Ultrasound-assisted extraction from defatted oat (Avena sativa L.) bran to simultaneously enhance phenolic compounds and β-glucan contents: Compositional and kinetic studies

Accepted Manuscript Ultrasound-assisted extraction from defatted oat (Avena sativa L.) bran to simultaneously enhance phenolic compounds and β-glucan contents: Compositional and kinetic studies Chao Chen, Li Wang, Ren Wang, Xiaohu Luo, Yongfu Li, Juan Li, Yanan Li, Zhengxing Chen PII:

S0260-8774(17)30469-7

DOI:

10.1016/j.jfoodeng.2017.11.002

Reference:

JFOE 9062

To appear in:

Journal of Food Engineering

Please cite this article as: Chao Chen, Li Wang, Ren Wang, Xiaohu Luo, Yongfu Li, Juan Li, Yanan Li, Zhengxing Chen, Ultrasound-assisted extraction from defatted oat (Avena sativa L.) bran to simultaneously enhance phenolic compounds and β-glucan contents: Compositional and kinetic studies, Journal of Food Engineering (2017), doi: 10.1016/j.jfoodeng.2017.11.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Highlights 1. Extraction of phenolics from defatted oat bran by ultrasound is suggested. 2. The first time to simultaneously investigate extraction of phenolics and β-glucan. 3. Free phenolics yield were significantly improved by high temperature.

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4. Ultrasonic extraction gave better phenolics yield in shorter time than maceration.

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5. The β-glucan yield was significantly increased by ultrasonic pretreatment.

ACCEPTED MANUSCRIPT Ultrasound-assisted extraction from defatted oat (Avena sativa L.) bran to simultaneously enhance

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phenolic compounds and β-glucan contents: compositional and kinetic studies

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Chao Chena, b, c, d, Li Wanga, b, c, d*, Ren Wanga, b, c, d, Xiaohu Luoa, b, c, d, Yongfu Lia, b, c, d, Juan Lia, b, c, d, Yanan

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Lia, b, c, d, Zhengxing Chena, b, c, d *

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State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China

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National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122,

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China

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School of Food Science and Technology, Jiangnan University, Wuxi 214122, China

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Collaborative Innovation Center for Food Safety and Quality Control in Jiangsu Province, Wuxi 214122,

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China

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Running title: Ultrasonic treatment enhance phenolics and β-glucan extraction yields of oat bran

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*

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School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.

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Corresponding author: Li Wang, Zhengxing Chen

Tel: +86-510-8591 7856;

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Fax: +86-510-8591 7856;

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E-mail: [email protected], [email protected]

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Abstract In this research, the influence of ultrasonic application and temperature on extraction yields of free,

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esterified, bound phenolics, and β-glucan from defatted oat bran was investigated. Ultrasonic-assisted

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extraction (UAE) and conventional extraction (CE) were performed at different temperature. Extracts

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kinetics were monitored by determining the total phenolic content (TPC), antioxidant capacity (ORAC), and

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total avenanthramides of free phenolic compounds by mathematical model. HPLC-DAD was used to

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identify and quantify the main phenolic compounds. The results suggested that phenolic extraction yields of

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UAE was faster and higher than that of CE for free phenolics, with fitting to mathematically model (MRPD

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< 6%) well, whereas the bound fractions decreased. Besides, the TPC, ORAC and total avenanthramides of

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free phenolics were significantly improved by increasing the temperature in both UAE and CE, whereas the

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bound were significantly decreased. β-Glucan yields pretreated in UAE were approximately 37% higher

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than that in CE.

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Keywords: Defatted oat bran; Ultrasonic-assisted extraction; Phenolic compounds; Antioxidant activity;

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Avenanthramides; β-Glucan

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1. Introduction Oat (Avena sativa L. and Avena nuda L.) is a member of the Gramineae family, and ranks seventh in

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world production within cereals and is one of the grains cultivated by humans for the longest time (Kellogg,

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1998). Oat bran is the edible, ground outer layer of oat kernel and contains many antioxidant components,

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including phytic acid, vitamin E, and many kinds of polyphenols, which have been reported to have high

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antioxidant activities in vitro and in vivo (Emmons and Peterson, 2000).

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Oat groat has the highest oil contents in cereal grains (Price and Parsons, 1975). Oat oil is interesting

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because of its low concentration of saturated fatty acids and high concentration of monounsaturated fatty

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acids (Zhou et al., 1999). Moreover, there have been oat bran oil products produced by cosmetics companies

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in China. However, large scale extraction of oat bran oil produces massive defatted oat bran meal. There are

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few researches focused on the potential significant value of defatted oat bran meal, which still include

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considerable number of active components with health benefits, such as β-glucan and phenolic compounds.

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The beneficial effects of oat bran are regarded to be mainly due to the β-glucan, which has been proven

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to be effective in reducing postprandial blood glucose level and serum cholesterol concentration (Tiwari and

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Cummins, 2009). To obtain high viscosity and concentration of β-glucan, oat bran meal needs to be treated

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for 30-120 minutes using 80% ethanol to obtain enzyme-deactivated oat bran flour (Wood et al., 1978).

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However, the ethanol extracts of oat bran produced in enzyme deactivation contain many phenolic

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compounds (Emmons and Peterson, 2000), which are typically discarded as waste. No researchers have

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simultaneously studied the extraction of β-glucan and phenolic compounds from oat bran. It is generally

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known that phenolics, which are rich in whole grains, have strong antioxidant characteristics (Emmons and

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Peterson, 2000). Most phenolic compounds are located in the outer bran layer of oats (Peterson, 2001).

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Several types of oat phenolic compounds have been identified, including phenolic acids and a unique source

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of soluble phenolics referred to as avenanthramides (AVs), which are only existed in oats (Collins, 1989).

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These avenanthramides accounts for the principal phenolic antioxidants in the oat bran (Dimberg et al.,

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1993). The most abundant ones are avenanthramide 2c (AV2c), avenanthramide 2f (AV2f), and

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avenanthramide 2p (AV2p). These three major AVs have been reported to show 10-30 times in vitro radical

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scavenging activity than other phenolics (Emmons and Peterson, 2000). Traditional liquid solvents extraction by maceration has been widely used to extract phenolics from

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oats. However, the main disadvantage of this conventional extraction (CE) method is the longtime to reach

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solid-solvent contact equilibrium and the low extraction efficiency (Kotovicz et al., 2014). High

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temperatures might improve the solubility and diffusion of phenolics, resulting in the increase of the

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extraction yield. A previous study indicated that the yields of phenolics in olive leaves were significantly

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improved by increasing temperature to up to 70 °C (Khemakhem et al. 2017). Using high temperatures does

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bring about a kinetic improvement of polyphenols, but it is sometimes limited by the fact that phenolics are

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sensitive to high temperatures. Therefore, it is important to choose an appropriate temperature range for the

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phenolics extraction. In recent years, in order to address these limitations, ultrasonic-assisted extraction

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(UAE) have been developed to increase the extraction rate of many phenol based natural products, including

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grape marc, wheat bran, coconut, and so on (Shirsath et al., 2012). The main benefits of UAE are particle

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size reduction, possible rupture of cell walls and overall mass transfer increase of intracellular content via

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hydration of plant tissue as well as cavitation bubble collapse (Gogate and Kabadi, 2009). There is no

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previous research evaluating the influence of ultrasonic treatment on antioxidant properties of oats. As

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mentioned in a recent review, some novel processing technologies like ultrasound assisted extraction and

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pressurized liquid extraction have the potential to efficiently extract various bioactives (phenolic compounds

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and β-glucan) from oats which have not been tried (Gangopadhyay et al., 2015).

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Generally, the extraction of β-glucan and phenolic compounds from oats were separately studied over

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the years (Wood et al., 1978; Collins, 1989; Emmons and Peterson, 2000; Peterson, 2001; Tiwari and

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Cummins, 2009). Thus, the aim of this study was to simultaneously evaluate the changes in phenolic profile,

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total phenolic contents, total AVs contents, antioxidant activity and β-glucan extraction rate of defatted oat

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bran after ultrasonic treatment, to evaluate the effects of different temperatures on extraction kinetics of

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polyphenols from defatted oat bran using both the conventional and ultrasound assisted extraction.

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2. Materials and methods

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2.1 Raw material Oats (Longyan 3, Avena sativa L) were collected from a local farm product market in the town of

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Guyuan City, the Ningxia Hui Autonomous Region, China. The dry and cleaned oats were processed in a

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mill (Landert-Motoren AG Company, Buelach, Switzerland) to collect the oat bran fraction. The final weight

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of oat bran was about 23% of the original hulless oats. The bran samples were grinded and stored at 4 °C

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until being used. Deionized water was used in all experiments.

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2.2 Chemicals and reagents

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Hexane, Folin-Ciocalteu reagent, ethanol, potassium phosphate dibasic (K2HPO4), potassium phosphate

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monobasic (KH2PO4), and sodium carbonate (Na2CO3) were purchased from Sinopharm Chemical Reagent

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Co., Ltd (Shanghai, China). 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-azobis

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(2-amidino-propane) dihydrochloride (AAPH), gallic acid, p-hydroxybenzoic acid, caffeic acid,

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protocatechuic acid, vanillic acid, gallic acid, ferulic acid, avenanthramide 2c, avenanthramide 2f, and

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avenanthramide 2p were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Acetonitrile and acetic

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acid were of HPLC grade and were obtained from Fisher Scientific Co. (Nepean, ON, Canada).

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2.3 Preparation of defatted oat bran

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One hundred grams of oat bran meal were passed through an 80-mesh sieve. After screening, the flour

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was mixed with 1 L of hexane for 16 h at room temperature performed by maceration. Subsequently, the

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mixture was centrifuged (15 min at 4000 × rpm), and then the residue was re-extracted three times following

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the same procedure. Finally, the residue was obtained and dried in a drying oven at 40°C for 10 h to remove

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any residual hexane and stored at 4°C prior to polyphenol extraction.

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2.4 Ultrasound assisted extraction (UAE) and conventional extraction (CE) for free and esterified

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phenolic compounds UAE experiments were performed using an ultrasonic cleaning bath (KH3200DB, Kunshan Ultrasonic

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instrument Co. Ltd, Suzhou, China) of internal dimensions 300 mm × 150 mm × 180 mm and tank capacity

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8 L approximately with ultrasonic power of 200-600 W and frequencies of 40 kHz, equipped with a heating

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system and digital temperature indicator, which used to keep the temperature constant. Ultrasound cleaning

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bath was also equipped with a powerful electric stirrer (JB300D, Shanghai Specimen and Model Factory,

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Shanghai, China). For CE the ultrasonic generator was switched off, while for UAE it was switched on.

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Both the UAE and CE procedure for free phenolic compounds were followed according to a method

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reported previously by Chen et al. (2016) with some modifications. Four grams of defatted oat bran were

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blended with 40 mL of 80% ethanol in a glass vessel. The glass vessel was instantly immersed in the

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ultrasonic bath for a preset time, equipped with a powerful electric agitator. All the experiments of extraction

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were carried out in glass vessels of known dimension (3.7 cm diameter and 7.5 cm height) placed in the

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bottom of ultrasonic bath at center position. Different temperatures were tested (20, 40, 50, 70 and 90 °C)

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supplying, in both UAE and CE, 100% of the total ultrasonic power of the system (600 W) as advised by

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Ahmad-Qasem et al. (2013) in order to accelerate the extraction rate. Extraction was performed till 25 min,

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taking samples at predetermined times (1, 5, 10, 15, 20 and 25 min) to measure the extraction kinetics.

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Following centrifugation (15 min at 4000 ×rpm), the supernatants were filtered through No. 2 Whatman

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filter paper, and the residue was re-extracted three times following the same method. The solvent was

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combined and evaporated under vacuum at 40°C using a rotary evaporator to remove organic solvent, then

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the water phase was freeze dried. Each freeze dried extract was resolubilized in 4 mL of methanol, filtered

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through a 0.22-µm nylon membrane, and stored at -20°C prior to analytical determinations. All the

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extraction experiments were carried out in triplicate.

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The esterified phenolic compounds was extracted from defatted oat bran according to the methods

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described by Bei et al. (2017). The esterified phenolic compounds was extracted from the above water phase

ACCEPTED MANUSCRIPT after the extraction of free phenolics by 100 mL ethyl acetate for three times. The water phase was

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resuspended in 80 mL of 2 M NaOH for 4 h at room temperature and acidified with appropriate amount of

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12 M HCl to pH 2.0. The hydrolysate was extracted by 100 mL of ethyl acetate for three times. After

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removal of the ethyl acetate by a rotary evaporator, the extract was resolubilized in 4 mL of methanol,

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filtered through a 0.22-µm nylon membrane, and stored at -20°C prior to analytical determinations.

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2.5 Extraction of bound phenolic compounds

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Bound phenolic compounds were extracted from the residue after removing free and esterified

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phenolics using the method described by Bei et al. (2017). The residue was first hydrolyzed with 100 mL of

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2 M NaOH for 4 h at room temperature. The mixture was then acidified with appropriate amount of 12 M

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HCl to pH 2.0. The remaining mixture was then extracted with 100 mL ethyl acetate for three times. After

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removal of the ethyl acetate by a rotary evaporator, the extract was resolubilized in 4 mL of methanol,

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filtered through a 0.22-µm nylon membrane, and stored at -20°C prior to analytical determinations. All the

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extraction experiments were carried out in triplicate.

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2.6 Determination of total phenolic content (TPC)

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The TPC of all samples were determined by the Folin–Ciocalteu method described by Singleton et al.

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(1998) and slightly modified by Adom and Liu (2002). Briefly, 50 µL of diluted sample was mixed with 50

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µL of Folin-Ciocalteu’s phenol reagent. After 6 min, 500 µL of Na2CO3 solution (7%, w/v) and 400 µL of

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deionized water were added. The reaction was performed in dark for 90 min and the TPC was measured by

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measuring the absorbance at 760 nm using a microplate reader (SH-1000, Corona Electric Co. Ltd,

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Lethbridge, Canada). A standard curve of gallic acid was prepared and the results were expressed in terms of

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gallic acid equivalent (mg GAE/100 g of dry matter).

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2.7 Determination of oxygen radical absorbance capacity (ORAC)

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ORAC was measured according to the protocols of Huang et al. (2002). Briefly, all the oat bran extracts

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were diluted 1000 times with phosphate buffer (75 mM, pH 7.4). The assay was conducted in 96-well

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standard (0, 6.25, 12.5, 25 and 50 µmol/L), and mixed with 200 µL of working fluorescein solution (0.96

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µM in phosphate buffer), which were incubated for 20 min at 37°C. Subsequently, 20 µL of AAPH (119 mM

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in phosphate buffer) was added to each 96-well. The fluorescence intensity (excitation/emission wavelength

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= 485/528 nm was measured using a Fluorescence microplate reader (Fluoroskan Ascent, Thermo Fisher

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Scientific, MA, USA) for 33 cycles every 2 min. The area under fluorescence curve (AUC) was calculated

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by KC4 3.0 software, which was then subtracted from the blank well to obtain the net AUC. A calibration

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curve was constructed by plotting the calculation differences of AUC and Trolox concentration (µM). This

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was used to obtain the ORAC values which were expressed as Trolox equivalent (µmol TE/g of dry sample).

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2.8 HPLC-DAD analyses

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The identification and quantification of the main phenolic compounds present in the CE and UAE

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extracts were conducted using an Agilent 1260 Series HPLC-DAD system (Agilent Technologies, Inc., Palo

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Alto, CA, USA) equipped with an automatic sampler, a quaternary pump, a column oven and a diode array

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detector (DAD). A Dionex Acclaim 120 C-18 analytical column (5 µm, 4.6 mm×150 mm) maintained at

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30°C was used. All samples and phenolic standards were dissolved in methanol and filtered through a

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0.22-µm nylon organic membrane prior to injection. Separation was performed through an optimized

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gradient program using 0.1% acetic acid (A) and acetonitrile (B), starting the sequence with 0% B and

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gradient program to obtain 17% B at 20 min, 25% B at 45 min, 60% B at 55 min, 0% B at 65 min. The flow

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rate was established at 0.5 mL/min and the injection volume was 10 µL. Chromatogram was monitored at

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280 nm.

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The separated phenolic compounds in the extracts were identified by their retention times with

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authentic standards. For the quantification, these standard compounds with known concentrations

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(3.125–100 µg/mL) were also used to obtain the external calibration curves. The results were expressed as

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mg/g dry weight (DW), including three parallel analyses.

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2.9 Mathematical models of free phenolics extraction kinetics A mathematical extraction kinetics model developed by Naik et al. (1989) was used to connect the TPC, ORAC, and total AVs of free phenolics:

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Y = Y∞· t / (B+ t)

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where Y represents the determined variables (TPC, ORAC or total AVs), Y ∞ the measured variable at

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equilibrium, B (min) the extraction time required to reach half of Y ∞, and t (min) the extraction time. The

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model parameters (B and Y∞) were determined using the Origin software version 8.0 (Origin Lab Co.,

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Northampton, MA, USA) by minimizing differences between the calculated and experimental Y. The

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performances of the models fitted to the actual values were statistically determined by coefficient of

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determination (R2) and mean relative percent deviation (MRPD), which were calculated using the following

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equations:

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where ya.i and yp.i are the actual and predicted values of the determined variables (TPC, ORAC or total AVs),

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respectively, y m is the mean value of determined variables, and n is the number of experimental runs.

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Recently, this model has also been used in many studies for ultrasound assisted extraction (Ahmad-Qasem et

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al., 2013; Khemakhem et al. 2017).

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2.10 Extraction of β-glucan

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Four oat bran residue samples after the ethanol extraction of free and esterified phenolic compounds for

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25 min were used to obtain the β-glucan (CE 20°C, UAE 20°C, CE 70°C, and UAE 70°C). Extraction of

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β-glucan was performed as reported by Asp et al. (1983). Oat bran meal was extracted in alkaline solution

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(pH = 10 with 0.1 M NaOH addition) for 1h at 70°C, then the starch was hydrolysed using heat-stable

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α-amylase in a 95°C water bath and the solution was acidified to remove proteins. After centrifugation, the

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residue was discarded and β-glucan was precipitated with aqueous 60% ethanol. Ethanol was slowly poured

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into solution with equilibrium and solution kept overnight at 4 °C. The supernatant was separated by

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centrifugation and β-glucan precipitate was collected freeze-dried.

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2.11 Determination of β-glucan content Oat bran β-glucan content in samples was determined using lichenase digestion (AOAC 995.16)

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method (Mccleary and Codd, 2010) with some modifications. This method allows high β-glucan content

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samples to be analyzed reliably. The samples were treated using isopropanol at room temperature overnight

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prior to analyses, then dried in a vacuum oven for 4 h at 80 °C. Then 0.2 mL of lichenase (10 U) and 8 mL

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of buffer were added prior to incubation. At the appropriate time, the solutions were diluted 2-4 times for

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treatment with β-glucosidase. Finally, the glucose produced was measured spectrophotometrically at 510

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nm.

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2.12 Statistical analyses

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The experimental data, expressed as mean ± SD, contained at least three replications per sample.

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Analyses of variance (ANOVA) and Tukey’s test were performed using SPSS statistics (version 22.0). The

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statistical differences was set at p = 0.05.

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3. Results and discussion

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3.1 Evolution of total phenolic content (TPC) during extraction

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TPC kinetic curves of free phenolics determined at different temperatures by CE and UAE are shown in

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Figs. 1A and Figs. 1B, respectively. Moreover, the fitting of mathematical model to experimental TPC

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values is also illustrated in Figs. 1, where experimental data are compared to the actual ones. The extraction

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kinetic curves were typically included a fast extraction step (first stage) and a slow extraction step (second

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stage). For the UAE method, more than 90% of TPC were extracted in first 5 min. After 10 min, the

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extraction rate reached a constant value close to equilibrium (Y∞), which indicated the process of extraction

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was mainly controlled by the first stage. The characteristics of first and second stages in the extraction can

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be determined by the radio of intact and broken cells after sample preparation (Chan et al., 2014). In

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ACCEPTED MANUSCRIPT addition, the calculated kinetic parameters of mathematical model are shown in Table 1. As shown, the

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coefficient of determination (R2) exceeded 0.977 and 0.996 for CE and UAE, respectively, and the mean

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relative percent deviation (MRPD) was lower than 6% and 3% for CE and UAE, respectively. These results

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indicated that the mathematical model fitted well to experimental results (Milić et al., 2013), especially for

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UAE.

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Moreover, as experimental data shown, the effect of temperature on extraction rate was significant

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(p=0.05). The TPC yields significantly increased when the temperature increased from 20 to 70 °C. For

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example, TPC yields measured at 70 °C were almost 2 times higher than that obtained at 20 °C for CE at 5

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min. High extraction temperature improved the diffusion and volatility coefficient for oat bran meal while

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enhancing the delivery of phenolics from oat bran cytoplasm. Additionally, the solvent viscosity decreased at

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higher temperatures, which may enhance the target phenolic compounds desorption from the cells, which

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eventually improves extraction efficiency (Yang and Wei, 2015). In addition, R0 and equilibrium contents

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(Y∞) for the TPC were significantly (p = 0.05) increased by improving the temperature from 20 to 70 °C.

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Therefore, the highest Y∞ was obtained using the temperature of 70 °C. It was interesting that the most

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significant difference was observed between 40 °C and 50 °C for both CE and UAE (p=0.05). It seems the

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temperature at 50 °C more likely to increase the solubilization and permeation to wash intracellular

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components out of oat bran cytoplasm. However, no significant differences in the extraction rates was

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observed between 70 °C and 90 °C for both CE and UAE (p=0.05). Similar results were also observed by

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Hemwimol et al. (2006) and Lou et al. (2010).

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Furthermore, the influence of both extraction method and temperature were described, at the lowest

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temperature (20 °C), there was no significant difference between CE and UAE for the first 1 min (p = 0.05).

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However, the TPC was almost 1.5 times higher in UAE than CE thereafter. As observed in Table 1 and Fig.

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1, the ultrasonic application created higher initial extraction rates (R0) in general terms. This fact could be

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due to the ultrasound influence on solubilization and release of polyphenol in the solvent, accelerating the

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temperature (70 °C). UAE extraction yields were just a little higher than that of CE at 25min. This result

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may be due to the reason that cavitational intensity typically reduces at higher temperatures beyond

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optimum and hence the intensification reduces. At lower temperature, the number of cavities generated is

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less but the intensity of their collapse is more, whereas at higher temperatures, higher vapor pressure causes

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formation of more number of cavities, but their collapse is less intense (Sutkar, 2009; Gogate, 2015).

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Figs. 1C and Figs. 1D showed the influence of extraction temperature on esterified phenolics (EP) and

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bound phenolics (BP) after free phenolics extraction at different temperatures and different time by CE (C)

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and UAE (D). A previous study indicated that esterified and bound phenolics predominated when analysing

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the TPC of different cereals, including oats (Adom and Liu, 2002). However, as shown in Figs. 1, the TPC

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of esterified and bound phenolics of defatted oat bran were much lower than free phenolics, especially for

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the esterified phenolics. This difference might be due to the degree of ripeness, oat varieties used, and

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environmental conditions (Xu and Chang, 2012). Moreover, the ultrasonic power, extraction temperature

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and time had no significantly effect on esterified phenolic contents (p=0.05). This result may be caused by

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the low contents of esterified phenolics in oat bran.

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As shown in Figs. 1C and Figs. 1D, the bound phenolic contents pre-treated by UAE were lower than

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that pre-treated by CE in most cases. For example, at the extraction temperatures of 40 °C, the TPC

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decreased from 45.62 ± 4.13 to 39.28 ± 1.42 mg GAE /100g with pretreatment of ultrasonics. During

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ultrasonic treatment, the bound phenolic contents was significantly affected by extraction time (p=0.05).

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When defatted oat bran powders were treated by UAE for 5, 15 and 25 min, the bound phenolic contents

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was significantly decreased with increasing treatment time (p=0.05). The decrease in the bound phenolics

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with ultrasonics indicated that the glycoside-bound phenolic acids could be cleaved by ultrasonic power and

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this might be attributed to the heating effect caused by cavitation influence. Furthermore, the bound phenolic

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yields significantly decreased when temperature increased from 20 to 70 °C for both CE and UAE. For

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ACCEPTED MANUSCRIPT instance, at 15 min of extraction, bound phenolic yields measured at 20 °C was almost 2 times higher than

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that obtained at 70 °C for UAE. Processing cereals with agitation heating and thermoplastic extrusion could

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release bound phenolic compounds due to the breaking of conjugated moieties. A previous study showed

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that high temperature cooking of yellow and white maize increased free ferulic acid and decreased bound

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ferulic acid forms in raw kernel (Mora-Rochin et al., 2010).

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3.2 Evolution of antioxidant capacity (ORAC) during extraction

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In this work, the effect of temperature and extraction methods on antioxidant capacity (ORAC) was

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also studied in the range of 20 to 90 °C, which are illustrated in Fig. 2. Moreover, the fitting of the

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mathematical model to experimental ORAC values of free phenolics and some kinetic parameters are shown

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in Figs. 2A, Figs. 2B, and Table 1. As shown, most of the results were similar to TPC assay. For example,

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the influence of temperature on antioxidant capacity was significant (p=0.05) from 20 to 70 °C for both CE

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and UAE. Nevertheless, there was a decrease of ORAC values as the temperature increased from 70 to

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90 °C, which was different from TPC assay. It seems that the highest temperature (90 °C) lead to an

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important degradation on other antioxidant ingredients. This result has already been observed in previous

288

literature, where there is negative effect of high temperature on antioxidant extraction processes (Zhang et

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al., 2009). Moreover, there was significant (p = 0.05) difference between CE and UAE at the lowest

290

temperature (20 °C) after 5 min, while the ORAC values in UAE were very close to that in CE at 70 °C.

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The parameters of mathematical model (Table 1) also confirmed the influence of temperature and

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extraction method on the kinetics of ORAC. The model used in this study fitted the ORAC extraction

293

kinetics well (MRPD < 10%), though not as well as TPC assay. The R0 for UAE experiments was almost

294

twice higher than the CE ones indicating the significant influence of ultrasound on antioxidant capacity.

295

Moreover, the differences among ORAC values identified at different temperatures were significant. For

296

example, the Y ∞ ranged from 23.91 at 20 °C to 38.27 at 70 °C for UAE.

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The antioxidant capacities of esterified and bound phenolics in oat bran were also evaluated by ORAC

ACCEPTED MANUSCRIPT assay. The ORAC assay showed a similar trend with TPC assay. For example, as shown in Fig. 2C and Fig.

299

2D, the UAE treatment and extraction temperature had no significantly effect on the antioxidant capacities

300

of esterified phenolics (p = 0.05). In addition, the antioxidant capacity of bound phenolics decreased with

301

increasing extraction temperature. For instance, at the extraction time of 10 min for UAE, the ORAC

302

activity of bound phenolics were decreased from 14.02 and 10.37 µmol/g when the extraction temperature

303

increased from 20 to 90 °C. Moreover, the ORAC values of bound phenolics pre-treated by UAE were lower

304

than that pre-treated by CE in most cases. This fact could be due to the ultrasound influence on release of

305

antioxidants in bound forms, accelerating the extraction process (Ahmad-Qasem et al., 2013).

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3.3 Identification and quantification of phenolic compounds by HPLC-DAD

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Free phenolics extracts obtained by UAE and CE at different temperatures (20 and 70 °C) and different

308

time (1, 5, 15 min) were analyzed using HPLC-DAD in order to identify and quantify the major phenolic

309

compounds in defatted oat bran (Table 2a). Fig. 3A presents the HPLC chromatograms of free phenolics of

310

defatted oat bran extracts obtained at 20 °C by UAE and CE for an extraction time of 25 min. The major

311

compounds identified in every defatted oat bran extract were p-hydroxybenzoic acid, caffeic acid,

312

protocatechuic acid, vanillic acid, gallic acid, ferulic acid, AV 2c, AV 2f, and AV 2p (Table 2a and Fig. 3A).

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The phenolic profile identified in this study was consistent with those found in previous researches, and AVs

314

were the most abundant phenolic compounds in all samples (Chen et al., 2015; Emmons and Peterson,

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2000).

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The sum of phenolic acids and AVs in UAE was higher than that in CE in all cases. Moreover, the

317

amount of phenolic acids and AVs extracted at 70 °C was generally higher compared to that extracted at

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20 °C. According to the ANOVA analysis, treating time, extraction methods and extraction temperature had

319

highly significant effect on the concentration of all tested phenolics at the level of p = 0.05. Additionally, the

320

degree of increase of different phenolic compounds content varied greatly. For example, after UAE

321

treatment for 15 min at 20 °C, the content of AV 2p increased from 73.07 to 121.55 µg/g, whereas that of

ACCEPTED MANUSCRIPT 322

protocatechuic acid only from 1.09 to 1.65 µg/g. This might due to the differences of total contents of

323

phenolic compounds in oats (Chen et al., 2015). Moreover, the contents of all the phenolic acids and AVs increased highly significantly (p = 0.05) after

325

treatment of high temperature (70 °C). After 1 min, the yields of p-hydroxybenzoic acid, vanillic acid,

326

protocatechuic acid, and AVs (2c, 2p, and 2f) at 70 °C were about 50%-100% higher than samples at 20 °C

327

for CE. Gallic acid and ferulic acid content significantly increased by approximately 200% and 300%,

328

respectively. After 5 min, the yields of ferulic acid, caffeic acid, and AVs (2c, 2p, and 2f) showed significant

329

increase additionally (p = 0.05) when compared to the low temperature extraction (20 °C) samples, but the

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increase of other phenolic compounds showed little additional change.

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The different patterns of extraction method showed that phenolic compounds studied in this research

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may interact with other components in distinct ways. For example, the contents of vanillic acid and

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protocatechuic acid were increased slightly by UAE but significantly by heating. However,

334

p-hydroxybenzoic acid, ferulic acid and AVs (2c, 2p, and 2f) were significantly released by either ultrasonic

335

extraction or high temperature treatment, indicating that they are combined to other components of oat

336

through a week bond that can be easily broken, such as encased by the network of starch granule (Chen et

337

al., 2015).

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Five phenolic acids, namely, gallic acid, caffeic acid, protocatechuic acid, p-coumaric acid and ferulic

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acid, were identified and quantified from esterified phenolic compound in oat bran (Table 2b). Gallic acid,

340

caffeic acid, p-coumaric acid and ferulic acid were also detected in esterified phenolics of fermented oats by

341

Bei et al. (2017). Moreover, the UAE treatment, extraction temperature and time had very little influence on

342

the esterified phenolic contents, which are consistent with the experimental results obtained by TPC and

343

ORAC assay. In addition, the bound phenolic compounds obtained by HPLC-DAD after free and esterified

344

phenolic compound extraction were also shown in Table 2c and Fig.3. As shown, most of hydroxycinnamic

345

acids in the oat bran (caffeic, p-coumaric, and ferulic acid) are present in bound forms. Specifically, the sum

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of caffeic, p-coumaric, and ferulic acid contents were 98.06±2.43 to 245.39±5.87 µg/g for the bound forms

347

while it is just 8.44 ± 0.15 to 24.66 ± 0.19 µg/g and 42.68 ± 3.42 to 62.60 ±5.01 for the free and esterified

348

forms, respectively. This result was consistent with Li et al. (2016). Moreover, the sum of the bound phenolic acid contents decreased significantly with high temperature

350

pretreatment. In addition, the content of individual phenolic acid also significantly decreased with high

351

temperature pretreatment, especially for p-coumaric acid and ferulic acid (Fig.3 and Table 2c). The effect of

352

UAE treatment was not as big as high temperature treatment on the decrease of bound phenolic acids. For

353

instance, after the UAE treatment for 15 min at 20 °C, the total phenolic acid contents decreased from

354

118.45 ± 2.08 µg/g to 110.64±5.64 µg/g compared to CE treatment. It is not difficult to comprehend that

355

ultrasonic treatment could accelerate the break of bound complexes since high ultrasonic power released

356

through implosion of acoustic cavitation bubble in the solvent medium, and a reaction of homogeneous

357

sonochemical reaction is usually partial in the cavitation region (Ahmad-Qasem et al., 2013). A previous

358

study revealed that alkaline hydrolysis might not necessarily release all bound phenolic compounds, and

359

using alkaline hydrolysis along with ultrasonication serves as an effective method in boosting the yield of

360

bound phenolics (Gonzales et al., 2014). Therefore, in this study, UAE pretreatment may lead to the release

361

of a certain amount of bound phenolics when compared to alkaline hydrolysis method alone.

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3.4 Extraction kinetics of AVs

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AVs were the most abundant phenolic compounds in oats and exhibited potent antioxidant activities in

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vitro and in vivo (Chen et al., 2007; Peterson, 2001). The antioxidant capacities of AVs was 10-30 times

365

higher than that of other phenolic compounds such as caffeic acid and vanillin (Dimberg et al., 1993). In

366

addition, AVs may interact with the cellular molecular and signaling pathways that control cellular responses

367

during inflammation. Sur et al. (2008) reported that at the concentrations as low as 1 ppb AVs could reduce

368

the release of proinflammatory cytokine, IL-8 and inhibited NF-kB activation in keratinocytes. Therefore, it

369

is important to determine the effect of extraction method (UAE and CE) and temperature (20, 40, 50, 70 and

ACCEPTED MANUSCRIPT 370

90 °C) on AVs extraction kinetics, which has not been reported previously as far as we are concerned. As shown in Table 1, the mathematical model fitted adequately to actual kinetics of AVs extraction for

372

both UAE and CE, since the R2 exceeded 0.975 and the MRPD was lower than 6%. Fig. 4 illustrate the good

373

fitness reached for the AVs kinetic. High temperatures might enhance the solubility and desorption of AVs,

374

and also intensified the rise of solvent diffusion into cells (Kaur et al., 2016). The effect of temperature was

375

more remarkable in the CE where a significant difference (p = 0.05) was found between 20 °C and 70 °C.

376

However, the Y∞ decreased when the extraction temperature went up to 90 °C, which was in accordance

377

with ORAC kinetics. Dimberg et al. (2008) reported that AVs, especially for AV 2c, diminished in a water

378

bath (95–98°C) at different PH conditions. As observed in Table 1 and Fig. 4, the UAE not only improved

379

the R0 and Y∞ of TPC and ORAC kinetics but also the AVs one. For example, compared with CE, UAE

380

reached Y∞ of 301.41 µg/g at 20°C while for CE it was only 217.24 µg/g. However, it only have a slightly

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influence on final AVs content (Y∞) when the temperature reached 70 °C.

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3.5 Extraction yield of β-glucan

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Four oat bran residue samples after ethanol extraction for 25 min were used to obtain the β-glucan (CE

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20°C, UAE 20°C, CE 70°C, and UAE 70°C) (data not shown). Results showed that the effect of ultrasonic

385

pretreatment on extraction yield of β-glucan was significant (p = 0.05). For example, in the UAE

386

pretreatment at 20°C, the extraction yield of β-glucan achieved was 5.73 %, which is an increase of 37% as

387

compared to CE. Whereas high temperature pretreatment showed no significant effect on extraction yield.

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For both CE and UAE, the extraction yield of β-glucan pretreated at 70°C was just slightly higher than that

389

pretreated at 20°C. This finding is in accordance with Wood et al. (1978).

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Among many approaches for the β-glucan extraction, UAE represents higher extraction efficiency since

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this method is an efficient, cost-effective, rapid, and simple technique which shortens the extraction time

392

when compared to conventional methods. Ultrasonic energy destroys cell wall and therefore, allows higher

393

penetration of water into cell walls of oat bran particles. Patist et al. (2008) and Bhaskaracharya et al. (2009)

ACCEPTED MANUSCRIPT believed that the generated energy from cavitation (air bubbles explosion) destructed the walls of cell and

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improved the release of cellular components such as polysaccharides. In a previous study, sonication time

396

had two opposite influence on the extraction yield. By increasing time to up to 5.5 min, β-glucan yield

397

increased significantly; while increasing the time to over 5.5 min, the negative effect on β-glucan yield was

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detected. (Sourki et al., 2017). These two different results indicated that a long-time ultrasonic treatment is

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more likely to disrupt the β-glucan chains.

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4. Conclusion

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Two extraction methods (CE and UAE) has been employed for better extraction of phenolic compounds

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and β-glucan from defatted oat bran. The results showed that UAE is more suitable to obtain a higher yield

403

of free phenolics with stronger antioxidant activities (ORAC) within shorter time as compared to CE method,

404

while the bound phenolic contents were decreased by UAE treatment. Moreover, the extraction kinetics of

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free phenolics in defatted oat bran were significantly improved by increasing extraction temperature whereas

406

the bound fractions were decreased. UAE improved the initial extraction rate (R0) but its effect on the

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evolution and the final values of free phenolic contents, antioxidant activities and AVs depended on the

408

temperature of extraction. Moreover, β-glucan yields pretreated in UAE were approximately 37% higher

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than that in CE at 20°C and 70°C.

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Acknowledgements Financial support for this research was provided by National Key R&D Program of China

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(2017YFD0401200 and 2017YFD0401100), Special Fund for Agro Scientific Research in the Public Interest

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(201303069), National Natural Science Foundation of China (NO. 31471616 and NO. 31501579),

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International S&T Cooperation Program of China (ISTCP), (2015DFA30540), and Postgraduate Research &

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Practice Innovation Program of Jiangsu Province (KYCX17_1409).

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ACCEPTED MANUSCRIPT Figure Captions

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Fig.1. Influence of extraction temperature on the total phenolic content (TPC) of free phenolics of defatted

520

oat bran obtained by CE (A) and UAE (B); The TPC of esterified phenolics (EP) and bound phenolics (BP)

521

after free phenolic compound extraction at different temperatures and different time by CE (C) and UAE

522

(D).

523

Fig.2. Influence of extraction temperature on the antioxidant capacity (ORAC) of free phenolics of defatted

524

oat bran obtained by CE (A) and UAE (B); The ORAC of esterified phenolics (EP) and bound phenolics (BP)

525

after free and esterified phenolic compound extraction at different temperatures and different time by CE (C)

526

and UAE (D).

527

Fig.3. Chromatograms at 280 nm of defatted oat bran extracts obtained at 20°C with an extraction time of

528

25min from HPLC-DAD. UAE (dotted line), CE (full line) (A); Chromatograms at 280 nm of bound

529

phenolic compounds obtained after free and esterified phenolic compounds extraction (25min) at 20°C

530

(dotted line) and 70°C (full line) (B).

531

Fig.4. Influence of extraction temperature on the total AVs of defatted oat bran obtained by CE (A) and UAE

532

(B), and comparison between CE and UAE performed at 20 °C and 70 °C (C).

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ACCEPTED MANUSCRIPT 534

Conflict of interest

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The authors declare no competing financial interest.

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A

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Fig.1. Influence of extraction temperature on the total phenolic content (TPC) of free phenolics of defatted

540

oat bran obtained by CE (A) and UAE (B); The TPC of esterified phenolics (EP) and bound phenolics (BP)

541

after free phenolic compound extraction at different temperatures and different time by CE (C) and UAE

542

(D).

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Fig.2. Influence of extraction temperature on the antioxidant capacity (ORAC) of free phenolics of defatted

547

oat bran obtained by CE (A) and UAE (B); The ORAC of esterified phenolics (EP) and bound phenolics (BP)

548

after free and esterified phenolic compound extraction at different temperatures and different time by CE (C)

549

and UAE (D).

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B

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Fig.3. Chromatograms at 280 nm of defatted oat bran extracts obtained at 20°C with an extraction time of

554

25min from HPLC-DAD. UAE (dotted line), CE (full line) (A); Chromatograms at 280 nm of bound

555

phenolic compounds obtained after free and esterified phenolic compounds extraction (25min) at 20°C

556

(dotted line) and 70°C (full line) (B).

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Fig.4. Influence of extraction temperature on the total AVs of defatted oat bran obtained by CE (A) and UAE

560

(B).

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ACCEPTED Table 1. Identified parameters of mathematical modelMANUSCRIPT for total phenolic content (TPC), antioxidant capacity (ORAC), and total avenanthramides (AVs) contents of free phenolics from defatted oat bran. Temperature

Total phenolic content (TPC)

(°C)

CE a

Y∞

R0

UAE

b

R2

MRPD (%)

Y∞

R0

R2

MRPD (%)

20°C

85.14

97.23

0.977

5.38

124.34

106.41

0.998

1.33

40°C

99.36

99.76

0.988

3.74

136.81

159.10

0.996

1.54

50°C

150.43

137.72

0.999

0.46

171.61

165.76

0.997

2.14

70°C

168.32

184.50

0.998

1.63

184.16

228.80

0.998

1.54

90°C

164.15

189.21

0.999

0.40

182.58

210.23

0.998

1.51

22.85

0.969

6.57

28.51

0.987

3.80

35.48

43.84

0.986

3.92

38.27

48.20

0.991

2.71

35.96

63.68

0.983

3.91

Antioxidant capacity (ORAC) 22.39

11.31

0.942

9.98

23.91

40°C

24.40

18.19

0.961

7.26

29.38

50°C

30.74

26.69

0.984

4.63

70°C

36.30

27.93

0.986

4.40

90°C

33.24

34.01

0.980

4.91

SC

20°C

RI PT

563 564

20°C

217.24

271.25

0.981

40°C

305.09

384.59

0.975

50°C

354.41

494.72

0.994

70°C

414.32

484.97

0.987

90°C

365.22

619.96

0.995

a

M AN U

Total avenanthramides (AVs) contents 5.12

301.41

359.48

0.999

0.72

5.50

325.52

391.14

0.989

5.07

2.55

369.46

525.02

0.996

2.20

3.42

444.67

573.41

0.997

2.18

2.24

389.46

594.14

0.999

1.33

EP AC C

565

TE D

The unit of Y∞ were expressed as mg/100g dm, µmol /g dm, and µg/g dm for TPC, ORAC, and total AVs, respectively . b The unit of R0 were expressed as mg/100g min, µmol /g min, and µg/g min for TPC, ORAC, and total AVs, respectively.

ACCEPTED MANUSCRIPT

566

Table 2a. Free phenolic compound contents obtained by HPLC-DAD at different temperatures (20 and

567

70 °C) and different time (1, 5, 15 min) from UAE and CE. Free phenolic

Temperature

compounda

(℃)

Extraction time (min) CE 1.00

Protocatechuic acid

Ferulic acid

Av 2c

Av 2p

Av 2f

Sum phenolic acids

Sum AVs

b

568

15.00

17.07±1.02ab

21.89±1.84c

14.34±0.52a

19.20±0.59bc

27.94±2.39d

70.00

42.19±2.24a

59.13±4.56b

65.34±1.67bc

47.21±3.74a

61.04±1.40bc

67.42±2.39c

20.00

9.21±0.64a

19.14±1.17b

21.96±0.74b

10.81±0.98a

28.00±1.46c

31.56±1.78d

70.00

14.31±0.56a

35.21±1.53b

40.65±1.90c

13.21±0.74a

38.01±1.08bc

40.81±1.09c

20.00

10.78±0.89a

18.59±0.35b

27.03±1.08c

13.64±0.41a

21.93±2.01b

32.14±1.71d

70.00

20.31±1.36a

32.13±2.57b

40.72±2.53c

19.31±1.16a

33.21±1.53b

39.43±2.01c

20.00

6.04±0.25b

4.52±0.07a

6.27±0.17b

6.35±0.04b

5.86±0.33b

8.08±0.01c

70.00

7.03±0.57b

8.31±0.44c

10.79±0.68d

6.01±0.58a

6.45±0.15ab

10.24±0.60d

20.00

1.02±0.04a

1.06±0.02ab

1.09±0.08ab

1.13±0.05ab

1.16±0.05b

1.65±0.06c

70.00

2.08±0.11a

2.35±0.07b

2.95±0.02c

2.01±0.03a

2.45±0.15b

3.25±0.12d

20.00

1.41±0.11a

1.43±0.07a

1.77±0.02ab

1.60±0.03ab

1.93±0.15b

4.20±0.12c

70.00

5.03±0.26a

7.56±0.34b

10.10±0.64c

7.42±0.24b

10.41±0.79c

14.32±0.49d

20.00

55.56±1.13a

59.00±3.09a

65.30±3.46b

56.69±5.62a

67.03±4.08b

76.58±1.49c

70.00

77.00±5.25a

107.00±6.00b

132.43±4.67c

79.00±1.32a

128.00±10.03c 143.43±8.79c

20.00

59.24±2.84a

69.95±6.08ab

73.07±5.36c

61.26±3.35ab

111.70±3.77d

70.00

95.02±4.43a

131.00±2.46b 150.57±11.46cd 99.00±6.79a

20.00

46.74±0.35a

51.01±3.25a

61.84±1.64b

47.55±3.44a

76.59±7.22c

70.00

74.00±2.36a

103.00±7.32b

131.70±3.96d

78.00±4.83a

124.00±3.74c 142.70±11.52e

20.00

42.25±2.53a

61.84±2.83c

80.03±5.27d

47.87±2.80b

78.09±1.37d

70.00

90.95±5.72a

144.69±2.27b 170.54±12.25cd 95.17±2.39a

RI PT

13.77±0.31a

165.5±7.87b

121.55±7.70d

142.00±4.88bc 161.57±12.43d 86.83±3.42d

105.59±8.89e

151. 57±7.38bc 175.47±8.02d

20.00

161.54±13.29a 179.96±7.93ab 200.21±14.88b

70.00

246.35±17.84a 341.03±23.52b 414.73±15.69c 256.55±23.69a 394.83±16.83c 448.84±25.07d

All compounds were quantified using authentic standards. Values with different letters within a row are significantly different (p < 0.05).

AC C

a

5.00

SC

Caffeic acid

1.00

M AN U

Vanillic acid

15.00

TE D

p-Hydroxybenzoic acid

5.00

20.00

EP

Gallic acid

b

UVE

255.32±9.00c 284.96±16.73d

ACCEPTED MANUSCRIPT

569

Table 2b. Esterified phenolic compound contents obtained by HPLC-DAD at different temperatures (20 and

570

70 °C) and different time (1, 5, 15 min) from UAE and CE. Extraction time (min)

(℃)

CE 1.00

Caffeic acid

Protocatechuic acid

p-Coumaric acid

Ferulic acid

Sum phenolic acids

a b

10.00

ND b

5.00

15.00

1.00

5.00

15.00

ND

0.42±0.02b

ND

ND

1.32±0.87c

1.89±0.78bc

2.04±0.18d

70.00

1.35±0.05a

1.93±1.24c

1.79±1.04b

1.46±0.89a

10.00

23.34±1.14a

23.42±1.03a

28.06±1.97c

25.12±1.78b

33.83±2.32e

31.75±2.08d

70.00

27.42±1.45b

28.45±2.42c

30.21±1.05d

26.29±2.43a

32.41±0.45e

34.66±2.42f

10.00

ND

1.93±0.04b

2.32±0.13c

0.43±0.02a

1.88±0.98b

2.68±0.05d

70.00

1.23±0.09b

2.46±0.14c

2.74±0.23d

1.04±0.07a

2.87±0.21de

3.01±0.12e

20.00

ND

ND

ND

ND

ND

0.94±0.05a

70.00

0.92±0.08a

1.69±0.04c

1.92±0.13d

1.35±0.15b

1.9±0.08de

2.31±0.17e

10.00

19.34±0.08a

22.34±0.23b

24.53±0.14c

20.41±0.15a

24.29±0.18c

24.52±0.14c

70.00

22.31±1.43ab 23.87±0.94abc 24.04±2.69abc 22.13±0.43a

24.51±1.34bc

25.63±2.42c

10.00

42.68±3.42a

47.69±2.54a

55.33±4.35b

45.96±3.09a

60.28±4.78bc

61.21±1.43c

70.00

53.23±3.44ab

58.4±2.94bc

60.7±1.45c

52.27±3.24a

63.58±4.87c

67.65±5.49d

RI PT

Gallic acid

UVE

SC

compound

Temperature a

M AN U

Phenolic

All compounds were quantified using authentic standards. Values with different letters within a row are significantly different (p < 0.05).

AC C

EP

TE D

571

ACCEPTED MANUSCRIPT

572

Table 2c. Bound phenolic compound contents obtained by HPLC-DAD after free and esterified phenolic

573

compound extraction at different temperatures (20 and 70 °C) and different time (1, 5, 15 min) from UAE

574

and CE. Temperature

Extraction time (min)

(℃)

CE 1.00

Caffeic acid

p-Coumaric acid

Ferulic acid

Sum phenolic acids

UVE

5.00

15.00

1.00

5.00

15.00

20.00

b

18.48±1.20c

15.67±0.68b

15.21±1.03b

13.71±0.93ab

14.53±1.12b

11.04±1.01a

70.00

14.35±1.17c

13.42±0.99c

13.04±1.03bc

10.34±0.79ab

9.98±0.53a

9.15±0.32a

20.00

90.13±5.43b

85.54±4.86ab

86.42±3.88ab

88.5±2.04ab

81.21±1.83a

80.43±4.95a

70.00

43.51±3.08b

40.52±4.87ab

38.52±2.94ab

41.53±1.83ab

39.53±1.09ab

37.03±2.43a

20.00

136.78±5.44c 121.24±1.45ab

118.45±2.08ab

125.94±7.43bc

123.24±3.75b

110.64±5.64a

70.00

61.24±2.45c

58.32±1.74b

57.13±3.87b

52.07±2.66a

51.88±1.68a

20.00

245.39±5.87d 222.45±4.35bc 220.08±10.45bc

228.15±2.09c

218.98±3.48b

202.11±4.98a

70.00

119.1±3.88d

109±2.87bc

101.58±0.89ab

98.06±2.43a

57.93±3.89b

111.87±3.68cd

RI PT

compound

a

SC

Bound phenolic

109.88±2.84bc

All compounds were quantified using authentic standards.

b

Values with different letters within a row are significantly different (p < 0.05).

AC C

EP

TE D

M AN U

a