Antioxidant property of water-soluble polysaccharides from Poria cocos Wolf using different extraction methods

International Journal of Biological Macromolecules 83 (2016) 103–110

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Antioxidant property of water-soluble polysaccharides from Poria cocos Wolf using different extraction methods Nani Wang, Yang Zhang, Xuping Wang, Xiaowen Huang, Ying Fei, Yong Yu, Dan Shou ∗ Department of Medicine, Zhejiang Academy of Traditional Chinese Medicine, Hangzhou, Zhejiang, China

a r t i c l e

i n f o

Article history: Received 17 September 2015 Received in revised form 19 October 2015 Accepted 12 November 2015 Available online 19 November 2015 Keywords: Polysaccharide Antioxidant property Response surface methodology

a b s t r a c t Poria cocos Wolf is a popular traditional medicinal plant that has invigorating activity. Water-soluble polysaccharides (PCPs) are its main active components. In this study, four different methods were used to extract PCPs, which include hot water extraction (PCP-H), ultrasonic-assisted extraction (PCP-U), enzyme-assisted extraction (PCP-E) and microwave-assisted extraction (PCP-M). Their chemical compositions and structure characterizations were compared. In vitro antioxidant activities were studied on the basis of DPPH radical, hydroxyl radical, reducing power and metal chelating ability. The results showed that PCPs were composed of mannose, glucose, galactose, and arabinose, and had typical IR spectra characteristics of polysaccharides. Compared with other PCPs, PCP-M had lower neutral sugar content, higher mannose content and higher uronic acid content. The molecular weight were determined as PCPE < PCP-M < PCP-U < PCP-H. PCP-M showed the strongest reducing power and highest scavenging abilities on hydroxyl and DPPH radicals, while PCP-U exhibited the lowest antioxidant activities. Response surface methodology was used to optimize the extraction yield of PCP-M by implementing the Box–Behnken design. Under the optimized conditions, the PCP-M yield was 9.95%, which was well in close agreement with the value predicted by the model. Overall, the microwave-assisted extraction was an effective and mild method for obtaining antioxidant polysaccharides from P. cocos Wolf. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Oxidation is an essential biological process in living organisms for the production of energy. It has been widely accepted that appropriate supplementation with exogenous antioxidant, which could scavenge the free radicals and retard the progress of many chronic diseases, may help reduce reactive oxygen species reduced diseases [1]. Nowadays, many synthetic antioxidants have been widely used. However, these antioxidants have potential hazards to health, such as carcinogenesis and liver damage [2]. Therefore, it is necessary to develop and utilized effective natural antioxidants to reduce the health risks. Recently, polysaccharides from various plants have been recognized as safe and effective antioxidants [3]. Poria cocos Wolf, called Fuling in China, is a popular medicinal mushroom that grows around the roots of pine trees in China, Japan, Korea, and North

∗ Corresponding author. Tel.: +86 571 88849089; fax: +86 571 88849089. E-mail addresses: [email protected] (N. Wang), [email protected] (Y. Zhang), [email protected] (X. Wang), [email protected] (X. Huang), [email protected] (Y. Fei), [email protected] (Y. Yu), [email protected] (D. Shou). http://dx.doi.org/10.1016/j.ijbiomac.2015.11.032 0141-8130/© 2015 Elsevier B.V. All rights reserved.

America. It is widely used to treat chronic gastritis, edema and emesis. The polysaccharides from P. cocos Wolf (PCPs) have attracted great attention for many years, due to their various biological functions such as inhibiting tumor growth and metastasis, and low toxicity [4]. Recent studies demonstrated that aqueous extracts of crude P. cocos Wolf exhibited antioxidant activities. Polysaccharide is the main important constituent of P. cocos Wolf. The most commonly used method for polysaccharides extraction is hot water extraction. The yield of this method largely depends on extraction time and temperature. Recently, other new methods are employed to extract polysaccharides, such as ultrasonic-assisted extraction [5] and microwave-assisted extraction [6]. However, different extraction efficiency by ultrasonic treatment, enzyme treatment, and microwave treatment is mainly attributed to the mechanical effect or catalytic action, which may influence the biological activities [7]. Up to date, there is little literature pertaining to the effects of extraction methods on the antioxidant properties of PCPs. In this work, we extracted four water soluble polysaccharides extracted from P. cocos Wolf via the method of hot water extraction, ultrasonicassisted extraction, enzyme extraction and microwave-assisted extraction. In vitro antioxidant activities of the four PCPs were estimated, including radical scavenging ability, reducing power and

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ferrous ion-chelating ability. High performance liquid chromatography (HPLC) and Fourier transform infrared (FT-IR) spectroscopy were employed to observe the preliminary structural characterization of PCPs. Furthermore, response surface methodology (RSM) was used to optimize the extraction process of PCPs. 2. Material and methods

measured with a modified carbazole method using d-glucuronic acid as the standard [13]. The total sugar contents of polysaccharide fractions were analyzed by the phenol-sulfuric acid method, using glucose as standard [14]. The linearity of the method was determined by analyzing a series of standard solutions at concentrations of glucose ranging from 0 to 0.02 ␮g mL−1 . The calibration curve (y = 35.68x + 0.025, r2 = 0.9991) was obtained by plotting peak area against analyte concentration.

2.1. Materials and chemicals P. cocos Wolf was purchased from Huadong Medicine Co. (Hangzhou, Zhejiang, China). Prepared drugs were then dried at 60 ◦ C for 3 h. The dried drugs were ground, screened through an 80 mesh sieve, and stored at room temperature in a vacuum-packed container before use. 1,1-diphenyl-2-picrylhydrazyl (DPPH) and cellulase (0.3 U mg−1 ) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of analytical grade from Sinopharm (Shanghai, China). 2.2. Preparation of polysaccharides The degreasing treatment was carried out according to the previous reports [3–5]. Briefly, dried P. cocos Wolf powder was immersed in petroleum ether at room temperature for 24 h. The solid residue was collected by filtration and the procedure was repeated twice in order to remove lipid material. The residue was air-dried at room temperature and then extracted by different previously reported methods [5,6,8,9] with some modifications. The PCPs extracted by hot water extraction, microwave-assisted extraction, enzyme extraction, and ultrasonic-assisted extraction were designated as PCP-H, PCP-M, PCP-E and PCP-U, respectively. PCP-H was extracted with hot water in a ratio (liquid to solid) of 20:1 (mL g−1 ) at 100 ◦ C for 2 h. PCP-M was extracted by the microwave-assisted method in a ratio (liquid to solid) of 20:1 (mL g−1 ) for 2 min in a CW 2000 microwave extractor (XT, Shanghai, China) at working frequency of 2450 MHz with 800 W. PCP-E was extracted by the enzyme method in a ratio (liquid to solid) of 20:1 (mL g−1 ) with 1.0% of cellulase concentration for 1 h. PCP-U was extracted by ultrasonic-assisted method in a ratio (liquid to solid) of 20:1 (mL g−1 ) with a power of 200 W in an ultrasonic bath for 1 h (KQ-400DB, Kunshan Ultrasonic Instrument Co., Kunshan, Jiangsu, China). After filtered, the residues were extracted again by the same method. Then, the extracts were filtered through gauze and centrifuged at 4000 rpm for 10 min to remove the water-insoluble materials. The volume of aqueous extract was concentrated under reduced pressure at 55 ◦ C to about 200 mL. The concentrated extracts were precipitated with four-fold volume of ethanol overnight at 4 ◦ C. The precipitate was collected by centrifugation (4000 rpm, 15 min, and 4 ◦ C) [10]. Then the precipitate was solubilized in deionized water, deprotenized with Sevage method (chloroform/butyl alcohol, 4:1 v/v). The aqueous phase was collected and freeze-dried to yield polysaccharides [11]. The polysaccharides yield (%) was calculated as follows: m1 Polysaccharide yield (%, w/w) = × 100 m0 where m1 is the weight of dried polysaccharide and m0 is the weight of P. cocos Wolf powder. 2.3. Characterization of polysaccharides 2.3.1. Component analysis The protein content was determined by the method of Bradford using bovine serum albumin as the standard [12]. Uronic acid was

2.3.2. Monosaccharide composition analysis The monosaccharide composition analysis followed previous reports with some modification [15]. Briefly, the polysaccharides were firstly hydrolyzed with 4.0 mol L−1 trifluoroacetic acid (100 ◦ C, 6 h), evaporated to dryness under reduced pressure. The hydrolysed polysaccharides or monosaccharide were dissolved in 1 mL distilled water. Then, the 400 ␮L of solution was transferred into a clean tube in which 400 ␮L of 0.5 mol L−1 methanol solution of 1-phenyl-3-methyl-5-pyrazolone (PMP) and 200 ␮L of 0.3 mol L−1 NaOH were added before. The mixture was allowed to react for 0.5 h at 70 ◦ C, then cooled to room temperature and was neutralized with 200 ␮L of 0.3 mol L−1 HCl. Then 4 mL of chloroform was added into the tube to perform a rotary extraction. The organic phase was abandoned to remove the excess reagents, and the extraction process was repeated three times. Finally, the aqueous phase was filtered through a 0.22 ␮m microporous filtering film and stored at 4 ◦ C before HPLC analysis. Chromatography analyses were performed using a Shimadzu LC-20 HPLC system comprising two pumps, a thermostated column compartment, a diode array detector and a manual injector with a 20 ␮L sample loop. In the HPLC analysis procedure, separation was achieved using a Shiseido Capcell Pak C18 column operated at 30 ◦ C with a flow rate of 1.0 mL min−1 . d-mannose, lrhamnose, d-glucose, d-arabinose, l-fucose and d-galactose were used as references. The mobile phase consisted of acetonitrile (A) and potassium phosphate buffer (B, 0.1 mol L−1 , pH 6.7) using the following gradient mode: 0–23 min, 82% B; 23–40 min, 82–72% B. UV detection was performed at 250 nm. 2.3.3. FT-IR FT-IR spectrum of the sample was determined using a Fourier transform infrared spectrophotometer (Thermo Scientific Nicolet iS10, USA). The sampler was grounded with spectroscopic grade potassium bromide powder and then pressed into a pellet for FT-IR measurement in the frequency range 4000–400 cm−1 . 2.3.4. Molecular weight The molecular weight of PCPs was determined by gel permeation chromatography [16]. Waters 515 HPLC system with a Waters 2410 differential refractive index detector was used. PCPs samples were dissolved in distilled water and filtered through membrane prior to HPLC analysis. 50 ␮L of solution was injected. Separation was achieved using a TOSOH BIOSEP G4000SWXL column at 40 ◦ C. The isocratic mobile phase was 0.1 mol L−1 NaNO3 aqueous solution. The dextran standards (500,000, 100,000, 60,000, 15,000, 4440 Da) were purchased from the Sigma (USA) were used to obtain the calibration curve. 2.4. Antioxidant activities 2.4.1. Scavenging of DPPH radicals Ability of polysaccharides to scavenge DPPH free radicals was determined using the method described by previous literature with some modification [17]. Briefly, 1 mL different concentration of polysaccharide in distilled water was thoroughly mixed with 2 mL of freshly prepared DPPH and 2 mL of methanol. Then the mixture was incubated at room temperature for 30 min. The sample

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absorbance was measured at 517 nm. Scavenging DPPH activity of butylated hydroxyl toluene (BHT) was carried out as the positive control. All the tests were carried out in triplicate. The scavenging activity of DPPH radicals was calculated according to the following equation: DPPH scavenging activity (%) =



1−

A1 A2



× 100

where A1 is the sample absorbance and A2 is the absorbance of the negative control.

a typical procedure, 50 ␮L of polysaccharide solution was mixed thoroughly with 30 ␮L of 0.2 mg mL−1 ferrous chloride solution. Then 70 ␮L of 2 mg mL−1 ferrozine solution was added to the reaction system. The mixture was shaken vigorously and maintained at room temperature for 10 min to make the reaction reach equilibrium. The absorbance of reaction mixture was then measured at 560 nm. Chelating Fe2+ ability of ethylene diamine tetraacetic acid (EDTA) was measured as the positive control. Chelating Fe2+ ability was evaluated employing the following equation: Chelating ability (%) =

2.4.2. Reducing power The reducing power was assayed according to the reported method with some modifications [18]. Briefly, 1 mL different concentration of polysaccharide in distilled water was mixed with 2.5 mL of phosphate buffer (0.2 mol L−1 , pH 6.6) and 2.5 mL of potassium ferricyanide (1.0%, w/v) in different test tubes. The mixtures were incubated for 20 min at 50 ◦ C. Then, 2.5 mL trichloroacetic acid in water (10%, w/v) was added to the mixtures and centrifuged at 5000 rpm for 10 min. 2.5 mL of the upper layer was mixed with 2.5 mL of distilled water and 0.5 mL of aqueous ferric chloride (FeCl3 ) solution (0.1%, w/v), and the absorbance was measured at 700 nm. The reducing Fe3+ power of BHT was carried out as the positive control. Reducing Fe3+ power was evaluated employing the following equation. Reducing power = A1 − A0 where A1 was the absorbance of a mixture solution of the sample and other reaction reagents, and A0 was the absorbance of the sample under identical conditions as A1 with water instead of FeCl3 solution. 2.4.3. Scavenging of hydroxyl radicals The ability of scavenging hydroxyl radicals was assayed according to the reported method with some modifications [19]. Briefly, 1 mL different concentration of polysaccharide in water was mixed with 1 mL of salicylic acid (9.0 mmol L−1 ) and 0.5 mL ferrous sulphate (9 mmol L−1 ). Then 1 mL hydrogen peroxide (8.8 mol L−1 ) was added before incubation at 25 ◦ C for 60 min. Ascorbic acid was used as positive control; distilled water was used as a blank control. The absorbance of the mixtures was measured at 510 nm. The scavenging hydroxyl radical activity of ascorbic acid (Vc) was used as positive control. Hydroxyl radical scavenging activity was calculated by the following equation: Hydroxyl radical scavenging activity (%) =



1−

(B1 − B2 ) B0



× 100

where B0 was the absorbance of the control group (water instead of test sample solution), B1 was the absorbance of the test group, and B2 was the absorbance of the sample only (water instead of H2 O2 solution). 2.4.4. Chelating Fe2+ ability assay The ability of chelating Fe2+ ability was estimated according to the method reported previously [20] with some modification. In

105



1−

(C1 − C2 ) C0



× 100

where C0 was the absorbance of reaction solution without tested samples, C1 was the absorbance of the test group and C2 was the absorbance of the sample only (water instead of ferrozine solution). 2.5. Statistical analyses All the experiments were carried out in triplicate, and the data were shown in means ± standard deviation (SD). Differences were considered significant at P < 0.05. 3. Results and discussion 3.1. Characterization of the polysaccharides The chemical compositions of PCP-H, PCP-M, PCP-E and PCP-U were shown in Table 1. The carbohydrate contents were different followed the order of PCP-M < PCP-E < PCP-H < PCP-U. PCP-M also had the highest contents of uronic acids. The difference might be related to the selective type of extraction method [8]. From Table 1, the four polysaccharides were consisted of mannose (Man), glucose (Glu), galactose (Gal) and arabinose (Ara). PCP-M had the highest content of mannose. In terms of molar ratio, glucose was major monosaccharide. These results revealed that these polysaccharides from P. cocos Wolf had different chemical compositions. Calculated Mw data of PCP-H, PCP-U, PCP-E and PCP-M were 21.5 kDa, 21.2 kDa, 10.6 kDa and 15.1 kDa, respectively. It suggested that the polysaccharide obtained from enzyme extraction had the lowest molecular weight. The possible reason might be that the enzyme degraded the polysaccharides to some degree. Wang et al. also found the molecular weight of polysaccharides obtained by enzyme extraction decreased [7]. The FT-IR spectra were used for determination of their structural features. As showed in Fig. 1, the FT-IR spectra of four polysaccharides were similar. The broad bands at 3388–3392 cm−1 indicated the O–H stretching vibration. The small band at 2927–2931 cm−1 represented the C–H stretching and bending vibrations. The peaks at 1615–1619 cm−1 and 1414–1420 cm−1 were attributed to asymmetric and symmetric stretching of C O [5]. Each polysaccharide has a specific band in the range of 1200–1000 cm−1 . This region was dominated by ring vibrations overlapped with stretching vibrations of C–O side groups and the C–O–C glycosidic band vibration [6]. From Fig. 1, the absorptions at 1075–1080 cm−1 suggested a pyranose form of sugars. Four polysaccharides displayed the

Table 1 Comparison of the chemical compositions of the four polysaccharides. Sample

PCP-E PCP-U PCP-H PCP-M a

Sugar component (%) Mannose

Galactose

Glucose

Arabinose

1.98 2.18 0.92 4.02

0.36 2.36 0.18 4.93

81.72 87.27 86.88 79.48

15.93 8.18 12.01 11.57

Value = mean ± SD.

Uronic acids (%)

Protein (%)

Carbohydrate (%)

7.96 ± 0.10a 6.81 ± 0.07 8.12 ± 0.11 8.96 ± 0.12

1.97 ± 0. 07 1.32 ± 0.10 1.20 ± 0.08 1.88 ± 0.09

80.07 ± 1.08 90.87 ± 2.25 85.68 ± 3.01 74.66 ± 2.45

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Fig. 1. FT-IR spectrum of PCP-H, PCP-M, PCP-E and PCP-U.

weak absorption at 887–891 cm−1 , which indicated that pyranoses existed in the ␤-configuration [11]. 3.2. Antioxidant activities 3.2.1. Reduce power The ion Fe3+ would active the reaction of lipid peroxidation in human body. Thus, the reducing Fe3+ power assay could be used to evaluate the potential antioxidant activity of polysaccharide [21]. Fig. 2A showed the reductive potential of PCP-M, PCP-U, PCP-H, and PCP-E increased with increase of polysaccharide concentration. Among these samples, reducing power decreased in an order of PCP-M, PCP-E, BHT, PCP-H, and PCP-U. The values of EC50 were shown in Table 2. Compared with that of BHT, as positive control, the reducing power of PCP-M and PCP-E was always higher at the tested concentrations. The results also showed PCP-M and PCP-E possessed stronger reducing power than BHT for their lower EC50 values. 3.2.2. Chelating metal ion ability The metal ions could be considered as oxidant substances, for their abilities to capture electrons [22]. Thus, metal chelating ability was recognized as a correlative activity to antioxidant. Ferrous ion chelating activities of polysaccharides at different concentration were shown in Fig. 2B. Chelating abilities of four polysaccharides climbed up as their concentrations increased. The chelating metal abilities were followed by PCP-M, PCP-E, PCP-H and PCP-U. The

EC50 values were listed in Table 2, and PCP-M showed significantly higher ability on ferrous ion chelating activity than other polysaccharides. The EC50 value of EDTA was 0.013 mg mL−1 . Higher EC50 values of PCPs demonstrated their comparatively weaker chelating ability than that of EDTA, but also desirable. 3.2.3. Scavenging activity of DPPH radical DPPH was a free radical compound, which has been widely used to evaluate the free radical scavenging ability of various samples [23]. Fig. 2C showed PCP-H, PCP-M, PCP-E and PCP-U had the scavenging activities of DPPH radical in a concentration dependent manner. When the PCP-M concentration of 2.0 mg mL−1 , scavenging DPPH activity reached 92.5%, which indicated that almost all of DPPH radicals in the solution had been cleaned up. At all our tested concentrations, the scavenging DPPH activities were always followed by PCP-M, PCP-E, PCP-H, and PCP-U. The EC50 values of polysaccharides for scavenging DPPH radical activity were summarized in Table 2. A lowest EC50 value of PCP-M showed this polysaccharide had stronger scavenging DPPH radical ability than that of BHT, as the positive control. 3.2.4. Scavenging activity of hydroxyl radical Hydroxyl radical could easily induce oxidative damage in cells, such as destroy the structures of most biomolecules, destruct the permeability of cell membranes, and induce tissue damage [24]. Fig. 2D showed the results of scavenging hydroxyl radical activities of polysaccharides. A positive correlation between polysaccharide

Table 2 EC50 values of P. cocos Wolf polysaccharides for antioxidant activity. Assay

Reducing power Scavenging OH activity Metal chelating ability Scavenging DPPH activity

EC50 (mg mL−1 ) PCP-H

PCP-M

PCP-E

PCP-U

Positive controla

2.308 0.4034 0.459 1.109

0.620 0.368 0.328 0.876

0.740 0.361 0.509 1.046

1.614 0.447 0.660 1.190

1.198 0.015 0.013 0.888

a The positive control for scaveng DPPH and reducing power assays was BHT, and positive control for chelating ability assays and scavenging OH activity was EDTA and Vc, respectively.

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107

Fig. 2. Antioxidant properties of PCP-H, PCP-M, PCP-E and PCP-U, (A) reducing power, (B) chelating ability, (C) DPPH radical scavenging effects and (D) OH radical scavenging ability.

concentration and the scavenging hydroxyl radical activity was observed in the studied concentration ranges. It was found that PCP-M always showed much better scavenging hydroxyl radical activity than that of other polysaccharides. Compared with other polysaccharides, an extremely low EC50 value of PCP-M for scavenging hydroxyl radical activity was shown in Table 2. Based on these evidences, PCP-M appears to be the most efficient antioxidant polysaccharide. The satisfactory scavenging hydroxyl radical activity demonstrated PCP could be utilized as a natural antioxidant instead of synthesized antioxidant. The data also indicated the antioxidant activity was closely related to the chemical characters. It might be rationally assumed that the stronger antioxidant activity of polysaccharides may be related to the relatively higher contents of uronic acid and protein. Besides, molecular weight about 10–20 kDa showed better antioxidant activity. This phenomenon was in accordance with the previous literatures [16,25]. Another possible reason was that the fraction of fungal polysaccharides exhibiting the higher antioxidant activity might have a low ratio of glucose [26]. Future study will be continued to explore the in vivo antioxidant activities of P. cocos Wolf polysaccharides using an animal model. 3.3. Optimization of PCP-M extraction 3.3.1. Single factor experimental analyses Extraction yield is affected by multiple independent variables. Therefore, developing an optimal method that can determine all the factors is necessary. The possibility of interactions between independent variables should be considered to determine the optimal extraction conditions. RSM is an effective statistical and mathematical technique for developing and optimizing complex processes [27].

To determine the optimal design ranges of extraction parameters for PCP-M preparation in RSM design, the single-factor experiments were conducted in this study. The effects of extraction factors were showed in Fig. 3. When the microwave power was increased from 200 W to 800 W, the PCP-M yield of polysaccharide was dramatically improved, as shown in Fig. 3(A). The accelerated yield was attributed by the heating mechanism of microwave. Microwave energy acted as an electromagnetic radiation that enhanced the loosening of the cell wall matrix rapidly, increased the penetration of the solvent into the plant matrix and led to the leaching of pectin during the microwave heating process [28]. In addition, this energy also transferred rapidly on the biomolecules, which generate molecular movement and heating on the extraction system quickly and improved the extraction efficiency [29]. However, when the power was higher than 800 W, the PCP-M yield decreased. The possible reason might be that high irradiation power provided superfluous energy to the solvent and matrix and disturbed molecular interactions [30]. These results proved the microwave power was an important factor influencing the extraction efficiency of polysaccharides. The effects of extraction time on PCP-M yields were presented in Fig. 3(B). As the time prolonged from 0.5 min to 2.0 min, the PCPM yield was increased. After that, PCP-M yield was decreased. It might be due to the fact that, the adsorption of microwave energy in the extraction system promoted the thermal accumulation of solution and led to the dissolution of polysaccharides into the solution until 2 min. However, the excessive time exposure under the microwave field might lead to degrading the polysaccharide chain molecules, affecting the extraction yield [28]. The similar phenomenon was reported in the extraction of other plant polysaccharides [6].

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Fig. 3. Effects of (A) microwave power, (B) extraction time, (C) ratio of liquid to solid, and (D) extraction number on PCP-M yield.

The effects of liquid to solid ratio were also studied, as showed in Fig. 3(C). When the ratio was set as 20:1 mL g−1 , the PCP-M yield reached the maximum. It might be explained that, the solvent could absorb microwave energy and enhanced the swelling of plant material, which led to easy release of polysaccharides into the surrounding medium. However, when the liquid to solid ratio was too high, the solution was saturated with the solute as well as higher solvent could decrease the absorption of microwave by the material, which was negatively affected the mass transfer rate [31]. Fig. 3(D) showed the effects of extraction number on PCP-M yield. When the plant material was extracted twice, the PCP-M yield got to the maximum. The yield showed a decreasing trend as extraction number increased. So the extraction number was not further optimized in the RSM experiment for two times was sufficient for PCP-M extraction.

3.3.2. RSM model building and statistical analyses Based on the above analyses of single factor experiments, RSM was used to optimize the extraction yield by implementing a three level-three factor Box–Behnken design. The experimental design matrix with observed values was listed in Table 3. A quadratic polynomial equation between PCP-M yield and the extraction variables was derived as follows: Y = 9.98 − 0.39X1 − 0.39X2 + 0.20X3 − 0.47X1 X2 − 0.19X1 X3 + 0.45X2 X3 − 2.28X12 − 2.40X22 − 2.39X32 where Y was the PCP-M yield (%), X1 , X2 and X3 were the coded values of the microwave power (W), extraction time (min) and liquid to solid ratio (mL g−1 ), respectively. The analysis of variance (ANOVA) of the four quadratic regression models was presented in Table 4. A high F-value and a low

Table 3 Box–Behnken design matrix and response values of PCP-M yields. Run

Microwave power (W, X1 )

Extraction time (min, X2 )

Liquid to solid ratio (mL g−1 , X3 )

Yield (%, Y)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1000(1) 800(0) 800(0) 800(0) 800(0) 600(−1) 1000(1) 800(0) 800(0) 600(−1) 1000(1) 800(0) 800(0) 600(−1) 600(−1) 1000(1) 800(0)

2(0) 3(1) 1(−1) 2(0) 2(0) 1(−1) 2(0) 1(−1) 2(0) 3(1) 1(−1) 2(0) 3(1) 2(0) 2(0) 3(1) 2(0)

10(−1) 30(1) 30(1) 20(0) 20(0) 20(0) 30(1) 10(−1) 20(0) 20(0) 20(0) 20(0) 10(−1) 30(1) 10(−1) 20(0) 20(0)

4.91 5.52 5.19 9.90 10.15 5.76 5.01 5.76 9.92 5.71 5.82 9.92 4.30 6.09 5.22 3.90 9.99

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Table 4 ANOVA for the quadratic polynomial model for PCP-M yields. Source

Sum of squares

d.f.

Mean square

F-value

P-value

Model Lack of fit R2 = 0.9980

82.93 0.12 Adj-R2 = 0.9955

9 3

9.21 0.04 Pred-R2 = 0.9951

397.95 3.67

<0.0001 (significant) 0.1207 (not significant)

and then exhibited a decreasing trend. Though the interaction of microwave power and liquid-to-solid ratio on PCP-M yield was significant (Fig. 4(B)), it had a negative effect on PCP-M yield for its minus cross product coefficient. Besides, the liquid-to-solid ratio showed a positive synergistic effect on PCP-M yield when couple with the extraction time, as showed in Fig. 4(C). The maximum predicted response was located on the peak of the 3D response surface. Considering the operating convenience of the extraction process, modified optimized extraction conditions for PCP-M extraction were as follows: microwave power at 780 W, extraction time at 2 min, liquid-to-solid ratio at 20 mL g−1 , and extraction number as two times. Under the optimized conditions, the experimental extraction yield of PCP-M was 9.95 ± 0.31%, which was agreed closely to the predicted value (10.02%). 4. Conclusions In the present study, the water-soluble and antioxidant polysaccharides (PCP-M, PCP-E, PCP-H, PCP-U) from P. cocos Wolf were obtained by four methods, including microwaveassisted extraction, enzyme extraction, hot water extraction and ultrasonic-assisted extraction. The four polysaccharides had the same monosaccharide compositions, but difference in the contents. Moreover, in vitro antioxidant activity studies showed that PCP-M possessed best antioxidant activity. It seemed that the polysaccharides with lower neutral sugar content, higher uronic acid content and lower molecular weight were found to have better antioxidant activities. The optimum process parameters for the PCP-M extractions were obtained by using RSM technique. The experimental yields were close to the predicted yield values. Thus, further works on purification, structure deduction and functions evaluation of polysaccharides from P. cocos Wolf are necessary, which would speed up the progress to make it an effective product. Acknowledgments The project was sponsored by the Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents, the Science and Technique Plan of Zhejiang (2014F50030) and the Science and Technique Plan of Traditional Chinese Medicine of Zhejiang province (2012ZB002).

Fig. 4. 3D response surface plot, X1 , X2 and X3 were the coded values of the microwave power (W), extraction time (min) and liquid to solid ratio (mL g−1 ), respectively.

P-value indicated the model was significant. Lack-of-fit was not significant for the RSM models at 95% confidence levels, which indicated that the models represented the data satisfactorily. The ANOVA showed that the proposed regression models for extraction yields were adequate with satisfactory R2 and adj-R2 values [32]. The 3D response surface plot was presented in Fig. 4. Fig. 4(A) graphed the effects of extraction time and microwave power on PCP-M yield and their interactions. The PCP-M yield augmented dramatically to the maximum as the microwave power increased,

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