Polysaccharide from Mesona chinensis: Extraction optimization, physicochemical characterizations and antioxidant activities

Accepted Manuscript Title: Polysaccharide from Mesona chinensis: Extraction optimization, physicochemical characterizations and antioxidant activities Authors: Lihua Lin, Jianhua Xie, Suchen Liu, Mingyue Shen, Wei Tang, Mingyong Xie PII: DOI: Reference:

S0141-8130(17)30295-7 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.040 BIOMAC 7206

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

21-1-2017 14-2-2017 6-3-2017

Please cite this article as: Lihua Lin, Jianhua Xie, Suchen Liu, Mingyue Shen, Wei Tang, Mingyong Xie, Polysaccharide from Mesona chinensis: Extraction optimization, physicochemical characterizations and antioxidant activities, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.03.040 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Polysaccharide from Mesona chinensis: extraction optimization, physicochemical characterizations and antioxidant activities

Lihua Lin, Jianhua Xie *, Suchen Liu, Mingyue Shen, Wei Tang, Mingyong Xie State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China

* Corresponding author: Professor Jianhua Xie, PhD, State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang, Jiangxi 330047, China. Tel: +86-791-88304347; Fax: +86-791-88304347; E-mail address: (J. Xie) [email protected]

Graphical abstract The polysaccharide from Mesona chinensis was extracted by response surface methodology and its physicochemical characterizations and antioxidant activities were investigated.

extraction

Polysaccharide from Mesona chinensis

Optimization of extraction conditions

Mesona chinensis

Physicochemical characterizations and antioxidant activities

1

Highlights • Optimal extraction conditions of MCP were obtained by response surface methodology. • Physicochemical characterizations of MCP were measured by HPGPC, FTIR and HPAEC. • MCP exhibited antioxidant activities in a concentration-dependent manner. • MCP might be a good candidate for further investigations of functional foods.

Abstract The optimization of extraction conditions, physicochemical characterizations and antioxidant activities of polysaccharide from Mesona chinensis (MCP) were investigated. Optimal extraction conditions of MCP with the highest yield of 7.05 ± 0.12% was obtained by response surface methodology (RSM). The physicochemical characterizations of MCP obtained at the optimal extraction conditions were detected by TU-1900 spectrophotometer, high performance gel permeation chromatography (HPGPC), high-performance anion exchange chromatography (HPAEC) and fourier transform infrared spectroscopy (FT-IR) as well, revealing that MCP was a heteropolysaccharide containing uronic acid (29.3±1.3%) and protein (10.4±0.8%), with an average molecular weight (Mw) of 1.45 × 106 Da, which mainly consisted of galactose (Gal) and glucose (Glc) in a molar ratio of 1.00:1.38. Furthermore, MCP exhibited considerable antioxidant potential

2

on scavenging hydroxyl (53.87±0.44%), superoxide anion (58.42±1.17%) and DPPH radicals (55.59±0.69%). Our results indicated that MCP might be a good candidate for further investigations of functional foods.

Abbreviations Used MCP, Polysaccharide from Mesona chinensis; HAE, Hot alkali extraction; DPPH, 1,1-Diphenyl-2-picrylhydrazyl; FTIR, Fourier transform infrared spectroscopy; Mw, Molecular weight; BBD, Box–Behnken design; RSM, Response surface methodology; HPGPC, high performance gel permeation chromatography; HPAEC, high-performance anion exchange chromatography; •OH, Hydroxyl radical; ANOVA, analysis of variance.

Keywords: Mesona chinensis; Polysaccharide; Physicochemical characterizations; Extraction; Antioxidant activities

1. Introduction Mesona chinensis also named Hsian-tsao, Herb Jelly and Mesona chinensis benth, is a yearly herbaceous plant containing a distinct flavor from Lamiaceae family [1]. It is generally planted in China and Southeast Asia like Indonesia, Vietnam and Burma [2], and has been extensively studied in the field of foods and pharmaceuticals for its biological functions like antioxidant, protective against heat-stroke, hepatoprotective

3

and anti-hypertensive [3]. In China, it has been used as an important medical and edible plant resource, and usually employed as food ingredients in the productions of herbal tea and jam-type dessert and edible gel [4]. The main constituents isolated from Mesona chinensis are polysaccharides, flavonoids, terpenoids polyphenols, etc. [5]. As the main component of Mesona chinensis, Mesona chinensis polysaccharide (MCP) has attracted a great deal of attention for its various biological activities such as anti-oxidant, immunoregulation, and anti-diabetics [6-7]. The addition of Mesona chinensis gum to casein film showed better mechanical properties and stronger antioxidant capacities than pure casein film, and the anti-oxidative effect provided by Mesona chinensis leaf gum was strongly concentration dependent [8]. In recent years, work has been done on the crude polysaccharide as well as the purified polysaccharide from Mesona chinensis [9-10]. However, few studies have been conducted for the extraction and characterizations of MCP that might result in low yield of polysaccharide, and high consumption of raw material, which may impede their productions and developments. There exist a variety of extraction methods to obtain polysaccharide, such as heating, boiling, or refluxing, and getting the best extraction conditions to gain the highest yield of polysaccharide is beneficial to the application and further study of plant resources [11]. Statistical technique and response surface methodology (RSM) are extensively employed for their superb combinations of extracting factors [12]. Compared with conventional approaches, Box–Behnken design (BBD) is more effective to conduct experiments which can simplify the intricacy of the experimental 4

tests required to assess multiple variables and their mutual effects [13]. Oxygen-derived free radicals are occasionally produced, and act a part as mediators of inflammation injury which might damage a number of biomacromolecules, such as nucleic acids, lipid membranes and amino acids in living bodies, leading to multifarious sickness and maladjustment, like diabetes, hypertension, infarction, rheumatoid arthritis, and atherosclerosis, etc. [14]. Investigating natural and low cytotoxicity antioxidants has great potential to overcome the carcinogenicity of synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) [15]. However, the information on extraction optimization by RSM and physicochemical characterizations of polysaccharide from Mesona chinensis are not available till date. Accordingly, a systematic and comprehensive research of extraction, physicochemical characterizations and antioxidant activities of MCP will provide useful information on the highly produced fruits. 2. Materials and methods 2.1 Materials and reagents Mesona chinensis were purchased from Xiaoshicheng, Ganzhou, Jiangxi, China. Mesona chinensis were air desiccated and milled into fine powder by a high speed disintegrator and passed through a 10-mesh sieve before extraction. DPPH, ascorbic acid, bovine serum albumin and the standard monosaccharides including glucose (Glc), mannose (Man), xylose (Xyl), rhamnose (Rha), arabinose (Ara), galactose (Gal), fructose (Fru), ribose (Rib) and fucose (Fuc) were purchased 5

from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents were of analytical grade and purchased from Shanghai Chemicals and Reagents Co. (Shanghai, China). The ultra-pure water was utilized from a Milli-Q water purification system (Millipore, Bedford, MA, USA). 2.2. Preparation and determination of MCP The dried Mesona chinensis were thoroughly sliced and ground into fine powder in a mill, then extracted with ten-fold 95% ethanol for 12 h to remove interference impurities and small lipophilic molecules like monosaccharide, disaccharide, oligosaccharide, fat acids and polyphenols [11]. The preprocessed samples were separated manually from the organic solvent through the nylon cloth (pore diameter: 38 μm), then dried. Each pretreated sample (5.0 g) and sodium carbonate solution at different liquid to solid ratios (5:1~30:1, mL/g) were put into 50 mL, 100 mL, 250 mL, 500 mL, 500 mL, 500 mL beaker, respectively. Then the extractive procedures were carried out at different temperatures (50~100 °C) in a JRY electric-heated thermostatic water bath at various sodium carbonate concentrations (1~6 mg/mL) for various time (0.5~3.0 h). The extracts were centrifuged at 4800 rpm for 10 min and then the supernatants were gathered. The supernatants were diluted with ultra-pure water and the polysaccharide content of MCP was determined by the phenol–sulfuric acid method [16]. The yield of polysaccharide (Y) in dried Mesona chinensis was calculated as: Y (%) = c×v/w×100%

(1)

Where c is the concentration of polysaccharide in the sample solution (mg/mL),

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v is the volume of sample solution (mL), and w is the mass of the dried sample (mg). After identifying the best extraction conditions, extraction procedure was conducted to obtain the dried MCP for latter physicochemical characterizations and antioxidant activities assays. After extraction, the extraction solutions were separated from insoluble residues by centrifugation (4800 rpm for 10 min, at 20 °C), followed by collected, the supernatants were concentrated with a rotary evaporator (EYELA OSB-2100, Tokyo, Japan) at 60 °C under vacuum, then precipitation with alcohol to a concentration of 80% (v/v) and kept at 4 °C overnight. The precipitate was collected by centrifugation at 4800 rpm for 10 min, and washed using 95% ethanol, 100% ethanol and acetone, respectively. After filtering and centrifuging, the precipitate was re-dissolved in ultra-pure water, and centrifuged at 9000 rpm for 15 min, then, the supernatant was further dialyzed for 36 h in natural water and 12 h in ultra-pure water (MW cut-off 14 kDa) before concentration under vacuum evaporator at 55 °C. Lastly, the precipitate was frozen at -20 °C overnight and lyophilized in vacuum freeze dryer (model ALPHA 2–4, Christ, Germany) to obtain the dried MCP. 2.3. Experimental design To confirm the variation range of extraction parameters, single factor experiment were carried out. Every test was performed in triplicates. Based on previous single factor experimental results, a four-variable, three-level experiments coded +1, 0 and －1 for high, middle and low value, respectively, and 29-run BBD were adopted for optimization, as shown in Table 1. The selected variables are numbered in accordance with the following equation: 7

= 1; 2; 3

(2)

Where xi is the numbered value of independent parameter; xi is the practical value of independent parameter; x0 is the real value of independent parameter at the center point; and △x is the step change value. Table 1 showed the matrix composed of 29 experimental runs, every test was implemented three times. Second-order polynomial model was employed for test results as mentioned above:

Where Y is the predicted response value; β0 is an intercept; βi, βii, and βij represent the regression coefficients of linear, quadratic, and interactive terms, respectively; Xi and Xj both are the numbered independent parameters (i≠j). Experimental results of BBD were analyzed by using Design-Expert software (Version 8.0.6.1, USA). The significance of every response was assessed by the means of analysis of variance (ANOVA). Three-dimensional (3D) response surface plots were adopted to analyze the interaction effects of every independent parameter on responses [17]. The significance of the regression coefficient was checked by using F-value and p-value, and the adequacies of models were assessed by employing the coefficient of determination (R2) and adjusted coefficient of determination (R2Adj). Finally, three validation experiments were carried out to check the optimized conditions. 2.4. Physicochemical characterizations of MCP 8

2.4.1. Determination of uronic acid and protein contents The uronic acid and protein contents of MCP obtained by the optimal extracting process were determined by the sulfate-carbazole method [18], and Bradford method [19], respectively, with a spectrophotometer (TU-1900 spectrophotometer, Beijing, China). 2.4.2. Homogeneity and molecular weight (Mw) analysis The homogeneity and Mw of MCP were determined by high performance gel permeation chromatography (HPGPC) according to our previous method [20], with T-series Dextran standards (Dextran T10, T40, T70, T2000 and Glc) as a standard. An Agilent 1260 HPLC system (Agilent, USA) collocated with an Ultrahydrogel TM linear column (300 mm × 7.8 mm), with refractive index detector and UV detector. 2.4.3. High-performance anion exchange chromatography (HPAEC) analysis for monosaccharide compositions of MCP Monosaccharide compositions of MCP were determined by high-performance anion exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD) according to the method as our previous description [21]. Briefly, the MCP was hydrolyzed with 2.0 M H2SO4 at 110 °C for 8 h. The supernatants (25 μL) were analyzed on a Dionex ICS-2500 system, coupled with PAD, and equipped with a Carbo PAC™ PA10 column (2.0 mm  250 mm). Retention times of monosaccharide standards (Fru, Ara, Glc, Xyl, Rha, Gal, Rib, Man and Fuc) were used to determine the monosaccharide compositions of MCP.

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2.4.4. Fourier transform infrared spectroscopy (FT-IR) analysis Infrared spectrum of MCP was measured by employing a Nicolet 5700 Fourier-transform infrared spectrophotometer (FTIR, Nicolet, USA) in the range of 4000-400 cm-1 at room temperature. The freeze-dried MCP sample (1.0 mg) was mixed with spectroscopic grade KBr powder at a ratio of 1:30, ground, then pressed into a 1 mm pellet by employing a stainless steel cup (diameter 10 mm) prior to FT-IR measurement [22]. 2.5. Antioxidant activities assays 2.5.1. Hydroxyl radical scavenging activities To study the hydroxyl radical scavenging ability of MCP, a widely known Fenton-pattern responding method was adopted with little modifications [23]. In brief, 1.0 mL of polysaccharide samples were blended with 1.0 mL FeSO4 (2 mM/L) and 1.0 mL salicylic acid–ethanol solution (6 mM/L). Subsequently, 1.0 mL H2O2 (6 mM/L) was added to the mixed solution before incubation at 37 °C in water bath for 30 min. After that, the mixture was immediately measured for absorbance at 510 nm on a UV-visible spectrophotometer; ascorbic acid was employed as a positive standard. Hydroxyl radical scavenging activity was counted by adopting the following formula: Scavenging activity (%) = [1 − (A s − Ab)/A c] × 100%

(4)

Where As is the absorbance of sample, Ab is the absorbance of sample using water instead of H2O2, and Ac is the absorbance of water instead of sample. 2.5.2. Superoxide anion scavenging activity The modified Marklund method was referenced to determine the superoxide 10

anion scavenging ability of MCP [24]. Briefly, 5.0 mL Tris-HCl buffer solution (50 mM/L, pH 8.2) and 0.5 mL sample solution were incubated at 25 °C in water bath for 20 min. 0.5 mL pyrogallol (3 mM/L) incubated at 25 °C was added quickly and the absorbance of the mixture was tested every 30s at 325 nm for 5 min, and the ascorbic acid was employed as a positive standard. The superoxide anion scavenging activity was counted adopting the formula: Superoxide anion scavenging activity (%) = (1 − As /Ac) × 100%

(5)

Where As is the absorbance of sample, Ac is the absorbance of water instead of sample. 2.5.3. DPPH radical scavenging activity The activity for scavenging DPPH radical of MCP was measured according to the literature with some modifications [25]. Simply, 2.0 mL DPPH (1 mM/L), 1.0 mL of samples and 1.0 mL deionized water were mixed in 10 mL tube. The compound was maintained at room temperature in the dark for 30 min, and the absorbance (A) was tested immediately at 517 nm. Ascorbic acid was employed as a positive standard. DPPH radical scavenging activity was counted according to Eq. (6). DPPH radical scavenging activity (%) = [1 − (As − Ab)/Ac] × 100%

(6)

Where As is the absorbance of sample, Ab is the absorbance of absolute ethyl alcohol instead of DPPH, and Ac is the absorbance of water instead of sample. 2.6. Statistical analysis All experimental data were expressed as means ± standard deviations (SD), Design-Expert (Version 8.0, Stat-Ease) and Origin Pro (version 8.0) software 11

(Stat-Ease Inc., Minneapolis, USA) were used for analyzing results, and p values less than 0.05 were considered statistically significant. 3. Results and discussion 3.1. Optimization of MCP extraction 3.1.1. Effects of extraction time, temperature, sodium carbonate concentration and ratio of extraction solvent to raw material on the yields of MCP The results of four parameters including extraction time, temperature, sodium carbonate concentration and ratio of extraction solvent to raw material in the extraction process of MCP were shown in Fig. 1. Different extraction time were employed, temperature, sodium carbonate concentration, and ratio of extraction solvent to raw material were set as 90 °C, 3 mg/mL and 20 mL/g, respectively. As showed in Fig. 1A, when extraction time was increased from 0.5 to 3.0 h, the yield of polysaccharide increased correspondingly and the highest extraction yield (6.27 ± 0.09%) was obtained at 2.0 h. An increased duration can strengthen the mass transmission so the release and diffusion of polysaccharide for entering into the solvent become easier and quicker. Nevertheless, excessive extraction time (>2.0 h) may lead to the degradation or conversion of the polysaccharide [26]. Therefore, extraction time varying from 1.5 h to 2.5 h was selected. The temperature of extraction was varied, while time, sodium carbonate concentration, and ratio of extraction solvent to raw material were maintained as 2.0 h, 3 mg/mL and 20 mL/g, respectively. When temperature was increased from 50 to 12

90 °C, the extraction yield of MCP gradually increased to 6.24 ± 0.05% (Fig. 1B), but over 90 °C, it dropped. Polysaccharide diffusion coefficient increases at higher temperature and promote polysaccharide diffusion in extraction solvent. Whereas, the structure of polysaccharide might be damage and degrade at high temperature. Besides, it also adds to the cost in terms of energy. Hence, extraction temperatures of 80 to 100 °C were chosen. MCP was a kind of acidic polysaccharide [10], sodium carbonate concentration is believed to be an important factor for the extraction of MCP. The influences of different sodium carbonate concentrations on the yield of MCP were investigated, extraction time, temperature, and ratio of extraction solvent to raw material were determined as 2.0 h, 90 °C, and 20 mL/g, respectively. As shown in Fig. 1C, the yield of MCP increased rapidly with increase of sodium carbonate concentration. Above 3 mg/ml of sodium carbonate, the yield increased slightly and produced some undesirable flavor, which would affect the subsequent production and application. Therefore, sodium carbonate concentrations range of 2~4 mg/mL were chosen. Different ratios of extraction solvent to raw material were applied and extraction time, temperature, and sodium carbonate concentration were determined as 2 h, 90 °C, and 3 mg/mL, respectively. As shown in Fig. 1D, as the ratio increased from 5 to 30 mL/g, the yield of polysaccharide also increased from 4.25% to 5.89% and reached a maximum at 15 mL/g, the yield achieved the highest value (6.41 ± 0.14%), then dropped at higher ratio. Higher ratios enabled higher concentration difference between extraction solvent and internal tissues of raw material, thus significantly facilitating 13

dissolution of polysaccharide and leading to an increase of polysaccharide yield [27]. However, excessive extraction solvent could assimilate cavitation energy from the extraction process, leading to a lower yield [28]. Therefore, 20 mL/g was chosen as the central point of BBD experiment. 3.1.2. Analysis of the predicted model Table 1 showed the variation range of four independent variables according to the single-factor data, and Table 2 displayed the design matrix and relevant results of RSM experiments. By using multiple regression analysis, the predicted response Y for the yield of MCP could be obtained by the following second-order polynomial equation: Y=6.76+0.040X1+0.24X2+0.16X3+0.16X4+0.12X1X2-0.15X1X3+0.21X1X4-0.045 2

2

2

2

X2X3-0.058 X2X4+0.022 X3X4-0.59 X1 -0.67 X2 -0.62 X3 -0.58 X4

(7)

Where Y is the predicted yield of MCP, and X1, X2, X3 and X4 are the coded parameters for extraction time, extraction temperature, sodium carbonate concentration and ratio of extraction solvent to raw material, respectively. Analysis of variance (ANOVA) was adopted with the purpose of finding out how different factors affected the yield of MCP, as displayed in Table 2, the predictive model was comprehensively evaluated. It was clearly observed that the model had a very high F-value (F = 15.65), implying that the quadratic regression equation was extremely significant [29]. Lower p-value indicates higher significance of the corresponding coefficient. Seen from Table 2, the p-value was lower than 0.0001, suggesting that the corresponding coefficient was highly significant and greatly 14

suitable for application in this extraction process. In addition, the linear coefficients of X2, X3 and X4, quadratic coefficients of X12, X22, X32 and X42 and cross product coefficients of X1X4 all had appreciable impacts (p﹤0.05). However, other coefficients like X1, X1X2, X1X3, X2X3, X2X4 and X3X4 were found to be non-significant. Meanwhile, F-value of the “Lack of Fit F-Value” was 0.94, indicating that the Lack of Fit had indistinctive relation with the pure error. Additionally, the determination coefficient (R2) had a high value of 0.9399, indicating that the four independent parameters just had 6.01% not accounted for by the model. Coefficient of variation (C.V.) had a relative low value (3.27), manifesting a preferable accuracy of the model and good reliance of the experimental data. 3.1.3. Analysis of response surface To better explain the relationships and interactions between these different parameters, three-dimensional response surface plots generated by Design-Expert software, were completely displayed in Fig. 2. It was worth mentioning that different shapes of the plots manifested different mutual effects among the variables, rounded contour plots denoting the effects of relevant parameters are insignificant, while elliptical contour indicates opposite [30]. Two parameters were maintained on the response every time, while the other two parameters were set at zero level. As demonstrated in Fig. 2A, the extraction yields (Y) of MCP under different extraction time (X1) and extraction temperature (X2), with sodium carbonate concentration (X3) and ratio of extraction solvent to raw material (X4) were determined at central levels. The yield (Y) of MCP increased quickly when extraction 15

time (X1) increased from 1.5 to 2.1 h, the same trend as the extraction temperature (X2) increased from 80 to 91.4 °C; but beyond 2.1 h and 91.4 °C, the yield (Y) of MCP decreased slightly. Additionally, the synergistic interactions of extraction time (X1) and extraction temperature (X2) were insignificant (p > 0.05). The same trends were described in Fig. 2B, of which Fig. 2B exhibited a similar plot. Meanwhile, the mutual effects of extraction time (X2) and ratio of extraction solvent to raw material (X4) on the yield of polysaccharide were depicted in Fig. 2C. The yield of MCP rapidly increased along with the extraction time (X2) increased from 1.5 to 2.5 h. With regard to longer extraction time (approximately 2.0 h) and higher extraction solvent to raw material (X4) (approximately 20 mL/g), maximum yield of MCP was deservedly gained. Additionally, the synergistic interactions of extraction time and extraction solvent to raw material on the yield were positive and significant, sustained by p-value less than 0.05. Therefore, great yield could be got under moderate extraction time and an appropriate level of extraction solvent to raw material should be chosen. What depicted in Fig. 2D were extraction time (X1) and ratio of extraction solvent to raw material (X4) determinated at zero levels, extraction temperature (X2) and sodium carbonate concentration (X3) were used to perform quadratic interactions on the yield (Y) of MCP. Nonetheless, Table 3 showed the interactions of extraction temperature (X2) and sodium carbonate concentration (X3) on MCP yield were insignificant (p > 0.05), which was in good agreement with the presented 3D-response surface plot. The similar trends were described in Fig. 2E and Fig. 2F. 16

According to the results and the analyses from Fig. 2, the highest yield of MCP was gained and the optimal variables were extraction time 2.03 h, extraction temperature 91.74 °C, sodium carbonate concentration 3.12 mg/mL, and ratio of extraction solvent to raw material 20.71 mL/g. Under these best conditions, the highest predicted yield of MCP was 6.80 %. 3.1.4. Verification To confirm the optimal parameters of the model equations, three verification tests were implemented under the best conditions as previously mentioned. Under the best conditions, maximum value of MCP was obtained (7.05±0.12%) and closed to the predicted value. Therefore, the experimental results indicated that the response model was accurate and suitable for optimizing the hot alkali extraction (HAE) process of MCP. 3.2. Physicochemical characterizations analyses 3.2.1. Contents of uronic acid and protein The uronic acid and protein contents of dried MCP prepared under the optimized extraction conditions were measured as 29.3 ± 1.3%, and 10.4 ± 0.8% (w/w), respectively. 3.2.2. Homogeneity and Mw of MCP MCP was detected by HPLC on UltrahydrogelTM linear column and a relative narrow and sharp symmetrical peak with elution time of about 13.3 min was observed, thus confirming its homogeneity (Fig. 3A). The average Mw of MCP was calculated to be approximately 1.45 × 106 Da according to the calibration equation derived from 17

linear regression of the calibration curve. Fig. 3A also showed that MCP had a highly strong response value at wave length of 280 nm with a single symmetrical peak. The peaks at 280 nm by UV detection also indicated that MCP contained protein. These results were in accordance with research results of protein determination of MCP. The peaks in UV-spectrum were the results of protein bound to the polysaccharide of Mesona chinensis and the polymer is a proteoglycan. This result was in accordance with the results of our previous study of the polysaccharides of Cyclocarya paliurus [21] and other studies in polysaccharides from leaves, flowers and seeds of green tea [31-32]. 3.2.3. Monosaccharide compositions of MCP A simple and rapid HPAEC-PAD method to analyze the composition of monosaccharides released from polysaccharide was developed previous [21]. HPAEC-PAD chromatogram profiles of standard monosaccharide mixture solution and hydrolysate of MCP were shown in Fig. 3B. These results showed that nine monosaccharides were detected and the peaks were also separated obviously. Peaks in Fig. 3B were identified by comparing the retention time of MCP with standards under the same conditions. Results showed that MCP was a kind of heteropolysaccharide composed of Gal and Glc in a molar ratio of 1.00:1.38, which were the mainly monosaccharides constructing the backbones sugar forms in MCP. It can be seen that Ara was also detected in MCP with a very small amount. 3.2.4. FT-IR spectroscopy analysis The MCP powder was implemented in the range of 4000－400 cm-1 in the FT-IR 18

spectra and different absorption bands were completely identified. As shown in Fig. 4, the broad band at 3422 cm−1 stands for the stretching vibration of O−H, which is common to all polysaccharides, whereas the signal at 2925 cm−1 is regarded as the stretching vibrations of C−H [15,33]. 1634 cm−1and 1396 cm−1 could be conformed to the carbonyl C=O vibrations in uronic acid and the carbonyl C−O stretching vibrations, respectively [34]. Absorption peaks at 1000–500 cm-1 primarily are owed to the variable angle vibration and bending vibration of alcohols including hydroxyl groups and benzene rings [35]. The absorption peaks at 841 cm−1 and 1017 cm−1 are indicative of α-configuration and α-1,6 linkage, respectively [36]. These results revealed that the MCP was a heteropolysaccharide containing uronic acid. 3.3. Antioxidant activities of MCP 3.3.1. Scavenging activities of hydroxyl radicals Hydroxyl radicals are extremely physiological reactive occasionally produced by immune action in living body [37]. Additionally, it can cause severe damage to tissue, cell, and neighboring biomolecules particularly on protein via multiple reactions with most biomacromolecules metabolism in living bodies [38]. The antioxidant mechanism is related to the supply of hydrogen provided by polysaccharide, which can combine with metal ions like Fe2+ and Cu2+ to stop the radical chain reactions [11]. Fig. 5A showed that the capacity to remove hydroxyl radicals of MCP exhibited a concentration-dependent pattern, and the highest scavenging rate was 54.36±1.56% at 1.6 mg/mL. The scavenging effects of ascorbic acid increased rapidly accompanied 19

by the increases of concentrations, however, when the concentration exceeded 0.80 mg/mL, it slowly reached to its maximum. These results proved that MCP was a potential scavenger by scavenging hydroxyl radicals. 3.3.2. Scavenging activities of superoxide radicals As a kind of precursors of singlet oxygen, superoxide anion is a resilient radical produced by various biologic and photochemical reactions [39]. Besides, superoxide anion radicals can react with numerous biomolecules to produce more reactive oxidative species, which can damage DNA and result in many diseases [27]. The scavenging activities of MCP were inferior than that of ascorbic acid, and it could be improved in higher concentration. The superoxide anion scavenging activity reached 36.07±1.31% at the concentration of 0.4 mg/mL, and then increased slightly. Lastly, the maximum scavenging rate (58.42±1.17%) of MCP was obtained when the concentration reached to 1.6 mg/mL, meanwhile, the scavenging rate of ascorbic acid was 93.34 ± 0.87% (Fig. 5B). Therefore, MCP showed a moderate scavenging ability on eliminating superoxide anion radicals. 3.3.3. Scavenging activities of DPPH radicals It is widely known that DPPH radicals, organic nitrogen radicals with the characteristic of stabilization, have an ultraviolet–visible absorption at 517 nm that can be faded by antioxidant for their proton-donating capacity [40]. As shown in Fig. 5C, the DPPH scavenging effects increased from 28.03±1.36% to 47.42±0.80% when the concentrations of MCP increased from 0.1 mg/mL to 0.8 mg/mL, then DPPH scavenging capacities of MCP were increased indistinctly, 20

reached to its maximum (55.59±0.69%) at 1.6 mg/mL, which showed considerable potential of MCP on scavenging DPPH free radicals. 4. Conclusions Polysaccharide from Mesona chinensis was extracted by HAE technology and the mutual effects of the variables were seriatim determined by employing RSM. The optimal extraction conditions were confirmed as follows: extraction time, 2.03 h; extraction temperature, 91.74 °C; sodium carbonate concentration, 3.12 mg/mL; and ratio of extraction solvent to raw material, 20.71 mL/g. Under these conditions, the highest yield was deservedly obtained as 7.05±0.12%. In addition, MCP possessed protein, a moderate uronic acid content, and with an average molecular weight (Mw) of 1.45 × 106 Da, which mainly consisted of galactose (Gal) and glucose (Glc) in a molar ratio of 1.00:1.38. FT-IR of MCP proved that it was a characteristic heteropolysaccharide. Besides, MCP possessed appreciable scavenging activities against DPPH, superoxide anion and hydroxyl radicals. Therefore, MCP could be developed as a potential antioxidant agent for application in the field of functional foods or medicine. These results may further supply theoretical basis for the extensive use of MCP in anti-oxidative products and pharmaceutical industries.

Conflict of Interest All authors have no personal or financial conflict of interest.

Acknowledgements 21

This work was financial supported by the Program of the National Natural Science Foundation of China (No. 21566024), and the Major Research Plan of the Natural Science Foundation of Jiangxi Province, China (No. 20152ACB21004).

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(B)

(A)

(C)

(D)

Fig. 1 Effects of different extraction parameters on the yields of MCP. (A) extraction time, (B) extraction temperature, (C) sodium carbonate concentration, (D) ratios of extraction solvent to raw material. Data represent means ± SD of three independent experiments.

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Fig. 2 3D-response surface plots (A–F) showing the effects of extraction time (X1), extraction temperatures (X2), sodium carbonate concentrations (X3), and ratio of extraction solvent to raw material (X4) on the extraction yields (Y) of MCP.

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Fig. 3 (A) HPGPC chromatogram profile of MCP in HPGPC on UltrahydrogelTM-500 column, with ultra-pure water as the eluent at a flow rate of 0.6 ml/min 0.1 mol/L, equipped with RI detection and UV detection. (B) HPAEC-PAD chromatogram profiles of standard monosaccharide and MCP. a: fucose (Fuc), b: rhamnose (Rha), c: arabinose (Ara), d: galactose (Gal), e: glucose (Glc), f: xylose (Xyl), g: mannose (Man), h: fructose (Fru), i: ribose (Rib).

30

Fig. 4 FT-IR spectra of MCP.

31

(B)

(A)

(C)

Fig. 5 Antioxidant effects of MCP. (A) scavenging effects of MCP on hydroxyl radicals, (B) scavenging effects of MCP on superoxide radicals, and (C), scavenging effects of MCP on DPPH radicals. Values were expressed as means ± SD (n = 3).

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Table 1 Levels and code of variable used for Box–Behnken design (BBD), and the observed responses for the extraction yields of MCP Independent variables Symbol Range and level -1 0 +1 Extraction time (h) X1 1.5 2.0 2.5 Extraction temperature (°C) X2 80 90 100 Sodium carbonate concentration (mg/mL) X3 2 3 4 Ratio of extraction solvent to raw material (mL/g) X4 15 20 25 Run Coded variable levels Extraction yield (%) X1 X2 X3 X4 Experimental * Predicted 1 2 80 4 20 5.58 5.43 2 2 90 3 20 6.85 6.76 3 2 90 4 15 5.46 5.53 4 1.5 90 3 15 5.43 5.60 5 2 90 4 25 5.72 5.89 6 2.5 90 4 20 5.69 5.60 7 2 90 2 15 5.39 5.26 8 2 90 3 20 6.85 6.76 9 1.5 80 3 20 5.39 5.35 10 2 80 2 20 5.09 5.02 11 2.5 90 2 20 5.31 5.58 12 2 90 2 25 5.56 5.53 13 2.5 100 3 20 5.82 5.90 14 2.5 90 3 15 5.22 5.26 15 2 100 3 15 5.86 5.64 16 2.5 80 3 20 5.12 5.18 17 2 100 2 20 5.39 5.59 18 2 90 3 20 6.60 6.76 19 2 100 4 20 5.70 5.82 20 2 90 3 20 6.83 6.76 21 2 90 3 20 6.68 6.76 22 2.5 90 3 25 6.12 6.00 23 2.5 90 2 20 5.82 5.58 24 2 100 3 25 6.01 5.85 25 2 80 3 15 4.98 5.05 26 2 80 3 25 5.36 5.48 27 1.5 90 3 25 5.48 5.50 28 1.5 90 4 20 5.94 5.82 29 1.5 100 3 20 5.60 5.58 * Experiments were performed in triplicate and the data were reported as means of three values. Table 2 Analysis of variance (ANOVA) for response surface quadratic model for extraction of polysaccharide from Mesona chinensis. Variables

Sum of squares

DF

Mean square 33

F value

p-value

Model 7.74 X1 0.017 X2 0.68 X3 0.26 X4 0.30 X1×X2 0.060 X1×X3 0.066 X1×X4 0.18 X2×X3 8.100E-003 X2×X4 0.013 X3×X4 2.025E-003 2 X1 2.06 2 X2 2.86 X32 2.32 2 X4 2.17 Residual 0.49 Lack of fit 0.31 Pure error 0.18 Cor total 8.23 2 R 0.9399 2 R adj 0.8799 2 R pred 0.7091 Adeq precision 12.864 C.V. % 3.27 a Significant (p < 0.05). b Not significant (p > 0.05).

14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 9 5 28

0.55 0.017 0.68 0.26 0.30 0.060 0.066 0.18 8.100E-003 0.013 2.025E-003 2.06 2.86 2.32 2.17 0.035 0.035 0.037

34

15.65 0.49 19.31 7.39 8.61 1.70 1.86 5.12 0.23 0.37 0.057 58.39 80.89 65.80 61.43

Prob. > F ＜0.0001a 0.4972 0.0006a 0.0167a 0.0109a 0.2133 0.1940 0.0401a 0.6393 0.5503 0.8142 ＜0.0001a ＜0.0001a ＜0.0001a ＜0.0001a

0.94

0.5585b