Comparison of different extraction methods for polysaccharides from Crataegus pinnatifida Bunge

Comparison of different extraction methods for polysaccharides from Crataegus pinnatifida Bunge

Journal Pre-proof Comparison of different extraction methods for polysaccharides from Crataegus pinnatifida Bunge Xing Chen, Hongbing Zhang, Wenqing ...

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Journal Pre-proof Comparison of different extraction methods for polysaccharides from Crataegus pinnatifida Bunge

Xing Chen, Hongbing Zhang, Wenqing Du, Liying Qian, Ying Xu, Yange Huang, Qingping Xiong, Hailun Li, Jun Yuan PII:

S0141-8130(19)37875-4

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.056

Reference:

BIOMAC 13837

To appear in:

International Journal of Biological Macromolecules

Received date:

29 September 2019

Revised date:

5 November 2019

Accepted date:

7 November 2019

Please cite this article as: X. Chen, H. Zhang, W. Du, et al., Comparison of different extraction methods for polysaccharides from Crataegus pinnatifida Bunge, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/ j.ijbiomac.2019.11.056

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© 2019 Published by Elsevier.

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Comparison of different extraction methods for polysaccharides from Crataegus pinnatifida Bunge Xing Chena,1, Hongbing Zhangb,1, Wenqing Dua, Liying Qiana, Ying Xua,c, Yange Huanga, Qingping Xionga, Hailun Lid*, Jun Yuana,* a

Jiangsu Key Laboratory of Regional Resource Exploitation and Medicinal Research,

b

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Huaiyin Institute of Technology, Huai'an 223003, China Pharmaceutical Department, Traditional Chinese Medicine Hospital of Zaozhuang,

National & Local Joint Engineering Research Center for Mineral Salt Deep

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c

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Zaozhuang, 277000, Shandong, PR China

Huai'an Second People's Hospital, Huai'an 223002, Jiangsu, PR China

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Utilization, Huaiyin Institute of Technology, Huai'an 223003, Jiangsu, PR China

* 1

Corresponding author: [email protected] (H. Li); [email protected] (J. Yuan). These authors contributed equally to this work.

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Abstract In this study, different extraction methods of polysaccharides from Crataegus pinnatifida Bunge (CPP) were compared by studying the extraction yield, structural characteristics and antioxidant activities. Firstly, polysaccharides were obtained using hot water extraction (CPPh), ultrasound assisted extraction (CPPu), enzyme assisted

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extraction (CPPe) and enzyme-ultrasound assisted extraction (CPPc), respectively. Meanwhile, the optimum extraction conditions of enzyme-ultrasonic assisted

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extraction were determined by response surface method (RSM). The extraction yields,

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structural characteristics and antioxidant activities were investigated and compared by

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visual photos, gas chromatography, ultraviolet-visible and Fourier-transform infrared

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spectroscopy. The results clearly showed that enzyme-ultrasonic assisted extraction

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possessed the highest extraction yield (10.39±0.04%). The molecular weight of CPPh was the highest while the other polysaccharides had no significant difference. Besides,

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the monosaccharide composition of CPPc, CPPh, CPPu and CPPe were similar but the molar percentages of monosaccharide were different. Finally, the results of antioxidant activities showed that CPPc exhibited the highest scavenging effect of superoxide radical and lipids inhibiting ability. In summary, enzyme-ultrasonic assisted extraction was a high-efficient and low-energy consumption method for CPP extraction. Keywords Polysaccharides from Crataegus pinnatifida Bunge; extraction methods; structural characteristics; antioxidant activity

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1 Introduction Polysaccharide is a kind of macro molecular carbohydrates composed of several monosaccharide units, and widely distributed in plants, algae, bacteria and animals. Studies on polysaccharide have been showed that it was the active component for lipid-decreasing [1] anti-oxidant [2], anti-tumor [3], and anti-inflammatory [4]

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properties. Recently, polysaccharides, extracted from plants, are favored by more and more researchers because of their wide sources and low toxicity.

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Crataegus pinnatifida Bunge is a member of Rosaceae family and widely

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distributed in Europe, East Asia and North America [5]. The fruit is not only a

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common edible fruit, but also a medicinal material with a long history in China. C.

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pinnatifida has drawn rising attention due to its active components and biological

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activities. Many studies have demonstrated its remarkable efficacy for diet-induced indigestion [5], antioxidant [6] and antiproliferative [7] activities. There are many

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bioactive ingredients in C. pinnatifida, such as flavonoids, polysaccharides and anthocyanin [8]. Among them, polysaccharides stand out for its rich contents and significant pharmacological activity. A suitable extraction method is important for extraction yield, structural properties and biological activities [9]. The extraction of plant polysaccharides required the breakage of the cell wall to improve the extraction efficiency. However, traditional hot water extraction could only do little damage to the cell walls. Ultrasound assisted extraction and enzyme assisted extraction could hydrolyze or mechanically destroy the cell wall [10], but the structures of polysaccharides could be

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destroyed by the rigorous extraction conditions of ultrasound assisted extraction [11]. In recent years, the use of enzymes to extract plant polysaccharides has been considered as a suitable method for its mildness and high effectiveness. In order to improve the extraction efficiency, enzyme assisted extraction was often combined with other methods, such as enzyme-ultrasound assisted extraction.

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Therefore, the purpose of this study was to evaluate the effects of different extraction methods on extraction yield, structural characteristics and antioxidant

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activities of polysaccharides from Crataegus pinnatifida Bunge (CPP). Firstly, on the

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basis of references and optimization of enzyme-ultrasound assisted extraction, CPP

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were extracted by hot water extraction (CPPh), ultrasound assisted extraction (CPPu),

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enzyme assisted extraction (CPPe) and enzyme-ultrasound assisted extraction (CPPc).

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Then, structural characteristics of these polysaccharides were investigated and compared by visual photos, gas chromatography (GC), high performance gel

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permeation chromatograph (HPGPC), ultraviolet-visible (UV) and Fourier-transform infrared (FT-IR) spectroscopy. Finally, the antioxidant activities, including the reducing power, ABTS+, DPPH and superoxide radical scavenging activity and lipid peroxidation inhibition activity, were determined to screen the four extraction methods. 2 Materials and Methods 2.1 Materials and reagents Hawthorn (Crataegus pinnatifida Bunge) was bought from the fruit market (Shandong, China) and identified by Dr. Qingping Xiong from Huaiyin Institute of

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Technology. Standard monosaccharides were bought from Sigma Chemical Co., Ltd. (St. Louis, MO, USA). Sodium chloride (NaCl) was purchased from Shanghai Jiuyi Chemical Reagent Co., Ltd. (Shanghai, China) and all other inorganic reagents of analytical grade were obtained from Nanjing Chemical Regents Co., Ltd. (Nanjing, China).

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2.2 Different extraction methods of CPP 2.2.1 Preparation of raw material

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Fresh C. pinnatifida was washed and cleaned to remove impurities. After pitted

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and cut into pieces, the fruit was dried at 60℃ until the water contents were below 12%

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according to the China Pharmacopeia (2010). Then, the flesh of C. pinnatifida was

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smashed using a high-speed disintegrator and screened to obtain the pre-treated

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powder (particle size: 0.25 mm).

2.2.2 Enzyme-ultrasound assisted extraction

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The dried powder was mixed with distilled water at a proper ratio of liquid-material, and a certain amount of cellulase was added to the solution subsequently. The mixture was heated at designated pH value, time, and temperature for in a water bath enzymatic reaction. Then the mixture was ultrasonic treated for a certain time. After extraction, the extract was centrifuged at 8000 rpm for 10 min. Finally, the supernatants were concentrated and lyophilized to obtain the crude polysaccharides of C. pinnatifida (CPPc). The contents of total carbohydrate were determined by the phenol-sulfuric acid method [12] with some modifications. The extraction yields of crude polysaccharides are calculated as follows:

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Polysaccharides yields (%) 

Polysaccharides contents of extraction (g) 100 Powder weight (g)

(Eq.1)

PH value, ultrasonic time, liquid-material ratio, enzymolysis time, enzymolysis temperature and enzyme amounts were selected as variables for single factor experiment, with extraction yields as dependent variables. On the basis of single factor experiment, a BBD-RSM test with three independent variables and three levels

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was used to evaluate the effects of main experimental factors on extraction yields of

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CPPc. The liquid-material ratio (X1), enzymolysis temperature (X2) and enzymolysis

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time (X3) were set as independent variables, while the yields of polysaccharides were

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taken as the response. The ranges of three factors were shown in Table 1, while each

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level was coded as +1, 0 and -1 for high value, center point and low value,

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respectively.

Table 1

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2.2.3 Hot water extraction

The extraction of CPP was carried out according to the method of Shi [13] with some modifications. The pre-treated powder (5 g) was mixed with distilled water (100 mL) and extracted under continuous stirring for 2 h at 90℃. Then, the solution was centrifuged at 4000 rpm for 10 min. After three runs of the repeated extraction, the supernatants were collected and then concentrated to 20 mL by a rotary evaporator. Three times volume of ethanol were added into the solution for the alcohol precipitation of the polysaccharides. The polysaccharides were dissolved in hot water and freeze-dried to obtain the crude polysaccharides of C. pinnatifida (CPPh). 2.2.4 Ultrasound assisted extraction

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The crude polysaccharides from C. pinnatifida were obtained based on ultrasound extraction according to Zhu [14] with some modifications. The powder (10 g) of C. pinnatifida was extracted with distilled water (200 mL) at 60℃ for 60 min using an ultrasonic cleaning apparatus (Geneng, G-100S, Guangzhou). The filtrates were centrifuged (4000 rpm, 10 min) and collected. After adding three volumes of

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ethanol, the supernatants were precipitated and the precipitates were collected. The polysaccharides were dissolved in proper amount of hot water and freeze-dried to

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obtain the crude polysaccharides of C. pinnatifida (CPPu).

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2.2.5 Enzyme assisted extraction

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Enzyme assisted extraction of CPP was performed by the method of Li [15] with

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some modifications. Briefly, 10 g flesh powder with 300 mL distilled water was

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mixed with cellulase (powder: cellulase=5% w/v). The enzymolysis reaction was carried out at 55℃ for 90 min. After centrifuged, the supernatant was collected and

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concentrated to 50 mL. Three times volume of ethanol were added into the concentrated solution and the precipitates were lyophilized to obtain the crude polysaccharides of C. pinnatifida (CPPe). 2.3 Comparison of structure 2.3.1 Determination of UV-vis and IR spectra CPP obtained by four different methods (10 mg) were dissolved in distilled water (100 mL), respectively. After phenol-sulfuric acid staining, the visual photographs were taken. Then, the UV-vis spectra of these sample solutions were determined on a UV-2450 spectrophotometer (Shimadzu Co., Kyoto, Japan).

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1 mg of the dried crude polysaccharides (CPPh CPPu, CPPe and CPPc) was mixed with 100 mg dried KBr, respectively, to prepare the tablet for IR experiment. The IR spectra was recoded at the wavenumber range of 4000-400 cm-1 by a Nicolet FT-IR spectrophotometer (Thermo Co., USA). 2.3.2 Determination of monosaccharide compositions

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The monosaccharide compositions of different crude polysaccharides were determined based on the method in our previous study with some modifications [16].

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Briefly, 5 mg sample was hydrolyzed with 4 mL trifluoroacetic acid (2 M) at 120℃

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for 2 h and the redundant TFA was removed with vacuum evaporation. Then,

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methanol (2 mL) was added in the sample and heated to dryness. After the hydrolyzed

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product was dissolved in pyridine (0.6 mL), hydroxylamine hydrochloride (10 mg)

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and inositol (5 mg) were added. The mixture was reacted in a water bath at 90℃ for 40 min, and acetylated with acetic anhydride (1 mL) for 30 min. A GC instrument

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(Agilent, 7890A, Santa Clara, CA, USA) with a fused silica capillary column (0.25 μm, 30 m × 0.32 mm) was used to determine the acetylated derivatives. The gas chromatographic conditions were performed referring to our previous study [16]. 2.3.3 Determination of molecular weight The molecular weights of CPPc, CPPh, CPPe and CPPu were analyzed on a high performance gel permeation chromatograph (HPGPC, Agilent 1260, USA), which was equipped with a TSK-GEL G3000 SWXL gel-filtration chromatographic column (7.5×300 mm, 0.5 μm, Tosoh Corp., Japan) and a refractive index detector (RID). The sample was filtered and the injection volume was 10 μL. The eluent was the

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phosphate buffer (0.01 M, pH 6.8) containing 0.1 mol/L Na2SO4 solution and the flow rate was 0.8 mL/min. Calibration curves were obtained from dextran with different molecular weight (3.62, 12.6, 70.8, 126, 289 and 489 kDa). 2.4 Comparison of antioxidant activity The antioxidant activities of CPPc, CPPh, CPPe and CPPu were assayed and

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compared. Briefly, the ABTS+, superoxide and DPPH radical scavenging activity were examined based on our previous method with some modifications [17-19]. The

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determination of lipid peroxidation inhibition activity and reducing power of these

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polysaccharides was according to our previous reports [19, 20]. In this paper, IC50

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value was used to evaluate the antioxidant activity of each polysaccharide.

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2.5 Statistical analysis

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All the data above were presented as a mean value, while each experiment was run in parallel for three times. The Design-Expert software version 11 was used for

when p<0.5.

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the experimental design. The differences of statistics were considered as significant

3 Results and discussion

3.1 Single-factor experimental analysis 3.1.1 Effect of pH value on the yields of CPPc The effects of different pH values (3, 4, 5, 6 and 7) on the yields of CPPc were displayed when the other factors remained constant. As shown in Fig.1(A), the contents of crude polysaccharides had an increase tendency with the pH value from 3 to 5 and reached its maximum at 5. Fig.1(a) showed that the yield of CPPc was less

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affected by pH value with the highest one (8.09%) at pH 5 and the lowest one (7.46%) at pH 3. That's could be due to the fact that the enzyme activity might be inhibited with too high or too low pH value, and pH 5 was chosen as the best conditions for following experiment [21]. 3.1.2 Effect of ultrasonic time on the yields of CPPc

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The extraction yields of CPPc under different ultrasonic time (10, 20, 30, 40 and 50 min) were displayed when the other factors were fixed. As shown in Fig.1(B),

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when ultrasonic time was 30 min, the contents of crude polysaccharides were the

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highest due to the darkest solution color and largest absorbance value. The ultrasonic

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time showed a positive effect on the yields of CPPc when it increased from 10 to 30

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min (Fig.1(b)). A decrease in extraction yield was also observed after the further

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increase of ultrasonic time. This might because that the long ultrasonic time could lead to the destruction of the product structure, hence the reduction of the extraction

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yield. Therefore, the optimal ultrasonic time was 30 min. 3.1.3 Effect of liquid-material ratio on the yields of CPPc To study the effects of different liquid-material ratio (15:1, 20:1, 25:1, 30:1 and 35:1 mL/g) on the extraction yields of CPPc, experiments were performed when the other factors remained constant. Fig.1(C) showed that with the darkest solution color and largest absorbance value, the solution at liquid-material ratio 30:1 mL/g had the highest CPPc contents. As shown in Fig.1(c), although the extraction yield increased as liquid-material ratio prolonged, the increase was not significant when the liquid-material ratio exceeded 30:1 mL/g. The extraction yield continued to increase

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from 7.27% to 10.51% with the increasing liquid-material ratio from 15:1 to 30:1 mL/g. This might be because high ratio of liquid to material, resulting in the increase of the concentration difference between plant cells and solvent and the diffusion speed of solute. Therefore, 30:1 mL/g was chosen for the following experiment. 3.1.4 Effect of enzymolysis time on the yields of CPPc

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The effect of enzymolysis time (30, 40, 50, 60 and 70 min) on the extraction yields of CPPc were carried out when the other factors were fixed. As shown in

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Fig.1(D) and, with the increase of enzymolysis time, the contents of crude

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polysaccharides increased continuously for the color of the sample was getting darker

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and the absorbance was getting higher. The results were consistent with the extraction

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yields in Fig.1(d). The extraction yield increased by 13.64% when enzymolysis time

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from 30 to 60 min, but only increased by 0.66% after enzymolysis time prolonged. This might because that long time enzymolysis reaction could cause the hydrolysis of

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polysaccharides. However, the extraction yields would not increase significantly when the substrate had been completely reacted by the enzyme. Therefore, 60 min was selected for further optimization. 3.1.5 Effect of enzyme amount on the yields of CPPc The effects of enzyme amount (1, 2, 3, 4 and 5%) on the yields of CPPc were investigated when the other three factors remained fixed. According to Fig.1(E) and Fig.1(e), with the increase of enzyme amount, both CPPc contents and extraction yields showed an increasing trend. However, the increase was not significant after the enzyme amount exceeded 3%. This might due to the enzyme amount would affect the

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degree of enzymatic hydrolysis. Considering the cost, the amount of enzyme was kept at 3% at later experiments. 3.1.6 Effect of enzymolysis temperature on the yields of CPPc The effects of enzymolysis temperature (30, 40, 50, 60 and 70℃) on the extraction yields of CPPc were investigated when other three factors were fixed. As

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shown in Fig.1(F), when enzymolysis temperature was 50℃, the color of the solution was the darkest and the absorbance was the highest, representing the highest crude

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polysaccharides contents of it. In Fig.1(f), the extraction yields increased from 9.17%

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to 13.15% with the enzymolysis temperature from 30℃ to 50℃ and peaked at 50℃.

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However, the extraction yields declined with further increasing temperature. This

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might due to the sacrifice of enzyme activity at high temperature. Therefore, 50℃

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was the most suitable enzymolysis temperature in this experiment. Fig.1

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3.2 Results of response surface experiment optimization 3.2.1 Establish model and statistical analysis of enzymatic extraction The optimized enzyme-ultrasonic extraction method was established by the RSM analysis of three factors and three levels. 17 runs tests were carried out randomly by BBD design and the results were shown in Table 2. The second-order polynomial equation was obtained to predict the yields of SCPs by regression analysis:

Y  60.7675  1.4244 X 1  1.44912 X 2  0.233575 X 3 0.012100 X 1 X 2  0.010700 X 1 X 3  0.003525 X 2 X 3

(Eq,2)

0.043590 X 12  0.019222 X 22  0.005373 X 32 where Y represents the yields of SCPs; X1, X2 and X3 represents liquid-material

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ratio, enzymolysis temperature, and enzymolysis time, respectively. Table 2 Analysis of variance (ANOVA) was summarized in Table 3. The model P-value (<0.0001) and F-value (43.23) represented that the model and its corresponding variables were highly significant [22]. The values of determination coefficient (R2)

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and adjusted R2 (Adj R2) were 0.9823 and 0.9596, indicating that the model fitting degree was good and only 1.77% variations could not be explained by the model.

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These also indicated the good consistency between experimental and predicted values

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[23]. In general, the P value was used for checking the significance of each regression

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coefficient. In this case, linear coefficients (X1, X2 and X3), the quadratic coefficients

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significant (P<0.05).

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(X1X2 and X1X3) and the quadratic term coefficients (X12, X22 and X32) were

Table 3

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3.2.2 Analysis of response surfaces

The three-dimensional (3D) response surface was a graphic technology to display the effects of each variable on the response values and their mutual interactions [24]. Fig.2(A) displayed the effects of enzymolysis temperature and liquid-material ratio on CPPc yields at fixed enzymolysis time (60 min). The extraction yields were elevated with the increase of enzymolysis temperature from 30℃ to 50℃ while decreased when the enzymolysis temperature exceeded 50℃. An increase of the extraction yields was observed with the liquid-solid ratio from 15 mL/g to 30 mL/g

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and peaked at 30 mL/g, too. However, the extraction yields declined with further increase. The effects of enzymolysis time and enzymolysis temperature on CPPc yields were shown in Fig.2(B). With enzymolysis time from 50 to 60 min and enzymolysis temperature from 40 to 50℃, a significant increase of extraction yield was observed.

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The yield peaked at 60 min and 50℃. As shown in Fig.2(C), the yield of CPPc increased rapidly with the increasing enzymolysis time from 50 to 60 min. When the

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liquid-material ratio was lower than 30 mL/g, the extraction yield increased with its

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increase, but when the liquid-material ratio exceeded 30 mL/g, the extraction yields

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decreased. When enzymolysis time and liquid-solid ratio reached 60 min and 30 mL/g,

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the extraction yield of CPPc reached its maximum.

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Fig.2

3.2.3 Variables optimization and models validation

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The extraction conditions of CPPc were optimized and determined as follows: enzymolysis time 66.36 min, liquid-solid ratio 30.89:1 mL/g and enzymolysis temperature 52.01℃. The predicted yields of CPPc was 10.50%. To verify the applicability of the model equation, the confirmatory experiment was carried out with liquid-solid ratio 30:1 mL/g, enzymolysis time 66 min, and enzymolysis temperature 52℃ and the experimental yields of CPPc was 10.39%. These data indicated that the model equation was reliable and accurate. 3.3 Comparison of CPP extracted by different methods 3.3.1 Characteristics of CPP extracted by different methods

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The extraction yields of CPP were compared and the results were shown in Fig.3. Fig.3(A) showed that CPPc contained the highest contents of carbohydrate with its darkest color and highest absorbance value. As shown in Fig.3(B), compared with traditional hot water extraction (5.88±0.19%), ultrasound-assisted extraction (7.47±0.05%) and enzyme-assisted extraction (9.59±0.16%), the yields of CPP by

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ultrasound-enzyme assisted extraction (10.39±0.04%) was the highest. Comparing with the extraction method of polysaccharides from C. pinnatifida reported by Zhang

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[25], the yield of ultrasound-enzyme assisted extraction method increased by 59.85%.

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As shown in Fig.3(C), the characteristic peaks of polysaccharides appeared in all

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of four polysaccharides. The broad and intense peak around 3400 cm-1 was observed

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due to the stretching vibration of O-H group. A weak band around 2950 cm-1 was a

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characteristic of C-H stretching. The strong peak around 1630 cm-1 was attributed to C=O asymmetric stretching vibration of carboxyl group [26]. The results of IR

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spectroscopy indicated that the four extraction methods did not cause damage to the characteristic functional groups and skeleton structures of crude polysaccharides [27]. Fig.3(D) showed the GC spectrogram of monosaccharides of CPPc, CPPh, CPPe and CPPu. These four crude polysaccharides were all consisted of rhamnose, xylose, glucose and galactose. The monosaccharide types of all polysaccharides were the same, which indicated that the extraction method had insignificant effect on the monosaccharide compositions [28, 29]. Molecular weight could affect the bioactivity of polysaccharides [27]. As shown in Fig.3(E), the molecular weight of CPPh was the highest (313.0 kDa) while CPPh,

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CPPe and CPPc were similar to each other. The long time in ultrasound and enzymolysis extraction could cause the break of polysaccharide chain, resulting in lower molecular weight [30]. Fig.3

3.4 Comparison of antioxidant activity

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3.4.1 Reducing power Generally speaking, the reduction power had a positive correlation with the

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antioxidant ability. Antioxidants could reduce the Fe3+ of potassium ferricyanide to

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Fe2+ and then react with ferric chloride to form a blue compound, which had

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maximum absorbance at 700 nm [31]. The absorbance value of the compound

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depended on the reduction ability of it. As shown in Fig.4(A), the reducing powder of

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the four polysaccharides increased significantly with the concentration increased from 0.1 mg/mL to 0.5 mg/mL. At a concentration of 0.5 mg/mL, the reducing abilities

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were 79.1%, 93.8%, 98.8% and 95% for CPPh, CPPe, CPPu and CPPc, respectively. However, Vc showed higher reducing power than them. As shown in Fig.4(F), the IC50 values of CPPc (0.108 mg/mL) and CPPu (0.107 mg/mL) was lower than others, representing better reducing power of them. 3.4.2 ABTS+ radical scavenging activity ABTS could be oxidized by reactive oxygen and formed the blue ABTS + radical, which revealed maximum absorption at 734 nm. The radical could be scavenged and faded when antioxidants existed [32]. The results of ABTS+ radical scavenging experiment were summarized in Fig.4(B). The scavenging activities increased with

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the increasing of the concentration of CPP concentrations. The reducing abilities of CPPh CPPu, CPPe and CPPc were 98.8%, 99.2%, 99.2% and 99.6% at a concentration of 0.5 mg/mL. The results demonstrated that CPP possessed a notable scavenging effect on ABTS+ radical. The scavenging activity of CPPu was the highest while CPPh was lowest among the four polysaccharides at the same concentrations. As shown in Fig.4(F), the IC50 value of CPPu (0.12 mg/mL) was the lowest among

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the four polysaccharides, indicating that its scavenging effect on ABTS+ was the best.

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3.4.3 DPPH radical scavenging activity

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DPPH radical was a stable free radical in organic solvent. It is dark purple in

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alcohol solution and had maximum absorbance at 517 nm. In the presence of free

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radical scavenging agents, the alcohol solution would fade. Fig.4(C) showed that the

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DPPH radical scavenging activities of the four crude polysaccharides were positively correlated with their concentration. When the concentration was 5 mg/mL, the

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scavenging activities of CPPh, CPPc, CPPu and CPPe were 69.5%, 84.1%, 87.4% and 82.9%, respectively. However, Vc showed higher reducing power than all polysaccharides. As shown in Fig.4(F), the IC50 value of CPPu was the lowest among all the polysaccharides, indicating that it exhibited the strongest DPPH radical scavenging activities. 3.4.4 Superoxide radical scavenging activity Superoxide radical, one of the precursors of the singlet oxygen and hydroxyl radicals, was closely related to aging and various diseases [33]. The results of superoxide radical scavenging effects on CPPh CPPu, CPPe and CPPc were shown in

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Fig.4(D). The scavenging activities increased when the concentration of polysaccharides increased from 1 mg/mL to 5 mg/mL. At a concentration of 5 mg/ml, the superoxide radical scavenging activities were 61.9%, 61.6%, 66.4% and 58.8% for CPPh CPPu, CPPe and CPPc, respectively. Apparently, the scavenging activity of CPPc was the lowest while CPPu was the highest at the same concentration. Fig.4(F)

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showed that the IC50 value was in the order of CPPu, CPPe, CPPh and CPPc. The data represented that the superoxide radical scavenging activities of CPPu was higher than

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3.4.5 Lipid peroxidation inhibition

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other polysaccharides, but lower than Vc.

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β-carotene was a polyene pigment that had maximum absorption at 470 nm and

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was easily oxidized to yellow. In the reaction medium, the oxidation of linoleic acid

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produced peroxides that could bleach beta carotene, hence reduced the absorbance of the solution over time. As shown in Fig.4(E), the bleaching rate of β-carotene varied

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due to the difference of antioxidant active ingredients and content in the reaction medium. Lipid peroxidation inhibition activities of the four crude polysaccharides were positively correlated with the concentration. At 0.5 mg/mL, the scavenging activities of CPPh CPPe, CPPu and CPPc were 96.6%, 82.0%, 93.6% and 83.0%, respectively. The results of IC50 values (Fig.4(F)) showed that CPPc and CPPu had better lipid peroxidation inhibition activities for their IC50 values were lower than CPPe and CPPh. By comparing the area of the four polysaccharides in the radar map (Fig.4(F)), their antioxidant activity could be preliminarily evaluated. The area of CPPc and

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CPPu was smaller than that of CPPh and CPPe, indicating that CPPc and CPPu had better antioxidant activities than others. Fig.4

4 Conclusion In this study, effects of CPPc, CPPh, CPPe and CPPu on extraction yield,

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structure characteristics and antioxidant activity were examined and compared. The crude polysaccharides were obtained after optimizing the enzyme-ultrasound assisted

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extraction conditions, and the optimal extraction conditions were the following:

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liquid-solid ratio 30:1 mL/g, enzymolysis time 66 min, enzymolysis temperature 52℃.

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Then, the results showed that different extraction methods had insignificant influence

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on the characteristic functional groups and monosaccharide composition of the

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polysaccharides. The crude polysaccharides obtained by the four methods were all mainly consisted of rhamnose, xylose, glucose and galactose. Meanwhile,

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enzyme-ultrasound assisted extraction possessed the highest extraction yield (10.39%) and medium molecular weight. Finally, the antioxidant activities of CPPu and CPPc were relatively high among the four polysaccharides. Therefore, enzyme-ultrasound assisted extraction could be selected as a suitable polysaccharides extraction method which would not do any harm for subsequent research on structure and chemical properties. Acknowledgment This work was supported by Jiangsu Key Laboratory of Regional Resource Exploitation and Medicinal Research (No. LPRK201705), and Jiangsu Provincial Key

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Laboratory of Palygorskite Science and Applied Technology (No. HPK201707). References [1] M. Yu, Y. Ji, Z. Qi, D. Cui, G. Xin, B. Wang, Y. Cao, D. Wang, Anti-tumor Activity of Sulfated Polysaccharides from Sargassum fusiforme, Saudi Pharm. J. 25 (2017) 464.

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Journal Pre-proof Table 1 Factors and levels of Box-Benhnken design test on CPPc extraction conditions Code levels

Independent variables

Symbols -1

0

+1

X1

25

30

35

Enzymolysis temperature (℃)

X2

40

50

60

Enzymolysis time (min)

X3

50

60

70

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Liquid-to-solid ratio (mL/g)

Journal Pre-proof Table 2 Box-Behnken experimental design and the response values for the yields of CPPc X1

X2

X3

Y

1

30

60

70

9.62±0.12

2

25

50

70

8.1±0.18

3

30

40

50

5.69±0.26

4

25

60

60

6.87±0.07

5

25

50

50

7.69±0.07

6

35

50

50

7.10±0.10

7

35

40

60

5.42±0.06

8

30

40

70

6.95±0.12

9

30

50

60

9.94±0.05

10

30

50

60

9.65±0.08

11

25

40

60

5.99±0.05

12

30

50

60

10.31±0.02

13

30

60

50

6.95±0.21

35

50

70

9.65±0.07

35

60

60

8.72±0.11

30

50

60

9.26±0.15

30

50

60

9.65±0.12

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Journal Pre-proof Table 3 ANOVA for response surface quadratic model Sum of

Mean

Source

df

p-value

Significance

Prob > F

level Significant a

F Value Square

41.54

9

4.62

43.23

<0.0001

X1

0.6272

1

0.6272

5.87

0.0458

X2

8.22

1

8.22

77.01

<0.0001

X3

5.93

1

5.93

55.58

0.0001

X1X2

1.46

1

1.46

13.71

0.0076

X1X3

1.14

1

1.14

10.72

0.0136

X2X3

0.4970

1

0.4970

4.66

0.0678

X1²

5.00

1

5.00

46.84

0.0002

X2²

15.56

1

15.56

145.73

<0.0001

X3²

1.22

1

1.22

11.38

0.0119

Residual

0.7473

7

Lack of Fit

0.1382

3

0.3026

0.8182

Pure Error

0.6091

Cor Total

42.29

R2adj C.V.%

0.9823 0.9596 4.04

a

Significant (p < 0.05).

b

Not significant (p > 0.05)

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R2

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Model

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squares

0.1068 0.0461 0.1523

not significant b

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Fig.1 Effect of different pH values (A and a), ultrasonic time (B and b), enzymolysis time (C and c), enzyme amount (D and d), enzymolysis temperature (E and e) and liquid-material ratio (F and f)

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on carbohydrate contents and the yields of CPPc.

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Fig.2 Response surface plots (A, B and C) showing the interaction effect of liquid-material

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ratio (X1), enzymolysis temperature (X2) and enzymolysis time (X3) on extraction yield.

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Fig.3 Visual photograph and UV-vis spectrogram after phenol-sulfuric acid staining (A), yields of different extraction methods (B), IR spectrogram (C), GC of monosaccharides (D) and HPGFC Chromatogram (E). Ⅰ-Ⅳ represented hot water extraction, ultrasonic assisted extraction, enzyme

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assisted extraction and enzyme-ultrasonic assisted extraction, respectively.

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Fig.4 Reducing power (A), scavenging effects on ABTS+ (B), superoxide (C), DPPH (D) radical and lipids peroxidation inhibition activity (E) of CPPh, CPPe, CPPu and CPPc, and the radar map

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of IC50 (F).

Journal Pre-proof Highlights 1. Four methods were used to extract polysaccharides from C. pinnatifida (CPP). 2. Enzyme-ultrasound assisted extraction method was optimized and had higher yield. 3. The structure characterization of CPP from different extraction were analyzed.

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4. The antioxidant activities of CPP from different extraction were compared.

Figure 1

Figure 2

Figure 3

Figure 4