Extraction condition optimization and effects of drying methods on physicochemical properties and antioxidant activities of polysaccharides from comfrey (Symphytum officinale L.) root

Accepted Manuscript Extraction condition optimization and effects of drying methods on physicochemical properties and antioxidant activities of polysaccharides from comfrey (Symphytum officinale L.) root

Hongmei Shang, Haizhu Zhou, Mengying Duan, Ran Li, Hongxin Wu, Yujie Lou PII: DOI: Reference:

S0141-8130(17)33563-8 https://doi.org/10.1016/j.ijbiomac.2018.01.198 BIOMAC 9030

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

15 September 2017 9 November 2017 30 January 2018

Please cite this article as: Hongmei Shang, Haizhu Zhou, Mengying Duan, Ran Li, Hongxin Wu, Yujie Lou , Extraction condition optimization and effects of drying methods on physicochemical properties and antioxidant activities of polysaccharides from comfrey (Symphytum officinale L.) root. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), https://doi.org/ 10.1016/j.ijbiomac.2018.01.198

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 Extraction condition optimization and effects of drying methods on physicochemical properties and antioxidant activities of polysaccharides from comfrey (Symphytum officinale L.) root Hongmei Shang a,b, Haizhu Zhou a,b, Mengying Duan a, Ran Li a, Hongxin Wu c, Yujie Lou a,b,* College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, PR

PT

a

Key Laboratory of Animal Nutrition and Feed Science of Jilin Province, Jilin Agricultural

SC

b

RI

China

University, Changchun 130118, PR China

Grassland Research Institute of CAAS, Hohhot 010010, PR China

MA

NU

c

The work was performed in College of Animal Science and Technology, Jilin Agricultural

D

University.

PT E

Address: No.2888 Xin-Cheng Street, Changchun 130118, Jilin, China.

CE

Correspondence to: Yujie Lou, College of Animal Science and Technology, Jilin Agricultural

AC

University, No.2888 Xin-Cheng Street, Changchun 130118, Jilin, China. Tel: +86-431-84532812. Fax: +86-431-84532812. E-mail: [email protected]

Running title: Extraction and Drying of Comfrey Polysaccharides

1

ACCEPTED MANUSCRIPT Extraction condition optimization and effects of drying methods on physicochemical properties and antioxidant activities of polysaccharides from comfrey (Symphytum officinale L.) root1 Hongmei Shang a,b, Haizhu Zhou a,b, Mengying Duan a, Ran Li a, Hongxin Wu c, Yujie Lou a,b,∗ College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, PR

PT

a

Key Laboratory of Animal Nutrition and Feed Science of Jilin Province, Jilin Agricultural

SC

b

RI

China

University, Changchun 130118, PR China

Grassland Research Institute of CAAS, Hohhot 010010, PR China

MA

NU

c

ABSTRACT

D

This study was designed to investigate the extraction conditions of polysaccharides from comfrey

PT E

(Symphytum officinale L.) root (CRPs) using response surface methodology (RSM). The effects of three variables including liquid-solid ratio, extraction time and extraction temperature on the

CE

extraction yield of CRPs were taken into consideration. Moreover, the effects of drying methods

AC

including hot air drying (HD), vacuum drying (VD) and freeze drying (FD) on the physicochemical properties and antioxidant activities of CRPs were evaluated. The optimal conditions to extract the polysaccharides were as follows: liquid–solid ratio (15 mL/g), extraction time (74 min), and extraction temperature (95 °C), allowed a maximum polysaccharides yield of 22.87%. Different drying methods had significant effects on the physicochemical properties of CRPs such as the

* Corresponding author at: College of Animal Science and Technology, Jilin Agricultural University, No.2888 Xin-Cheng Street, Changchun 130118, PR China. E-mail address: [email protected] (Y. J. Lou). 2

ACCEPTED MANUSCRIPT chemical composition (contents of total polysaccharides and uronic acid), relative viscosity, solubility and molecular weight. CRPs drying with FD method showed stronger reducing power and radical scavenging capacities against DPPH and ABTS radicals compared with CRPs drying with HD and VD methods. Therefore, freeze drying served as a good method for keeping the

PT

antioxidant activities of polysaccharides from comfrey root.

RI

Keywords:

SC

Symphytum officinale L. polysaccharides Extraction condition

NU

Drying method

MA

1. Introduction

Comfrey (Symphytum officinale L.), a perennial herb belonging to Boraginacae, has long been

D

used in folk medicine for the treatment of traumatism, bedsore, sprain and catagma [1]. Comfrey

PT E

has potent effects of anti-inflammatory, acesodyne, granulation promoting and anti-exudation [2]. The pharmacodynamics of comfrey is due to some critical chemical components in root, such as

CE

ureidohydantoin, rosmarinic acid and muco-polysaccharides [3].

AC

Polysaccharides are an important class of biological macromolecules. Recently, there is a growing interest in polysaccharides due to their biological activities, such as oxidation resistance, antiviral action, anti-fatigue, immunoregulation, hypoglycemic activity, and antilipidemic effect [4,5]. The extraction efficiency of polysaccharides from comfrey root has great signification for the development and utilization of this raw plant. However, there are few studies have been focused on the polysaccharides from comfrey root. Extraction polysaccharides with hot water is the most common way used in food industry due to its easy accessibility and security [6]. The response

3

ACCEPTED MANUSCRIPT surface methodology (RSM) is widely applied to obtain the optimization extracting conditions of polysaccharides. The relationship among several variables can be investigated by RSM methodology. In addition, drying methods have a great influence on the physicochemical properties and

PT

bioactivities of polysaccharides [7]. Several methods such as hot air drying, vacuum drying and

RI

freeze drying have been used for the drying process of polysaccharides. The most common drying

SC

method used in food processing industry is hot air drying due to its easy accessibility and low cost. However, serious physicochemical changes of the dried polysaccharides may appear during hot air

NU

drying process [8]. The oxidization of the dried materials can be prevented during vacuum drying

MA

process. Nevertheless, the structure damage of the dried products may occur under the high temperature of vacuum drying [9]. The bioactivities of the dried polysaccharides could be

D

maximally protected under the vacuum and freeze condition during freeze drying process [10].

PT E

However, the cost of freeze drying is high because of the energy consumption during the long drying time needed [8]. Therefore, the selection of drying methods is important for the maintaining

CE

of physicochemical characteristics and bioactivities of polysaccharides.

AC

Antioxidants are a class of chemical substances that reduce free radicals and inhibit oxidation directly or indirectly. The oxidative stress of body may be alleviated by exogenous supplement of antioxidants. However, some synthetic antioxidants showed potential adverse effects, such as liver injury and carcinogenesis, especially administration of synthetic antioxidants over the course of long-term [11]. Thus, exploration of safe and natural antioxidants to resist oxidative stress has become a research hotspot in recent years. Many research findings showed plant polysaccharides had the strong antioxidant activities, for instance, Dioscorea hemsleyi and Lycium barbarum

4

ACCEPTED MANUSCRIPT polysaccharides exhibited good reducing power and DPPH radical scavenging activity [12,13], and Amomum villosum polysaccharides possessed strong superoxide radical scavenging activity and reductive potential [14]. However, there are few studies have been focused on the antioxidant activity of polysaccharides from comfrey root (CRPs). In order to further clarify the activities of

PT

CRPs, the in vitro antioxidant activities of CRPs were evaluated in this study.

RI

Therefore, the present research was designed to study the water extraction conditions

SC

(liquid–solid ratio, extraction time and extraction temperature) of CRPs using RSM. In addition, the influences of different drying methods (hot air drying, vacuum drying and freeze drying) on the

NU

physicochemical properties and antioxidant activities of CRPs were determined to seek the potential

MA

drying techniques. The in vitro antioxidant activities of CRPs were evaluated on the base of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity,

2. Materials and methods

PT E

ferric reducing power.

D

2,2-azino-bis-3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity and

CE

2.1. Materials and chemicals

AC

Fresh comfrey root was harvested in Jilin Agricultural University (Changchun, China). The plants were in bloom stage and approximately 1 m in height. The roots were cut into 5 mm slices before drying in a drier (101-2-BS, Shanghai Yuejin Medical Instrument co., LTD, Shanghai, China) at 50 °C, then the dried root slices were ground into powders, and passed through a 1 mm sieve prior to extraction of the polysaccharides. The chemical reagents including DPPH radical, ABTS radical and vitamin C were obtained from Sigma-Aldrich (St. Louis, USA). All other chemicals used were analytical grade and bought from local suppliers.

5

ACCEPTED MANUSCRIPT 2.2. Preparation of polysaccharides The lipids of comfrey root powder were removed with 85% (v/v) ethanol for 24 h. The insoluble residue was dried in a dryer (101-2-BS, Shanghai Yuejin Medical Instrument co., LTD, Shanghai, China) at 50 °C and prepared for the extraction of polysaccharides with distilled water according to

PT

the designed conditions. The aqueous extract was separated through gauze with six layers. The

RI

filtrate was centrifuged at 3000 rpm for 15 min. Then, the supernatant was concentrated to a 1/4

SC

volume of the primary supernatant using a rotary evaporator under reduced pressure at 60 °C. The starch fraction in the concentrated solution was removed by α-amylase at 60 °C. Subsequently, the

NU

concentrated solution was precipitated with 4 volumes of absolute ethanol for 12 h at 4 °C. The

MA

precipitate was obtained by centrifugation (3000 rpm for 15 min), and washed 3 times with absolute ethanol. Then the precipitate was dissolved with deionized water, deproteinated by the Seveg

D

reagent (4:1 of chloroform: normal butanol, v/v) [5], and dialyzed in a dialysis bag (MWCO 1400

PT E

Da, Union Carbide). The CRPs was obtained by lyophilization. The extraction yield (%) was calculated using the weight of CRPs and the weight of samples powder following the equation as

CE

described by Wang et al. [15].

AC

2.3. Single-factor design

The influences of liquid-solid ratio (5, 10, 15, 20, 25 and 30 mL/g), extraction time (40, 50, 60, 70, 80 and 90 min), and extraction temperature (50, 60, 70, 80, 90 and 100 °C) on the CRPs extraction yield were studied by single-factor experiment. Each sample was extracted with distilled water according to the procedure mentioned above (2.2). The extraction yields of different groups were compared by one-way analysis of variance (ANOVA) using SPSS 17.0 (SPSS Inc., Chicago, IL).

6

ACCEPTED MANUSCRIPT 2.4. RSM design The appropriate ranges of liquid–solid ratio (X1), extraction time (X2) and extraction temperature (X3) were obtained according to the single-factor experiment. The Box–Behnken design (BBD, Design-Expert 8.0.6 Trial, State-Ease, Inc., Minneapolis, USA) with 3 variables of 3 levels each on

PT

the extraction yield of CRPs (Y) were performed. The whole design was consisted of 17 experiment

RI

runs carried out in random order, and contained 5 central points to estimate the repeatability of the

SC

experiment method. The 3 levels (low, intermediate and high values) of each variable were encoded as −1, 0 and +1, respectively (Table 1). For predicting the optimized extraction conditions, a

NU

second-order polynomial equation (1) as follow was used to fit the relationship between variables

3

3

i 1

i 1

2

Y  A0   Ai X i   Aii X i   2

3

MA

and response (CRPs yield).

A X X

i 1 j  i 1

ij

i

j

(1)

PT E

D

where Y is the predicted response (CRPs yield); Xi and Xj are the variables; Xij is the interaction term; Xi2 is the quadratic term, and A0, Ai, Aii and Aij are the constant coefficient, the linear

CE

coefficient, the squared coefficient, and the interaction coefficient of two variables, respectively. 2.5. Drying procedure of CRPs

AC

CRPs were extracted under the optimum extraction condition, and were dried using three different methods (hot air drying, vacuum drying and freeze drying) with the thickness of 0.2 cm until the weight of CRPs became constant. Hot air drying (HD) was carried out in an electric heating air-blowing drier (101-2-BS, Shanghai Yuejin Medical Instrument co., LTD, Shanghai, China) at 50 °C for 6.5 h. Vacuum drying (VD) was carried out in a vacuum drying oven (DZF, Shanghai Longyue Instrument Equipment co., LTD, Shanghai, China) at 50 °C for 5.5 h. Freeze drying (FD) was carried out in a vacuum freeze dryer (SCIENTZ-12N, Ningbo Scientz 7

ACCEPTED MANUSCRIPT Biotechnology co., LTD, Ningbo, China) at −70°C for 8 h. The CRPs obtained by hot air drying, vacuum drying and freeze drying were named as HD-CRPs, VD-CRPs and FD-CRPs, respectively. 2.6. Physicochemical properties of CRPs 2.6.1. Chemical composition analysis of CRPs

PT

The total polysaccharides content of CRPs was determined by phenol-sulfuric acid method

RI

using D-glucose as standard (y = 0.0143 x - 0.0322, R2 = 0.9931) [16]. Uronic acid content of CRPs

SC

was measured by m-hydroxybiphenyl assay using glucuronic acid as reference material (y = 0.0127 x - 0.0309, R2 = 0.9948) [17]. Protein content of CRPs was measured by Bradford’s method using

NU

bovine serum albumin as standard (y = 0.0054 x - 0.0031, R2 = 0.9969) [18]. Sulfate radical content

0.0027 x + 0.0191, R2 = 0.9926) [19].

MA

was determined by barium chloride-gelatin nephelometry using potassium sulfate as standard (y =

D

2.6.2. Moisture, pH and relative viscosity determinations of CRPs

PT E

The moisture content of the CRPs was measured according to the method of Kong et al. [7]. The relative viscosity of CRPs (10 mg/mL) to deionized water was determined using a rotation

CE

viscometer (NDJ-8S, Shanghai Jitai Electronic Technology co., LTD, Shanghai, China) at 25°C.

AC

The pH of the CRPs (2 mg/mL) was measured using a pH meter (PHSJ-5, Shanghai Yidian Scientific Instrument co., LTD, Shanghai, China). 2.6.3. Solubility test of CRPs The solubility of CRPs was determined in distilled water. Briefly, the CRPs sample (0.1 g) was placed into distilled water (50 mL) in a beaker, and then the beaker was placed in a water bath (20, 40, 60, 80, 100°C) with a digital mixer (JJ-5, Medical Instrument Factory of Jintan City, Jintan,

8

ACCEPTED MANUSCRIPT China). The CRPs solution was stirred (150 rpm/min) until it was completely dissolved, and the length of dissolved time was recorded. 2.6.4. Molecular weight distribution determination of CRPs Before determination the molecular weight distribution of polysaccharides obtained by three

PT

drying methods, the CRPs was preliminarily purified by using DEAE-52 cellulose (3.5 cm × 20 cm)

RI

column, and eluted first with distilled water, then with a linear gradient (0 mol/L to 1 mol/L) of

SC

sodium chloride solution at a flow rate of 2 mL/min. The eluent (8 mL/tube) was collected automatically, and the polysaccharides content of each tube was monitored by the phenol-sulfuric

NU

acid method [16]. The fraction (one fraction eluting with distilled water) containing carbohydrates

MA

were collected and concentrated to determine the molecular weight of CRPs. The molecular weight of CRPs was determined by gel filtration chromatography on a Sepharose

D

CL-6B column (2.6 cm × 100 cm) using distilled water as the eluant at a flow rate of 0.9 mL/min.

PT E

The eluent (4.05 mL/tube) was collected and monitored for carbohydrate content using phenol-sulfuric acid method [16]. Series of dextran (T-10, T-40, T-70, T-500) were used as standards

CE

for calibration.

AC

2.6.5. Monosaccharide composition analysis of CRPs The monosaccharide composition of CRPs obtained by three drying methods was analyzed using high performance liquid chromatographic (HPLC) by following methods as described previously [20,21] with some modifications. The CRPs sample (2 mg) was hydrolyzed with 0.5 mL trifluoroacetic acid (TFA, 2 mol/L) in a sealed flask fulfilled with N2 at 120 °C for 2 h. After hydrolysis, the excess TFA in the system was removed by repeated co-evaporation with ethanol at 45 °C. Subsequently, dry hydrolysate samples of CRPs or monosaccharide standard (Sigma, St.

9

ACCEPTED MANUSCRIPT Louis, MO, USA) were added 0.5 mL methanol solution of 1-phenyl-3-methyl-5-pyrazolone (PMP, 0.5 mol/L) and 0.5 mL aqueous solution of NaOH (0.3 mol/L) for derivatization at 70 °C for 30 min. Then, the mixture solution was centrifuged at 10 000 rpm for 5 min. The supernatant was mixeded with 0.05 mL HCl (0.3 mol/L), the reaction mixture was extracted with chloroform for three times

PT

to remove the excess PMP. The aqueous layer was filtered through a 0.22 μm membrane and

RI

analyzed by a Shimadzu 2010AHT HPLC system (SHIMADZU, Kyoto, Japan) equipped with an

SC

UV detector (245 nm). Amethyst C18 column (4.6 mm × 250 mm, 5 μm, Sepax, Delaware, USA) was used and the column oven was kept at 25 °C. The injection volume was 10 μL. The mobile

NU

phase was a mixture of phosphate buffered saline (PBS, 0.1 mol/L, pH 7) and acetonitrile (80:20,

MA

v/v). The flow rate was 1 mL/min.

2.6.6. Complex formation with Congo Red of CRPs

D

The CRPs sample (1.0 mg) was dissolved in deionized water (2.0 mL), and then aqueous Red

PT E

(2.0 mL, 80 μmol/L) in 0.001 M NaOH was added to the CRPs solution. Aqueous solution of NaOH (4.0 mol/L) was added to the mixture till the final concentration of NaOH in the mixture was

CE

in the range from 0 to 0.5 mol/L. The maximum absorption wavelength (λ max) of the reaction

AC

solution was measured using a spectrophotometer (752, Shanghai Xianke Spectrophotometer Instrument co., LTD, Shanghai, China). 2.7. In vitro antioxidant activities of CRPs 2.7.1. DPPH radical scavenging activity assay The DPPH radical scavenging activity of CRPs was determined by following method as a previous description [22] with some modifications. Briefly, 3.0 mL of different concentration (0.05–1 mg/mL) of CRPs in water was mixed with 1.0 mL of 0.1mM DPPH solution in ethanol,

10

ACCEPTED MANUSCRIPT respectively. The mixture was placed in the dark environment at room temperature for 30 min. After incubation, the absorbance of mixture was determined at 517 nm. Vitamin C was used as the positive control. The DPPH radical scavenging activity of CRPs was calculated using the formula (2) below:

[ A0  ( A1  A2 )] 100 A0

PT

DPPH radical scavenging activity (%) 

(2)

RI

where A0 was the absorbance of the control group (3.0 mL distilled water and 1.0 mL of DPPH). A1

SC

was the absorbance of the test group. A2 was the absorbance of the polysaccharides sample (3.0 mL

2.7.2. ABTS radical scavenging activity assay

NU

CRPs water solution and 1.0 mL of ethanol).

MA

The ABTS radical scavenging activity of CRPs was evaluated with a previous procedure [23] with minor modifications. The ABTS radical cation was produced by the mixture of 5 mL of ABTS

PT E

D

solution (7 mM) and 1 mL of K2S2O8 aqueous solution (15 mM) for 24 h in the darkness at 20 °C. After incubation, the ABTS+ solution was diluted with the deionized water to obtain an absorbance

CE

of 0.70 (±0.02) at 734 nm. Then, 0.75 mL of various concentrations (0.25–4 mg/mL) of CRPs solution was reacted with 3 mL of the ABTS+ solution, respectively. The mixture was reacted for 15

AC

min at 20 °C. Then the absorbance at 734 nm was determined. Vitamin C was served as the positive reference reagent. The ABTS radical scavenging activity of CRPs was calculated by the equation (3) below:

ABTS radical scavenging activity (%) 

[ A0  ( A1  A2 )] 100 A0

(3)

11

ACCEPTED MANUSCRIPT where A0 was the absorbance of the control group (containing all the reagents but the CRPs solution was instead with distilled water). A1 was the reaction result of CRPs. A2 was the results of all the reagents except the ABTS solution was instead with distilled water. 2.7.3. Ferric reducing power

PT

The ferric reducing power of CRPs was determined with previous studies [24,25] with minor

RI

modifications. Vitamin C was served as the positive reference reagent. Different concentrations of

SC

the CRPs solutions (1.5 mL, 1–6 mg/mL) were mixed with 1.5 mL of sodium phosphate buffer (0.2 M, pH 6.6) and 1.5 mL of K3[Fe(CN)6] (1%, w/v), respectively. The mixture was reacted at 50°C

NU

for 20 min. After incubation, the mixture was cooled quickly, and then was added with 1.5 mL of

MA

Cl3CCOOH (TCA, 10%, w/v). The mixture was centrifuged at 3000 rpm for 10 min. After centrifugation, 1.5 mL of the centrifugate was mixed with 1.5 mL of deionized water and 0.3 mL of

D

FeCl3 (0.1%, w/v). After 10 min, the absorbance of the reaction mixture was determined at 700 nm

PT E

against a blank. The reducing power of CRPs was calculated by the equation (4) below: Ferric reducing power  A1  A2

(4)

CE

where A1 was the reaction result of CRPs. A2 was the results of all the reagents except the FeCl3

AC

solution was instead with distilled water. 2.8. Statistical analysis

All experiments were performed at least in triplicate. Analyses of all samples were run in triplicate and averaged. The result values were presented as mean±standard deviation. The one-way ANOVA in SPSS 17.0 for Windows (SPSS Inc., Chicago, IL) was performed for statistical analysis. Duncan post hoc tests were performed when significant differences were found, P < 0.05 was considered to be significant, and P < 0.01 was considered to be highly significant. The

12

ACCEPTED MANUSCRIPT influences of CRPs doses on the determination indicators in vitro antioxidant activity assay were determined using curve estimation for linear and quadratic terms. 3. Results and discussion 3.1. Single-factor experimental evaluation

PT

3.1.1. Influence of liquid-solid ratio on the yield of CRPs

RI

The effects of liquid-solid ratio (5, 10, 15, 20, 25 and 30 mL/g) on the yield of CRPs were

SC

investigated when the extraction temperature and time were 90 °C and 60 min, respectively (Fig. 1A). As the liquid-solid ratio improved from 5 to 15 mL/g, the CPRs yield increased from 16.32%

NU

to 22.17%, and the CPRs yield of 15 mL/g was significant higher than those of 5 and 10 mL/g (P <

MA

0.05). However, when the liquid-solid ratio continued to increase from 20 to 30 mL/g, there were no significant increases of CPRs yield compared to the yield under the liquid-solid ratio of 15 mL/g

D

(P > 0.05). The reason might be due to the difference of concentration between the exterior solvent

PT E

and the interior tissue of comfrey root [26]. A higher liquid-solid ratio significantly improved the dissolution of polysaccharides from the interior tissue of material, and caused an increase of CRPs

CE

yield. Nevertheless, along with the enhancement of liquid-solid ratio, the diffusion distance of

AC

polysaccharides from the intra-tissue raised, resulting in an insignificant increase of CRPs yield [27]. Therefore, the liquid-solid ratio of 15 mL/g was chosen as the sufficient ratio to extract CRPs. 3.1.2. Influence of extraction time on the yield of CRPs The effects of extraction time (40, 50, 60, 70, 80 and 90 min) on the yield of CRPs were investigated, and the extraction temperature and liquid-solid ratio were set as 90 °C and 15 mL/g, respectively. As presented in Fig. 1B, the extraction yield of CRPs significantly increased from 15.76% to 22.19% along with the increase of extraction time from 40 to 70 min (P < 0.05).

13

ACCEPTED MANUSCRIPT However, the CRPs yield decreased when the extraction time was 80 and 90 min. The reason might be due to the hydrolysis of CRPs under the condition of long extraction time and certain temperature [28]. Therefore, the extraction time of 70 min was selected as the sufficient time to extract CRPs.

PT

3.1.3. Influence of extraction temperature on the yield of CRPs

RI

To evaluate the influence of extraction temperature (50, 60, 70, 80, 90 and 100 °C) on the yield

SC

of CRPs, the extraction time and liquid-solid ratio were maintained at 70 min and 15 mL/g, respectively. As presented in Fig. 1C, when the temperature increased from 50 to 90 °C, the yield of

NU

CRPs was raised from 14.81% to 22.25%, and the CRPs yield of 90 °C was significantly higher

MA

than those of 50, 60, 70 and 80 °C (P < 0.05). However, a further rise of temperature slightly lowered the extraction yield of CRPs compared to 90 °C (P > 0.05). That might be due to the

D

structural degradation of CRPs under too high extraction temperature [29]. Thus, the extraction

PT E

temperature of 90 °C was selected as the sufficient temperature to extract CRPs. 3.2. Optimization of extracting conditions by Box-Behnken design

CE

3.2.1. Prediction model and statistical analysis

AC

According to the single-factor experimental evaluation above, 3 variables and 3 levels were chosen to optimize the extraction condition of CRPs (Table 1). Based on the BBD, 17 runs were performed to investigate the extraction condition of CRPs. The experiment parameters and the CPRs yield (%) of the 17 runs were presented in Table 1. The CRPs yields were in the range of 16.15% to 22.75%. Multiple regression analysis was performed with the experiment values, and the response model Y was achieved by the second-order polynomial equation (5) as follow: Y (%)  254.56  3.96 X1  3.05 X 2  2.86 X 3  0.014 X1 X 2  0.021X1 X 3  0.005 X 2 X 3  0.030 X12  0.022 X 22  0.015 X 32

(5) 14

ACCEPTED MANUSCRIPT where Y is the CRPs yield; X1, X2 and X3 are the variables of liquid-solid ratio, extraction time and extraction temperature, respectively. F-test and ANOVA analysis were performed by BBD to estimate the significance and fitness of the regression model (Table 2). The results showed that the regression model for CRPs yield was

PT

significant (P < 0.05). The determination coefficient (R2) of the model was 0.9807, indicating that

RI

the agreement between the experiment data and predicted values was satisfactory. In addition, the

SC

adjusted determination (adj-R2) value (0.9560) represented most of the CPR yield variation could be predicted by the regression model. The coefficient of variation (C.V.) decides the reproducible

NU

character of the model, and the value should be lower than 5.00% [30]. The C.V. was 2.29% in the

MA

present study, suggesting that the standard deviation as a percentage of the mean for CRPs yield. Base on the experimental values, the lack of fit P-value was more than 0.05, indicating that the lack

D

of fit was insignificant relating to the pure error.

PT E

The significance of coefficients could be check by the P-values, and a smaller P-value indicated a more significant of the corresponding coefficient. As presented in Table 3, the linear coefficients

CE

(X1, X2 and X3), the cross-product coefficient (X1X3) and the quadratic term coefficients (X22 and X32)

AC

were found to be highly significant (P < 0.01). The cross-product coefficient (X1X2) and the quadratic term coefficient (X12) were found to be significant (P < 0.05). The other term coefficient (X2X3) was not significant (P > 0.05). The results indicated that liquid–solid ratio (X1), extraction time (X2), and extraction temperature (X3) were significant single parameters affecting the extraction yield of CRPs.

15

ACCEPTED MANUSCRIPT 3.2.2. Response surface plot The effects of individual variables and the interactions between two variables on the CRPs yield could be shown in the form of 3-dimensional (3D) response surface graphs and their corresponding 2-dimensional (2D) contour graphs. These graphical representations are visual interpretation of the

PT

regression model. Two test variables were described in the 3D and 2D surface graphs while another

RI

variable were set at the 0 level. The patterns (circular or elliptical) of the 2D contour graphs showed

SC

the statistical significance of the mutual interactions between two tested variables. The contour plot with circular shape means that the interaction between the two variables is not significant, while the

NU

contour plot of elliptical or saddle shapes suggests that the interaction between the two variables is

MA

significant [31]. As presented in Fig. 2, two interactions among the variables (liquid-solid ratio and extraction time, and liquid-solid ratio and extraction temperature) were significant (P < 0.05).

D

Nevertheless, the interaction between extraction time and extraction temperature was not significant

PT E

(P > 0.05).

3.2.3. Optimization of regression model

CE

Base on the Design-Expert software, the optimal conditions for the extraction of CRPs were

AC

liquid–solid ratio 14.68 mL/g, extraction time 73.70 min, and extraction temperature 95.13 °C, allowed a maximum predicted CRPs yield of 23.00%. Considering the convenience during actual operation, the actual conditions were modified slightly: liquid–solid ratio 15 mL/g, extraction time 74 min, and extraction temperature 95 °C. Three verify tests were performed and the extraction yield of CRPs was found to be 22.87±0.46 % (n=3), which was in good agreement with the predicted CRPs yield of 23.00%. Therefore, the regression model was adequate in predicting the optimum extraction conditions of CRPs.

16

ACCEPTED MANUSCRIPT 3.3. Physicochemical properties of CRPs obtained by different drying methods 3.3.1. Yield and chemical composition of CRPs Drying methods is very important due to the yields and bioactivities of polysaccharides are significantly influenced by the type of drying and drying temperature used. The yields and chemical

PT

composition of the three CRPs were listed in Table 3. Polysaccharides yields of HD-CRPs,

RI

VD-CRPs and FD-CRPs were 21.53%, 21.65% and 22.81%, respectively. The yield of FD-CRPs

SC

was significantly higher than those of HD-CRPs and VD-CRPs (P < 0.05). The bioactivities of polysaccharides are affected by many factors, such as their chemical

NU

composition and molecular structure [32]. Therefore, the contents of chemical composition

MA

including total polysaccharides, uronic acid, protein and sulfate radical in CRPs samples were analyzed and shown in Table 3. The contents of total polysaccharides, uronic acid, protein and

D

sulfate radical in HD-CRPs, VD-CRPs, and FD-CRPs were increased from 55.43% to 58.95%,

PT E

1.18% to 1.56%, 3.36% to 3.68%, and 1.51% to 1.67%, respectively. The yield and contents of total polysaccharides, uronic acid and sulfate radical of FD-CRPs were significantly higher than those of

CE

HD-CRPs and VD-CRPs (P < 0.05). These might due to the degeneration of the chemical

AC

constituents at the temperature of 50 °C during hot air drying and vacuum drying. It was reported that there was a significant correlation between the radical scavenging activity and the uronic acid content of tea polysaccharides [33]. 3.3.2. Moisture, pH and relative viscosity of CRPs The moisture contents, pH and relative viscosity of the three CRPs were shown in Table 3. There were no significant differences in moisture contents of HD-CRPs (10.53%), VD-CRPs (10.64%) and FD-CRPs (10.87%), as well as pH of HD-CRPs (7.13), VD-CRPs (7.13) and

17

ACCEPTED MANUSCRIPT FD-CRPs (7.14) (P > 0.05). The property of low moisture contents of CRPs is a good fit for large scale application in food industry. However, significant differences were found in relative viscosity of HD-CRPs (17.50), VD-CRPs (21.77) and FD-CRPs (33.82) (P < 0.05). The relative viscosity of FD-CRPs was significantly higher than those of HD-CRPs and VD-CRPs (P < 0.05).

PT

3.3.3. Solubility of CRPs

RI

The solubility of CRPs was shown in Fig. 3. The dissolved time for CRPs was gradually

SC

reduced as the temperature increasing. At the temperature of 20 °C, the time for FD-CRPs to dissolve was significantly lower than those of polysaccharides obtained by the other two drying

NU

methods (P < 0.05). The dissolved time of FD-CRPs was also shorter than those of HD-CRPs and

MA

VD-CRPs at 40, 60, 80 and 100 °C. The reason might be due to that the looser structure of FD-CRPs made it easier to combine with water during the process of re-dissolving.

D

3.3.4. Molecular weight distribution of CRPs

PT E

The application and bioactivity of polysaccharides are related to their chemical composition, structure, molecular weight and chain conformation. The chromatographic profiles (Fig. 4) showed

CE

the molecular weight distribution of CRPs. As shown in Fig. 4A, when CRPs was dried by hot air,

AC

there was one distinct group with the molecular weight of 7042.63 Da. In Fig. 4B, VD-CRPs showed two distinct groups with the molecular weights of 189.06 × 104 Da and 6202.02 Da, respectively. In Fig. 4C, when CRPs was freeze dried, molecular weight distribution showed one distinct group with the molecular weight of 5461.75 Da. There was an apparent difference in the molecular weight of CRPs dried with different methods. The reason might be due to the presence of intermolecular aggregation and the different drying conditions.The molecular weights of VD-CRPs and HD-CRPs were higher than FD-CRPs, which indicated that the polysaccharides molecules were

18

ACCEPTED MANUSCRIPT easy to aggregate at the high temperature condition. This might be due to the drying process that removed part of the hydration layer, which disrupted the polysaccharides structure and caused aggregation [34]. It was reported that drying methods could significantly affect the structure and the crystallinity of polysaccharides [10]. The X-ray diffraction curves of Hohenbuehelia serotina

PT

polysaccharides obtained from freeze vacuum-drying (FD-HSP) possessed the weakest intensity

RI

among the three polysaccharides dried by air-drying, hot air-drying and freeze vacuum-drying,

SC

suggesting that a lesser degree of crystallinity was existed in FD-HSP, the reason might be due to the higher temperature could accelerate the aggregation process of the polysaccharides [10]. The

NU

Bletilla striata polysaccharides obtained by vacuum-drying showed larger degree of crystallinity

MA

than polysaccharides dried with vacuum freeze-drying, which might be related to the slower decomposition of the former observed in thermal analysis and larger aggregation process of this

D

derivative [7]. The molecular weight of FD-CRPs was smaller than those of VD-CRPs and

PT E

HD-CRPs. It was reported that the polysaccharides with small molecular weight usually showed more significant biological activities in the body compared with the large ones, because the relative

CE

low molecular weight polysaccharides could freely pass through biological membranes without the

AC

coercion of the immune system including cell membrane and nuclear membrane [10]. 3.3.5. Monosaccharide composition of CRPs The monosaccharide analysis showed that HD-CRPs, VD-CRPs and FD-CRPs were composed of galacturonic acid, glucose, galactose and arabinose (Fig. 5). The molar ratios of galacturonic acid, glucose, galactose and arabinose in HD-CRPs were 1, 15.27, 3.41 and 2.37. VD-CRPs consisted of galacturonic acid, glucose, galactose and arabinose with the molar ratios of 1, 15.17, 3.42 and 2.71. FD-CRPs was composed of galacturonic acid, glucose, galactose and arabinose with the molar

19

ACCEPTED MANUSCRIPT ratios of 1, 15.05, 3.51 and 2.34. The monosaccharide analysis results indicating the homogeneity of the polysaccharides obtained from comfrey root by hot air, vacuum and freeze drying methods. 3.3.6. Complex formation with Congo Red of CRPs The bioactivities of polysaccharides are affected by their conformation [32]. The

PT

monosaccharide composition of the polysaccharides is one of the factors determining the

RI

conformation of polysaccharides [34]. The conformation information of polysaccharides can be

SC

simply and rapidly analyzed by the Congo red assay. A biopolymer can be formed between Congo red and polysaccharides with the triple-helix conformation and exhibit a large red shift in λmax

NU

compare to the Congo red solution. When the biopolymer is exposed in the alkaline condition of

MA

enough strength, the helix conformation of polysaccharides is destroyed and the λmax of the biopolymer declines. The influences of NaOH concentrations on the λ max of the Congo red-CRPs

D

complexes were shown in Fig. 6. No significant shift in λmax could be observed for the three CRPs

PT E

obtained by different drying methods, suggesting that there was no existence of triple-helix conformation in CRPs. By using complex formation with Congo red, Rout et al. [35] confirmed the

CE

existence of triple helix in β-D-glucan. Mao et al. [36] found that a heteropolysaccharide could not

AC

form a triple-helix structure. CRPs were heteropolysaccharide that consisted of four types of monosaccharides.

3.4. Antioxidant activities of CRPs obtained by different drying methods 3.4.1. Scavenging activity on DPPH radical The scavenging activity on DPPH radical assay has been widely used to determine the antioxidant capacity of samples due to its easy accessibility and the stability of DPPH radical [37]. The scavenging abilities of HD-CRPs, VD-CRPs and FD-CRPs on DPPH radical were shown in

20

ACCEPTED MANUSCRIPT Fig. 7A. The results indicated the DPPH radical scavenging activities of the three CRPs increased quadratically (P＜0.05) as the concentration of polysaccharides ranged from 0.05 to1 mg/mL. At each concentration, FD-CRPs showed higher scavenging activity against DPPH radical compared to HD-CRPs and VD-CRPs, and there were significant differences of DPPH radical scavenging

PT

abilities between FD-CRPs and HD-CRPs when the determined concentration of polysaccharides

RI

was more than 1 mg/mL (P＜0.05). Taking the differences among the chemical composition

SC

(contents of total polysaccharides, uronic acid and sulfate radical) of HD-CRPs, VD-CRPs and FD-CRPs into consideration, a conclusion can be made that different drying methods indeed

NU

affected the DPPH radical scavenging activities of polysaccharides. The maximum DPPH radical

MA

scavenging activity of FD-CRPs reached 85.83% of vitamin C, which indicated that it was possible to prepare CRPs with strong DPPH radical scavenging activity through freeze drying technology.

D

3.4.2. Scavenging activity on ABTS radical

PT E

The ABTS scavenging activity assay was shown to be simple and quick in operation, and has been extensively used to evaluate the antioxidant activity of biological samples [38]. The effects of

CE

drying methods on ABTS radical scavenging ability of CRPs were shown in Fig. 7B. The activities

AC

of the three CRPs toward ABTS radical increased quadratically (P＜0.05) with the CRPs levels increasing. The scavenging powers of FD-CRPs were significantly higher than those of HD-CRPs at the concentration from 0.25 to 3.5 mg/mL (P＜0.05). The reason might be due to the higher polysaccharides yield and the higher contents of total polysaccharides, uronic acid and sulfate radical in FD-CRPs. It was reported that the hydroxyl groups in polysaccharides played an important role in ABTS radical scavenging activity [10]. However, oxidation reaction of the hydroxyl groups could easily occur under the aerobic condition. The polysaccharides were dried

21

ACCEPTED MANUSCRIPT under aerobic condition during hot air drying. Therefore, the reason that HD-CRPs possessed the lowest ABTS radical scavenging activity among the three CRPs might be related to the oxidation of the hydroxyl groups. No significant difference between HD-CRPs and VD-CRPs on the scavenging activity of ABTS radical was found in the present study (P > 0.05). At the concentration of 4

PT

mg/mL, the scavenging activity of FD-CRPs was 98.72±2.36%, which suggested that FD-CRPs

RI

had significant scavenging activity on ABTS radical.

SC

3.4.3. Ferric reducing power

Ferric reducing power could be recognized as a significant symbol to evaluate the potential

NU

antioxidant activity of natural products [39]. The reducing capability of the three CRPs was

MA

presented in Fig. 7C. The reducing powers of the three CRPs showed well linear connection (R2 > 0.99, P < 0.05) to the test concentration. The reducing power of FD-CRPs was significantly higher

D

than that of HD-CRPs at each concentration point (P＜0.05). There were no significant differences

PT E

of reducing power between HD-CRPs and VD-CRPs (P > 0.05). In the concentration range from 1 to 6 mg/mL, the reducing power of FD-CRPs increased from 0.186 to 0.835, while the reducing

CE

power of vitamin C was 1.712 at 1 mg/mL. The three CRPs showed significant lower reducing

AC

power than vitamin C at the doses of all detecting concentration (P < 0.05). However, it was reported that the reducing power of polysaccharides from garlic [40] and Hohenbuehelia serotina [10] were about 0.500 at the concentration of 10 mg/mL. These results indicated that FD-CRPs had potential antioxidant activities of reducing power. In general, the radical scavenging activities of polysaccharides were due to their electron or hydrogen donating abilities. The uronic acid groups existing in the polysaccharides could trigger the hydrogen atom of the anomeric carbon [41]. Therefore, the highest radical scavenging activities of

22

ACCEPTED MANUSCRIPT FD-CRPs among the three CRPs in this study might be related to its higher content of uronic acid in the chemical composition. It was reported that the ferric reducing powers of polysaccharides were related to their molecular weights [15]. The polysaccharides with smaller molecular weights showed better ferric reducing powers than the polysaccharides with larger molecular weights due to

PT

the more exposed reducing ends in the former kinds of polysaccharides [42]. Therefore, the highest

RI

ferric reducing power of FD-CRPs might be related to its lower molecular weight among the three

SC

CRPs. 4. Conclusions

NU

RSM was performed to optimize the extractive conditions of the polysaccharides from comfrey

MA

root. The results showed that the 3 test variables including liquid-solid ratio, extraction time and extraction temperature had significant influences on the extraction yield of CRPs (P < 0.05).

D

Quadratic regression model was used to predict responses and it had satisfactory fit to the actual

PT E

values. A maximum yield of CRPs of 22.87 % was obtained under liquid–solid ratio 15 mL/g, extraction time 74 min, and extraction temperature 95 °C. In addition, different drying methods had

CE

significant effects on the antioxidant activities and physicochemical properties of CRPs such as the

AC

chemical composition (contents of total polysaccharides, uronic acid and sulfate radical), relative viscosity and solubility. Compared with the CRPs obtained by hot air-drying and vacuum drying, FD-CRPs presented significant higher ferric reducing power and scavenging activities against DPPH and ABTS radicals (P < 0.05). The results suggested that freeze drying method served as a good method for the preparation of polysaccharides from comfrey root. Further works on the antioxidant activity mechanism of CRPs are in progress.

23

ACCEPTED MANUSCRIPT Conflict of interest The authors report no declarations of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31601972)

AC

CE

PT E

D

MA

NU

SC

RI

PT

and the Project funded by China Postdoctoral Science Foundation (No. 2017M621224).

24

ACCEPTED MANUSCRIPT References [1] D.B. Smith, B.H. Jacobson, Effect of a blend of comfrey root extract (Symphytum officinale L.) and tannic acid creams in the treatment of osteoarthritis of the knee: randomized, placebo-controlled, double-blind, multiclinical trials, J. Chiropr. Med. 10 (2011) 147−156.

PT

[2] B. Grube, J. Grünwald, L. Krug, C. Staiger, Efficacy of a comfrey root (Symphyti offic. radix)

RI

extract ointment in the treatment of patients with painful osteoarthritis of the knee: Results of a

SC

double-blind, randomised, bicenter, placebo-controlled trial, Phytomedicine 14 (2007) 2−10. [3] P. Andres, R. Brenneisen, J.T. Clerc, Relating antiphlogistic efficacy of dermatics containing

NU

extracts of symphytum-officinale to chemical profiles, Planta Med. 55 (1989) 643–644.

MA

[4] M.C. Kang, S.Y. Kim, Y.T. Kim, E.A. Kim, S.H. Lee, S.C. Ko, W.A.J.P. Wijesinghe, K.W. Samarakoon, Y.S. Kim, In vitro and in vivo antioxidant activities of polysaccharide purified

D

from aloe vera (Aloe barbadensis) gel, Carbohydr. Polym. 99 (2014) 365–371.

PT E

[5] L.J. Han, Y.R. Suo, Y.J. Yang, J. Meng, N. Hu, Optimization, characterization, and biological activity of polysaccharides from Berberis dasystachya Maxim, Int. J. Biol. Macromol. 85 (2016)

CE

655–666.

AC

[6] Y.Q. Jing, Y.Z. Gao, W.F. Wang, Y.Y. Cheng, P. Lu, C. Ma, Y.H. Hu, Optimization of the extraction of polysaccharides from tobacco waste and their biological activities, Int. J. Biol. Macromol. 91 (2016) 188–197. [7] L.S. Kong, L. Yu, T. Feng, X.J. Yin, T.J. Liu, L. Dong, Physicochemical characterization of the polysaccharide from Bletilla striata: Effect of drying method, Carbohydr. Polym. 125 (2015) 1–8. [8] L.P. Fan, J.W. Li, K.Q. Deng, L.Z. Ai, Effects of drying methods on the antioxidant activities of

25

ACCEPTED MANUSCRIPT polysaccharides extracted from Ganoderma lucidum, Carbohydr. Polym. 87 (2012) 1849–1854. [9] Q.S. Zhao, B.T. Dong, J.J. Chen, B. Zhao, X.D. Wang, L.W. Wang, S.H. Zha, Y.C. Wang, J. H. Zhang, Y.L. Wang, Effect of drying methods on physicochemical properties and antioxidant activities of wolfberry (Lycium barbarum) polysaccharide, Carbohydr. Polym. 127 (2015)

PT

176–181.

RI

[10] X.Y. Li, L. Wang, Y. Wang, Z.H. Xiong, Effect of drying method on physicochemical

SC

properties and antioxidant activities of Hohenbuehelia serotina polysaccharides, Process Biochem. 51 (2016) 1100–1108.

NU

[11] Muriel P, Rivera-Espinoza Y. Beneficial drugs for liver diseases, J. Appl. Toxicol. 28 (2008)

MA

93–103.

[12] C.C. Zhao, X. Li, J. Miao, S.S. Jing, X.J. Li, L.Q. Huang, W.Y. Gao, The effect of different

D

extraction techniques on property and bioactivity of polysaccharide from Dioscorea hemsleyi,

PT E

Int. J. Biol. Macromol. 102 (2017) 847–856. [13] R.F. Yang, C. Zhao, X. Chen, S.W. Chan, J. Y. Wu, Chemical properties and bioactivities of

CE

Goji (Lycium barbarum) polysaccharides extracted by different methods, J. Funct. Foods 17

AC

(2015) 903–909.

[14] Y.J. Yan, X. Li, M.J. Wan, J.P. Chen, S.J. Li, M. Cao, et al., Effect of extraction methods on property and bioactivity of water-soluble polysaccharides from Amomum villosum, Carbohydr. Polym. 117 (2015) 632–635. [15] W. Wang, X.Q. Wang, H. Ye, B. Hu, L. Zhou, S. Jabbar, X.X. Zeng, W.B. Shen, Optimization of extraction, characterization and antioxidant activity of polysaccharides from Brassica rapa L, Int. J. Biol. Macromol. 82(2016) 979–988.

26

ACCEPTED MANUSCRIPT [16] M. Dubois, K.A. Gilles, J.K. Hamilton, P. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (1956) 350−356. [17] N. Blumenkrantz, G. Asboe-Hansen, New method for quantitative determination of uronic acids, Anal. Chem. 54 (1973) 484–489.

PT

[18] M.M. Bradford, A rapid and sensitive method for the quantification of microgram quantities of

RI

protein, utilizing the principle of protein–dye binding, Anal. Chem. 72 (1976) 248–254.

polysaccharides, Biochem. J. 84 (1962) 106–110.

SC

[19] K.S. Doigson, R.G. Price, A note on the determination of the ester sulfate content of sulfated

NU

[20] Y.Y. Chai, M. Zhao, Purification, characterization and anti-proliferation activities of

MA

polysaccharides extracted from Viscum coloratum (Kom.) Nakai, Carbohydr. Polym. 149 (2016) 121–130.

D

[21] Z.P. Ye , W. Wang, Q.X. Yuan, H. Ye, Y. Sun, H.C. Zhang, et al., Box–Behnken design for

PT E

extraction optimization, characterization and in vitro antioxidant activity of Cicer arietinum L. hull polysaccharides, Carbohydr. Polym. 147 (2016) 354–364.

CE

[22] C.S. Kumar, M. Sivakumar, K. Ruckmani, Microwave-assisted extraction of polysaccharides

682–693.

AC

from Cyphomandra betacea and its biological activities, Int. J. Biol. Macromol. 92 (2016)

[23] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Rad. Bio. Med. 26 (1999) 1231–1237.

27

ACCEPTED MANUSCRIPT [24] Y.S. Jing, J.H. Zhu, T. Liu, S.X. Bi, X.J. Hu, Z.Y. Chen, L.Y. Song, W.J. Lv, R.M. Yu, Structural characterization and biological activities of a novel polysaccharide from cultured Cordycep smilitaris and its sulfated derivative, J. Agr. Food Chem. 63 (2015) 3464–3471. [25] L.H. Tan, D. Zhang, B. Yu, S.P. Zhao, J.W. Wang, L. Yao, W.G. Cao, Antioxidant activity and

PT

optimization of extraction of polysaccharide from the roots of Dipsacus asperoides, Int. J. Biol.

RI

Macromol. 81 (2015) 332–339.

SC

[26] Q. Zheng, D.Y. Ren, N.N. Yang, X.B. Yang, Optimization for ultrasound-assisted extraction of polysaccharides with chemical composition and antioxidant activity from the Artemisia

NU

sphaerocephala Krasch seeds, Int. J. Biol. Macromol. 91 (2016) 856–866.

MA

[27] Z. Ying, X.X. Han, J.R. Li, Ultrasound-assisted extraction of polysaccharides from mulberry leaves, Food Chem. 127 (2011) 1273–1279.

D

[28] J.C. Liu, S. Miao, X.C. Wen, Y.X. Sun, Optimization of polysaccharides (ABP) extraction from

PT E

the fruiting bodies of Agaricus blazei Murill using response surface methodology (RSM), Carbohydr. Polym. 78 (2009) 704–709.

CE

[29] K.Thirugnanasambandham, V. Sivakumar, J.P. Maran, Microwave-assisted extraction of

AC

polysaccharides from mulberry leaves, Int. J. Biol. Macromol. 72 (2015) 1–5. [30] Y. Zhang, H.X. Wang, P. Wang, C.Y. Ma, G.H. He, M. Ramim, T. Rahman, Optimization of PEG-based extraction of polysaccharides from Dendrobium nobile Lindl. and bioactivity study, Int. J. Biol. Macromol. 92 (2016) 1057–1066. [31] Z.G. Liu, J. Dang, Q.L. Wang, M.F. Yu, L. Jiang, L.J. Mei, Y. Shao, Y.D. Tao, Optimization of polysaccharides from Lycium ruthenicum fruit using RSM and its anti-oxidant activity, Int. J. Biol. Macromol. 61 (2013) 127–134.

28

ACCEPTED MANUSCRIPT [32] Z. Wu, Effect of different drying methods on chemical composition and bioactivity of finger citron polysaccharides, Int. J. Biol. Macromol. 76 (2015) 218–223. [33] H.X. Chen, M. Zhang, Z.S. Qu, B.J. Xie, Antioxidant activities of different fractions of polysaccharide conjugates from green tea (Camellia Sinensis), Food Chem. 106 (2008)

PT

559–563.

RI

[34] L.S. Ma, H.X. Chen, W.C. Zhu, Z.S. Wang, Effect of different drying methods on

SC

physicochemical properties and antioxidant activities of polysaccharides extracted from mushroom Inonotus obliquus, Food Res. Int. 50 (2013) 633–640.

NU

[35] D. Rout, S. Mondal, I. Chakraborty, S.S. Islam, The structure and conformation of a

MA

water-insoluble (1→3)-, (1→6)-β-D-glucan from the fruiting bodies of Pleurotus florida, Carbohyd. Res. 343 (2008), 982–987.

D

[36] C.F. Mao, M.C. Hsu, W.H. Hwang, Physicochemical characterization of grifolan: Thixotropic

PT E

properties and complex formation with Congo Red, Carbohydr. Polym. 68 (2007), 502–510. [37] S.L. Xiong, A.L. Li, N. Huang, F. Lu, D.B. Hou, Antioxidant and immune regulatory activity

CE

of different polysaccharide fractions from tuber of Ophiopogon japonicas, Carbohydr. Polym.

AC

86 (2011) 1273–1280.

[38] L.C. Wu, H.W. Hsu, Y.C. Chen, C.C. Chiu, Y.I. Lin, J.A. Ho, Antioxidant and antiproliferative activities of red pitaya, Food Chem. 95 (2006) 319–327. [39] Y.Q. Xu, F. Cai, Z.Y. Yu, L. Zhang, X.G. Li, Y. Yang, G.J. Liu, Optimisation of pressurised water extraction of polysaccharides from blackcurrant and its antioxidant activity, Food Chem. 194 (2016) 650–658.

29

ACCEPTED MANUSCRIPT [40] S.K. Pan, S.J. Wu, Cellulase-assisted extraction and antioxidant activity of the polysaccharides from garlic, Carbohydr. Polym. 111 (2014) 606–609. [41] J.L. Wang, H.Y. Guo, J. Zhang, X.F. Wang, B.T. Zhao, J. Yao, Y.P. Wang, Sulfated modification, characterization and structure-antioxidant relationships of Artemisia sphaerocephala

PT

polysaccharides, Carbohydr. Polym. 81 (2010) 897–905.

RI

[42] H.M. Qi, T.T. Zhao, Q.B. Zhang, Z.E. Li, Z.Q. Zhao, R. Xing, Antioxidant activity of different

SC

molecular weight sulfated polysaccharides from Ulva pertusa Kjellm (Chlorophyta), J. Appl.

AC

CE

PT E

D

MA

NU

Phycol. 17 (2005) 527–534.

30

ACCEPTED MANUSCRIPT

Table 1 Box-Behnken experimental design and results of CRPs yields. CRPs yields (%) Liquid–solid ratio (X1)

Extraction time (X2)

Extraction temperature

Run (min)

Predicted

value

value

(X3) (°C)

0(90)

16.64

16.64

2

1(20)

-1(60)

0(90)

19.42

19.52

3

-1(10)

1(80)

0(90)

20.90

20.81

4

1(20)

1(80)

0(90)

20.81

20.81

5

-1(10)

0(70)

17.37

17.05

6

1(20)

0(70)

-1(80)

21.03

20.62

7

-1(10)

0(70)

1(100)

21.41

21.83

8

1(20)

0(70)

1(100)

20.81

21.13

9

0(15)

-1(60)

-1(80)

16.15

16.46

10

0(15)

1(80)

-1(80)

17.79

18.2

11

0(15)

-1(60)

1(100)

18.53

18.12

12

0(15)

1(80)

1(100)

22.15

21.84

13

0(15)

0(70)

0(90)

22.75

22.43

14

0(15)

0(70)

0(90)

22.25

22.43

15

0(15)

0(70)

0(90)

22.02

22.43

16

0(15)

0(70)

0(90)

22.39

22.43

17

0(15)

0(70)

0(90)

22.74

22.43

D

MA

-1(80)

PT E CE AC

RI

-1(60)

SC

-1(10)

NU

1

PT

(mL/g)

Actual

31

ACCEPTED MANUSCRIPT

Table 2 F-test and ANOVA analysis for the response surface quadratic model. Sum of squares

DF

Mean square

F value

P value

Model

77.13

9

8.57

39.58

<0.0001**

X1

4.14

1

4.14

19.13

0.0033**

X2

14.87

1

14.87

X3

13.98

1

13.98

X1X2

2.06

1

X1X3

4.56

1

X2X3

0.98

1

X12

2.33

1

X22

21.19

1

X32

9.88

Residual

1.52

Lack of fit

1.11

Pure error

0.4

R2

78.64

64.58

<0.0001**

2.06

9.53

0.0176*

4.56

21.07

0.0025**

0.98

4.54

0.0707

2.33

10.74

0.0135*

21.19

97.86

<0.0001**

1

9.88

45.65

0.0003**

7

0.22

3

0.37

3.69

0.1200

4

0.1

D

MA

NU

SC

RI

<0.0001**

16

0.9807

Adj R2

0.9560

C.V%

2.29

*

68.69

PT E

CE

AC

Cor total

PT

Source

P values < 0.05 were considered to be significant.

**

P values < 0.01 were considered to be highly significant.

32

ACCEPTED MANUSCRIPT

Table 3 The yields and chemical composition of CRPs obtained by different drying methods. HD-CRPs

VD-CRPs

FD-CRPs

Polysaccharides yield (%)

21.53 ± 0.36 b

21.65 ± 0.22 b

22.81 ± 0.31 a

Total polysaccharides content (%)

55.43 ± 0.38 c

56.90 ± 0.30 b

58.95 ± 0.82 a

Protein content (%)

3.36 ± 0.09 b

3.48 ± 0.20 ab

3.68 ± 0.14 a

Uronic acid content (%)

1.18 ± 0.07 b

Sulfate radical content (%)

1.51 ± 0.05 b

Moisture content

10.53 ± 0.82

pH

7.13 ± 0.03

1.56 ± 0.10 a

1.55 ± 0.04 b

1.67 ± 0.04 a

10.64 ± 0.70

10.87 ± 0.92

7.13 ± 0.04

7.14 ± 0.06

21.77 ± 0.06 b

33.82 ± 1.41 a

SC

1.33 ± 0.03 b

NU

MA

17.50 ± 0.19 c

Relative viscosity

RI

PT

Samples

D

Values are expressed as mean ± standard deviation (n = 3). Means within a row with different superscripts differ

AC

CE

PT E

significantly (P＜0.05).

33

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7