.; McClements, D. J.; Liu, J., Separation and characterization of polyphenolics from underutilized byproducts of fruit production (Choerospondias axillaris peels): inhibitory activity of proanthocyanidins against glycolysis enzymes. Food & function 2015. 15. Li, Q.; Chen, J.; Li, T.; Liu, C.; Liu, W.; Liu, J., Comparison of bioactivities and phenolic composition of Choerospondias axillaris peels and fleshes. Journal of the Science of Food and Agriculture 2015. 16. Ordonez, A.; Gomez, J.; Vattuone, M., Antioxidant activities of Sechium edule (Jacq.) Swartz 27



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710

Figure Captions

711

Figure 1 Antioxidant activities of the PAs extract isolated from C. axillaris peels

712

were determined by (A) DPPH; (B) ABTS and (C) FRAP assays. For each assay, a

713

positive control using ascorbic acid.

714 715

Figure 2 Cellular antioxidant activity of the PAs extract isolated from C. axillaris

716

peels. Peroxyl radical-induced oxidation of DCFH to DCF in Caco-2 cells and the

717

inhibition of oxidation by the purified PAs extract over time, using the protocol

718

involving no PBS wash between antioxidant and ABAP treatments (A, C) and the

719

protocol with a PBS wash (B, D) to remove antioxidants in the medium not associated

720

with cells. Ascorbic acid was used as a positive control.

721 722

Figure 3 Effects of the purified PAs extract on endothelial cell migration. (A)

723

Representative photographs of 1 µM SU5416 and PAs extract-treated HUVECs at

724

times 0 h and 18 h. (B) Quantitative analysis of effects of purified PAs extract on

725

endothelial cell migration. Data represent means ± standard error (SE) of three

726

independent experiments. Symbols indicate significant differences between control-

727

untreated and treated cells (*p

Proanthocyanidins, Isolated from Choerospondias axillaris Fruit Peels

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