Extraction optimization, characterization and antioxidant activity in vitro of polysaccharides from mulberry (Morus alba L.) leaves

Accepted Manuscript Title: Extraction optimization, characterization and antioxidant activity in vitro of polysaccharides from mulberry (Morus alba L.) leaves Author: Qingxia Yuan Yufeng Xie Wei Wang Yuhua Yan Hong Ye Saqib Jabbar Xiaoxiong Zeng PII: DOI: Reference:

S0144-8617(15)00337-9 http://dx.doi.org/doi:10.1016/j.carbpol.2015.04.028 CARP 9859

To appear in: Received date: Revised date: Accepted date:

10-2-2015 3-4-2015 7-4-2015

Please cite this article as: Yuan, Q., Xie, Y., Wang, W., Yan, Y., Ye, H., Jabbar, S., and Zeng, X.,Extraction optimization, characterization and antioxidant activity in vitro of polysaccharides from mulberry (Morus alba L.) leaves, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.04.028 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.

Extraction optimization, characterization and antioxidant activity in vitro of

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polysaccharides from mulberry (Morus alba L.) leaves

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Qingxia Yuana, Yufeng Xiea, Wei Wangb, Yuhua Yana, Hong Yea, Saqib Jabbara,c,

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Xiaoxiong Zenga,*

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a

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210095, Jiangsu, China

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b

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Agricultural University, Nanjing 210095, Jiangsu, China

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b

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an

College of Food Science and Technology, Nanjing Agricultural University, Nanjing

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College of Life Sciences and Laboratory Center of Life Sciences, Nanjing

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Institute of Food Science and Nutrition, University of Sargodha, Sargodha, Pakistan



Corresponding author: Fax: +86 25 84396791; E-mail address: [email protected] (X. Zeng). 1

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Abstract

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Extraction optimization, characterization and antioxidant activity in vitro of

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polysaccharides from mulberry leaves (MLP) were investigated in the present study.

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The optimal extraction conditions with an extraction yield of 10.0 ± 0.5% for MLP

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were determined as follows: extraction temperature 92 oC, extraction time 3.5 h and

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ratio (v/w, mL/g) of extraction solvent (water) to raw material 34. Two purified

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fractions, MLP-3a and MLP-3b with molecular weights of 80.99 and 3.64 KDa

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respectively, were obtained from crude MLP by chromatography of DEAE-Cellulose

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52 and Sephadex G-100. Fourier transform-infrared spectroscopy revealed that crude

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MLP, MLP-3a and MLP-3b were acidic polysaccharides. Furthermore, crude MLP

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and MLP-3a had more complicated monosaccharide compositions, while MLP-3b had

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a relatively higher content of uronic acid. Crude MLP, MLP-3a and MLP-3b exhibited

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potent

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1,1-diphenyl-2-picrylhydrazyl,

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Fe2+

chelating

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power

and hydroxyl,

scavenging

activities

superoxide

on and

2,2'-azinobis-(3-ethyl-benzothiazolin-6-sulfonic acid) radicals. The results suggested that MLP could be explored as natural antioxidant. Keywords: Mulberry leaf; Polysaccharide; Extraction; Characterization; Antioxidant

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1. Introduction Mulberry (Morus alba L.), a multipurpose agro-forestry plant that belongs to the

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family of Moraceae, is widely distributed in tropical, subtropical and temperate areas

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(Agarwal & Kanwar, 2007). It is commonly used as a silkworm (Bombyx mori L.) diet,

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alternative medicine in China and Japan and a kind of tea due to its low toxicity and

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good therapeutic performance (Chung, Kim, Kim, & Kwon, 2013; Jia, Tang, & Wu,

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1999; Nookabkaew, Rangkadilok, & Satayavivad, 2006; Wang, Fang, Ma, & Zhang,

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2014; Zhong, Furne, & Levitt, 2006). It has been reported that mulberry contains a lot

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of bioactive compounds including polysaccharides, 1-deoxynojirimycin, moracin,

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chlorogenic acid, rutin, flavonol glycosides and anthocyanins, which are associated

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with its biological functions such as anti-obesity, anti-diabetes, anti-oxidation,

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anti-inflammation and anti-atherosclerosis (Harauma et al. 2007; Hunyadi, Martins,

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Hsieh, Seres, & Zupkó, 2012; Katsube et al., 2006; Katsube, Tsurunaga, Sugiyama,

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Furuno, & Yamasaki, 2009; Kimura, Nakagawa, Kubota, Kojima, & Goto, 2007; Li et

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cr

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al., 2011; Peng et al., 2011; Yang, Wang, Wang, & Zhang, 2012; Yatsunami, Ichida, & Onodera, 2008; Zhang et al., 2014a). Mulberry leaves polysaccharides (MLP), the main active components of mulberry

leaves, have been attracted increasing attention as other herb polysaccharides, due to

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their multiple biological activities such as anti-diabetic, anti-tumor, anti-inflammatory

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and immunostimulatory effects (Li, Chen, Wang, Tian, & Zhang, 2010; Li et al., 2011;

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Wang, Li, & Jiang, 2010; Yan, Wang, & Wu, 2014; Yang, Zhao, Yang, & Ruan, 2008;

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Yang et al., 2012; Zhang et al., 2010, 2014a). Besides, it has been reported that many

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polysaccharides including MLP have potential antioxidant activities (Scartezzini &

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Speroni, 2000; Wang et al, 2013). However, the reports on the antioxidant activity of

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MLP are relatively insufficient (Samavati & Yarmand, 2013; Wang, Li, & Jiang,

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2010). It is well known that the biological functions including antioxidant activity of

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polysaccharides are intimately related to their structure features such as chemical

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components, molecular weight, monosaccharide composition and glycosidic linkage

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(You et al., 2013; Zeng, Zhang, & Jia, 2014). Accordingly, a comprehensive study of

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purification, characterization and antioxidant activities of MLP will provide useful

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information on the relationship between antioxidant activity and structure features.

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Furthermore, in order to study and explore MLP better, an efficient technique for the

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extraction of MLP is necessary. For polysaccharide extraction, hot water extraction

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technology is still the main and classic method due to its convenience, low cost and

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high extraction yield (Passos & Coimbra, 2013; Wei et al., 2010). However, the

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existing water extraction method for MLP needs long extraction time and several

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extraction cycles (Samavati & Yarmand, 2013). Therefore, we report here the extraction, optimization, characterization and antioxidant activity in vitro of MLP. Firstly, response surface methodology (RSM) based on a Box-Behnken design (BBD) was applied to optimize the extraction conditions, and the resulting extraction

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conditions were used to prepare crude MLP through water extraction and ethanol

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precipitation. The crude MLP was then purified by ion-exchange chromatography of

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DEAE-52 cellulose and size exclusion chromatography of Sephadex G-100, and the

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crude MLP and its purified fractions were further characterized via chemical analysis,

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high performance liquid chromatography (HPLC) and Fourier transform-infrared

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(FT-IR) spectroscopy. Finally, the antioxidant activities in vitro of crude MLP and its

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purified fractions were investigated. To the best of our knowledge, it is the first report

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on the antioxidant activities of the purified fractions of MLP.

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2. Materials and methods

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2.1. Materials and chemicals

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The mulberry leaves were collected from the mulberry plantation in Bozhou

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(Anhui, China), washed with top water, air dried at room temperature and ground into

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fine powder. DEAE-52 cellulose, Sephadex G-100, mannose (Man), arabinose (Ara),

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galactose (Gal), galacturonic acid (GalA), glucose (Glc), glucuronic acid (GlcA),

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ribose (Rib), xylose (Xyl), 3-methyl-1-phenyl-2-pyrazolin-5-one (PMP), nitroblue

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tetrazolium (NBT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), phenazine methosulphate

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(PMS),

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nicotinamide

adenine

dinucleotide

(NADH),

ferrozine,

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[2,2'-azinobis-(3- ethyl-benzothiazolin-6-sulfonic acid)] diammonium salt (ABTS) and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rhamnose (Rha) and fucose (Fuc) were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). All other chemicals used were of analytical grade.

2.2. Extraction of polysaccharides

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In order to defat and remove most of the monosaccharides, oligosaccharides,

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pigments and other small molecules, the powder of mulberry leaves was treated with

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85% ethanol twice at room temperature for 24 h. The resulting residue was dried and 5

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used for next extraction. The dried pretreated sample was extracted with designed

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extraction temperature, extraction time and ratio of extraction solvent (deionized

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water) to raw material. The resulting solution was filtered, mixed with a triple volume

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of absolute ethanol and kept overnight. The precipitates were collected by

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centrifugation at 4000 rpm for 15 min, washed with absolute ethanol and acetone and

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dried, affording the crude MLP. The extraction yield was calculated according to the

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following formula:

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W1  100 W0

Extraction yield (%) 

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where W1 and W0 are the weights of crude MLP and pretreated sample respectively.

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2.3. Experimental design of RSM

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Effects of extraction parameters including extraction temperature, extraction time,

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extraction cycles and ratio of water to raw material on the yields of MLP were

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investigated by single-factor tests (data not shown). Accordingly, three major factors

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(extraction temperature, extraction time and ratio of water to raw material) were chosen and their proper ranges were determined based on the preliminary experimental results. Furthermore, a three-level, three-variable BBD was applied to determine the optimal levels of extraction variables including the extraction temperature (X1), extraction time (X2) and ratio of water to raw material (X3) for the

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extraction of MLP. For statistical calculation, the variables were coded according to

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the equation:

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Xi 

i  0  i

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where Xi is the coded value of independent variable, χi is the actual value of the

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independent variable, χ0 is the actual value of the independent variable at the central

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point, and Δχi is the step change of the variable. Table 1 shows the range of

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independent variables and their levels.

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The whole design consisted of 17 experimental runs, including 12 factorial points

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and 5 axial points. The 5 axial points were used to allow for estimation of a pure error

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sum of squares. The experiments were carried out in random order, and the

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experimental data (Table 1) were fitted to the following second-order polynomial

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mode

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Y   0   i X i    i i X i2  

3

i 1

i 1

2

3



i 1 j  i 1

Xi X j

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ij

where Y, extraction yield of MLP, is the predicted response; β0, βi, βii and βij are the

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regression coefficients for intercept, linear, quadratic and interaction terms,

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respectively; Xi and Xj are the independent variables (i ≠ j).

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2.4. Purification of crude MLP

Crude MLP was dissolved in deionized water and loaded onto a DEAE-52

cellulose column (2.6 × 50 cm). Then, the column was stepwise eluted with 0, 0.1, 0.3 and 0.5 M sodium chloride (NaCl) solution at a flow rate of 60 mL/h. Three completely separated fractions, MLP-1, MLP-2 and MLP-3, were collected by

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checking the absorbance at 490 nm by using the phenol-sulphuric acid method

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(Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). As the main fraction, MLP-3 was

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loaded onto a column (1.6 × 100 cm) of Sephadex G-100 and the column was eluted

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with 0.2 M NaCl solution at a flow rate of 15 mL/h. The elution was checked as 7

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described above. As a result, two purified fractions were collected, dialyzed and

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lyophilized, affording MLP-3a and MLP-3b, respectively.

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2.5. Characterization of MLP

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2.5.1. Determination of contents of carbohydrate, protein, uronic acid, sulfuric

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radical and total polyphenols

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The contents of carbohydrate in crude MLP and its purified fractions were

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determined by the phenol-sulphuric acid method (Dubois et al., 1956) using glucose

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as the standard. The content of protein was determined according to the reported

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method (Bradford, 1976) using bovine serum albumin as the standard. The content of

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uronic acid was determined according to the method of Blumenkrantz and

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Asboe-Hansen (1973) using galacturonic acid as the standard. The content of sulfate

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radical was determined according to the reported method (Doigson & Price, 1962).

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The content of total polyphenols was estimated by the Folin-Ciocalteu colorimetric

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method (Li, Nie, Xie, & Li, 2014) using gallic acid (GA) as the standard, and it was

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expressed as mg GA equivalent (GAE) per 100 mg dry sample. 2.5.2. Determination of homogeneity and molecular weight The homogeneity and molecular weight of MLP-3a and MLP-3b were determined

by high-performance gel permeation chromatography (HPGPC) using an Agilent 1200

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series (Agilent Technologies, Santa Clara, CA, USA) apparatus equipped with a

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Shodex OH-pak SB-804 HQ column (8 × 300 mm). The column was eluted with 0.2

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M NaCl solution at a flow rate of 0.5 mL/min. The HPLC system was pre-calibrated

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with D-series dextran standards (D-2, D-3, D-4, D-5, D-6, D-7 and D-8).

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2.5.3. Analysis of monosaccharide composition The monosaccharide composition of MLP was analyzed according to the reported

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method (Dai et al., 2010) with some modifications. The polysaccharide solution (100

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L, 5 mg/mL) was hydrolyzed with 100 L 4 M trifluoroacetic acid (TFA) at 120 oC

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for 2 h. After hydrolysis, the excess TFA was removed by the addition of methanol

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and evaporated at reduced pressure. The hydrolysate was dissolved with deionized

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water (100 L) and mixed with 100 L 0.6 M NaOH. Then, 100 L of the resulting

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mixture was labeled with PMP by adding 100 L of 0.5 M methanol solution of PMP

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and incubating at 70 oC for 100 min. After being cooled to room temperature, the

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resultant solution was neutralized by adding 50 L of 0.3 M HCl and evaporated to

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dryness. The residue was dissolved in 1.0 mL deionized water, and the excess PMP

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was leached with chloroform for three times. The aqueous layer was filtered through a

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0.45 m membrane and analyzed by an Agilent 1100 HPLC system equipped with

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photodiode array detector. The chromatographic conditions were as follows: column,

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Eclipse Plus C18 (4.6 × 250 mm, 5 m, Agilent); column temperature, 30 oC; mobile phase, a mixture of phosphate buffered saline (PBS, 0.1 M, pH 6.7) and acetonitrile in a ratio of 83: 17 (v/v); flow rate, 1.0 mL/min; detector wavelength, 245 nm. The injection volume was 20 L. In similar manner, the monosaccharide standards were

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PMP-labeled and analyzed by HPLC.

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2.5.4. FT-IR spectrometric analysis

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The

dried crude

MLP and

its

purified fractions

were mixed

with

spectroscopic-grade potassium bromide powder, ground and pressed into pellets for

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FT-IR measurement. FT-IR spectra were recorded with a Nicolet 6700 FT-IR

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spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) at the frequency

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range of 4000-400 cm−1.

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2.6. Assay of antioxidant activity in vitro of MLP

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2.6.1. Assay of DPPH radical scavenging activity

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The DPPH radical scavenging activity was measured by the method of previous

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report (Shimada, Fujikawa, Yahara, & Nakamura, 1992) with slight modifications.

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Briefly, MLP was dissolved in deionized water to afford a series of concentrations

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(0.0625, 0.125, 0.25, 0.5, 1.0, 2.0, 3.0 and 4.0 mg/mL). Then, 50 L of sample

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solution, 25 L of DPPH-ethanol solution (0.4 mM) and 100 L of deionized water

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were mixed in a 96-well plate. The mixture was kept at room temperature in the dark

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for 30 min, and the absorbance (Abs) at 517 nm was measured by a microplate reader

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(BioTek Instruments Inc., Winooski, VT, USA). Ascorbic acid was used as positive

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control. DPPH radical scavenging activity was calculated by the following formula:

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DPPH radical scavenging activity (%) = [1 − (Abs1 − Abs2)/Abs0] × 100 where Abs0 is the Abs of the control (deionized water instead of sample), Abs1 is the Abs of the sample, and Abs2 is the Abs of the sample under identical conditions as Abs1 with ethanol instead of DPPH-ethanol solution. 2.6.2. Assay of hydroxyl radical scavenging activity

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The hydroxyl radical scavenging activity of MLP was determined according to the

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reported method (Liu, Luo, Ye, & Zeng, 2012). Briefly, the reaction mixture contained

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50 L ferrosin (0.75 mM), 75 L phosphate buffer (0.15 M, pH 7.4), 50 L FeSO4

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(0.75 mM), 50 L sample solution and 50 L H2O2 (0.01%, w/v). After incubation at

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37 oC for 30 min, the Abs at 536 nm was measured. Ascorbic acid was used as

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positive control. The scavenging activity was calculated using the following equation:

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Hydroxyl radical scavenging activity (%) = [(Abs2 − Abs0) − (Abs1 − Abs0)] × 100

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where Abs0 is the Abs of the control (deionized water instead of sample), Abs1 is the

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Abs of the deionized water instead of H2O2 and sample, and Abs2 is the Abs of the

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sample.

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2.6.3. Assay of superoxide radical scavenging activity

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The scavenging ability on superoxide radical was measured by the previously

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described method (Bi et al., 2013; Robak & Gryglewski, 1988) with minor

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modifications. The reaction mixture contained 50 L of sample solution, 50 L of

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NBT solution (156 M), 50 L of NADH solution (156 M) and 50 L of PMS

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solution (60 M). The mixture was incubated at 25 oC for 5 min, and the Abs at 560

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nm was determined. Ascorbic acid was used as positive control. The superoxide

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radical scavenging activity was calculated according to the formula below: Superoxide radical scavenging activity (%) = [1 − (Abs1 − Abs2)/Abs0] × 100 where Abs0 is the Abs of the control (deionized water instead of sample), Abs1 is the Abs of the sample, and Abs2 is the Abs of the sample under identical conditions as

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Abs1 with 0.1 M phosphate buffer instead of NBT solution.

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2.6.4. Assay of ABTS radical scavenging activity

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The ABTS radical scavenging activity of MLP was measured by ABTS radical

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cation decolorization assay (Re et al., 1999; Xie, Hu, Wang, & Zeng, 2014). Briefly,

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ABTS solution (7 mM) was oxidized with 4.95 mM of potassium persulphate for 12 h

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in the dark at room temperature. The ABTS+ solution was then diluted with PBS (0.2

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M, pH 7.4) to an Abs of 0.70 ± 0.02 at 734 nm, and 200 µL of the resulting ABTS+

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solution was mixed with 20 L of sample solution. The mixture was kept for 6 min at

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room temperature, and the Abs at 734 nm was then measured. The ABTS radical

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scavenging effect was calculated as follows:

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ABTS radical scavenging activity (%) = [1 − (Abs1 − Abs2)]/Abs0 × 100

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where Abs0 is the Abs of the control (deionized water instead of sample), Abs1 is the

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Abs of the sample, and Abs2 is the Abs of the sample only (PBS instead of ABTS+

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solution).

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2.6.5. Assay of Fe2+ chelating activity

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The Fe2+ chelating ability of MLP was measured according to the reported method

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(Decker & Welch, 1990). Briefly, 50 L sample solution was mixed with 2.5 L of

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ferrous chloride (FeCl2) solution (5.0 mM), 10 L of ferrozine solution (5 mM) and

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137 L of deionized water. The mixture was incubated for 10 min at room temperature, and then the Abs at 562 nm was measured. EDTA was used as positive control. The Fe2+ chelating activity was calculated by the following equation: Fe2+ chelating activity (%) = [1 − (Abs1 − Abs2)/Abs0] × 100

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where Abs0 is the Abs of the control (deionized water instead of sample), Abs1 is the

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Abs of the sample, and Abs2 is the Abs of the sample only (deionized water instead of

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FeCl2 solution).

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2.7. Statistical analysis

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The Design-Expert software version 8.0.6 was used for the experimental design

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and data analysis of RSM. The results of the antioxidant assay are reported as mean ±

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SD of three replicates. The data were statistically analyzed by one-way analysis of

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variance (ANOVA) procedure with SPSS software version 19.0 (Chicago, IL, USA),

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followed by the Duncan test. P-value of less than 0.05 was regarded as significant.

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3. Results and discussion

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3.1. Optimization of extraction parameters

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3.1.1. Predicted model and statistical analysis

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The experimental design along with the extraction yields is shown in Table 1, and

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the data were analyzed by Design-Expert software. As a result, a second-order

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polynomial equation describing the correlation between the MLP yield and the test

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variables was obtained as follows:

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Y = −454.60135 + 10.05870X1 + 4.80828X2 − 0.17728X3 − 0.02612X1X2 +

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0.00683X1X3 + 0.01416X2X3 − 0.05583X12 − 0.43506X22 − 0.00718X32 where Y represents the extraction yield of MLP, X1, X2 and X3 represent extraction temperature (℃), extraction time (h) and ratio (v/w, mL/g) of water to raw material, respectively.

The ANOVA, lack-of-fit and the adequacy of the model are indicated in Table 2.

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The model F-value of 82.47 implied that the model was significant, indicating that

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there was only a 0.01% chance that the model F-value could occur due to noise. The

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determination coefficient (R2) and the adjusted determination coefficient (adj-R2)

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were 0.9907 and 0.9786, respectively, which showed a good agreement between the 13

Page 13 of 46

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experimental and the predicted values of the MLP yield with goodness-of-fit of the

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regression equation. The P-value is used as a tool to check the significance of each coefficient, which

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in turn may indicate the pattern of the interactions between the variables. The smaller

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the P-value is, the more significant the corresponding coefficient is. Table 2 shows

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that the linear coefficients (X1, X2 and X3), quadratic term coefficients (X12, X22 and

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X32) and cross product coefficients (X1X3) were significant on extraction yield of MLP

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due to P-value  0.05. The results also suggested that, among the independent

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variables, the extraction temperature was the most significant parameter affecting the

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yield of MLP followed by ratio of water to raw material and extraction time.

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3.1.2. Response surface plot and contour plot

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The 3D response surface and 2D contour plots are provided as the graphical

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representations of a regression equation. They show the type of interactions between

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two tested variables and the relationship between responses and experiment levels of

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each variable. In the present study, the response surface and contour plots (Fig. 1) were obtained by using Design-Expert. Figure 1A and 1B show the effects of extraction temperature (X1), extraction time(X2) and their reciprocal interaction on extraction yield when the ratio of water to raw material (X3) was fixed at level 0. The

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X1 and X2 demonstrated quadratic effects on the extraction yield. The yield of MLP

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increased to the maximum value with increase of extraction temperature but it then

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decreased slightly with the further increase of extraction temperature. The extraction

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time showed a similar effect on the yield of MLP. Generally, higher extraction

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temperature and longer extraction time can promote the dissolution of polysaccharides

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from plant tissues. However, too high extraction temperature and too long extraction

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time may result in degradation of polysaccharides and hence decrease the

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polysaccharides yield (Samavati & Yarmand, 2013). Figure 1C and 1D show the

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quadratic effects of X1 and X3 on the extraction yield when X2 was fixed at level 0.

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The elliptical contour plot as shown in Fig. 1D indicated that the mutual interactions

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between the extraction temperature and ratio of water to raw material were significant.

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The extraction yield of MLP increased with the increases of extraction temperature

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and ratio of water to raw material from 85 to 93.27 oC and 20 to 39.56 mL/g, but it did

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not further increase when extraction temperature and ratio of water to raw material

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were over 93.27 oC and 39.56 mL/g. The result is in agreement with that of previous

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reported (Chen, Li, Li, Jin, & Lu, 2015). Figure 1E and 1F show the effects of X2 and

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X3 and their reciprocal interaction on the extraction yield when X1 was fixed at level 0.

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The maximum yield of MLP could be achieved when extraction time and ratio of

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water to raw material were in the range of 2.78-3.95 h and 29.27-38.36 mL/g, respectively. However, the extraction yield of MLP decreased when extraction time and ratio of water to raw material were over 3.95 h and 38.36 mL/g. This phenomenon could be explained that the polysaccharides will be completely dissolved

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in the extraction solvent and partial of polysaccharides may be hydrolyzed due to

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longer extraction time. The result is in agreement with that reported by Qiao et al.

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(2009).

311

3.1.3. Optimization of extracting parameters and validation of the model

15

Page 15 of 46

By employing the Design-Expert software, the optimal conditions for MLP

313

extraction were extraction temperature 91.52 oC, extracting time 3.53 h, and ratio (v/w,

314

mL/g) of water to raw material 34.09. In the optimal conditions, the maximum

315

predicted yield of MLP was 10.1%. For operation convenience, the optimal

316

parameters were determined as follows: extraction temperature 92 oC, extracting time

317

3.5 h, and ratio (v/w, mL/g) of water to raw material 34. To ensure that the predicted

318

result was not biased toward the practical value, experimental rechecking was

319

performed using the deduced optimal conditions. A mean value of 10.0 ± 0.5% (n = 3)

320

was obtained from actual experiments, which demonstrated the validation of the RSM

321

model and indicating that the model was adequate for the extraction of MLP. The

322

optimal extraction conditions with high yield were more efficient than that reported

323

(Samavati & Yarmand, 2013).

324

3.2. Purification and characterization of MLP

326 327 328 329

cr

us

an

M

d

te

Crude MLP was prepared using the optimal extraction conditions. Then, the crude

Ac ce p

325

ip t

312

MLP was separated by a DEAE-52 cellulose chromatography column, affording three independent elution peaks of MLP-1, MLP-2 and MLP-3 (Fig. 2A). The third fraction of MLP-3, the main fraction of crude MLP, was further purified with a Sephadex G-100 gel permeation chromatography column, resulting in two fractions of MLP-3a

330

and MLP-3b (Fig. 2B).

331

3.2.1. Contents of carbohydrate, protein, uronic acid, sulfuric radical and total

332

polyphenols in MLP

333

Table 3 shows the contents of carbohydrate, protein, uronic acid, sulfuric radical

16

Page 16 of 46

and total polyphenols in crude MLP, MLP-3a and MLP-3b. The carbohydrate contents

335

in crude MLP, MLP-3a and MLP-3b were 52.09%, 89.74% and 37.20%, respectively.

336

The contents of protein in crude MLP, MLP-3a and MLP-3b were 2.16%, 0.83% and

337

0.22%, respectively. Among all the polysaccharides tested, MLP-3b contained the

338

highest contents of uronic acid. The contents of total polyphenols for MLP-3a and

339

MLP-3b (0.17 and 0.16 mg GAE/100 mg respectively) were much lower than that

340

(1.93 mg GAE/100 mg) for crude MLP, indicating that most of the polyphenols in

341

crude MLP was removed during the purification.

342

3.2.2. Molecular weight of MLP

an

us

cr

ip t

334

The homogeneity and molecular weights of MLP-3a and MLP-3b were analyzed

344

by HPLC. Both MLP-3a and MLP-3b gave a single and symmetrical peak, indicating

345

that they were homogeneous. The average molecular weights of MLP-3a and MLP-3b

346

were estimated to be 80.99 kDa and 3.64 KDa (Table 3), respectively, according to the

347

calibration curve. The molecular weight of MLP-3b was lower than those of

349 350 351 352

d

te

Ac ce p

348

M

343

polysaccharides from mulberry leaves as reported, 8 KD (Zhang et al., 2014b), 15 KDa (Xia et al., 2008), 24.898 and 61.131 KDa (Ying, Han, & Li, 2011), 55.7062 KDa (Jia, Zhang, Lan, Yang, & Sun, 2013) and 550 KDa (Katayama et al., 2008), which indicating that MLP-3b was a novel polysaccharide from mulberry leaves. 3.2.3. Monosaccharide composition of MLP

353

The results of monosaccharide compositions of crude MLP and its purified

354

fractions (MLP-3a and MLP-3b) determined by HPLC are shown in Table 3. It was

355

found that both crude MLP and MLP-3a were composed of Man, Rha, GlcA, GalA,

17

Page 17 of 46

Glc, Gal, and Ara, but the molar ration was quite different. The molar ratio of Man,

357

Rha, GlcA, GalA, Glc, Gal, and Ara for crude MLP was 0.51: 5.13: 2.23: 3.02: 2.13:

358

2.95: 2.55, while it was 0.77: 4.53: 0.81: 1.21: 3.47: 12.55: 11.14 for MLP-3a. For

359

MLP-3b, the molar ratio of Rha, GlcA, GalA, Gal and Ara was 1.57: 0.20: 6.10: 1.27:

360

0.89, and Man and Glc were not detected. These results indicated that the crude MLP

361

and its purified fractions were acidic polysaccharide. In addition, the monosaccharide

362

composition and molar ratio of MLP were different from those of the earlier reports

363

for mulberry leaf polysaccharides (Katayama et al., 2008; Xia et al., 2008). As

364

reported, a pectic polysaccharide from mulberry leaves was comprised of Rha, Ara,

365

Xyl, Glc, Gal and GalA in a molar ratio of 5: 4: 1: 2: 6: 38 (Xia et al., 2008).

366

Katayama et al. (2008) separated and characterized a main polysaccharide from

367

mulberry leaves, which was composed of Rha, Gal, Glc, GalA and GlcA in a molar

368

ratio of 1: 0.2: 0.5: 2.3: 1.5. The difference might be related to the extraction,

369

purification and the raw material (mulberry leaf). It has been reported that different

371 372 373

cr

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an

M

d

te

Ac ce p

370

ip t

356

results for compositional components of tea polysaccharides were given in various reports due to different tea raw material or purification process (Nie & Xie, 2011). 3.2.4. FT-IR spectrum

The FT-IR spectra of crude MLP and its purified fractions are shown in Fig. 3.

374

Broad and strong absorption bands around 3400 cm−1 for C-H stretching vibrations

375

and 2939 cm−1 for C-H stretching vibrations were observed in the FT-IR spectra of

376

crude MLP and its purified fractions. The absorption peaks at 1604.48 cm−1 and

377

1421.3 cm−1 for crude MLP, 1734.8 cm−1 and 1641.1 cm−1 for MLP-3a, 1738.6 cm−1

18

Page 18 of 46

and 1610.6 cm−1 for MLP-3a were attributed to the stretching vibrations of ester

379

carbonyl groups (C=O) and carboxylic groups (COO−) (Li et al., 2013; Santhiya,

380

Subramanian, & Natarajan, 2002; Zhao, Yang, Yang, Jiang, & Zhang, 2007),

381

respectively, which indicated that crude MLP, MLP-3a and MLP-3b were acidic

382

polysaccharides. The results are consistent with the analytical results of

383

monosaccharide compositions for crude MLP, MLP-3a and MLP-3b as mentioned

384

above. The absorbance peaks at 1414.91 cm−1 for MLP-3a and 1415.46 cm−1 for

385

MLP-3b were associated with the stretching of the pectin methyl ester group (−OCH3),

386

which suggested that some of the uronic acids in polysaccharides were esterified

387

(Al-Sheraji et al., 2007). A characteristic peak at around 894 cm−1 was found in crude

388

MLP, MLP-3a and MLP-3b, indicating the existence of -glycosidic bonds in the

389

three polysaccharides (Coimbra, Gonçalves, Barros, & Delgadillo, 2002; Yang et al.,

390

2006).

391

3.3. Antioxidant activity in vitro of MLP

393 394 395

cr

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M

d

te

Ac ce p

392

ip t

378

3.3.1. DPPH radical scavenging activity DPPH free radical is a stable free radical with a characteristic absorption at 517

nm, which will decrease significantly on exposure to proton-donating substance (Yamaguchi, Takamura, Matoba, & Terao, 1998). Accordingly, it has been widely

396

used to evaluate the antioxidant activity of natural antioxidants. In the present study,

397

the scavenging ability of MLP on DPPH free radicals was examined and the results

398

are shown in Fig. 4A. The scavenging rates of MLP and Vc increased with the

399

increase of sample concentration. At a concentration of 4.0 mg/mL, the DPPH free

19

Page 19 of 46

radical scavenging activities for crude MLP, MLP-3a, MLP-3b and Vc were 68.21%,

401

44.96%, 60.17% and 91.16%, respectively. Compared with purified fractions, the

402

crude MLP exhibited relative higher (P  0.05) DPPH radical scavenging activity,

403

which might be partly attributed to its relative higher content of polyphenols (Table 3).

404

The scavenging abilities of MLP-3a and MLP-3b might mainly come from the

405

polysaccharides due to their relatively low contents of total polyphenols. It has been

406

reported that the antioxidant activity of polysaccharides was related to their molecular

407

weight, uronic acid content, degree of sulfation, type of monosaccharide and

408

glycosidic linkage (Liu et al., 2010; Melo, Feitosab, Freitasa, & de Paula, 2002; Sun,

409

Wang, Li, & Liu, 2014; Wang, Chang, Stephen Inbaraj, & Chen, 2010; Zeng, Zhang,

410

& Jia, 2014). MLP-3b displayed higher antioxidant activity than MLP-3a, which

411

might be due to the lower molecular weight and higher content of uronic acid of

412

MLP-3b (Asker, Mahmoud, & Ibrahim, 2007; Chen, Zhang, Qu, & Xie, 2008). But

413

the exact mechanism is unclear.

415 416 417

cr

us

an

M

d

te

Ac ce p

414

ip t

400

3.3.2. Hydroxyl radical scavenging activity Hydroxyl radical, which is well known as one of the most reactive free radical,

can react with almost the biomacromolecules in living cells and induce severe damage to the adjacent biomolecules (Spencer et al., 1994). Therefore, the hydroxyl radical

418

scavenging activities of MLP and its purified fractions were investigated. As shown in

419

Fig. 4B, all MLP samples and Vc exhibited scavenging activity on hydroxyl radicals

420

in a dose-dependent manner. At a concentration of 4.0 mg/mL, the scavenging

421

abilities of crude MLP, MLP-3a, MLP-3b and Vc were 88.85%, 57.89%, 68.12% and

20

Page 20 of 46

92.44%, respectively. Notably, crude MLP showed similar (P > 0.05) scavenging

423

activity as Vc. The results indicated that the crude MLP exhibited strong scavenging

424

activity and its purified fractions had moderate scavenging activities. It has been

425

reported that the hydroxyl scavenging ability is related to the number of hydroxyl or

426

amino groups in polysaccharide (Guo et al., 2013). This might be explained by the

427

factor that crude MLP would provide more active hydroxyl groups than its purified

428

fractions as crude MLP contained much higher content of polyphenols.

429

3.3.3. Superoxide radical scavenging activity

an

us

cr

ip t

422

Superoxide radical is a long lifetime radical that is generated by numerous

431

biological and photochemical reactions (Banerjee, Dasgupta, & De, 2005; Dahl &

432

Richardson, 1978). Although superoxide radical is a relatively weak oxidant, it can

433

further interact with other molecules to generate more strong and reactive oxidative

434

species such as singlet oxygen and hydroxyl radicals, and cause oxidative tissue

435

damage and various diseases. Therefore, the superoxide radical scavenging activity of

437 438 439

d

te

Ac ce p

436

M

430

MLP was measured by the PMS/NADH-NBT system in the present study. The results demonstrated that MLP exhibited dose-dependent superoxide radical scavenging capacity at concentrations ranging from 0.0625 to 2.0 mg/mL (Fig. 4C). At the concentration of 2.0 mg/mL, the superoxide radical scavenging activities for crude

440

MLP, MLP-3a, MLP-3b and Vc were 84.47%, 71.91%, 75.25% and 99.53%,

441

respectively. Notably, all the MLP samples had strong superoxide radical scavenging

442

activity. The possible mechanism of polysaccharide for scavenging superoxide anion

443

is reported to be associated with the dissociation energy of O-H bond, that is, the more

21

Page 21 of 46

electron withdrawing groups such as carboxylic groups and aldehyde groups attached

445

to polysaccharide, the weaker the dissociation energy of O-H bond (Lin, Wang, Chang,

446

Inbaraj, & Chen, 2009; Jin et al., 2012). The present result showed that MLP-3b

447

displayed higher scavenging activity than MLP-3a, which might be partly due to that

448

of MLP-3b with higher content of carboxylic groups (Sun, Liu, & Kennedy, 2010),

449

which is in agreement with the result of uronic acid content. The crude MLP with

450

highest scavenging ability might be partly due to its reduction activity, which might

451

be related to the higher polyphenols content (Wang et al, 2010).

452

3.3.4. ABTS radical scavenging activity

an

us

cr

ip t

444

The ABTS radical cation has often been used in the evaluation of total antioxidant

454

activity of single compounds and complex mixtures of various origins (body fluids,

455

foods, beverages, plant extracts). In the present study, the scavenging ability of MLP

456

on ABTS free radical was determined and the results are shown in Fig. 4D. The

457

scavenging activities of all the samples increased in a concentration-dependent

459 460 461

d

te

Ac ce p

458

M

453

manner, and all the samples exhibited strong radical scavenging activities at higher doses. At the dose of 4.0 mg/mL, the scavenging abilities of crude MLP, MLP-3a, MLP-3b and Vc were 99.33%, 79.81%, 90.47% and 100.00%, respectively. Therefore, MLP had an appreciable scavenging activity on ABTS free radical. It has been

462

reported that the antioxidant activity obtained by ABTS assay was positively

463

correlated to that obtained by DPPH radical assay (Floegel, Kim, Chung, Koo, &

464

Chun, 2011; Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, & Byrne, 2006). In

465

our present study, however, the scavenging ability of MLP by ABTS assay was higher

22

Page 22 of 46

than that by DPPH assay. The reason might be due to that ABTS assay is more

467

suitable for evaluating hydrophilic antioxidant while DPPH assay is more applicable

468

for evaluating hydrophobic antioxidant (Floegel et al, 2011).

469

3.3.5. Fe2+ chelating activity

ip t

466

Iron is essential in the human body for respiration and the activity of a large range

471

of enzymes, oxygen transport and redox reactions (Hsu, Coupar, & Ng, 2006).

472

However, iron is an extremely reactive metal, which can accelerate the reaction of

473

oxidation of important biological molecules (Liu, Wang, Xu, & Wang, 2007).

474

Therefore, the ferrous ion chelating activity of MLP was investigated and the results

475

are shown in Fig. 4E. The Fe2+ chelating activity of MLP was correlated well with

476

increase of concentration. At a concentration of 4.0 mg/mL, the Fe2+ chelating

477

activities of crude MLP, MLP-3a, MLP-3b and Vc were 92.96, 51.33, 87.76 and

478

99.91%, respectively, indicating that crude MLP and MLP-3b had potent Fe2+

479

chelating activity. It has been reported that compounds with metal ion chelating

481 482 483

us

an

M

d

te

Ac ce p

480

cr

470

activity often own functional groups such as –OH, –SH, –COOH, –PO3H2, C=O, –NR2, –S– and –O– (Jiang et al., 2014; Liu et al., 2010). The strong Fe2+ chelating activity of crude MLP and MLP-3b might be partially due to the high contents of –COOH and C=O groups in their structures (Table 3). However, the exact

484

mechanisms should be investigated further.

485

4. Conclusion

486

In the present study, the optimal extraction parameters for MLP with a extraction

487

yield of 10.0 ± 0.5% were determined as follows: extraction temperature of 92 oC, 23

Page 23 of 46

extraction time of 3.5 h, ratio of water to raw material of 34 mL/g and extraction

489

cycles of two times. Two fractions of polysaccharides with the average molecular

490

weights of 80.99 and 3.64 kDa (MLP-3a and MLP-3b, respectively) were obtained

491

from crude MLP by chromatography of DEAE-Cellulose 52 and Sephadex G-100.

492

The crude MLP and MLP-3a were composed of Man, Rha, GluA, GalA, Glu, Gal, and

493

Ara in the molar ratio of 0.51: 5.13: 2.23: 3.02: 2.13: 2.95: 2.55 and 0.77: 4.53: 0.81:

494

1.21: 3.47: 12.55: 11.14, respectively, while MLP-3b was composed of Rha, GluA,

495

GalA, Gal and Ara in a molar ratio of 1.57: 0.20: 6.10: 1.27: 0.89. The results along

496

with the FT-IR spectra demonstrated that the crude MLP, MLP-3a and MLP-3b were

497

acidic polysaccharides. Furthermore, the crude MLP and its purified fractions

498

exhibited potent antioxidant activity in vitro, specifically superoxide scavenging

499

activity, ABTS radical scavenging activity and Fe2+ chelating power. The results

500

suggested that the mulberry leaves can be utilized much better by using the optimal

501

extraction parameters obtained in the present study and MLP could be explored as a

503 504

505

cr

us

an

M

d

te

Ac ce p

502

ip t

488

natural antioxidant for use in medicine or functional foods. Further studies on the mechanism of antioxidant activity, precise chemical structures of the purified fractions and other biological functions of MLP are in progress.

Acknowledgements

506

This work was supported by the National Natural Science Foundation of China

507

(No.31201454), the Fundamental Research Funds for the Central Universities

508

(KYZ201218) and a Project Funded by the Priority Academic Program Development

509

of Jiangsu Higher Education Institutions. 24

Page 24 of 46

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669

properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion.

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700 701 702 703

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733

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745 746 747

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d

M

an

us

cr

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752

36

Page 36 of 46

Figure Captions

756

Fig. 1. Response surface plots (A, C and E) and contour plots (B, D and F) showing

757

the effects of variables (X1, extraction temperature; X2, extraction time; X3, ratio of

758

water to material) and their mutual effects on the extraction yield of MLP.

759

Fig. 2. Stepwise elution curve of crude MLP on DEAE-cellulose column (A) and

760

elution curve of polysaccharides fraction (MLP-3) on Sephadex G-100 column (B).

761

Fig. 3. FT-IR spectra of crude MLP (A), MLP-3a (B) and MLP-3b.

762

Fig. 4. Scavenging effects on DPPH radical (A), hydroxyl radical (B), superoxide

763

radical (C) and ABTS radical (D) and Fe2+ chelating activity (E) of crude MLP,

764

MLP-3a and MLP-3b.

Ac ce p

te

d

M

an

us

cr

ip t

755

37

Page 37 of 46

765

Table 1

766

Box–Behnken design matrix and the response values for the yield of MLP Temperature (oC)

Ratio of water to raw

Time (h)

yield (%)

Code X1

X2

Code X2

X3

Code X3

1

95

1

2

−1

30

0

8.5

2

90

0

2

−1

20

−1

8.2

3

90

0

3

0

30

0

10.1

4

90

0

3

0

30

0

5

95

1

3

0

40

6

85

−1

3

0

20

7

90

0

2

−1

40

8

90

0

4

1

40

9

90

0

3

0

30

cr 9.8 9.3

us

1

ip t

X1

6.8

1

8.7

1

9.5

0

9.9

30

0

7.7

30

0

9.8

20

−1

8.4

−1

30

0

6.9

0

20

−1

7.5

0

30

0

9.7

an

−1

85

−1

4

1

90

0

3

0

12

90

0

4

1

13

85

−1

2

14

95

1

3

15

90

0

3

16

85

−1

3

0

40

1

7.3

17

95

1

4

1

30

0

8.9

te

d

M

10 11

Ac ce p

767

Polysaccharide

material (mL/g)

Run

38

Page 38 of 46

768

Table 2

769

ANOVA for response surface quadratic model of MLP extraction Sum of squares

df

Mean square

F-value

P-Value

Model

18.92

9

2.10

82.47

< 0.0001 a

X1

3.66

1

3.66

143.49

< 0.0001 a

X2

0.60

1

0.60

23.51

X3

1.92

1

1.92

75.34

X1X2

0.068

1

0.068

2.65

X1X3

0.47

1

0.47

18.40

X2X3

0.0019 a

< 0.0001 a

cr

0.1475 b 0.0036 a 0.1175 b

1

0.081

3.19

8.19

1

8.19

321.37

< 0.0001 a

X 22

0.80

1

0.80

31.25

0.0008 a

X 32

2.17

1

2.17

85.02

< 0.0001 a

Residual

0.18

7

0.025

Lack of fit

0.096

3

0.032

Pure error

0.082

4

0.021

Cor Total

19.10

16

5% significance level.

b

Not significant relative to the pure error.

an

0.3315 b

Ac ce p

te

d

a

1.55

M

R2 = 0.9907, adjusted R2 =0.9786

us

0.081

2

X1

770 771 772

ip t

Source

39

Page 39 of 46

Table 3

774

Preliminary characterization of crude MLP, MLP-3a and MLP-3b Crude MLP

MLP-3a

MLP-3b

Carbohydrate (%)

52.09 ± 1.10

89.74 ± 0.31

37.20 ± 0.59

Protein (%)

2.16 ± 0.02

0.83 ± 0.03

0.22 ± 0.02

Uronic acid (%)

32.45 ± 0.92

6.53 ± 0.13

65.29 ± 1.53

Sulfuric radical (%)

1.73 ± 0.10

1.40 ± 0.02

1.22 ± 0.02

Total polyphenols (mg GAE/100 mg)

1.93 ± 0.02

0.17 ± 0.01

0.16 ± 0.01

Molecular weight (KDa)

-

80.99

3.64

Man

0.51

0.77

Rha

5.13

4.53

GluA

2.23

0.81

GalA

3.02

1.21

Glu

2.13

Gal

2.95

Ara

2.55

cr

Item

us

Monosaccharide composition (mol)

1.57

0.20

an

6.10

3.47

1.27

11.14

0.89

M

12.55

te

d

- Not detected.

Ac ce p

775

ip t

773

40

Page 40 of 46

(B)

(C)

(D)

(F)

776 777

Ac ce p

te

(E)

d

M

an

us

cr

ip t

(A)

Fig. 1

41

Page 41 of 46

cr

ip t

(A)

us

778

779 Fig. 2

Ac ce p

780

te

d

M

an

(B)

42

Page 42 of 46

ip t cr te

d

M

an

us

Fig. 3

Ac ce p

781 782

43

Page 43 of 46

cr

ip t

(A)

us

783

784

Ac ce p

(C)

te

d

M

an

(B)

785

44

Page 44 of 46

cr

ip t

(D)

us

786

789

Fig. 4

Ac ce p

787 788

te

d

M

an

(E)

45

Page 45 of 46

Highlights

790

► The hot water extraction of MLP was optimized by a Box-Behnken design.

791

► The maximum extraction yield of MLP was 10.0 ± 0.5%.

792

► Crude MLP and its purified fractions (MLP-3a and MLP-3b) were acidic

793

polysaccharides.

794

► MLP-3b had a relatively high content of uronic acid.

795

► Crude MLP, MLP-3a and MLP-3b exhibited potent antioxidant activity in vitro.

us

cr

ip t

789

Ac ce p

te

d

M

an

796

46

Page 46 of 46