Extraction optimization, characterization and antioxidant activity of polysaccharide from Gentiana scabra bge

International Journal of Biological Macromolecules 93 (2016) 369–380

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Extraction optimization, characterization and antioxidant activity of polysaccharide from Gentiana scabra bge Zhenyu Cheng a , Yuewei Zhang a , Haiyan Song b , Hongli Zhou a , Fangli Zhong a,∗ , Haobin Hu c , Yu Feng a a

School of Chemistry & Pharmaceutical Engineering, Jilin Institute of Chemical Technology, Jilin, 132022, China Laboratory of Test Center, Jilin Agricultural Science and Technology College, Jilin, 132101 China, China c College of Chemistry & Chemical Engineering, Longdong University, Qingyang, 745000, China b

a r t i c l e

i n f o

Article history: Received 10 June 2016 Received in revised form 15 August 2016 Accepted 22 August 2016 Available online 24 August 2016 Keywords: Smashing tissue extraction Characterization Antioxidant activity

a b s t r a c t In this study, optimization of smashing tissue extraction (STE), preliminary chemical characterization and antioxidant activity in vitro of crude polysaccharides (CPS) from Gentiana scabra bge (G. scabra) were investigated. The optimal extraction conditions were determined as follows: sample particle size of 80 mesh, solid/liquid ratio of 1:34, extraction voltage of 157.09 V and extraction time of 130.38 s. Under these conditions, the extraction yield of CPS had reached 15.03 ± 0.14% (n = 3). Chemical composition analysis indicated CPS was mainly composed of mannose, rhamnose, galacturonic acid, glcose, galactose, arabinose and fucose in a molar ratio of 1.00:9.89:51.59:35.37:38.06:99.13:21.34, respectively. The average molecular weight of CPS was estimated to be 3.8 × 104 Da. In addition, the potential antioxidant activity of CPS extracted by STE were demonstrated by DPPH radical scavenging assay, superoxide anion radical scavenging assay, hydroxyl radical scavenging assay and ferric reducing power assay. Overall, this study provided an effective extraction technique for G. scabra polysaccharides which would be explored as a promising natural antioxidant agent applied in functional foods or medicines. © 2016 Elsevier B.V. All rights reserved.

1. Introduction In recent years, more and more attention has been casted on polysaccharides obtained from natural Chinese herbal medicine in the fields of biochemistry and pharmacology due to their significant pharmacological actions [1]. Many previous studies have suggested that plant polysaccharides possessed a variety of biological activities, such as antidiabetic [2], antioxidant [3,4], anti-cancer [5], antimicrobial [6], antitumor [7] and immunological activities [8,9]. With the development of immunology, cytobiology and molecular biology, search for plant-derived medicine with low cytotoxicity and few side effects has become an important branch of biomedicine [10]. Therefore, discovery and evaluation of novel and safe polysaccharides obtained from plants have become a very popular research topic. The genus Gentiana of the family Gentianaceae comprises over 400 species that are widely distributed in alpine habitats of temperate regions of Asia, Europe, and America. Some species also

∗ Corresponding author. E-mail address: [email protected] (F. Zhong). http://dx.doi.org/10.1016/j.ijbiomac.2016.08.059 0141-8130/© 2016 Elsevier B.V. All rights reserved.

thrive in Northwest Africa, Eastern Australia, and New Zealand [11]. Gentiana scabra, which was officially listed in the Chinese Pharmacopoeia as “Longdan” [12] is one of the most famous traditional Chinese herbal medicines. It had been reported to be effective in treating liver dysfunction, anorexia, inflammation, and indigestions [13]. Current research on G. scabra mainly focuses on gentiopicroside, which had been considered as the primary chemical constituents for the pharmacological actions [14–17]. The latest reports on G. scabra, however, have indicated that polysaccharide is another main bioactive component with potent antioxidant and immunological activities [10,18]. In terms of extraction technique of polysaccharides from G. scabra, as far as we are aware, the literatures have been scare. Furthermore, among the few reported methods, basic hydrolysis [19], maceration and hot water reflux extraction [20,21] were mostly used methods which usually needed longer extraction time, large volumes of solvent and high extraction temperature and caused degradation of target ingredient, low extraction efficiency and high cost in the production of industrial process [22]. New approaches including microwave-assisted method [23] and ultrasound-assisted method [24] have been reported to extract polysaccharides from G. scabra during the last few years, which have improved the extraction technology significantly since it min-

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imized the solvent consumption and increased the extraction yields of target compound [25]. MAE and UAE, however, still have the disadvantages including long extraction time and inhomogeneous heating [7]. Therefore, a novel and efficient technique which could avoid the disadvantages mentioned above is an urgent need for the development of extraction of polysaccharides from G. scabra. A new developed method named smashing tissue extraction (STE) had become an emerging and potential technology in the pharmaceutical industry owing to its obvious advantages including environment-friendly technique, lower energy consumption, simplified manipulation and especially higher extraction yield within a very short extraction time (finished an extraction successfully within seconds or minutes at most) in the last few years [26–28]. Moreover, the degradation chance of the CPS can also be reduced obviously since STE is commonly performed at room temperature. Compared with other classical methods [29,30], STE had been characterized as the easiest and fastest extraction technique for biological components derived from plant tissues, these advantages can be explained and indicated by the basic principle of STE as follows: based on high velocity mechanical shearing power and ultra-dynamic molecular percolation techniques, the roots, stems, leaves, flowers, fruits and other parts of various medical plant was crushed into crude powder within a few seconds or minutes in the presence of proper extraction solvents at room temperature, the target ingredients reached the balance of internal and external tissues rapidly and subsequently be separated from the mixtures by filtration [30,31]. Therefore, STE can be developed as an effective method for extracting CPS. To the best of our knowledge, there is no study on extraction of G. scabra polysaccharides by the use of STE. Response surface methodology (RSM) originated from the report of Box and Wilson [32] was an effective statistical method which had been widely used to optimize the complex process due to the advantages of reduction of extraction time, medical plant materials and cost [33,34]. In addition, the effects of single factors as well as possible interactions between the independent variables on the index (desirable response) can be acquired from the accurate analytical results of RSM [35–37]. In the present study, the extraction conditions, preliminary characterization and antioxidant ability in vitro of CPS in G. scabra were investigated. Firstly, the STE process was optimized by RSM based on Box-Behnken statistical design and further compared with another three common extraction techniques including HRE, MAE and UAE. Then, CPS was characterized by chemical analysis, monosaccharide composition, determination of molecular weight, ultraviolet spectroscopy (UV), high performance liquid chromatography and Fourier transform-infrared spectroscopy (FT-IR). Finally, the antioxidant activity of CPS in vitro was evaluated by investigating the superoxide anion radical scavenging activity, hydroxyl radical scavenging activity, DPPH radical scavenging activity and ferric reducing ability.

2. Materials and methods 2.1. Materials and reagents Raw material of G. scabra was collected from Huadian city, Jilin province, China in October in 2014. The samples were identified as G. scabra based on the morphologic appearance, microscopic and physiochemical analyses according to the latest Chinese Pharmacopoeia [12]. After being dried at room temperature, the samples were ground by a grinder machine and passed trough 40, 60, 80, 100 and 120-mesh screen, respectively, and then were stored in a refrigerator at 4 ◦ C until used. Phenol of analytical grade was bought from Beijing Dingguo Bio-Tech Co., Ltd. (Beijing, China);

DPPH was bought from Sigma-Aldrich (St. Louis, USA); the standard of d-glucose (purities ≥ 99.5) was purchased from Amresco Inc. (American); petroleum ether, ethanol, phenol, sulfuric acid and other chemicals in the studies were of the highest quality commercially available from local suppliers (Jilin, China). 2.2. Pretreatment of samples In order to avoid the effects of some impurities such as some colored compounds, monosaccharide, oligosaccharides and some soluble small molecule materials contained in G. scabra on the determination of polysaccharide, the powder of G. scabra was firstly degreased with petroleum ether (boiling point: 60–90 ◦ C) in the soxhlet apparatus for 12 h, subsequently the residue separated from petroleum by filtration was extracted with 80% ethanol for 4 h according to our recent study [38] as well as the relative previous reports [39,40]. Finally, ethanol was removed by distillation and the insoluble residue was collected and dried at 45 ◦ C, and then stored at 4 ◦ C until use. 2.3. Sample preparation Four kinds of extraction methods, including STE, hot water reflux extraction (HRE), microwave-assisted extraction (MAE) and ultrasonic-assisted extraction (UAE) have been employed to extract CPS in the present study. However, In consideration of the main objective of developing an effective and novel STE method in this paper, only STE conditions were investigated systematically. The extraction parameters of STE were optimized by RSM on the basis of investigating single factors including solid-liquid ratio, particle size of medicinal material sample, extraction voltage and extraction time. And the rest three reference methods mentioned above were all conducted purely according to the published literatures [20,23,24] on extracting polysaccharides from G. scabra with a slight modification, i.e. the extraction parameters used in UAE, MAE and HRE were all originate from the previous reports. 2.3.1. Smashing tissue extraction STE was carried out on a smashing tissue extractor (JHBE-50S, Zhengzhou, China). According to the experimental design in this study, 1.0 g of the pretreated sample mentioned in Section 2.2 was accurately weighed and put into a 200 mL extraction cell, after a certain amount of distilled water was added, the mixture was extracted under a definite voltage at room temperature for some time. After the extraction was performed, the slurry was centrifuged at 3000 r/min for 15 min to gain the supernatant; the insoluble residue was added to small amount of distilled water and handled again as mentioned above. The incorporated supernatant was concentrated to one-fifth of the initial volume at 60 ◦ C in a rotary evaporator under reduced pressure and added four times of dehydrate ethanol; subsequently, the mixtures was stirred vigorously and kept for 24 h at 4 ◦ C for precipitation [41,42]. Finally, the precipitate was collected by vacuum filter. After being washed by dehydrated alcohol and acetone for three times, the precipitate was removed protein by the method of Sevag [43] and then dried at 50 ◦ C under reduce pressure until its weight remained constant. All experiments were performed in triplicate. 2.3.2. Hot water reflux extraction HRE was conducted purely according to the study reported by reference [20] with a slight modification. 1.0 g of the pretreated sample was added to 27.0 mL distilled water and heated by electric stove maintained at 100 ◦ C for 3 h, the extraction was finished when the extraction was carried out twice. The resulting mixture was centrifuged at 3000 r/min for 15 min, and the following procedures

Z. Cheng et al. / International Journal of Biological Macromolecules 93 (2016) 369–380

were operated as described in Section 2.3.1. All experiments were performed in triplicate. 2.3.3. Microwave-assisted extraction According to the previous literature [23] on extraction of polysaccharides from G. scabra, 1.0 g of the pretreated sample was accurately weighed into a 150 mL flat-bottomed flask which contained 27.0 mL distilled water, then the flat-bottomed flask was sealed using ground stopper and irradiated at the microwave power of 450 W for 4 min maintained at 100 ◦ C. After microwave irradiation, the flat-bottomed flask was removed from apparatus and cooled to room temperature. The water extraction solutions as well as the following procedures were all handled as described in Section 2.3.1. All experiments were performed in triplicate. 2.3.4. Ultrasonic-assisted extraction In the UAE experiments, an ultrasonic cleaner was used as the ultrasonic generator, to which the constant working frequency was 45 kHz and the power was 250 W on the scale of 0–100%. The extraction of polysaccharides was carried out as described in relative reference [24] with minor modification. 1.0 g of the pretreated sample was weigh accurately and put into 120 mL of distillation water contained in a round-bottom flask, then the flask was partially immersed into the ultrasonic bath and treated at the ultrasonic power of 180 W maintained at 50 ◦ C. The water extraction solutions as well as the following procedures were all handled as described in Section 2.3.1. All experiments were performed in triplicate. 2.4. Analytical methods The content of polysaccharides was calculated according to the phenol-sulfuric method using d-Glucose as a standard [44], The yield (%) of polysaccharide was calculated with Eq. (1) [41]. Polysaccharide yield (%) =

Weight of dried CPS (g) × 100 Weight of sample (g)

(1)

2.5. Experimental design 2.5.1. Single factor experimental design The effects of extraction voltage (90, 110, 130, 150, 170 and 190 V), extraction time (30, 60, 90, 120, 150 and 180 s), particle size of sample (40, 60, 80, 100 and 120 mesh) and ratio of sample to liquid (g/mL) (1:10, 1:20, 1:30, 1:40 and 1:50) on the yield (%) of polysaccharide were firstly studied by designing single factors. In each experiment, the factor under evaluation was changed while the other factors remained unchanged. 2.5.2. Box-Behnken design On the basis of single factor experiments results, RSM was used to further optimize the STE conditions, in which three independent variables (X1 , extraction voltage; X2 , extraction time and X3 , amount of extraction solvent) at three levels [7] were analyzed. The detailed range and levels of the independent variables were presented in Table 1. Taking the evaluation of pure error sum of squares into consideration [45], five replicates at the center of the design were performed in the complete design consisted of 17 experimental points. Furthermore, the experiment was performed in a random to realize the minimization of the effects of unexpected variables [46]. Data from the BBD were fitted to a quadratic polynomial model by using the Design-Expert software of 8.0.6, the following quadratic Eq. (2) can be right for explaining the model. Y = ˇ0 +

3  i=1

ˇiXi +

3  i=1

2

ˇiiXi +

3 3  

ˇijXiXj

i=1 j=i+1

(2)

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Where Y was the yields of polysaccharides (the dependent variables), ˇ0 was the constant coefficient, ˇi , ˇii , and ˇij were the coefficients which represented the linear, quadratic, and crossproduct effects of the factors including X1 , X2 , and X3 , respectively, where X1 was extraction voltage; X2 was extraction time and X3 was ratio of sample to liquid (g/mL). 2.6. Scanning electron microscopy (SEM) analysis In order to investigate the effects of extraction methods on the microstructure of G. scabra and reveal the extraction mechanism, the morphological changes of crude material before and after extraction of polysaccharide by different techniques were observed according to the previous reports [8,38], in which the residue was collected by centrifugation and dried at 50 ◦ C. The medicinal material samples used for investigation were fixed on the silicon wafer and sputtered with gold using an ion sputter coater. The shape and the surface characters of the samples scanned under high vacuum conditions at an accelerating voltage of 15.0 KV were recorded on the scanning electron microscope (Quanta-200, FEI Ltd., The Netherlands). 2.7. Analysis of polysaccharides characterization 2.7.1. Analysis of contents of total sugars, sulfate, protein and uronic acid To characterize the chemical properties of CPS, its physiochemical properties were determined in this study. The total sugar content was determined by the phenol–sulfuric acid method using d-glucose as the standard [47]. The protein content was analyzed by the method described by Bradford [48] in which bovine serum albumin was selected as the standard. The sulfate radical content was estimated as reported in the previous literature [49]. The uronic acid content was determined by the carbazole–sulfuric acid method [50]. 2.7.2. Analysis of monosaccharide composition The monosaccharide composition of CPS was analyzed using reversed-phase high performance liquid chromatography after pre-column derivatization according to the method reported in the previous literature [34] with slight modification. 5.0 mg of CPS was hydrolyzed with 2 mol/L trifluoroacetic acid at 100 ◦ C for 6 h. Excessive acid was removed by co-distillation with methanol after hydrolysis. The dry hydrolysate was firstly dissolved in 100 ␮L of 0.3 mol/L NaOH, and then was added to 120 ␮L of 0.5 mol/L methanol solution of 1-phenyl-3-methyl-5-pyrazolone (PMP) at 75 ◦ C for 90 min. The resulting mixtures were added to 100 ␮L of 0.3 mol/L HCl, vigorously shaken and added to 4 mL of trichloromethane. Subsequently, the resulting product was centrifuged at 3000 r/min for 5 min, the supernatant containing the labeled carbonhydrate was filtered through 0.22 ␮m PES membranes (MSI, Westborough, MA, USA) and 20 ␮L of the filtrate was injected into the C18 column (Hypersil, 4.6 × 250 mm, 5 ␮m). The mobile phase was a mixture composed of 0.1 mol/L KH2 PO4 (pH,10)–acetonitrile (87:13). The flow rate was 1.0 mL/min and column temperature was 30 ◦ C. Sugar identification was conducted by comparison with reference sugars (mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactosel, xylose, arabinose). 2.7.3. Determination of molecular weights The average molecular weights of polysaccharides obtained from HRE, MAE, UAE and STE were all identified by highperformance gel-permeation chromatography (HPGPC) on a Waters high performance liquid chromatograph system (Waters Corporation, USA) equipped with a Waters 2410 refractive index detector (RID). Two UltrahydrogelTM Linear columns

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Table 1 The results of code experiment and predicted value for RSM design based on BBD. No.

X1

X2

X3

Voltage(V)

Extraction time(s)

Amount of solvent (g/mL)

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

0 1 0 1 0 −1 1 0 0 1 −1 0 0 −1 0 0 −1

0 0 1 −1 −1 0 0 0 0 1 −1 −1 0 1 0 1 0

0 1 1 0 −1 −1 −1 0 0 0 0 1 0 0 0 −1 1

150 170 150 170 150 130 170 150 150 170 130 150 150 130 150 150 130

120 120 150 90 90 120 120 120 120 150 90 90 120 150 120 150 120

30 40 40 30 20 20 20 30 30 30 30 40 30 30 30 20 40

(300 mm × 7.8 mm) were employed as the separation medium, which were connected in series. The columns were maintained at 40 ◦ C and eluted with 0.1 M NaNO3 solution at a flow rate of 0.8 mL/min, and the injection volume was 50 ␮L. Column calibration was carried out using standards dextrans (six different molecular weights: T10, T40, T70, T100, T380 and T500). Calibration curve of Log Mw (molecular weight) of standard dextrans against their retention time (Rt ) was acquired (Log Mw = −0.496Rt + 6.195, R2 = 0.9958). 2.7.4. IR and UV spectrometric analysis The organic functional groups of CPS were identified and recorded by a Nicolet 6700 Fourier transform infrared Spectrometer (Thermo Co., USA) using KBr disks method. Briefly, samples were dried at 35 ∼ 44 ◦ C in vacuum over P2 O5 for 48 h, ground with spectroscopic grade potassium bromide (KBr) powder and then pressed into 1 mm pellet for FT-IR spectral measurement within 4000–400 cm−1 . UV spectra of CPS was recorded with a UV-2450 Spectrophotometer (Shimadzu Co., Kyoto, Japan). 2.8. Antioxidant activity assay 2.8.1. DPPH radical scavenging assay Antioxidants have abilities to seize the free-radical chain of oxidation and form stable free radicals, which would prevent further oxidation. DPPH had been used extensively as a free radical to evaluate reducing substances. In this paper, the DPPH free radical scavenging ability of CPS was measured according to the previously described method [51,52] with a slight modification. Briefly, 2.0 mL DPPH· solution (0.01 mmol/L in dehydrate alcohol) was added to 2.0 mL of CPS solution with six different concentrations (0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg/mL), the mixtures were shaken vigorously and incubated at 25 ◦ C for 30 min in darkness, and the absorbance of resulting mixtures was recorded at 517 nm. Vc was used as positive control. In addition, DPPH radical scavenging activities of CPS extracted by HRE, MAE and UAE were also determined at the concentration of 1.0 mg/mL. The DPPH radical scavenging activity was calculated according to Eq. (3): A0 − (A1 − A2 ) Scavenging rare (%) = × 100 A0

(3)

Where A0 was the absorbance of blank samples (2.0 mL ethanol plus 2.0 mL DPPH solution), A1 was the test group absorbance (2.0 mL samples solution plus 2.0 mL DPPH solution) and A2 was the control group absorbance (2.0 mL samples and 2.0 mL ethanol).

Extraction yield (%) Experimental

Predicted

15.43 13.70 14.49 13.23 10.69 9.75 12.76 13.89 14.43 14.03 9.82 11.76 14.29 12.51 15.16 10.96 13.03

14.64 13.90 14.50 13.14 10.68 9.55 12.87 14.64 14.64 13.81 10.04 11.65 14.64 12.61 14.64 11.07 12.92

2.8.2. Hydroxyl radical scavenging assay The hydroxyl radicals scavenging assay was determined based on the literature reported by Wu [51] with minor modifications. Reaction mixtures (8.0 mL) consisted 2.0 mL of CPS (extracted by STE) solution with six different concentrations (1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 mg/mL), 0.6 mL 6 mM H2 O2 , 0.6 mL 6 mM FeSO4 , 0.6 mL 6 mM salicylic acid in Tris–HCl buffer solution (pH 7.4), the reaction mixtures were incubated at 37 ◦ C in a water bath for 30 min, Vc was used as positive control. The absorbance was observed and recorded at 510 nm. In addition, hydroxyl radical scavenging activities of CPS extracted by HRE, MAE and UAE were also determined at the concentration of 1.0 mg/mL. The hydroxyl radicals scavenging rate was expressed as Eq. (4): Scavenging rate (%) =

A0 − (A1 − A2 ) × 100 A0

(4)

Where A0 was the absorbance of blank (distilled water instead of sample solution), A1 was the absorbance of sample and A2 was the absorbance of sample under identical conditions as A1 (distilled water instead of salicylic acid solution). 2.8.3. Assay of superoxide anion radical scavenging activity Superoxide anion radical was generated in the system of pyrogallol’s auto oxidation in an alkalescent condition. The scavenging effects of polysaccharides on superoxide anion radical (O2• ) was investigated according to the previous report [52,53] with a minor modification. Briefly, CPS solutions with different concentrations (0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg/mL) were mixed with 50 mM Tris–HCl buffer (pH 8.2) and incubated at 25 ◦ C for 20 min. Subsequently, 25 mM pyrogallol at the same temperature was added to the mixture and the reaction proceeded for 5 min. Finally, 1 mL of 8 mM HCl was quickly added to terminate the reaction, Vc was used as positive control. The absorbance of the mixture was determined at 299 nm. Furthermore, superoxide anion radical scavenging activities of CPS extracted by HRE, MAE and UAE were also determined at the concentration of 1.0 mg/mL. The superoxide anion scavenging activity was calculated using Eq. (5): Scavenging activity (%) =

A0 − A1 × 100 A0

(5)

Where A 0 was the absorbance without sample and A1 was absorbance with sample. 2.8.4. Measurement of reducing power The reducing power of CPS was determined based on the method described by Zhen et al. [53] with minor modification. 2.0 mL of

Z. Cheng et al. / International Journal of Biological Macromolecules 93 (2016) 369–380

the CPS solution with six different concentrations (0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg/mL), 2.0 mL of 0.2 mol/L phosphate buffer (pH 6.6) and 2.0 mL of potassium ferricyanide (1%, w/v) were mixed and incubated at 50 ◦ C in water bath for 20 min, followed by addition of 2.5 mL 10% trichloroacetic acid. Afterwards, the mixture was centrifuged at 3000 r/min for 10 min. Subsequently, 2.0 mL of the supernatant obtained from each incubated mixture was mixed with 2.0 mL of deionized water and 0.4 mL of fresh ferric chloride (FeCl3 , 0.1%, w/v) in a test tube. After a 10 min reaction at 50 ◦ C in a water bath, the absorbance of the resulting solution was measured at 700 nm against as the blank (water instead of the CPS solution). Vc was used as positive control. Absorbance of the reaction mixture indicated the reduction capability of sample, and the reducing power can be determined as Eq. (6) Reducingpower = A1 −A2

(6)

Where A1 was the absorbance of the sample and A2 was the absorbance of the sample under identical conditions as A1 with water instead of FeCl3 solution. 2.9. Statistical analyses All data were expressed as means ± standard deviations (SD) of three replicated determinations. Statistical analysis was performed by one-way analysis of variance (ANOVA) using the SPSS 13.0 (version 19.0, SPSS Inc.), and P-Values of less than 0.05 were regarded as statistically significant. The STE model was also analyzed using the ANOVA and DesignExpert 8.0.6 (Stat-Ease Inc., Minneapolis, USA). The significance of all terms in the polynomial was statistically tested by evaluating the F-value at a probability (p) of 0.001, 0.01 or 0.05. The quality of the response surface model was expressed by the determination coefficient (R2 ), lack of fit, adjusted determination coefficient (Adj-R2 ), predicted determination coefficient (Pre-R 2 ), coefficient of variation (C.V.) and adequate precision. The fitted polynomial equation was expressed as surface to visualize the relationship between response and each factor. 3. Result and discussion 3.1. Optimization of single extraction conditions 3.1.1. Effects of extraction time on extraction yield of CPS Extraction time played a very important role in the extraction process of CPS from plant materials, which had a significant influence on the extraction efficiency of target components as well as energy consumption [30]. In order to evaluate the effects of extraction time on extraction yield, six different extraction time (30, 60, 90, 120, 150 and 180 s) were tested, with the ratio of material to solvent, extraction voltage and particle size of sample fixed at 1:40 (g/mL), 110 V and 60 mesh, respectively. As shown in Fig. 1A, the extraction yield of CPS increased from 6.44% to 11.97% as extraction time was lengthened from 30 s to 120 s. And when the treatment time continued to increase, the extraction yield almost no longer changed. Therefore, extraction time of 90–150 s was selected for further optimization in the following studies. 3.1.2. Effects of material-solvent ratio on extraction yield of CPS Ratio of material to solvent was also an important factor for extraction of CPS from plant materials. Small amount of extraction solvent may cause incomplete immersion of raw material into the extraction solvent, resulting in decrease of mass transfer and lower extraction yields of target components [7]. While a large amount of solvent can not only cause complex procedures and unnecessary wasting, but also bring about a lower contact probability between

373

medicinal materials with small particle size and broken cutter of smashing tissue extractor employed in this study, resulting in the decrease of shear probability of cutting head and final incomplete extraction of CPS from G. scabra. To evaluate effects of the amount of solvent on extraction yield, a series of extractions were performed with different solid/liquid ratios (1:10, 1:20, 1:30, 1:40 and 1:50 g mL−1 ), with the extraction time, extraction voltage and particle size of sample fixed at 120 s, 110 V and 60 mesh, respectively. Fig. 1B revealed that the average extraction yield increased obviously when material-solvent ratio was changed from 1:10 to 1:30, but the extraction yield of CPS decreased when solid/liquid was fixed at 1:40. Therefore, the ratios of material to solvent between 1:20 to 1:40 were used for further optimization experiments. 3.1.3. Effects of extraction voltage on extraction yield of CPS The extraction voltage was also an important factor that affected the extraction yield of CPS, for the higher of the extraction voltage, the higher of the rotational speed of the inside cutting head which was fixed into the extractor. To be specific, the shearing power between the inside and outside cutting head can be increased dramatically when the voltage was maintained at a high level, and thus the broken efficiency of medicinal material as well as the permeation rate of target compounds between cell tissue and extraction solvent were further accelerated. In order to examine the effects of voltage on extraction yield of CPS exactly, the extractions with six different voltages (90, 110, 130, 150, 170 and 190 V) were investigated, with the extraction time, material-solvent ratio and particle size of sample fixed at 120 s, 1:30 and 60 mesh, respectively. Fig. 1C revealed the extraction yield increased rapidly as the extraction voltage increased from 90 V to 150 V, and achieved 15.62% at 150 V. However, the extraction yield of CPS decreased slightly when voltage continued to increase. This result was almost in agreement with the findings of our previous report on extracting lignan with STE method [31]. Thus, the voltage of 130–170 V was used for further optimization experiments. 3.1.4. Effects of particle size of sample on extraction yield of CPS The air-dried G. scabra were ground and passed through No. 40, 60, 80, 100 and 120 meshes, respectively, with the extraction time, material-solvent ratio and extraction voltage fixed at 120 s, 1:30 and 150 V. As shown in Fig. 1D, the extraction yield increased rapidly with the increase of mesh, and achieved a maximum of 16.82% when sample sieved 80 meshes were employed. Our previous study [31] had indicated these results may be concerned with the increase of surface area of medicinal material, and higher permeability or diffusivity of solvent into sample material. However, no significance difference can be observed when the particle size was changed from 80 to 120 meshes, taking the difficulty of subsequent filtration into consideration, medicinal material powder sieved with 80 meshes was determined as the optimum particle in the present study. 3.2. Optimization of extraction conditions of CPS by RSM 3.2.1. Statistical analysis and model fitting RSM was an effective and powerful approach for the optimization of complex process since it offered the advantages of saving time, space and raw material when compared to classical single factors experiments [41]. The RSM employed in this study involved three individual variables with three levels based on BBD, in which five replicates at the center point were conducted to investigate the inherent variability and process stability, i.e. the pure error sum of squares can be obtained finally. The 17 different experimental conditions and the response (extraction yield of CPS) including experimental and predicted values were shown in Table 1. Using multiple regression analysis on the experimental data, the response

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Fig. 1. Effects of different extraction parameters on extraction yield of polysaccharides. (A: Extraction time, s; B: Ratio of material to solvent, g/mL; C: Extraction voltage, V; D: Particle size of sample, mesh).

Table 2 Analysis of variance for the fitted regression model. Source

df

SS

MS

F

P

Pr > F

Model X1 X2 X3 X1 X2 X1 X3 X2 X3 X1 2 X2 2 X3 2 Residul Lack of Fit Pure error Cor Total

9 1 1 1 1 1 1 1 1 1 7 3 4 16

48.97 9.27 5.27 9.72 0.89 1.37 0.016 3.83 6.99 7.98 1.87 0.24 1.62 50.84

5.44 9.27 5.27 9.72 0.89 1.37 0.016 3.83 6.99 7.98 0.27 0.081 0.41

20.40 34.74 19.74 34.46 3.35 5.13 6.37 14.36 26.22 29.90

<0.0003 0.0006 0.0030 0.0005 0.1100 0.0579 0.0395 0.0068 0.0014 0.0009

**

0.20

0.8915

N.S

** ** **

N.S. N.S. * ** ** **

N.S. Not significant. * Significant at 0.05 level. ** High significant at 0.01 level.

Y (extraction yield of CPS) can be characterized by the following second-order polynomial Eq. (7): Y = 14.64 − 1.08X 1 + 0.81X 2 + 1.10X 3 − 0.47X 1 X2 − 0.69X 1 X3 − 0.61X 2 X3 − 0.95X 1 2 − 1.29X 2 2 − 1.38X 3 2

(7)

Where Y was the extraction yield of CPS, X1 , X2 and X3 represented voltage (V), extraction time (s) and amount of extraction solvent (mL/g), respectively. The statistical significance of the regression equation was evaluated using the F-test and p-value. The results of analysis of variance (ANOVA) for the response surface quadratic model were shown in

Table 2. As can be seen in Table 2, the high F-value (48.97) and low P-value less than 0.0003 of model implied the regression equation was ideal and the model was highly statistically significant. The credibility analysis of the regression equation were presented in Table 3, from which we can know the value of determination coefficient (R2 ) of 96.33% was close to 1.0, indicating the correlation between actual and predicted values was satisfactory. The adjusted determination coefficient (Adj-R2 ) of 91.61% indicated that 91.61% of the extraction yield of CPS could be illustrated by the model. Meanwhile, a relatively low value of coefficient of variation (C.V) suggested a better reliability of the experiments values, and thus

Z. Cheng et al. / International Journal of Biological Macromolecules 93 (2016) 369–380 Table 3 The credibility analysis of the regression equation. Index marka

Extraction yield of CPS

Std. dev Mean C.V.% PRESS R-Squared Adjust R-Squared Pred R-Squared Adequacy Precision

0.52 12.94 3.99 6.43 0.9633 0.9161 0.8735 12.860

a

The results were obtained from the Design Expert 8.0.6 software.

C.V. value of 3.99% in the current paper showed the accuracy and the general availability of the polynomial model were adequate. Significance of the model was also determined by lack-of-fit test which could measure the failure of the model by means of presenting the data in experiment domain at points which were not included in the regression, as shown in Table 3, the F-value of 0.20 and p-value of 0.8915 suggested the lack-of-fit was not significant. The P-value was adopted as a tool for evaluation of the significance of each coefficient, which in turn may imply the pattern of the interactions between the variables. The smaller the P value was, the more significant of the corresponding coefficient was. As can be seen from Table 2, the lineal coefficient (X1 , X2 and X3 ) and quadratic term coefficient (X1 2 , X2 2 and X3 2 ) were highly significant with small P values (P < 0.01), the coefficient of X2 X3 was significant with P value less than 0.05, indicating all the three variables investigated in this study were high significant, the interactions of X2 X3 was significant. The interaction terms of X1 X2 and X1 X3, however, were found to be non-significant for the P value was above 0.05. Analysis of the regression equation using Design-Expert software allowed us to generate contour plots and three-dimensional maps (Fig. 2) to visualize the interaction between any two independent variables and dependent variable while the other one variable remained at zero. 3.2.2. Verification of the predictive model The optimal parameter values of the factors for obtaining maximal yield of CPS (15.19%) according to Eq. (7) could be predicted as follows: extraction time of 130.38 s; extraction voltage of 157.09 V and material/solvent ratio of 1:34.02. It was of great need to test the accuracy and reliability of the model equation for predicting an optimum response value. Taking the precision of extraction equipment into account, the confirmatory experiment was carried out under the following procedure: 1.0 g of the dried G. scabra powder passed through 80-mesh sieve was accurately weighed and added to 34 mL of distilled water, then extracted under the voltage of 158 V for 131 s at room temperature. The actual extraction yield of CPS had reached 15.03 ± 0.14% (n = 3), which was very close to the predicted value, indicating the predicted model was accordant with the actual value and thus can be used for the prediction of extraction yield of CPS G. scabra successfully. 3.3. Comparison of different extraction method In this study, the STE method for extraction of polysaccharides from G. scabra was compared with three other techniques including HRE, MAE and UAE. As can be seen from Table 4, the extraction yield of CPS (15.03 ± 0.14%) obtained from STE was the highest among the four methods, followed by MAE with an extraction yield of 12.56 ± 0.18%. The lowest extraction yield of 10.41 ± 0.27% can be found when UAE was employed, which was also lower than that of HRE (11.12 ± 0.37%). Table 4 also exhibited the average molecular weight and scavenging rate of CPS on DPPH radical, superoxide anion radical and

375

hydroxyl radical. According to the calibration curve of the standards, the average molecular weight of polysaccharides extracted by HRE, UAE, MAE and STE were estimated to be 4.82 × 104 Da, 4.73 × 104 Da, 3.39 × 104 Da and 3.87 × 104 Da, respectively. At the concentration of 1.0 mg/mL, the polysaccharides extracted by the above four methods all exhibited potential antioxidant ability, but the DPPH radical scavenging rate (55.12%–69.38%), superoxide anion radical scavenging rate (28.33%–37.13%) and hydroxyl radical scavenging rate (8.69%–10.24%) were all different. However, all the three radical scavenging activities of CPS increased in the following order: HRE < UAE < MAE < STE. These results implied the antioxidant capability of polysaccharides was affected significantly by the extraction method, and too high or too low molecular of polysaccharides was not beneficial to its antioxidant ability, only the polysaccharides with appropriate molecular weight possessed the higher antioxidant activities. Therefore, G. scabra polysaccharides obtained from STE appeared to be the most effective and potential antioxidant which would be expected to be extensively utilized as a natural antioxidant instead of synthesized antioxidant in the future. Compared with the three reference extraction methods mentioned above, STE gave the highest extraction yield with the highest antioxidant ability in the shortest period of extraction time. In addition, the operation of STE was conducted at room temperature without heating. Therefore, STE can not only save a lot of time and energy as compared to HRE and UAE, but also can bring higher yield and stronger antioxidant ability of CPS than MAE, which demonstrated STE was an efficient extraction approach. The higher extraction yield could be obtained since this novel technique combined all advantages of smashing, stirring and vibration together with the proper solvent, which produced the disruptions of tissues and cell walls and thus resulted in a greater contact area between solid and liquid phase, better access of solvent to valuable components [29]. 3.4. Analysis of microscopic changes Extraction yield of CPS extracted by different methods were related to the extraction characteristics, and thus the effects of the extraction methods on raw material were investigated. SEM analysis was taken advantage to observe the morphology changes of the sample residue as well as the untreated samples. Parenchyma was undamaged and cells were completely nubbly when untreated sample was observed. However, an increasing level of cell damage and parenchyma disorganization increased in the order of HRE < UAE < MAE can be found obviously, which were completely consistent with our recent study on extracting polysaccharides from Schisandra chinesis Baill [38] as well as other relative report [7]. Most importantly, the level of cell walls damage of sample treated by STE were compared with that of MAE in this study, a more significant impact on the structural changes caused by STE can be observed easily, indicating that STE were superior to HRE, UAE as well as MAE. STE combined the techniques of smashing, soaking, stirring and vibration, making the parenchyma of material ultrathin and disorganized. Therefore, the chemical components within the cell can be released easily into the surrounding solvents more than the other methods. 3.5. Characterization of CPS In the present study, after G. scabra raw material was defatted by petroleum and removed some colored materials by 90% ethanol [34], the CPS was prepared using STE method based on the optimal extraction conditions, deproteinization, centrifugation, ethanol precipitation and dryness. Then, CPS was preliminary characterized by chemical analysis, Molecular weight, HPLC, UV and

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Fig. 2. Response surface plots (3-D) showing the effects of variables (X1 : Extraction voltage, V; X2 : Extraction time, s; X3 : Ratio of material to solvent, g/mL) on the response Y. Table 4 Comparisons of the extraction yield, average molecular weigh and antioxidant activityb of CPS extracted by different methods. Method

STE

Extraction voltage/V Microwave irradiation time/min Extraction temperature/◦ C Particle size of sample/mesh Extraction time/min Extraction times Ultrasonic power/W Microwave power/W Ratio of material to water g/mL Extraction yields of CPS (%)a Average molecular weight/Da DPPH radical scavenging activity/% Hydroxyl radical scavenging activity/% Superoxide anion radical scavenging activity/%

157.09

a b

UAE

25 80 2.17 1

50 80 60 1 180

1:34 15.03 ± 0.14 3.87 × 104 69.38 10.24 37.13

1:60 10.41 ± 0.27 4.73 × 104 60.24% 8.76 29.21

MAE

HRE

4 100 80 4 1

100 80 180 2

450 1:27 12.56 ± 0.18 3.39 × 104 62.09 9.97 35.47

1: 27 11.12 ± 0.37 4.82 × 104 55.12 8.69 28.33

Extraction yield of CPS (%) = mean ± S.D. (n = 3) and RSD were all lower than 3.5%. Concentrations of polysaccharides extracted by four methods were all 1.0 mg/mL.

Table 5 The chemical compositions of CPS. Component

CPS

Content (%) Sugar

Protein

Sulfate

Uronic acid

41.33%

1.28%

33.38%

17.86%

FT-IR. The chemical compositions of CPS were shown in Table 5, indicating that the contents of sugar, protein, sulfate radicals and uronic acid were 41.33%, 1.28%, 17.86% and 33.38%, respectively. Notably, CPS had relatively high content of sulfate radical (17.86%). The monosaccharide composition analyses were shown in Fig. 3. HPLC analysis showed that CPS mainly consisted of mannose, rhamnose, galacturonic acid, glcose, galactose, arabinose and fucose in a molar ratio of 1.00: 9.89: 51.59: 35.37: 38.06: 99.13: 21.34, respectively. Fig. 3 For determination of homogeneity and molecular weigh of CPS, the HPLC system was employed. As shown in Fig. 4, the

HPLC profile of CPS was presented as a single and symmetrical peak, which suggested that CPS extracted by STE was homogeneous polysaccharides. Base on the calibration curve of dextran standards, the average molecular weight of CPS was estimated to be 3.87 × 104 Da. Fig. 4 The UV spectra of CPS were shown in Fig. 5. No significant absorption at 260–280 nm can be found in the UV spectrum, indicating no presence or only a slight amount of protein contained in CPS. This finding was consistent with the results obtained from chemical analysis of CPS, as can be seen from Table 5, the content of protein was only 1.28%. Fig. 5 The IR spectrum was shown in Fig. 6. The strong and broad band around 3434 cm−1 was attributed to the O H stretching vibration, and the band around 2912 cm−1 and 1384 cm−1 were due to C H stretching vibration and bending vibration, respectively. Two distinct stretching peaks at 1743 cm−1 and 1630 cm−1 were related to the stretching vibrations of C O and COO− , which suggested the presence of uronic acids in CPS from G. scabra [34]. In addition, the two stretching peaks around 1015 cm−1 and

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Fig. 5. UV spectra of CPS from G. scabra.

Fig. 6. FT-IR spectrum of CPS from G. scabra.

3.6. In vitro antioxidant activities of CPS Fig. 3. HPLC analysis of monosaccharide composition of derivatives of nine standards (A) and CPS (B) in G. scabra, respectively (Man, mannose; Rha, rhamnose; GlcA, glucuronic acid; GalA, galacturonic acid; Glc, glcose; Gal, galactose; Xyl, xylose; Arab, arabinose; Fuc, fucose).

3.6.1. Scavenging activity to DPPH radicals DPPH radical was one of the few relatively stable free radicals and had been extensively employed to estimate the free radical scavenging activities of antioxidants such as polysaccharides. When DPPH radicals were reduced by accepting an electron or hydrogen in the presence of antioxidant compound, a noticeable change in color from violet to yellow can be found and evaluated at 517 nm [51]. Therefore, the antioxidant ability of a substance can be expressed as its scavenging ability on DPPH free radical [53,55]. Fig. 7A described the DPPH scavenging ability of CPS extracted by STE and ascorbic acid (Vc). As can be seen, both CPS and Vc showed an obvious antioxidant activity in a concentration-dependent manner. DPPH radical scavenging rate of CPS significantly increased from 17.80% to 80.81% as the CPS concentration increased from 0.2 to 1.2 mg/mL, and DPPH radical scavenging rate of Vc dramatically increased from 22.11% to 95.81% during the same concentration range. These results indicated both Vc and CPS had significant DPPH radical scavenging ability. DPPH radical scavenging ability was affected by many factors, some literatures have reported the reason may be related with the relatively higher contents of uronic acid [56], which possessed strong hydrogen-donating ability and can form non-radical DPPH H by combing with DPPH [3].

Fig. 4. HPGPC chromatogram of CPS extracted by STE.

1107 cm−1 indicated the presence of C O C and C O H [54], indicating the presence of pyranose [55]. Overall, these results indicated that CPS exhibited the typical absorption peaks of plant polysaccharide.

3.6.2. Scavenging activity to hydroxyl radical Hydroxyl radicals, which were well known as the most reactive free radical, can easily cross cell membranes and readily react with almost all the bio-macromolecules including carbohydrates, lipids, proteins and DNA in cells and resulting in aging, cancer, and several diseases [57]. Therefore, the removal of hydroxyl radical was extremely important to protect living systems [33]. Fig. 7B

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Fig. 7. Antioxidant ability of CPS determined by DPPH radical scavenging ability (A), Hydroxyl radical scavenging ability (B), Superoxide anion scavenging ability (C) and Ferric reducing antioxidant power (D) .

showed that the hydroxyl free radical scavenging effects of CPS sample obtained from STE and Vc increased with the increase of concentration, and the scavenging rate of CPS was 29.53% at 5.5 mg/mL. When in all the investigated concentration range from 3.0 to 5.5 mg/mL, CPS exhibited a relatively poor hydroxyl radical scavenging capacity. However, the hydroxyl radical scavenging rate of Vc reached 94.31% at the concentration of 3.0 mg/mL. These findings demonstrated that the CPS extracted by STE possessed the ability to scavenge hydroxyl radical, but it was lower than that of Vc. The possible mechanism on scavenging ability of polysaccharides in this process may be associated with the number of active hydroxyl. The addition of electron donating substituent increased radical scavenging activity because of increasing electron density on the heterocyclic ring of the carbons. Low activity may relate to the formation of strong intermolecular and intramolecular hydrogen bonds, resulting in inhibition of the reactivity of hydroxyl in the polymer chains [58].

3.6.3. Scavenging activity to superoxide anion radicals Superoxide anion radical, one kind of weak oxidant and a source of free radicals formed in most organisms, can degrade continuously to produce other active ROS such as hydrogen peroxide, signal oxygen and hydroxyl radical by attack other biological molecules, and thus it can directly damage DNA and membrane of cell and further induce pathological incidents [3,59]. Therefore, it was of great need to remove superoxide anion radical.

Superoxide radical can be generated by pyrogallol auto oxidation in which the colored compound named tangerine phenol can be produced [60] and lead to a color change from purple to yellow, and thus the content of superoxide anion radicals and the antioxidant activity of the sample can be evaluated by observing the absorbance. In this study, the scavenging effects of Vc and CPS sample obtained from STE with the concentrations ranged from 0.2–1.2 mg/mL were shown in Fig. 7C. The scavenging ability on superoxide anion radicals also followed a dose-dependent manner at all the tested concentrations. The scavenging rate increased from 5.07% to 46.84% as the polysaccharide concentration increased from 0.2 mg/mL to 1.2 mg/mL, indicating the CPS obtained from STE have definite ability to scavenge superoxide anion radicals. Compared with CPS obtained from STE, Vc apparently showed higher scavenging rate (95.36%) at 1.2 mg/mL. Previous study had indicated the mechanism of scavenging superoxide anion may be related with the dissociation energy of O H bond, i.e. the higher the number of electron withdrawing groups attached to polysaccharide was, the weaker the energy of O H bond was. Superoxide anion radicals scavenging ability can be attributed to the presence of some electrophilic groups such as keto or aldehyde group, which facilitated liberation of hydrogen from O H bond and thus stabilized superoxide anion. The scavenging activity had been confirmed by the result of IR in Fig. 6. On the other hand, previous literature had showed the sulfate content had significant effect on the antioxidant activity [61]. Hence, the high content of sulfate (33.38%) in

Z. Cheng et al. / International Journal of Biological Macromolecules 93 (2016) 369–380

CPS from G. scabra exhibited a stronger antioxidant activity and potential scavenging ability on superoxide radical. 3.6.4. Ferric reducing activity The Ferric reducing activity assay based on the ability of antioxidant to reduce Fe3+ to Fe 2+ was served as an indicator of natural products potential antioxidant activity. Fig. 7D illustrated the reducing power of Vc and CPS extracted by STE also followed a dosedependent manner at all the tested concentrations. As indicated in Fig. 7D, the ferric reducing power of CPS increased remarkably within the increasing concentration ranged from 0.2 to 1.2 mg/mL. But compared with Vc (1.92) at the concentration of 1.2 mg/mL, CPS extracted by STE exhibited relatively lower activity of 0.99. Therefore, CPS extracted by STE possessed definite reducing capacity, but it was lower than that of Vc. It had been reported reducing power of plant polysaccharides was often associated with the capacity of reacting with given precursors of peroxide. 4. Conclusion In the present study, an effective and advisable smashing tissue extraction approach has been investigated systematically to extract polysaccharide from G. scabra, in which the extraction conditions was optimized by RSM on the basis of single factors including extraction voltage, amount of extraction solvent, particle size of sample and extraction time. The highest extraction yield had reached 15.03 ± 0.14% (n = 3), which was agreed closely with the predicted yield of 15.19% under the optimal STE conditions as follows: extraction time of 130.38 s; extraction voltage of 157.09 V and raw material/solvent ratio of 1:34. Compared to other extraction techniques including MAE, UAE and HRE, the novel STE technique proposed in this study not only gained the highest extraction yield of CPS within the shortest extraction time, but also obtained polysaccharides with the highest scavenging ability against DPPH radicals, hydroxyl radical and superoxide anion radical. Chemical compositions of CPS suggested the G. scabra polysaccharides contained sugar, slight protein, high content of sulfate radicals and uronic acid. The results of HPGPC indicated CPS obtained from STE was homogeneous polysaccharides and its average molecular weight was approximately 3.87 × 104 Da. Monosaccharide composition analysis suggested CPS extracted by STE mainly consisted of mannose, rhamnose, galacturonic acid, glcose, galactose, arabinose and fucose. Moreover, the polysaccharides exhibited strong antioxidant activity in vitro. Thus, this study provided a new and highly efficient method for extraction of G. scabra polysaccharides which could be explored as a natural antioxidant. Acknowledgement This work was supported by the Foundation of Science and Technology Research Projects (2015033), and Youth Foundation of Jilin Province (20160520130JH), Jilin, China. References [1] Y.X. Sun, J.C. Liu, J.F. Kennedy, Extraction optimization of antioxidant polysaccharides from the fruiting bodies of Chroogomphis rutilus (Schaeff.: Fr.) O.K. Miller by Box-Behnken statistical design, Carbohydr. Polym. 82 (2010) 209–214. [2] T. Zhao, G.H. Ma, M. Zhang, F. Li, Y. Zou, Y. Zhou, W. Zheng, D.H. Zheng, Q. Li. Yang, X.Y. Wu, Anti-diabetic effects of polysaccharides from ethanol-insoluble residue of Schisandra chinensis (Turcz) baill on alloxan induced diabetic mice, Chem. Res. Chin. Uni. 29 (2013) 99–102. [3] Z.Y. Zhao, Q. Zhang, Y.F. Li, L.L. Dong, S.L. Liu, Optimization of ultrasound extraction of Alisma orientalis polysaccharides by response surface methodology and their antioxidant activities, Carbohydr. Polym. 119 (2015) 101–109.

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