Microwave-assisted extraction releases the antioxidant polysaccharides from seabuckthorn (Hippophae rhamnoides L.) berries

International Journal of Biological Macromolecules 123 (2019) 280–290

Contents lists available at ScienceDirect

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

Microwave-assisted extraction releases the antioxidant polysaccharides from seabuckthorn (Hippophae rhamnoides L.) berries Enwei Wei a,c,1, Rui Yang a,1, Hepeng Zhao a, Penghui Wang a, Suqing Zhao a, Wanchen Zhai c, Yang Zhang b,⁎, Hongli Zhou a,⁎ a b c

Jilin Engineering Research Center for Agricultural Resources and Comprehensive Utilization, Jilin Institute of Chemical Technology, Jilin 132022, PR China School of Biology and Food Engineering, Changshu Institute of Technology, Changshu 215500, PR China School of Pharmaceutical Sciences, Jilin University, Changchun 130012, PR China

a r t i c l e

i n f o

Article history: Received 24 July 2018 Received in revised form 22 September 2018 Accepted 12 November 2018 Available online 13 November 2018 Keywords: Seabuckthorn berries Polysaccharide Microwave-assisted extraction Microwave power Antioxidant activity

a b s t r a c t Seabuckthorn berries are rich in various bioactive components and used as a traditional medicine for a long time. Until now, little information is available for the extraction of polysaccharides from seabuckthorn berries (PSB) by linking antioxidant activity and microwave power. In this study, microwave-assisted extraction, characterization, in vitro and in vivo antioxidant activities of PSB were explored. The maximum PSB extraction yield of 0.264 ± 0.005% was obtained under the optimal conditions as follows: microwave power 600 W, extraction time 6 min, liquid to material ratio 10: 1 mL/g, and extraction temperature 85 °C. Meanwhile, effects of microwave power on antioxidant activity of PSB was investigated and found that microwave at power of 600 W can facilitate the release of antioxidant PSB in a high yield. The main monosaccharides of PSB were Rha, Man, Glu, and Gal at a molar ratio of 1.00: 6.89: 1.62: 13.52, UV and FT-IR analysis coupled with molecular weight determination further indicated that PSB is a polydisperse polysaccharide. Moreover, PSB obtained under the optimal conditions equally exerted in vivo antioxidant activity through decreasing malonaldehyde and protein carbonyls and increasing superoxide dismutase and glutathione. © 2018 Published by Elsevier B.V.

1. Introduction Seabuckthorn (Hippophae rhamnoides L.), a member of the Elaeagnaceae family naturally distributed in Europe and Asia, is belonged to the thorny and deciduous shrub with fixing nitrogen [1,2]. Owing to its drought, wind and sand resistance, seabuckthorn can survive in saline alkali land, so it is widely used in soil and water conservation and has high ecological value [3]. Various parts of seabuckthorn, particularly berries are known to be used in Chinese, Mongolian and Tibetan traditional medicines and to possess diverse activities, such as antimicrobial, antioxidant, and dermatological effects [4,5]. Seabuckthorn berries are rich in vitamins, phenolic compounds and carotenoids, and these bioactive components may contribute to the aforementioned pharmacological effects [6,7]. Recently, Zhang et al. [8] found that polysaccharides from seabuckthorn berries (PSB) can protect against carbon tetrachloride-induced hepatotoxicity in mice through their antioxidant and anti-inflammatory activities, indicating that the extraction process of PSB is worthy to be further ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Zhang), [email protected] (H. Zhou). 1 These authors contributed equally to this article.

https://doi.org/10.1016/j.ijbiomac.2018.11.074 0141-8130/© 2018 Published by Elsevier B.V.

researched. However, to the best of our knowledge, there is little information on PSB extraction. Several methods have been developed for the extraction of polysaccharides from natural sources. The principle of method selection is to keep the inherent characters of polysaccharides unchanged during processing. Due to the fact that most plant polysaccharides are present in cell walls, thus the first step of polysaccharides extraction is to facilitate the release of intracellular polysaccharides [9]. Microwave-assisted extraction (MAE) is an emerging technology commonly applied to the extraction of nature-based bioactive constituents [10–13]. The microwave energy can penetrate the plant materials and reach the inner glandular, trichomes and vascular systems, resulting in a sudden rise in temperature inside the materials. This heat can accelerate the vaporization of volatile materials, raising the intracellular pressure and finally causing the rupture of the cell walls to release the active components, which will be then trapped by the relatively cold surrounding medium and dissolved in it [14]. Compared with traditional extraction methods, MAE can enhance the extraction yield of targeting compounds more efficiently and is also more environmental friendly owing to the reduced consumption of solvents and energy [15]. MAE has been proved to be efficient for the extraction of antioxidant polysaccharides from different kinds of natural resources including Auricularia auricular [16], Flammulina velutipes [17], Ascophyllum nodosum [18], and

E. Wei et al. / International Journal of Biological Macromolecules 123 (2019) 280–290

281

Chuanminshen violaceum [19]. However, some extraction parameters, such as microwave power significantly affects the stability of target compounds, leading to the decrease of activities [20–22]. It is therefore important to grope for the balance between extraction yield and activity via controlling the conditions of MAE. In present study, with the aim to explore the balance of extraction yield and antioxidant activity as well as the relationship between key extraction parameter of MAE and activity. Effects of microwave power on the in vitro antioxidant activity of PSB were first investigated according to the results of single-factor experiment. Then the extraction process was optimized with response surface methodology (RSM) using extraction yield as response value. After characterization, the in vivo antioxidant capacities of obtained PSB under the optimal conditions were further confirmed.

10 g of the mashed juice and pomace were added in distilled water at different liquid-to-solid ratio (5: 1–25: 1 mL/g), and put into a microwave extraction apparatus (MAS-II Plus, Sineo Microwave, Shanghai, China). The extraction parameters including microwave power, time, and temperature were set to 500–700 W, 4–12 min, and 75–95 °C, respectively. After filtration, the filtrate was concentrated to about 20 mL under vacuum. The concentrated solution was placed in beaker, mixed with 4-fold amount of 95% ethanol to precipitate crude PSB at 4 °C for 12 h. The crude PSB was collected by centrifugation and grounded into powders after freeze-drying. Polysaccharides content in PSB was determined according to the phenol‑sulfuric acid method by using glucose as reference standard [24]. The standard curve and PSB extraction yield were expressed as follows:

2. Materials and methods


 A ¼ 12:644C−0:00714 R2 ¼ 0:99904

2.1. Materials and chemicals Seabuckthorn berries were collected from a new variety Changbai Mountain No. 27 in Seabuckthorn Planting Base, Jiao he, Northeast China, and authenticated by Prof. Guangshu Wang, School of Pharmaceutical Sciences, Jilin University, Changchun, China. Most of the chemicals used in the experiment of in vitro antioxidant activity were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China) including 1, 1-diphenyl-2-picrylhydrazyl (DPPH), phenanthroline, ferrous sulfate (FeSO4), potassium ferricyanide, trichloroacetic acid, ferric chloride (FeCl3), ferrozine, vitamin C (VC), and ethylene diamine tetraacetic acid (EDTA). Other reagents such as trifluoroacetic acid (TFA), hydroxylamine hydrochloride, pyridine, acetic anhydride, chloroform, n-butanol, and ethanol were obtained from Sigma Aldrich Chemical Co., Ltd. (St. Louis, MO, USA). DEAE (diethyl amino ethyl) cellulose was from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Standard sugars including D‑glucose, D‑galactose, D‑arabinose, D‑rhamnose, D‑fructose, D‑xylose and D‑mannose were mixed were obtained from Sino-pharm Chemical Reagent Co., Ltd. (Shanghai, China). T-series Dextran standards including T-10, T-40, T-70, T-100, and T500 were purchased from National Institutes of Food and Drug Control (Beijing, China). Reagent kits for the determination of antioxidant biomarkers including malonaldehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and protein carbonyls (PCO) were provided by Jiancheng Bioengineering Institute (Nanjing, China).

2.2. Experimental animals Male ICR mice with SPF grade (weighing 20 ± 2 g, aged 4 weeks) were purchased from the Experimental Animal Center of Jilin University (Approval No. SCXK (Ji) 2014–0004, Changchun, China). Mice were allowed free access to water and food and kept in polypropylene cages. The feeding conditions were as follows: temperature, 20 ± 2 °C; relative humidity, 60 ± 10%; light/dark regime, 12 h. All the animal experiments were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978), and approved by the Animal Care and Welfare Committee of Jilin Institute of Chemical Technology.

ð1Þ

where A-absorbance; C-polysaccharides content (mg/mL); the linear range was from 0.02 to 0.06 mg/mL. PSB extraction rate ð%Þ ¼ C  V  10−3 =M  100

ð2Þ

where C-polysaccharides content (mg/mL); V-volume of filatrate (mL); M-weight of seabuckthorn juice and pomace (g). 2.3.2. Heat reflux extraction (HRE) Ten grams of seabuckthorn juice and pomace were added into 100 mL of distilled water to be extracted at 85 °C for 6 min. After cooling, the mixture was filtered. The filtrate was concentrated and mixed with 95% ethanol to precipitate crude PSB. 2.4. Single-factor experimental design for PSB extraction Four single factors were selected to assess their effects on PSB extraction yield, and their ranges were determined via single-factor experiments, including microwave power (500–700 W), extraction time (4–12 min), liquid-to-material ratio (5: 1–25: 1 mL/g) and extraction temperature (75–95 °C). In each experiment, one factor was changed when other factors were kept constant. Then, in order to evaluate the effect of microwave power on the antioxidant activity of PSB, different microwave powers (550–650 W) were used to extract PSB by fixing liquidto-material ratio of 10: 1, extraction temperature of 85 °C, and extraction time of 6 min. Four in vitro antioxidant models including hydroxyl radical, DPPH radical, reducing power to ferric iron, and ferrous ionchelating capacity were applied to investigate the antioxidant activities of PSB using VC and EDTA as positive controls, respectively. Experimental procedures of the in vitro antioxidant evaluation can be seen in Section 2.11. All experiments were performed in triplicate. 2.5. Optimization of experimental design Based on the results of single-factor experiments, the MAE of PSB was optimized by using RSM. A four-variable and three-level BBD comprising 29 runs was applied at the center point using extraction yield as response value (Table 1). Table 1 The code and level of factors selected for the trials.

2.3. Extraction of PSB 2.3.1. Microwave-assisted extraction (MAE) PSB was prepared by using MAE based on the reported method with some modifications [23]. An appropriate amount of fresh seabuckthorn berries were collected, washed, crushed in a mortar and degreased with petroleum ether to form the final seabuckthorn juice and pomace. Then

Independent variable

Microwave power (W, X1) Extraction time (min, X2) Liquid-to-solid ratio (mL/g, X3) Extraction temperature (°C, X4)

Level -1

0

1

550 4 5: 1 80

600 6 10: 1 85

650 8 15: 1 90

282

E. Wei et al. / International Journal of Biological Macromolecules 123 (2019) 280–290

Regression analysis was applied for the experimental data and fitted to the following second-order polynomial equation: Y ¼ β0 þ

4 X i¼1

βi X i þ

4 X

βii X 2i

i¼1

3 X 4 X

βii X i X j

i¼1 j¼iþ1

where Y-response function; β0-intercept; βi, βii and βij-coefficients of linear, quadratic, and interactive terms, respectively; Xi and Xj-coded independent variables. The experimental design, analysis and prediction were conducted by using a Design-Expert software 8.0.6.1 (Stat-Ease, Minneapolis, MN, USA). 2.6. Scanning electron microscope (SEM) analysis The raw materials of pomace of seabuckthorn berries and the residues after extraction of PSB by different methods were respectively observed by SEM. The samples were immobilized on a silicon wafer and sputtered with gold in a thickness of 100 nm under reduced pressure. The microstructure of samples were studied using a scanning electron microscope (Quanta-200, FEI Ltd., The Netherlands) at a 20 kV accelerating voltage. 2.7. Purification of crude PSB Crude PSB was deproteinized using the Sevag reagent (chloroform: nbutanol = 4: 1, v/v) [25]. After removal of the Sevag reagent, the solution was dialyzed (MD10, Viskase, Darien, IL, USA) in distilled water for 72 h and precipitated again by 80% ethanol. After being centrifuged, the precipitate was dissolved in distilled water and subjected to a DEAE cellulose column (eluent-0.5 mol/L NaCl; flow velocity-3 mL/min). The elute was collected and lyophilized to obtain the purified PSB, whose content of polysaccharides was determined by phenol‑sulfuric acid method [24]. 2.8. Molecular weights analysis The molecular weight of purified PSB was analyzed by using highperformance size-exclusion chromatography (HPSEC) equipped with a

refractive index detector (RID) [26]. 20 μL of sample solution (2.0 mg/mL) was passed through a 0.45 μm- microporous filtering film and injected into a HPLC (Elite P230IIHPLC, Elite analytical instruments Co. Ltd., Dalian, China) equipped with a Shodex sugar KS-804 column (8.0 mm × 300 mm) (Showa Denko, Tokyo, Japan), and a RID (RI2000, Schambeck SFD GmbH, Bad Honnef, Germany). Data was recorded and processed by a N2000 GPC chromatographic work station (Surwit Technology Inc., Hangzhou, China). The chromatographic conditions were as follows: mobile phase, ultrapure water; flow rate, 1.0 mL/min; column temperature, 50 °C; RID temperature, 35 °C; run time, 30 min. Molecular weight of PSB was estimated according to the calibration curve obtained from T-series Dextran standard of known molecular weight (10,000, 40,000, 70,000, 100,000, and 500,000). 2.9. Monosaccharide composition analysis Twenty milligrams of purified PSB was added into a reactor, dissolved in 4 mL of 2 M TFA and kept at 120 °C for 3 h to hydrolyze the polysaccharides into monosaccharides. To prepare the standard monosaccharide mixture, 10 mg of each monosaccharide including D‑glucose, D‑galactose, D‑arabinose, L‑rhamnose, D‑fructose, D‑xylose and D‑mannose were mixed. Then the mixture was reacted with hydroxylamine hydrochloride (12 mg) and pyridine (0.5 mL) and heated at 90 °C for 30 min. After cooling, the reaction solution was mixed with 0.5 mL of acetic anhydride and heated at 90 °C for another 30 min. After that, the solution was dried by a rotary evaporator, dissolved in 3 mL of chloroform and 0.2 mL of distilled water, and filtered twice with microporous membrane. Finally, 10 μL of mixed samples were prepared and analyzed by a GC-2010 Shimadzu gas chromatograph (Shimadzu Technologies, Kyoto, Japan), equipped with a flame ionization detector and a Rtx-5MS column (30 m × 0.32 mm × 0.25 μm). Carrier gas: hydrogen at a flow rate of 2 mL/min; Temperature of vaporizer chamber: 280 °C; Programmed column temperature: from 60 °C to 120 °C at 4 °C/min, to 200 °C at 6 °C/min, and to 280 °C at 6 °C/min [27,28]. Sugar identification was identified by their characteristic retention times. Purified PSB was derivatized the same as the standard monosaccharides.

Fig. 1. Effects of microwave power (a), extraction time (b), liquid-to-material ratio (c), and extraction temperature (d) on the extraction yield of PSB. Data were expressed as the means ± SD (n = 3).

E. Wei et al. / International Journal of Biological Macromolecules 123 (2019) 280–290

2.10. Ultra violet (UV) and fourier transform infrared (FT-IR) spectroscopy analysis Polysaccharide solution at concentration of 1 mg/mL was scanned by a UV–vis spectrophotometer (L5S, INESA Analytical Instrument Co. Ltd., Shanghai, China) between 200 and 800 nm [29]. The purified PSB was ground with KBr and pressed into pellets, then the IR spectrum was determined by using a FTIR-650 Fourier transform infrared spectrophotometer (Gangdong Sci. & Tech. Development Co., Ltd., Tianjin, China) in the range of 4000–400 cm−1 [30]. 2.11. In vitro antioxidant activity assay 2.11.1. DPPH radical-scavenging assay Experiments were carried out according to the methods reported by Li et al. [31] and Chang et al. [32] with some modifications. In single-factor experiment, 2 mL of crude PSB solution (0.05 mg/mL) was mixed with 2 mL of DPPH ethanol solution (0.5 mmol/L), and the absorbance at 517 nm after maintenance in the dark at room temperature for 30 min was measured (As). Reaction system without DPPH was applied as normal control (Ac), and system without tested sample was used as blank solution (A0). VC at concentration of 0.05 mg/mL was taken as positive control. In section of extraction method comparison, the concentrations of tested sample were replaced with 0.01, 0.02, 0.03, 0.04, and 0.05 mg/mL. DPPH radical−scavenging rate ð%Þ ¼ ðAs −Ac Þ  100=A0

ð3Þ

283

2.11.2. Hydroxyl radical-scavenging assay Hydroxyl radical-scavenging assay was carried out based on the method reported by You et al. [33] with some modifications. In singlefactor experiment, 20 μL of crude PSB solution (0.05 mg/mL) was mixed with 40 μL of 2.0 mmoL/L phenanthroline and 40 μL of PBS (pH = 7.4), followed by addition of 20 μL of 0.75 mmoL/L FeSO4. After being mixed, 20 μL of 0.12% H2O2 was added and incubated at 37 °C for 60 min, then the absorbance at 536 nm was determined (As). The other two systems without H2O2 and tested samples were used as normal control (Ac) and blank solution (A0), respectively. VC at concentration of 0.05 mg/mL was served as positive control. In section of extraction method comparison, the concentrations of tested sample were replaced with 0.01, 0.02, 0.03, 0.04, and 0.05 mg/mL. Hydroxyl radical−scavenging rate ð%Þ ¼ ðAs −A0 Þ  100=ðAc −A0 Þ ð4Þ

2.11.3. Reducing power assay According to the test method [34], the experimental program was slightly modified. In single-factor experiment, 10 μL of crude PSB solution (0.05 mg/mL) was mixed with 25 μL of PBS buffer (pH 6.6) and 25 μL of 1% potassium ferricyanide solution and incubated at 50 °C for 20 min. After being cooled, 25 μL of 10% trichloroacetic acid solution was added and centrifuged at 3000 rpm for 10 min. 2.5 mL of supernatant was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% FeCl3 solution, then the absorbance was measured at 700 nm. VC at concentration of 0.05 mg/mL was served as positive control. In

Fig. 2. Effects of microwave power on the in vitro antioxidant activities of PSB. (a) DPPH radical-scavenging activity; (b) Hydroxyl radical (•OH)-scavenging activity; (c) Reducing power; (d) Ferrous ion (Fe2+)-chelating ability. Data was expressed as the means + SD (n = 3). Different symbols indicate statistically significant differences, ∗P b 0.05, ∗∗P b 0.01 compared with VC. VC, vitamin C; EDTA, ethylene diamine tetraacetic acid; MP, microwave power.

284

E. Wei et al. / International Journal of Biological Macromolecules 123 (2019) 280–290

Table 2 Box-Behnken design and observed responses. Run Independent variable

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Response (PSB extraction yield, %)

X1

X2

X3

X4

600 (0) 550 (−1) 550 (−1) 550 (−1) 600 (0) 550 (−1) 650 (1) 550 (−1) 600 (0) 600 (0) 600 (0) 650 (1) 600 (0) 600 (0) 600 (0) 600 (0) 600 (0) 600 (0) 550 (−1) 600 (0) 650 (1) 650 (1) 650 (1) 600 (0) 600 (0) 600 (0) 600 (0) 650 (1) 600 (0)

6 (0) 6 (0) 6 (0) 6 (0) 4 (−1) 8 (1) 4 (−1) 4 (−1) 6 (0) 6 (0) 8 (1) 6 (0) 4 (−1) 6 (0) 4 (−1) 6 (0) 6 (0) 8 (1) 6 (0) 4 (−1) 6 (0) 6 (0) 6 (0) 6 (0) 6 (0) 8 (1) 6 (0) 8 (1) 8 (1)

10:1 (0) 10:1 (0) 5:1 (−1) 15:1 (1) 10:1 (0) 10:1 (0) 10:1 (0) 10:1 (0) 10:1 (0) 15:1 (1) 10:1 (0) 10:1 (0) 5:1 (−1) 5:1 (−1) 10:1 (0) 15:1 (1) 10:1 (0) 5:1 (−1) 10:1 (0) 15:1 (1) 15:1 (1) 10:1 (0) 5:1 (−1) 10:1 (0) 5:1 (−1) 10:1 (0) 10:1 (0) 10:1 (0) 15:1 (1)

85 (0) 80 (−1) 85 (0) 85 (0) 90 (1) 85 (0) 85 (0) 85 (0) 85 (0) 90 (1) 90 (1) 90 (1) 85 (0) 90 (1) 80 (−1) 80 (−1) 85 (0) 85 (0) 90 (1) 85 (0) 85 (0) 80 (−1) 85 (0) 85 (0) 80 (−1) 80 (−1) 85 (0) 85 (0) 85 (0)

0.264 0.167 0.138 0.151 0.185 0.124 0.160 0.135 0.251 0.170 0.168 0.176 0.151 0.181 0.153 0.171 0.261 0.132 0.146 0.137 0.158 0.165 0.166 0.253 0.137 0.187 0.257 0.156 0.159

was measured at 562 nm (As). Distilled water instead of tested sample was used as normal control (Ac). Distilled water instead of ferrozine solution was served as blank control (A0). EDTA at concentration of 0.05 mg/mL was served as positive control. Ferrous ion−chelating rate ð%Þ ¼ ½ðAc −ðAs –A0 Þ  100=Ac

ð5Þ

2.12. In vivo antioxidant activity assay

section of extraction method comparison, the concentrations of tested sample were replaced with 0.01, 0.02, 0.03, 0.04, and 0.05 mg/mL.

Thirty mice were randomly divided into six groups (5 mice in each group): normal control (NC), positive control (PC), model control (MC), and three PSB-treated groups. Mice in PSB-treated groups were orally administered with purified PSB in doses of 100 (low dose, LD), 200 (medium dose, MD), and 400 mg/kg BW (high dose, HD) once a day for 30 consecutive days, mice in NC and MC were orally dosed with equal volume of distilled water, and mice in PC were orally treated with VC in a dose of 200 mg/kg BW [36]. On the 30th day, after being fasted for 12 h, all the mice except ones in NC were orally administered with 50% ethanol to induce oxidative stress in a dose of 12 mL/kg BW. Six hours later, animals were anesthetized by intraperitoneal injection of pentobarbital sodium in a dose of 50 mg/kg BW, blood samples were harvested from orbit and centrifuged at 4 °C, 4000 rpm for 10 min to obtain serum for the determination of MDA and SOD. After being euthanized with carbon dioxide, the livers were immediately dissected from animals, homogenized in physiological saline, and centrifuged at 4 °C, 4000 rpm for 10 min to prepare supernatant for the determination of PCO and GSH. The determination of MDA, SOD, GSH and PCO levels were conducted according to the methods described in the kits instruction (Jiancheng Bioengineering Institute, Nanjing, China). 2.13. Statistical analysis

2.11.4. Ferrous ion-chelating assay The ferrous ion-chelating assay was conducted according to the method of Wang et al. [35] with minor modifications. 1 mL of crude PSB solution (0.05 mg/mL) was mixed with 1 mL of 0.1 mmol/L FeSO4 solution and 1 mL of ferrozine solution (dissolved in methanol, 0.25 mmol/L) and incubated in the dark for 10 min, then the absorbance

Experimental data were expressed as means± or +SD (standard deviation). Statistics analysis was conducted by using SPSS19.0 software (SPSS Inc., Chicago, USA). The t-test was used to evaluate the significance of distances between two means, and the Levene's test was applied to detect the homogeneity of variances, if homogeneous, oneway ANOVA was operated.

Table 3 ANOVA for response surface quadratic model. Source Model X1 X2 X3 X4 X1 X2 X1 X3 X1 X4 X2 X3 X2 X4 X3 X4 X21 X22 X23 X24 Residual Lack of fit Pure error Cor total R2 a b

Sum of squares

DFa

Mean square

0.047 1.200 × 10−3 2.521 × 10−6 1.449 × 10−4 1.841 × 10−4 1.482 × 10−5 1.134 × 10−4 2.560 × 10−4 4.202 × 10−4 6.502 × 10−4 5.062 × 10−4 0.020 0.020 0.019 8.033 × 10−3 1.220 × 10−3 1.103 × 10−3 1.168 × 10−3 0.049 0.9749

14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 10 4 28

3.389 × 10−3 1.200 × 10−3 2.521 × 10−6 1.449 × 10−4 1.841 × 10−4 1.482 × 10−5 1.134 × 10−4 2.560 × 10−4 4.202 × 10−4 6.502 × 10−4 5.062 × 10−4 0.020 0.020 0.019 8.033 × 10−3 8.713 × 10−5 1.103 × 10−4 2.920 × 10−5

Degree of freedom. ⁎P b 0.05 significant; ⁎⁎P b 0.01 highly significant; n.s. means not significant.

P-value

Significanceb

38.89 13.77 0.029 1.66 2.11 0.17 1.30 2.94 4.82 7.46 5.81 230.68 225.23 221.96 92.20

b0.0001 0.0023 0.8674 0.2181 0.1681 0.6863 0.2730 0.1086 0.0454 0.0162 0.0303 b0.0001 b0.0001 b0.0001 b0.0001

** ** n.s. n.s. n.s. n.s. n.s. n.s. * * * ** ** ** **

3.78

0.1060

n.s.

Adjusted R2

0.9499

F-value

E. Wei et al. / International Journal of Biological Macromolecules 123 (2019) 280–290

3. Results and discussion 3.1. Single-factor experiment 3.1.1. Effects of microwave power, extraction time, liquid-to-solid ratio and extraction temperature on the extraction yield of PSB As shown in Fig. 1a, the extraction yield gradually rose with the increase of microwave power from 500 W to 600 W, maximized at

285

600 W and then gradually decreased with the increase of microwave power. The glycosidic linkage of PSB may be destroyed with the increase of microwave power, which decreased the content of polysaccharides [37]. The extraction yield gradually rose within 4 min to 6 min and reached the highest level after 6 min of extraction and then decreased at 10 min, which could be due to partial degradation [38], indicating the best extraction time was 6 min (Fig. 1b). The extraction yield of PSB increased obviously within the liquid-to-solid ratio of 5: 1–10:

Fig. 3. Response surface plots for the microwave-assisted extraction of polysaccharides from seabuckthorn berries (PSB). (a) Extraction time vs. Microwave power; (b) Liquid-to-solid ratio vs. Microwave power; (c) Extraction temperature vs. Microwave power; (d) Liquid-to-solid ratio vs. Extraction time; (e) Extraction temperature vs. Extraction time; (f) Extraction temperature vs. Liquid-to-solid ratio.

286

E. Wei et al. / International Journal of Biological Macromolecules 123 (2019) 280–290

Table 4 Comparison of PSB extraction using MAE and HRE. Method

MAE HRE

Microwave powder (W)

600 −

Liquid-to-solid ratio (mL/g)

10: 1 10: 1

Extraction time (min)

6 6

Extraction temperature (°C)

80 80

PSB extraction yield (%)

0.264 ± 0.005⁎⁎ 0.207 ± 0.006

IC50 a (mg/mL)

IC50 b (mg/mL)

EC50 C (mg/mL)

Hydroxyl radical

DPPH radical

Reducing power

0.016 ± 0.002⁎⁎ 2.691 ± 0.048

0.016 ± 0.001⁎⁎ 0.239 ± 0.016

0.148 ± 0.004⁎⁎ 1.868 ± 0.028

IC50, half maximal inhibitory concentration; EC50, median effect concentration. Data were expressed as means ± SD. ⁎⁎ P b 0.01 highly significant versus HRE.

1 mL/g, maximized at the ratio of 10: 1 mL/g and then gradually decreased (10: 1–25: 1 mL/g) (Fig. 1c), which may be due to the fact that a higher liquid-to-solid ratio could cause a lower density and viscosity of the extraction solvent and facilitate polysaccharide dilution in water [39]. With the extraction temperature from 75 °C to 85 °C, the PSB extraction yield increased (Fig. 1d). The extraction temperature influenced the PSB extraction yield, which maximized at 85 °C and decreased between 85 °C and 95 °C, indicating the optimal extraction temperature was 85 °C. The reason was that rising temperature may also affect the structural stabilities of targeting polysaccharides [38]. 3.1.2. Effects of microwave power on the in vitro antioxidant activities of PSB Considering the fact that microwave irradiation may have some damaging effects on bioactive components, inducing the decline of activity [40]. Effects of microwave power on the in vitro antioxidant activities of PSB were further investigated based on the results of singlefactor experiments. As shown in Fig. 2a–d, the antioxidant capacity of PSB increased with the increase of microwave power from 550 W to 600 W. For instance, after being heated at 600 W, the. OH-scavenging rate of PSB significantly increased from 42.68 ± 1.83% to 89.15 ± 2.44%. The. OH-scavenging capacity and reducing power of PSB at 600 W were significantly higher than those of VC. In contrast, when compared with EDTA, even at 600 W, Fe2+-chelating ability of PSB still significantly decreased, which was consistent with the general findings that Fe2+-chelating ability of polysaccharides was weaker than that of EDTA [41–44]. These findings may be attributed to the presence of functional groups present in polysaccharides and EDTA. In polysaccharides, hydroxyl group is the functional group, by contrast, nitrogencontaining and carboxyl groups are the functional groups in EDTA, which may exert more powerful Fe2+-chelating ability than that of hydroxyl group [45]. The antioxidant capacity of PSB decreased with the increase of microwave power from 600 W to 650 W, which was similar to the outcomes obtained from single-factor experiment (Fig. 1a), where the PSB yield increased with the increase of microwave power from 500 W to 600 W, maximized at 600 W and then decreased with the increase of microwave power, indicating that the declined antioxidant activity of PSB could be caused by the decreased content of polysaccharides due to the cleavage of glucosidic bond induced by the enhanced microwave power. Therefore, microwave irradiation at

600 W could facilitate the release of antioxidant polysaccharides from seabuckthorn berries in a high yield. 3.2. Model fitting and statistical analysis In this study, according to the results of single-factor experiments, four independent factors including microwave power (X1), extraction time (X2), liquid-to-solid ratio (X3), and extraction temperature (X4) were optimized by RSM using PSB extraction yield as the dependent variable in the BBD. 29 experimental combinations and response values were listed in Table 2, which showed that PSB extraction yield ranged from 0.124% to 0.264% and the maximum extraction yield (0.264%) was observed in the condition of X1 = 600 W, X2 = 6 min, X3 = 10: 1 mL/g, and X4 = 85 °C. These results were fitted with the following second-order polynomial equation: Y ¼ 0:26 þ 0:01X1 þ 0:000458X2 þ 0:00348X3 þ 0:00392X4 þ 0:00193X1 X2 −0:00533X1 X3 þ 0:008X1 X4 þ 0:01X2 X3 −0:013X2 X4 −0:011X3 X4 −0:056X1 2 −0:055X2 2 −0:055X3 2 −0:035X4 2 where Y-PSB extraction yield (%), X1-microwave power (W), X2extraction time (min), X3-liquid-to-solid ratio (mL/g), and X4extraction temperature (°C). Results of the analysis of variance (ANOVA) for the response surface quadratic model were exhibited in Table 3. Low P-value (b0.0001) of the model indicated that regression equation was ideal. Determination coefficient (R2 = 0.9749) suggested that 97.49% of the experimental variables could be explained by the obtained model. At the same time, Pvalue of the lack of fit was found to be 0.106, N0.05, implying nonsignificance compared with the pure error. Consequently, the obtained model was proved to be suitable for the analysis and prediction of PSB extraction. In this model, the linear parameter X1 was highly significant (P b 0.01), while other linear parameters including X2, X3, and X4 were not significant (P N 0.05). The interaction parameters X1X2, X1X3, and X1X4 were not significant (P N 0.05), but X2X3, X2X4, and X3X4 was significant (P b 0.05). The quadratic parameters X12, X22, X32 and X42 were all highly significant (P b 0.01).

Fig. 4. SEM images of untreated raw material of pomace of seabuckthorn berries (a), sample extracted by MAE (b) and HRE (c).

E. Wei et al. / International Journal of Biological Macromolecules 123 (2019) 280–290

Contour and 3D response surface plots can be generated by DesignExpert software to visualize the interaction between variables. The relationship between PSB extraction yield and any two independent variables (with the other factor was set at level zero) was shown in Fig. 3.

Table 5 GC data of monosaccharides present in standard mixture and PSB sample. Monosaccharide

3.3. Verification of predictive model After a series of linear fitting and response surface analysis, the optimum extraction parameters obtained from Design-Expert software were as follows: microwave power 604.66 W, extraction time 6 min, liquid to material ratio 10.1:1 mL/g, and extraction temperature 85.31 °C, where the theoretical extraction yield of PSB was 0.258%. Considering the output power of microwave apparatus and convenience of practical operation, the optimum extraction parameters were corrected to be: microwave power 600 W, extraction time 6 min, liquid to material ratio 10: 1 mL/g, and extraction temperature 85 °C. Under these conditions, the actual extraction yield of PSB was 0.264 ± 0.005%, with relative error 2.33%. The actual value was consistent with the prediction, thus the model could better predict PSB extraction. 3.4. Comparison of MAE with HRE Moreover, PSB extraction by MAE was compared with HRE. As shown in Table 4, the yield of PSB extracted by MAE was significantly (P b 0.01) higher than that extracted by HRE, and the in vitro antioxidant capacities of PSB extracted by MAE were significantly (P b 0.01) stronger than that extracted by HRE, suggesting that microwave could facilitate the release of PSB and the microwave power of 600 W could be proper for the retention of antioxidant activity, which was in compliance with the results of single-factor experiments. 3.5. SEM analysis With the aim of elucidating the underlying mechanism of extraction, effects of MAE and HRE on the microstructure of samples were further investigated by SEM. As can be seen from Fig. 4a, regarding untreated raw material, the parenchyma was nubbly, rugged and rambling, no destroy of cell walls can be found. After being processed by HRE (Fig. 4b), the surface of parenchyma seemed to be organized integrally, but any noticeable damages on the microstructure cannot be observed. Unlike HRE-treated material, the residual obtained from MAE process presented the illustration of parenchyma disorganization (Fig. 4c), it can be seen that the cells were damaged seriously and the texture of tissue seemed to be thinner than that processed by HRE, which may be due to the sudden increases of internal pressure and temperature induced by

287

L-Rhamnose (Rha) D‑Xylose

(Xyl)

D‑Mannose D‑Glucose

D‑Galactose D‑Fructose a b c

(Man)

(Glu) (Gla)

(Fru)

Standard mixture Fig. 5 Q1

PSB sample Fig. 5 Q2

Peaka

tRc (min)

Peakb

tRc (min)

Peak area

Molar ratio

1 2

27.944 28.541

1 –

28.160 –

236,144 –

1.00 –

3

32.084

3

32.028

1,608,572

6.89

4

32.221

4

32.203

378,336

1.62

5

32.604

5

32.557

3,157,241

13.52

6

35.848

–

–

–

–

Refer to Fig. 5 Q1. Refer to Fig. 5 Q2. Refer to retention time.

microwave irradiation [46]. These results indicated that MAE was superior to HRE, and the absorbed energy of microwave could promote the ruptures of cell wall, thereby facilitating the release of PSB. 3.6. Purification of crude PSB After deproteinization, the deproteinized rate was 91.23% and the purity of polysaccharides was 63.84%. After purification by DEAE cellulose, the purity of polysaccharides reached 89.16%. 3.7. Molecular weights analysis With the advances in polysaccharides research, scientists have proved that molecular weights of polysaccharides may be negatively correlated to their antioxidant activities [47,48]. In present work, according to the calibration based on dextran standard, PSB had three peaks with molecular weights of 968,297 Da, 177,802 Da, and 33,601 Da accounted for 20.7%, 8.1%, and 71.2%, respectively. These results indicated that PSB belongs to polydisperse polysaccharide with a broad distribution of molecular weights. Moreover, the antioxidant activities of PSB may be chiefly attributed to the subunit that possesses the lowest molecular weight (33,601 Da), due to the fact that content of this subunit was 71.2% with the highest value. 3.8. Structural analysis 3.8.1. Monosaccharide composition analysis Polysaccharides are usually composed of different monosaccharides linked each other through glucosidic bond [49]. After hydrolysis of glycosidic linkages by trifluoroacetic acid and derivatization, the

Fig. 5. GC chromatograms of standard mixture (Q1) and PSB (Q2). Rha, L‑Rhamnose; Xyl, D‑Xylose;

Man, D‑Mannose; Glu, D‑Glucose; Gal, D‑Galactose; Fur, D‑Fructose.

Fig. 6. UV scanning spectra of PSB.

288

E. Wei et al. / International Journal of Biological Macromolecules 123 (2019) 280–290

et al. studied the relationship between monosaccharide composition and antioxidant activities using the polysaccharides from thirteen different boletus mushrooms. They noted that galactose (Gal) content shows significant correlations with DPPH radical-scavenging capacity and reducing power [50]. In present investigation, Gal was the most abundant monosaccharide present in PSB, which may provide larger contributions than other monosaccharides.

Fig. 7. The FT-IR spectra of PSB.

monosaccharide composition of PSB was analyzed using GC. As shown in Fig. 5 and Table 5, in PSB sample, four peaks were found to match with those in standard mixture compared with the tR values of standard mixture, suggesting that PSB mainly comprises Rha, Man, Glu, and Gal at a molar ratio of 1.00: 6.89: 1.62: 13.52. In addition to molecular weight, monosaccharide composition also affects the antioxidant capacities of natural polysaccharides. Zhang

3.8.2. UV and FT-IR analysis From Fig. 6, we can see that in the range of 200–800 nm, the UV scanning spectra of PSB appeared a downward trend, which was in accordance with the general characters of polysaccharides, and any significant peaks were not observed, indicating that the amount of protein and nucleic acid impurities had been reduced to the lowest level [29]. Fig. 7 exhibited the FT-IR spectra of PSB, which showed strong absorption peaks at 3437.2 cm−1 (OH stretching vibration) and 2925.6 cm−1 (C\\H stretching vibration), respectively, indicating typical vibrations of the glucose unit [51]. In addition, the peak at 1725.3 cm−1 is the C_O stretching vibration, ~1626.5 cm−1 is the C_C asymmetric stretching vibration, and ~1065.4 cm−1 is assigned to the C-OH stretching vibration [15]. The FT-IR spectroscopy revealed that PSB possesses the structural characteristics of polysaccharide. 3.9. In vivo antioxidant of PSB In the interest of exploring the in vivo antioxidant profiles of PSB, some antioxidant biomarkers including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and protein carbonyls

Fig. 8. Effects of PSB on (a) MDA, (b) SOD, (c) GSH, and (d) PCO. Data were expressed as means + SD (n = 10). PSB, Polysaccharides from seabuckthorn berries; NC, Normal control; MC, Model control; PC, Positive control (VC in a dose of 200 mg/kg); LD, Low dose (PSB in a dose of 100 mg/kg); MD, Medium dose (PSB in a dose of 200 mg/kg); HD, High dose (PSB in a dose of 400 mg/kg). Symbols indicate statistically significant differences, ^P b 0.01, ^^P b 0.01 as compared with NC group; ∗∗P b 0.01 as compared with MC group; #P b 0.05, ##P b 0.01 as compared with PC group.

E. Wei et al. / International Journal of Biological Macromolecules 123 (2019) 280–290

(PCO) were chosen to investigate the antioxidant activities in an ethanol-induced oxidative stress model mouse. 3.9.1. Effects of PSB on MDA MDA is the most common final metabolite of lipid peroxidation, its production is in proportion to the level of oxidative damage [52]. As shown in Fig. 8a, with the increase of PSB dose, the MDA content in PSB-treated groups decreased. Significant difference (P b 0.01) was observed between NC and MC groups. When compared with MC, statistical difference (P b 0.01) were found in PC and all the PSB-treated groups. The MDA content in HD was 7.93 ± 1.76 mmol/L with a lowest value, 2.9-fold lower than that in MC (P b 0.01), and 1.3-fold lower than that in PC (but no statistical difference was found). 3.9.2. Effects of PSB on SOD SODs are oxidant scavenger enzymes that comprise three isozymes, i.e. SOD-1, SOD-2, and SOD-3, which display different enzymatic properties and biolocalizations, but elicit the same scavenging capacities against superoxide through dismutation reaction [53]. As shown in Fig. 8b, the SOD activities increased with the increase of PSB dose. When compared with NC, the SOD activity in MC significantly (P b 0.05) decreased, but those in PC and PSB-treated groups significantly (P b 0.01) raised compared with MC. Besides, the SOD activities in PSB-treated groups were all significantly (P b 0.01) lower than that in PC. 3.9.3. Effects of PSB on GSH GSH is an endogenous antioxidant that consists of glutamic acid, cysteine, and glycine. It can react with free radicals to change them into a lower reaction state [54]. As shown in Fig. 8c, the GSH level increased in a dose dependent manner (P b 0.01) in PSB dose ranging from 100 to 200 mg/kg, then decreased in a dose dependent manner (P b 0.01) ranging from 200 to 400 mg/kg. There was significant (P b 0.05) difference between NC and MC. When compared with MC, significant (P b 0.01) differences in GSH content were found in PC and MD groups. The GSH contents in LD and HD groups were significantly (P b 0.01, P b 0.05) lower than that in PC, and the GSH content in MD was 48.33 ± 6.75 mg/gprot with a highest level, higher than that in PC (37.24 ± 10.05 mg/gprot), but no statistical difference was noted. 3.9.4. Effects of PSB on PCO PCO is an irreversible form of protein metabolism, which is more stable than MDA and usually formed earlier than other biomarkers [55]. As shown in Fig. 8d, PCO content in MC was significantly (P b 0.01) higher than that in NC. Significant (P b 0.01) differences in PCO were observed in PC and all PSB-treated groups compared with MC. PCO was reduced in a dose-dependent manner (P b 0.01), the lowest content was 0.061 ± 0.043 nmol/mgprot in HD, which was still lower than that in PC (0.118 ± 0.026 nmol/mgprot), but no statistical difference was noted was found. These results implied that PSB equally elicits antioxidant activity against oxidative stress induced by ethanol in mice. The underlying mechanism may involve in decreasing MDA and PCO as well as increasing SOD and GSH. As for GSH, the effects of PSB presented a trend of “first rise and then drop” with the increase of dose, which may be due to the fact that high dose of PSB could reduce GSH either by decreasing the activities of GSH synthetases or by increasing the activities of GSH breakdown enzymes [56]. The exact mechanisms will be needed further study to explore. 4. Conclusion The results of present investigation demonstrated that microwave plays an important role in the release of antioxidant PSB. Under the optimal conditions of MAE, the extraction yield, IC50 values towards hydroxyl and DPPH radicals as well as EC50 value of reducing power

289

were all significantly superior to those obtained from HRE, which were further confirmed by the observation of SEM. The subunit with lower molecular weight and a monosaccharide of Gal may mainly contribute to the antioxidant activities of PSB, but the exact relationship between structure and antioxidant activity will be needed to intensively study. Furthermore, in an ethanol-induced oxidative stress mouse model, the PSB acquired from the optimal conditions of MAE equally showed antioxidant activity in the dose ranging from 100 to 400 mg/kg BW. Its underlying mechanisms may relate to the decrease of MDA and PCO as well as the increase of SOD and GSH. Herein, present work will play a certain guiding role in the extraction and research of antioxidant PSB in industrial production. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements This work was supported by the Program of Jilin Provincial Development and Reform Commission [grant number 2017C044]; and the Program of Jilin Municipal Science and Technology Bureau [grant number 201750254]. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2018.11.074. References [1] A. Rousi, The genus Hippophaë L. A taxonomic study, Ann. Bot. Fenn. 8 (3) (1971) 177–227. [2] S. Geetha, M. Sai Ram, S.S. Mongia, V. Singh, G. Ilavazhagan, R.C. Sawhney, Evaluation of antioxidant activity of leaf extract of Seabuckthorn (Hippophae rhamnoides L.) on chromium(VI) induced oxidative stress in albino rats, J. Ethnopharmacol. 87 (2–3) (2003) 247–251. [3] F.S. Yang, M.M. Cao, H.E. Li, X.H. Wang, C.F. Bi, Simulation of sediment retention effects of the single seabuckthorn flexible dam in the Pisha sandstone area, Ecol. Eng. 52 (3) (2013) 228–237. [4] V.B. Guliyev, M. Gul, A. Yildirim, Hippophae rhamnoides L.: chromatographic methods to determine chemical composition, use in traditional medicine and pharmacological effects, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 812 (1–2) (2004) 291–307. [5] A. Zeb, Important therapeutic uses of sea buckthorn (Hippophae): a review, J. Biol. Sci. 4 (5) (2004) 687–693. [6] X. Gao, M. Ohlander, N. Jeppsson, L. Bjork, V. Trajkovski, Changes in antioxidant effects and their relationship to phytonutrients in fruits of sea buckthorn (Hippophae rhamnoides L.) during maturation, J. Agric. Food Chem. 48 (5) (2000) 1485–1490. [7] D. Rosch, A. Krumbein, C. Mugge, L.W. Kroh, Structural investigations of flavonol glycosides from sea buckthorn (Hippophae rhamnoides) pomace by NMR spectroscopy and HPLC-ESI-MS(n), J. Agric. Food Chem. 52 (13) (2004) 4039–4046. [8] W. Zhang, X. Zhang, K. Zou, J. Xie, S. Zhao, J. Liu, H. Liu, J. Wang, Y. Wang, Seabuckthorn berry polysaccharide protects against carbon tetrachloride-induced hepatotoxicity in mice via anti-oxidative and anti-inflammatory activities, Food Funct. 8 (9) (2017) 3130–3138. [9] L. Shi, Bioactivities, isolation and purification methods of polysaccharides from natural products: a review, Int. J. Biol. Macromol. 92 (2016) 37–48. [10] S. Tsubaki, K. Oono, M. Hiraoka, A. Onda, T. Mitani, Microwave-assisted hydrothermal extraction of sulfated polysaccharides from Ulva spp. and Monostroma latissimum, Food Chem. 210 (2016) 311–316. [11] J.L. Liu, L.Y. Li, G.H. He, Optimization of microwave-assisted extraction conditions for five major bioactive compounds from Flos Sophorae Immaturus (cultivars of Sophora japonica L.) using response surface methodology, Molecules 21 (3) (2016) 296. [12] V. Mandal, S. Dewanjee, S.C. Mandal, Microwave-assisted extraction of total bioactive saponin fraction from Gymnema sylvestre with reference to gymnemagenin: a potential biomarker, Phytochem. Anal. 20 (6) (2009) 491–497. [13] Y. Zhai, S. Sun, Z. Wang, J. Cheng, Y. Sun, L. Wang, Y. Zhang, H. Zhang, A. Yu, Microwave extraction of essential oils from dried fruits of Illicium verum Hook. f. and Cuminum cyminum L. using ionic liquid as the microwave absorption medium, J. Sep. Sci. 32 (20) (2009) 3544–3549. [14] M. Desai, J. Parikh, P.A. Parikh, Extraction of natural products using microwaves as a heat source, Sep. Purif. Rev. 39 (1–2) (2010) 1–32. [15] C. Chen, B. Zhang, Q. Huang, X. Fu, R.H. Liu, Microwave-assisted extraction of polysaccharides from Moringa oleifera Lam. leaves: characterization and hypoglycemic activity, Ind. Crop. Prod. 100 (2017) 1–11.

290

E. Wei et al. / International Journal of Biological Macromolecules 123 (2019) 280–290

[16] W.C. Zeng, Z. Zhang, H. Gao, L.R. Jia, W.Y. Chen, Characterization of antioxidant polysaccharides from Auricularia auricular using microwave-assisted extraction, Carbohydr. Polym. 89 (2) (2012) 694–700. [17] Z. Zhang, G. Lv, W. He, L. Shi, H. Pan, L. Fan, Effects of extraction methods on the antioxidant activities of polysaccharides obtained from Flammulina velutipes, Carbohydr. Polym. 98 (2) (2013) 1524–1531. [18] Y. Yuan, D. Macquarrie, Microwave assisted extraction of sulfated polysaccharides (fucoidan) from Ascophyllum nodosum and its antioxidant activity, Carbohydr. Polym. 129 (2015) 101–107. [19] H. Dong, Q. Zhang, Y. Li, L. Li, W. Lan, J. He, H. Li, Y. Xiong, W. Qin, Extraction, characterization and antioxidant activities of polysaccharides of Chuanminshen violaceum, Int. J. Biol. Macromol. 86 (2016) 224–232. [20] Z. Zhao, P. Liu, S. Wang, S. Ma, Optimization of ultrasound, microwave and Soxhlet extraction of flavonoids from Millettia speciosa Champ. and evaluation of antioxidant activities in vitro, J. Food Meas. Charact. 11 (4) (2017) 1–12. [21] H. Shang, S. Chen, R. Li, H. Zhou, H. Wu, H. Song, Influences of extraction methods on physicochemical characteristics and activities of Astragalus cicer L. polysaccharides, Process Biochem. 73 (2018) 220–227. [22] L. He, X. Yan, J. Liang, S. Li, H. He, Q. Xiong, X. Lai, S. Hou, S. Huang, Comparison of different extraction methods for polysaccharides from Dendrobium officinale stem, Carbohydr. Polym. 198 (2018) 101–108. [23] H. Rostami, S.M. Gharibzahedi, Microwave-assisted extraction of jujube polysaccharide: optimization, purification and functional characterization, Carbohydr. Polym. 143 (2016) 100–107. [24] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (3) (1956) 350–356. [25] G.L. Huang, Q. Yang, Z.B. Wang, Extraction and deproteinization of mannan oligosaccharides, Z. Naturforsch. C 65 (5–6) (2010) 387–390. [26] Y. Sun, J. Tang, X. Gu, D. Li, Water-soluble polysaccharides from Angelica sinensis (Oliv.) Diels: preparation, characterization and bioactivity, Int. J. Biol. Macromol. 36 (5) (2005) 283–289. [27] J. Ma, L. Adler, G. Srzednicki, J. Arcot, Quantitative determination of non-starch polysaccharides in foods using gas chromatography with flame ionization detection, Food Chem. 220 (2017) 100–107. [28] H.P. Zhao, Y. Zhang, Z. Liu, J.Y. Chen, S.Y. Zhang, X.D. Yang, H.L. Zhou, Acute toxicity and anti-fatigue activity of polysaccharide-rich extract from corn silk, Biomed. Pharmacother. 90 (2017) 686–693. [29] Q. Liu, X. Ge, L. Chen, D. Cheng, Z. Yun, W. Xu, R. Shao, Purification and analysis of the composition and antioxidant activity of polysaccharides from Helicteres angustifolia L, Int. J. Biol. Macromol. 107 (Pt B) (2018) 2262–2268. [30] Y. Qu, C. Li, C. Zhang, R. Zeng, C. Fu, Optimization of infrared-assisted extraction of Bletilla striata polysaccharides based on response surface methodology and their antioxidant activities, Carbohydr. Polym. 148 (2016) 345–353. [31] X. Li, A. Zhou, Y. Han, Anti-oxidation and anti-microorganism activities of purification polysaccharide from Lygodium japonicum in vitro, Carbohydr. Polym. 66 (1) (2006) 34–42. [32] W.C. Chang, S.C. Kim, S.S. Hwang, B.K. Choi, H.J. Ahn, Y.L. Min, H.P. Sang, S.K. Kim, Antioxidant activity and free radical scavenging capacity between Korean medicinal plants and flavonoids by assay-guided comparison, Plant Sci. 163 (6) (2002) 1161–1168. [33] L. You, M. Zhao, R.H. Liu, J.M. Regenstein, Antioxidant and antiproliferative activities of loach (Misgurnus anguillicaudatus) peptides prepared by papain digestion, J. Agric. Food Chem. 59 (14) (2011) 7948–7953. [34] A.F.M.S. Ud-Daula, F. Demirci, K.A. Salim, B. Demirci, L.B.L. Lim, K.H.C. Baser, N. Ahmad, Chemical composition, antioxidant and antimicrobial activities of essential oils from leaves, aerial stems, basal stems, and rhizomes of Etlingera fimbriobracteata (K.Schum.) R.M.Sm, Ind. Crop. Prod. 84 (2016) 189–198. [35] T. Wang, R. Jónsdóttir, H. Liu, L. Gu, H.G. Kristinsson, S. Raghavan, G. Ólafsdóttir, Antioxidant capacities of phlorotannins extracted from the brown algae Fucus vesiculosus, J. Agric. Food Chem. 60 (23) (2012) 5874–5883.

[36] Q. Zhang, N. Li, G. Zhou, X. Lu, Z. Xu, Z. Li, In vivo antioxidant activity of polysaccharide fraction from Porphyra haitanesis (Rhodephyta) in aging mice, Pharmacol. Res. 48 (2) (2003) 151–155. [37] Z.Y. Zhu, F. Dong, X. Liu, Q. Lv, F. Yingyang, L. Liu, T. Chen, Z. Wang, Y. Zhang Wang, Effects of extraction methods on the yield, chemical structure and anti-tumor activity of polysaccharides from Cordyceps gunnii mycelia, Carbohydr. Polym. 140 (2016) 461–471. [38] Ophélie Fliniaux, Cyrielle Corbin, Aina Ramsay, Sullivan Renouard, Vickram Beejmohun, Microwave-assisted extraction of herbacetin diglucoside from flax (Linum usitatissimum L.) seed cakes and its quantification using an RP-HPLC-UV system, Molecules 19 (2014) 3025–3037. [39] C. Chen, L.J. You, A.M. Abbasi, X. Fu, R.H. Liu, Optimization for ultrasound extraction of polysaccharides from mulberry fruits with antioxidant and hyperglycemic activity in vitro, Carbohydr. Polym. 130 (2015) 122–132. [40] S. Kowalski, Changes of antioxidant activity and formation of 5hydroxymethylfurfural in honey during thermal and microwave processing, Food Chem. 141 (2) (2013) 1378–1382. [41] E. Vamanu, Biological activities of the polysaccharides produced in submerged culture of two edible Pleurotus ostreatus mushrooms, J Biomed Biotechnol 2012 (2012) 565974. [42] W. Li, J. Ji, X. Chen, M. Jiang, X. Rui, M. Dong, Structural elucidation and antioxidant activities of exopolysaccharides from Lactobacillus helveticus MB2-1, Carbohydr. Polym. 102 (2014) 351–359. [43] Z. Zhang, J. Jin, L. Shi, Antioxidant activity of the derivatives of polysaccharide extracted from a Chinese medical herb (Ramulus mori), Food Sci. Technol. 14 (2) (2008) 160–168 International Tokyo. [44] S. Li, L. Wang, C. Song, X. Hu, H. Sun, Y. Yang, Z. Lei, Z. Zhang, Utilization of soybean curd residue for polysaccharides by Wolfiporia extensa (Peck) Ginns and the antioxidant activities in vitro, J. Taiwan Inst. Chem. Eng. 45 (1) (2014) 6–11. [45] D.P. Naughton, M. Grootveld, EDTA bis-(ethyl phenylalaninate): a novel transition metal-ion chelating hydroxyl radical scavenger with a potential anti-inflammatory role, Bioorg. Med. Chem. Lett. 11 (19) (2001) 2573–2575. [46] T. Yoshida, S. Tsubaki, Y. Teramoto, J. Azuma, Optimization of microwave-assisted extraction of carbohydrates from industrial waste of corn starch production using response surface methodology, Bioresour. Technol. 101 (20) (2010) 7820–7826. [47] K.C. Siu, L. Xu, X. Chen, J.Y. Wu, Molecular properties and antioxidant activities of polysaccharides isolated from alkaline extract of wild Armillaria ostoyae mushrooms, Carbohydr. Polym. 137 (2016) 739–746. [48] Q. Ge, J.W. Mao, X.Q. Guo, Y.F. Zhou, J.Y. Gong, S.R. Mao, Composition and antioxidant activities of four polysaccharides extracted from Herba Lophatheri, Int. J. Biol. Macromol. 60 (2013) 437–441. [49] A. Zong, H. Cao, F. Wang, Anticancer polysaccharides from natural resources: a review of recent research, Carbohydr. Polym. 90 (4) (2012) 1395–1410. [50] L. Zhang, Y. Hu, X. Duan, T. Tang, Y. Shen, B. Hu, A. Liu, H. Chen, C. Li, Y. Liu, Characterization and antioxidant activities of polysaccharides from thirteen boletus mushrooms, Int. J. Biol. Macromol. 113 (2018) 1–7. [51] C. Li, X. Fu, F. Luo, Q. Huang, Effects of maltose on stability and rheological properties of orange oil-in-water emulsion formed by OSA modified starch, Food Hydrocoll. 32 (1) (2013) 79–86. [52] H. Esterbauer, R.J. Schaur, H. Zollner, Chemistry and biochemistry of 4hydroxynonenal, malonaldehyde and related aldehydes, Free Radic. Biol. Med. 11 (1) (1991) 81–128. [53] D. Hu, E. Klann, E. Thiels, Superoxide dismutase and hippocampal function: age and isozyme matter, Antioxid. Redox Signal. 9 (2) (2007) 201–210. [54] T.M. Bray, C.G. Taylor, Tissue glutathione, nutrition, and oxidative stress, Can. J. Physiol. Pharmacol. 71 (9) (1993) 746–751. [55] D. Weber, M.J. Davies, T. Grune, Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: focus on sample preparation and derivatization conditions, Redox Biol. 5 (2015) 367–380. [56] W. Zhao, J.J. Li, S.Q. Yue, L.Y. Zhang, K.F. Dou, Antioxidant activity and hepatoprotective effect of a polysaccharide from Bei Chaihu (Bupleurum chinense DC), Carbohydr. Polym. 89 (2) (2012) 448–452.