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Abstract
antioxidants Article Extraction Optimization and Evaluation of the Antioxidant and -Glucosidase Inhibitory Activity of Polysaccharides from Chrysanthemum morifolium cv. Hangju 1 ,y 1 ,y 2 ,y 1 1 Xiaoyan Hou , Xia Huang , Jianlong Li , Guangyang Jiang , Guanghui Shen , 1 1 1 1 1 1 Shanshan Li , Qingying Luo , Hejun Wu , Meiliang Li , Xingyan Liu , Anjun Chen , 3 1 , Meng Ye and Zhiqing Zhang * College of Food Science, Sichuan Agricultural University, Ya’an 625014, China; houxiaoyan106@outlook.com (X.H.); xiahuang129@outlook.com (X.H.); GuangyangJ@outlook.com (G.J.); shenghuishen@163.com (G.S.); lishanshan.812@163.com (S.L.); cherry12112009@163.com (Q.L.); hejunwu520@163.com (H.W.); lml05@163.com (M.L.); LXY05@126.com (X.L.); anjunc003@163.com (A.C.) College of Biomass Science and Engineering, Sichuan University, Chengdu 610000, China; jlli999@foxmail.com College of Forest, Sichuan Agricultural University, Chengdu 610000, China; yemeng5581@163.com * Correspondence: zqzhang721@163.com; Tel.: +86-0835-288-2403 y These authors contributed equally to this work. Received: 6 December 2019; Accepted: 7 January 2020; Published: 9 January 2020 Abstract: In order to evaluate the antioxidant and -glucosidase activities of polysaccharides from Chrysanthemum morifolium cv. Hangju (CMPs), the response surface methodology was applied to optimize the parameters for extraction progress of CMPs by ultrasound, with heat reflex extraction (HRE) performed as the control. The di erence in the physicochemical properties of polysaccharides obtained by the two methods were also investigated. The maximum yields (8.29 0.18%) of polysaccharides extracted by ultrasonic assisted extraction (UAE) were obtained under the optimized conditions of ultrasonic power 501 W, extraction time 19 min, and ratio of liquid-to-raw material 41 mL/g. Polysaccharides extracted by UAE possessed lower protein contents (2.56%) and higher uronic acids contents (7.08%) and low molecular weight fractions than that by HRE. No significant di erences were found in monosaccharide composition and Fourier transform infrared (FT-IR) spectra of polysaccharides extracted by UAE and HRE, while polysaccharides by UAE possessed stronger antioxidant and -glucosidase inhibitory activities. Therefore, UAE was an ecient way to obtain CMPs. Keywords: Chrysanthemum morifolium cv. Hangju; polysaccharides; ultrasonic assisted extraction; antioxidant activity; -glucosidase inhibitory activity 1. Introduction Oxidative stress, which can attack healthy cells and make their function and structure to be lost, is usually considered to be caused by reactive oxygen species (ROS) [1]. It is reported that more than 100 diseases such as Alzheimer ’s disease, arteriosclerosis, and cancer are associated with oxidative stress [1,2]. Antioxidants are compounds or systems that can inhibit or delay the oxidation progress and play a significant role in antioxidant defense [3]. The functions of antioxidants such as lowering oxidative stress and reducing DNA and cell damage have been documented [1–4]. It has been stated that some exogenous antioxidants like vitamin C, vitamin E, and phenolics in plants can also perform the activity of endogenous antioxidative defense [5]. Antioxidants 2020, 9, 59; doi:10.3390/antiox9010059 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, 59 2 of 17 Chrysanthemum morifolium cv. Hangju is a plant belonging to the Compositae family [6]. It is a traditional Chinese herb, which is famous for its capacity to reinforce kidney, tonify spleen, and improve vision and has been used in folk medicine as a tea or drug for thousands of years [7,8]. Up to now, bioactive compounds such as lignans, phenolic glycosides, and flavonoids have been isolated from C. morifolium flowers [9,10]. Furthermore, these ingredients make the traditional herb exhibit numerous health benefits including antioxidant, anti-inflammatory, neuroprotective, and anti-HIV activity [10–15]. However, polysaccharides in C. morifolium flowers have rarely been studied. Only a few studies about C. morifolium polysaccharides (CMPs) have been reported [11–14]. Zheng et al. [13] studied a water soluble polysaccharide from C. morifolium and found that it showed excellent antioxidant activity. Moreover, a polysaccharide from the same material reported in another study exhibited anti-angiogenic activity. According to Tao et al. [11], polysaccharides from C. morifolium positively a ected the short-chain fatty acids’ intestinal production and could prominently ameliorate colitis in rats. Polysaccharides, presenting in almost all organisms, are important functional biological macromolecules because of their significant benefit to human health such as antioxidant, antidiabetic, immunopotentiation, antitumor, anti-inflammatory, and hypoglycemic activities [16–18]. More and more studies have shown evidence that polysaccharides have the capacity of scavenging free radicals and may be potential natural antioxidants. The biological activities of polysaccharides are mostly related to their physicochemical properties [19]. The conditions for extracting polysaccharides such as the extraction method, extraction time, or temperature and the ratio of solid-to-liquid are important due to their great impact on the structures and properties of polysaccharides. Heat reflux extraction (HRE) is the most common method for polysaccharides extraction. In general, HRE involves in a long extraction time and high temperature, causing the degradation of polysaccharides and decreasing their physiological activity [20]. Recently, ultrasonic assisted extraction (UAE) has become popular for its high eciency, low consumption of energy, and high automation [20,21]. The cavitation e ect of ultrasonic assisted extraction promotes the release of bioactive compounds from plant cells [22,23]. Moreover, a number of literature works suggested that polysaccharides extracted by UAE showed higher antioxidant activity than that of HRE [24–26]. Thus, UAE is a promising technique for polysaccharides’ extraction. In the present work, the process of polysaccharides extraction by UAE was optimized using response surface methodology (RSM) with HRE as the control. Moreover, the physicochemical prosperities, antioxidant activity, and -glucosidase inhibitory activity of polysaccharides obtained by the two methods were investigated and compared. The aim of the study is to reveal more information about the e ect of extraction methods on the physicochemical properties and physiological activities of CMPs and provide a novel idea for full utilization of C. morifolium and improve its economic value. 2. Materials and Methods 2.1. Plant Material and Chemicals C. morifolium flowers were purchased from Tongxiang Shine Herb Health products Co., Ltd., (Jiaxing, China). The plant material was powdered and stored in a sealed bag until use. 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), trifluoroacetic acid (TFA), 1-phenyl-3-methyl-5- pyrazolone (PMP), trichloroacetic acid (TCA), butyl hydroxyanisole (BHA), 4-nitrophenyl- -D- glucopyranoside (pNPG), and -glucosidase (biological reagent, 50 U/mg) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acarbose, 1,10-phenanthroline, bovine serum albumin (BSA), and monosaccharide standards (glucose, fructose, rhamnose, galactose, xylose, arabinose) were purchased from J & K Scientific Co., Ltd. (Beijing, China). All other chemicals used were of analytical grade. Antioxidants 2020, 9, 59 3 of 17 2.2. Extraction of Polysaccharides from C. morifolium 2.2.1. Single Factor Analysis The dried C. morifolium powder (5.0 g) was firstly refluxed with petroleum (50 mL) for 2 h and then 80% (v/v) ethanol (10 mL) for another 2 h to remove small molecular weight ingredients and lipids. The UAE was performed in an ultrasonic processor (JY99-IIDN, 1.8 kW, Xinzhi Biotechnology Co., Ltd., Ningbo, China). The influence of extraction time (5, 10, 15, 20, and 25 min), ultrasonic power (120, 240, 360, 480, and 600 W), and the ratio of liquid-to-solid (20, 30, 40, 50, and 60 mL/g) on the yield of CMPs was evaluated. The extracted polysaccharides (CMP-U i.e., C. morifolium polysaccharides obtained by UAE) were freeze dried and stored at 20 C for further study. 2.2.2. RSM Optimization RSM was employed to obtain the optimum conditions based on the above work. A three level Box–Behnken design (BBD) with three variables (extraction time X , ultrasonic power X , and ratio of 1 2 liquid-to-solid X ) were performed to optimize the extraction process. The complete design consisted of seventeen experimental points, and the variables and their levels with both coded and actual values are shown in Table 1. A quadratic polynomial model was fitted to correlate the response variable to the independent variables. The quadratic polynomial equation is generally as follows: 3 3 2 3 X X X X Y(%)= + X + X + X X (1) 0 i i i j i j j i i i=1 i=1 i=1 j=i+1 where Y (%) is the predicted response variable, , , , and are the regression coecients of the 0 i j ij model, linearity, square, and interaction, respectively, and X and X are the independent variables. i j The data of this experiment were analyzed by multiple regression analysis through the least squares method. Analysis of variance (ANOVA) was applied to evaluate the observed data and the regression coecients of linear, quadratic, and interaction terms, and the e ects of the variables were generated and investigated. The yield of polysaccharides (Y, %) is calculated as follows: Weight of dried crude extraction Y(%, w/w) = 100 (2) Weight of C. mori f olium powder Table 1. Box–Behnken design matrix and the response values for the yield of CMPs by UAE. Levels on Independent Factors Extraction Run Yield (%) X (min) X (W) X (mL/g) 1 2 3 1 20 (0) 480 (0) 40 (0) 8.29 2 25 (1) 480 (0) 30 ( 1) 5.28 3 20 (0) 480 (0) 40 (0) 8.36 4 25 (1) 360 ( 1) 40 (0) 6.88 5 20 (0) 600 (1) 30 ( 1) 5.23 6 20 (0) 360 ( 1) 30 ( 1) 6.46 7 20 (0) 480 (0) 40 (0) 8.20 8 15 ( 1) 480 (0) 50 (1) 6.50 9 20 (0) 360 ( 1) 50 (1) 5.56 10 20 (0) 600 (1) 50 (1) 7.22 11 25 (1) 480 (0) 50 (1) 7.31 12 15 ( 1) 600 (1) 40 (0) 7.63 13 15 ( 1) 360 ( 1) 40 (0) 6.75 14 20 (0) 480 (0) 40 (0) 8.29 15 25 (1) 600 (1) 40 (0) 6.89 16 20 (0) 480 (0) 40 (0) 8.11 17 15 ( 1) 480 (0) 30 ( 1) 7.13 X : extraction time; X : ultrasonic power; X : ratio of liquid-to-solid. 1 2 3 Antioxidants 2020, 9, 59 4 of 17 2.2.3. Heat Reflux Extraction HRE was carried out as the control to estimate the eciency of UAE. The conditions for HRE involved an electric heater (SXKW, 500 mL, 0.3 kW, Beijing Ever Bright Medical Treatment Instrument Co., Ltd., Beijing, China) according to the optimized UAE. In brief, 5.0 g of pre-treated C. morifolium powder were mixed with 205 mL distilled water; the extraction temperature was set at 85 C and the extraction time 2.5 h. The polysaccharides obtained by HRE (CMP-H) were freeze dried and stored at 20 C for further use. 2.3. Determination of Chemical Composition and Molecular Weights The contents of protein and uronic acids in CMPs were determined using Bradford’s method with BSA as a standard [27] and the carbazole-sulfuric acid method [28], respectively. Molecular weight analysis of CMPs was performed using high performance gel permeation chromatography (HPGPC). In brief, the samples were first dissolved in deionized water. After passing through a 0.45 m filter, the dissolved samples were applied to two Ultrahydrogel Linear columns (300 mm 7.8 mm i.d.) in series; the columns were eluted with 0.1 M NaNO . The flow rate was 0.9 mL/min, and the column temperature was 45 C. The separation of CMPs was performed by a Waters 1525 high performance liquid chromatography (HPLC) system equipped with a Waters 2414 refractive index detector. A calibration curve was obtained using T-series dextran standards whose molecular weights ranged from 1 kDa to 2000 kDa. 2.4. Determination of Monosaccharide Composition A method based on that of Wu et al. [29] was modified. The samples were first hydrolyzed with 4 M trifluoroacetic acid (100 C, 6 h) to generate monosaccharides. Then, the solution was evaporated to dryness. The hydrolyzed polysaccharides (4 mg) were dissolved in 20 mL deionized water. Then, 400 L of the solution were transferred to a centrifuge tube (2 mL) containing 200 L of 0.3 M NaOH; 400 L of 0.5 M methanol solution of 1-phenyl-3-methyl-5-pyrazolone (PMP) were added to the mixture and incubated at 70 C for 0.5 h. After cooling to room temperature, 200 L of 0.3 M HCl and 4 mL chloroform were sequentially added to the tube. The organic solvent layer (lower layer) was removed, and the procedure was repeated thrice. All the supernatants were collected, mixed, and filtered through a 0.22 m membrane filter before HPLC analysis. The monosaccharide compositions of CMPs were analyzed using an Agilent 1260 series HPLC system (Agilent, Palo Alto, CA, USA). A C column (4.6 mm 50 mm, 1.8 m; Zorbax Eclipse Plus, Agilent) was employed and eluted with a mixture of acetonitrile and phosphate bu er (20:80). The flow rate was 1 mL/min, and the UV detection was set at 250 nm at 30 C. In addition, the injection volume was 10 L, and the analysis time for each sample was 25 min. Six standard monosaccharides including galactose, arabinose, rhamnose, glucose, and fructose were used as the references after being derivatized by PMP. 2.5. Fourier Transform Infrared Spectrum Analysis FT-IR analysis was employed to identify the organic functional groups of the CMPs by a spectrophotometer (Nicolet iS5, Thermo Fisher Scientific, Waltham, MA, USA). One milligram of CMPs extracted by di erent methods was ground with 100 mg dried KBr power, and the spectra were recorded at 4000 to 400 cm . 2.6. Antioxidant Activities of C. morifolium Polysaccharides 2.6.1. DPPH Free Radical Scavenging Activity The DPPH free radical scavenging activity was performed according to a previous method with a slight modification [13]. CMPs solutions were prepared in deionized water to a final concentration Antioxidants 2020, 9, 59 5 of 17 of 1.0, 2.0, 3.0, 4.0, and 5.0 mg/mL. One milliliter of the CMPs solutions was mixed with 3 mL DPPH solutions (0.1 mM in ethanol). After incubation for 30 min at 30 C, the absorbance was recorded at 517 nm. Butyl hydroxyanisole (BHA) was used as the positive control. The DPPH scavenging activity was calculated as follows: DPPH scavenging activity (%) = 1 100 (3) where A is the absorbance of a mixture of DPPH and the CMP solution; A is the absorbance of the s 0 DPPH solution mixed with absolute ethanol. The results are also presented as an IC factor that represents the concentration of the sample that inhibits 50% of DPPH radicals. 2.6.2. Hydroxyl Radical Scavenging Activity The hydroxyl radicals scavenging assay was performed using a method described by Chen et al. [19] with a slight modification. CMPs solutions were prepared in deionized water to various concentrations (1.0, 2.0, 3.0, 4.0, and 5.0 mg/mL). Fifty microliters of CMPs solutions were firstly mixed with 50 L of 3 mM 1,10-phenanthroline in a 96 well microtiter plate. Then, 50 L of 3 mM FeSO and 50 L of 0.01% aqueous H O were added to each well. The reaction system was covered with aluminum foil 2 2 and incubated at 37 C for 1 h with shaking. The absorbance value was recorded at 536 nm. BHA was used as the positive control. The hydroxyl radical scavenging activity was estimated by the following equation: h i Hydroxyl radicals scavenging activity (%) = A A /(A A ) 100 (4) sample control blank control where A is the absorbance of the sample at 536 nm; A is the absorbance of the control that sample control contained a mixture of 1,10-phenanthroline, FeSO , and H O ; A is the absorbance of the blank 4 2 2 blank solution in the absence of H O . 2 2 2.6.3. Ferrous Chelating Activity Ferrous chelating activity was measured using a method described before [18]. CMPs solutions were prepared in deionized water to di erent concentrations (1.0, 2.0, 3.0, 4.0, and 5.0 mg/mL). Then, 0.1 mL FeCl (2 mM), 0.15 mL ferrozine (5 mM), and 0.55 mL methanol were mixed with the samples (1.0 mL). The mixture was vortexed and incubated at room temperature for 10 min. The absorbance was recorded at 562 nm. EDTA was used as the positive control. The ferrous chelating activity of CMPs was calculated as follows: Ferrous chelating activity (%) = 1 100 (5) where A is the absorbance of the CMPs; A is the absorbance of the positive control. s 0 2.6.4. Reducing Power A method based on that of Jing et al. [30] was modified to evaluate the reducing power of CMPs. CMPs solutions were prepared in deionized water to di erent concentrations (1.0, 2.0, 3.0, 4.0, and 5.0 mg/mL). Zero-point-one milliliters of K [Fe(CN) ] (1%, w/v, prepared in 20 mM PBS (pH 6.8)) 3 6 were added in 0.1 mL of sample solutions, followed by incubating at 50 C for 20 min. Then, 0.1 mL trichloroacetic acid (TCA, 10%, w/v) were added and vortexed. After centrifugation at 3000 g for 10 min, the supernatant (0.1 mL) was mixed with 0.1 mL of distilled water and 20 L of FeCl (0.1%, w/v). After incubation at the temperature for 30 min, the absorbance was recorded at 700 nm. BHA was used as the positive control. Antioxidants 2020, 9, 59 6 of 17 2.7. Alpha-Glucosidase Inhibition Assay A method based on that of Wang et al. [31] was modified to evaluate the -glucosidase inhibitory ability of CMPs. Alpha-glucosidase was prepared in PBS (0.1 M, pH 6.9) to a final concentration of 1 U/mL, and CMPs were prepared in deionized water to final concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg/mL. One hundred microliters of the Alpha-glucosidase solution were mixed with 50 L of the sample, and the mixture was then incubated at 25 C for 10 min in a 96 well plate. After that, 50 L of 4-nitrophenyl- -D-glucopyranoside (pNPG, 5 mM, prepared in 0.1 M PBS) were added, and the mixture was incubated again at 25 C for 5 min. The absorbance was determined at 405 nm before and after the last incubation. Acarbose was used as the positive control. The -glucosidase inhibitory activity can be expressed as follows: Inhibition (%) = 1 DA /DA 100 (6) sample control where A is the absorbance of the sample and A is that of the control. sample control 2.8. Statistical Analysis Statistical analysis was performed using SPSS software (Version 20.0, IBM SPSS, Armonk, NY, USA). All the experiments were carried out three times, and the data are expressed as the means the standard deviation. Their statistical significance of di erence was determined with Tukey’s multiple comparison test. A value of p < 0.05 was considered statistically significant. 3. Results and Discussion 3.1. Single Factor Assessment 3.1.1. E ect of the Ratio of Liquid-to-Solid on the Yield of CMP The e ect of the ratio of liquid-to-solid on the yield of CMPs is shown in Figure 1a. It was suggested that the yield of CMP increased significantly in the range of 20–40 mL/g and reached a peak of 8.28% at 40 mL/g. However, there was no obvious increase when the ratio of liquid-to-solid continued to rise above 40 mL/g. This meant that a higher ratio of raw material (>40 mL/g) resulted in no higher yield, indicating that the ratio of liquid-to-solid higher than 40 mL/g was not necessary. 3.1.2. E ect of Ultrasonic Power on the Yield of CMP The e ect of ultrasonic power on the yield of CMP is shown in Figure 1b. It was obvious that the yield increased with the increasing ultrasonic power from 120 W to 480 W and reached a maximum of 8.04% at 480 W. Ultrasonic power higher than 480 W caused the decrease of CMP yield which, may be due to the reason that higher ultrasonic power would result in degradation of polysaccharides [32]. Thus, the suitable ultrasonic power for the BBD design was from 360 to 600 W. 3.1.3. E ect of Extraction Time on the Yield of CMP The yield of CMP under di erent extraction time is shown in Figure 1c. An obvious increase of CMP yield was observed from 5 to 20 min. However, the yield of CMP exhibited a decreasing trend when the extraction time further increased. It was reported that longer extraction time in the ultrasonic extraction might induce the degradation of polysaccharides and decrease the yield. The results were in correspondence with those of Maran et al. [33] and Guo et al. [24], both of whom declared that 20 min of ultrasonic treatment was sucient enough for polysaccharides extraction. Therefore, 20 min of extraction time was chosen as the central point of the BBD design. Antioxidants 2020, 9, 59 7 of 17 Antioxidants 2020, 9, x FOR PEER REVIEW 7 of 18 (a) (b) (c) Figure Figure 1. 1. E Effe ect ct of three indep of three independent endent variabl variables es on the yield on the yield of CMP-U. of CMP-U. (a() aRatio of liqui ) Ratio of liquid-to-sol d-to-solid; id; (b) (bultrasonic po ) ultrasonic pow wer; and ( er; and c () c e ) extraction xtraction tim time. e. CMP-U CMP-U:: C C.. morifolium morifolium poly polysaccharides saccharides obtained obtained byby ultrasonic assisted extraction. ultrasonic assisted extraction. 3.2. Extraction and Optimization of CMP by RSM 3.2. Extraction and Optimization of CMP by RSM 3.2.1. Model Fitting 3.2.1. Model Fitting According to the single factor experiments, a total of seventeen runs of the BBD experiment was According to the single factor experiments, a total of seventeen runs of the BBD experiment was performed to optimize the UAE. Three independent variables including extraction time X , ultrasonic performed to optimize the UAE. Three independent variables including extraction time X1, ultrasonic power X , and the ratio of liquid-to-solid X were optimized, and the corresponding results are shown 2 3 power X2, and the ratio of liquid-to-solid X3 were optimized, and the corresponding results are shown in Table 1. The final equation obtained in terms of coded factors is described as below: in Table 1. The final equation obtained in terms of coded factors is described as below: 2 2 2 2 2 2 Y = 8.25 0.21X + 0.17X + 0.31X 0.22X X + 0.66X X + 0.72X X 0.39X 0.83X 1.31X (7) 2Y = 8.25 − 0.21X1 + 0.17X 2 + 0.31X − 3 0.22X X 1 + 2 0.66X X1 + 3 0.72X X 2 − 30.39X − 0.83X − 1.31X (7) 1 2 3 2 1 2 3 1 2 1 3 2 3 1 2 3 ANOVA is shown in Table 2. The model F-value (133.87) and p-value (<0.0001) indicated that ANOVA is shown in Table 2. The model F-value (133.87) and p-value (<0.0001) indicated that the model was significant. Furthermore, the adjusted determination coefficient value (Radj = 0.9868) the model was significant. Furthermore, the adjusted determination coecient value (R = 0.9868) adj also confirmed the high significance of the model. The determination coefficient (R ) was 0.9942, also confirmed the high significance of the model. The determination coecient (R ) was 0.9942, indicating that only 0.58% of the total variance was not explained by the model. In addition, the linear indicating that only 0.58% of the total variance was not explained by the model. In addition, the linear 2 2 coefficients (X1, X2, and X3), interaction terms (X1X2, X1X3, and X2X3) and quadratic terms (X1 , X2 , 2 2 coecients (X , X , and X ), interaction terms (X X , X X , and X X ) and quadratic terms (X , X , 1 2 3 1 2 1 3 2 3 1 2 and X3 ) were significant (p < 0.05), implying that these items could significantly affect the yield of and X ) were significant (p < 0.05), implying that these items could significantly a ect the yield of CMP. The p-value (0.2312) and F-value (2.20) of the lack of fit for the model demonstrated that it was CMP. The p-value (0.2312) and F-value (2.20) of the lack of fit for the model demonstrated that it was not significant relative to the pure error, which indicated that the model was credible. The low value not significant relative to the pure error, which indicated that the model was credible. The low value of of the coefficient of variation (CV, 1.68%) indicated the similarity between predicted and the coecient of variation (CV, 1.68%) indicated the similarity between predicted and experimental experimental values, suggesting that the model had a high degree of reliability. values, suggesting that the model had a high degree of reliability. Antioxidants 2020, 9, x FOR PEER REVIEW 8 of 18 Table 2. Analysis of variance (ANOVA) testing the fitness of the regression equation. Antioxidants 2020, 9, 59 8 of 17 Source Sum of Squares Df Mean Square F-Value p-Value Model 17.03 9 1.89 133.87 <0.0001 Table 2. Analysis of variance (ANOVA) testing the fitness of the regression equation. X1 0.34 1 0.34 24.07 0.0017 X2 0.22 1 0.22 15.40 0.0057 Source Sum of Squares Df Mean Square F-Value p-Value X3 0.78 1 0.78 54.81 0.0001 Model 17.03 9 1.89 133.87 <0.0001 X1X2 0.19 1 0.19 13.38 0.0081 X 0.34 1 0.34 24.07 0.0017 X X1X3 1. 0.22 77 11 0.221.77 15.40125.11 <0.00 0.005701 X 0.78 1 0.78 54.81 0.0001 3 X2X3 2.09 1 2.09 147.68 <0.0001 X X 0.19 1 0.19 13.38 0.0081 1 2 2 X1 0.63 1 0.63 44.71 0.0003 X X 1.77 1 1.77 125.11 <0.0001 1 3 X2 2.87 1 2.87 202.68 <0.0001 X X 2.09 1 2.09 147.68 <0.0001 2 3 X3 7.20 1 7.20 509.09 <0.0001 X 0.63 1 0.63 44.71 0.0003 Residual 0.099 7 0.014 X 2.87 1 2.87 202.68 <0.0001 X 7.20 1 7.20 509.09 <0.0001 3 Lack of fit 0.062 3 0.021 2.20 0.2312 Residual 0.099 7 0.014 −3 Pure error 0.037 4 9.350 × 10 Lack of fit 0.062 3 0.021 2.20 0.2312 Cor total 17.31 16 Pure error 0.037 4 9.350 10 R 0.9942 Cor total 17.31 16 Adjusted R 0.9868 R 0.9942 0.9868 Adjusted RCV % 1.68 CV % 1.68 3.2.2. Response Surface Analysis and Verification of the Model 3.2.2. Response Surface Analysis and Verification of the Model The 3D response surface and 2D contour plots revealed the interaction among the variables and The 3D response surface and 2D contour plots revealed the interaction among the variables and the response. As shown in Figure 2b, the contour plot was elliptical, suggesting that the mutual the inter response. actions be As tween ex showntr in act Figur ion teim 2e b, athe nd ul contour trasonic plot power were sign was elliptical, suggesting ificant. A simi that lathe r tre mutual nd was interactions between extraction time and ultrasonic power were significant. A similar trend was found for extraction time and the ratio of liquid-to-solid (Figure 2d) and ultrasonic power and the found ratio of for liextraction quid-to-solid time (Fi and gure the 2f). ratio Theof opt liquid-to-solid imum parame (Figur ters obt e 2a d) ine and d from ultrasonic the above exp power and erimen thet ratio of liquid-to-solid (Figure 2f). The optimum parameters obtained from the above experiment were as follows: extraction time 18.90 min, ultrasonic power 501.36 W, and ratio of liquid-to-solid wer 41.13 mL/g. e as follows: Under extraction the optim time ized con 18.90d min, ition, th ultrasonic e maximum power predicted 501.36 W y ,iand eld o ratio f CMP w of liquid-to-solid as 8.31%. The 41.13 mL/g. Under the optimized condition, the maximum predicted yield of CMP was 8.31%. The verification assays were conducted under the optimized conditions, and the actual extraction yield verification was 8.29 ± 0. assays 18%, wh wer ich e conducted was in corrunder espondence with the optimized the predic conditions, ted value. and the actual extraction yield was 8.29 0.18%, which was in correspondence with the predicted value. In the present work, HRE was conducted to evaluate the superiority of UAE in extracting polys Inaccha the pr ride from esent work, C. morifolium HRE was . CM conducted P obtainto ed by evaluate HRE the (7.25 superiority ± 0.10%, da oftaUAE not s in hown) extracting at the polysaccharide from C. morifolium. CMP obtained by HRE (7.25 0.10%, data not shown) at the optimized conditions was lower than that of UAE. Moreover, the UAE procedure took less time (19 optimized min) comconditi pared with ons was HRlower E (2.2 h than ). Pow thateof r consum UAE. Mor ptieover on of the , the UAE two m pre ocedur thods w e took as 0.less 57 kWh time (19 for UAE min) compared with HRE (2.2 h). Power consumption of the two methods was 0.57 kWh for UAE and 0.66 and 0.66 kWh for HRE. Therefore, UAE was a less time consuming and more efficient way to kWh extraction C for HRE. MTher Ps. efore, UAE was a less time consuming and more ecient way to extraction CMPs. (a) (b) Figure 2. Cont. Antioxidants 2020, 9, 59 9 of 17 Antioxidants 2020, 9, x FOR PEER REVIEW 9 of 18 Extraction yield (%) 6.97644 40 5 6.97644 15 17 19 21 23 25 Extraction time (min) (c) (d) Extraction yield (%) 6.97644 8 45 40 5 6.97644 480 30 35 420 360 420 480 540 600 Ratio of liquid to solid (mL/g) Ultrasounic power (W ) 30 360 Ultrasounic power (W) (e) (f) Figure Figure 2. 2. Re Response sponse su surface rface p plots lots (left (left) ) and and contour contour plots plots ( (right) right) showing showing the the interacti interactive ve effe e ects cts of of di erent variables on the yield of CMP-U. (a,b) Ultrasonic power and extraction time; (c,d) ratio of different variables on the yield of CMP-U. (a,b) Ultrasonic power and extraction time; (c,d) ratio of liquid-to-solid and extraction time; (e,f) ratio of liquid-to-solid and ultrasonic power. liquid-to-solid and extraction time; (e,f) ratio of liquid-to-solid and ultrasonic power. 3.3. Physicochemical Properties of CMPs 3.3. Physicochemical Properties of CMPs As shown in Table 3, the extraction yields of CMP-U (8.29 0.18%) were higher than those of As shown in Table 3, the extraction yields of CMP-U (8.29 ± 0.18%) were higher than those of CMP-H (7.25 0.10%) due to mechanical fluctuation and ultrasonic cavitation e ect [34]. Besides, CMP-H (7.25 ± 0.10%) due to mechanical fluctuation and ultrasonic cavitation effect [34]. Besides, CMP-U had a higher content of uronic acid than CMP-H, which was determined to be 7.08 0.25% CMP-U had a higher content of uronic acid than CMP-H, which was determined to be 7.08 ± 0.25% and 1.61 0.10%, respectively. It was reported that uronic acid is of great importance to the biological and 1.61 ± 0.10%, respectively. It was reported that uronic acid is of great importance to the biological activities of polysaccharides [35]. The protein content in CMP-U and CMP-H was 2.56 0.08% and activities of polysaccharides [35]. The protein content in CMP-U and CMP-H was 2.56 ± 0.08% and 3.36 0.09% according to the Bradford method. Therefore, in terms of extraction yield, UAE is a 3.36 ± 0.09% according to the Bradford method. Therefore, in terms of extraction yield, UAE is a method that used less power and had shorter time and lower temperature. He et al. [36] found that method that used less power and had shorter time and lower temperature. He et al. [36] found that Polyporus umbellatus polysaccharides with higher uronic acids content exhibited higher antioxidant Polyporus umbellatus polysaccharides with higher uronic acids content exhibited higher antioxidant activity. In another work, it was reported that the amount of uronic acids could influence the antioxidant activity. In another work, it was reported that the amount of uronic acids could influence the capacity and free radicals scavenging activity of Astragalus membranaceus polysaccharides [37]. antioxidant capacity and free radicals scavenging activity of Astragalus membranaceus polysaccharides [37]. Extraction yield (%) Ratio of liquid to solid (mL/g) Ratio of liquid to solid (mL/g) Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 18 Antioxidants 2020, 9, 59 10 of 17 Table 3. Physicochemical properties of C. morifolium polysaccharides obtained by UAE and HRE. Items CMP-U CMP-H Table 3. Physicochemical properties of C. morifolium polysaccharides obtained by UAE and HRE. a b Yield (%) 8.29 ± 0.18 7.25 ± 0.10 Items CMP-U CMP-H b a Protein content (%) 2.56 ± 0.08 3.36 ± 0.09 a b a b Yield (%) 8.29 0.18 7.25 0.10 Uronic acid (%) 7.08 ± 0.25 1.61 ± 0.10 b a Protein content (%) 3.36 0.09 2.56 0.08 Constituent monosaccharides and molar ratios a b Uronic acid (%) 7.08 0.25 1.61 0.10 Glucose 1 1 Constituent monosaccharides and molar ratios Fructose 0.011 0.007 Glucose 1 1 Rhamnose 0.040 0.052 Fructose 0.011 0.007 Galactose 0.065 0.095 Rhamnose 0.040 0.052 Xylose 0.113 0.214 Galactose 0.065 0.095 Arabinose 0.184 0.045 Xylose 0.113 0.214 CMP-H: C. morifolium poly Arabinose saccharides obtained by 0.184 hot reflux extractio 0.045 n; CMP-U: C. morifolium polysaccharides obtained by ultrasonic assisted extraction; different letters (a, b) in superscript for CMP-H: C. morifolium polysaccharides obtained by hot reflux extraction; CMP-U: C. morifolium polysaccharides a,b obtained by ultrasonic assisted extraction; di erent letters ( ) in superscript for each index denote a significant each index denote a significant difference (p < 0.05). di erence (p < 0.05). HPLC results indicated that both CMP-U and CMP-H contained galactose, rhamnose, glucose, and fructose, but in different proportions (shown in Table 3 and Figure 3). The monosaccharide HPLC results indicated that both CMP-U and CMP-H contained galactose, rhamnose, glucose, and composition showed that glucose (76.40% and 69.08, respectively) was the dominant sugar for CMP- fructose, but in di erent proportions (shown in Table 3 and Figure 3). The monosaccharide composition U and CMP-H. Moreover, the molar ratios of glucose, fructose, rhamnose, galactose, xylose, and showed that glucose (76.40% and 69.08, respectively) was the dominant sugar for CMP-U and CMP-H. arabinose in CMP-U were 1:0.011:0.04:0.065:0.113:0.184, and those in CMP-H were Moreover, the molar ratios of glucose, fructose, rhamnose, galactose, xylose, and arabinose in CMP-U 1:0.007:0.052:0.095:0.214:0.045. were 1:0.011:0.04:0.065:0.113:0.184, and those in CMP-H were 1:0.007:0.052:0.095:0.214:0.045. Figure 3. HPLC chromatogram of monosaccharides in CMPs. MD: mixed standard of monosaccharides; Figure 3. HPLC chromatogram of monosaccharides in CMPs. MD: mixed standard of 1: fructose; 2: rhamnose; 3: glucose; 4: galactose; 5: xylose; 6: arabinose; CMP-H: C. morifolium monosaccharides; 1: fructose; 2: rhamnose; 3: glucose; 4: galactose; 5: xylose; 6: arabinose; CMP-H: C. polysaccharides obtained by hot reflux extraction; CMP-U: C. morifolium polysaccharides obtained by morifolium polysaccharides obtained by hot reflux extraction; CMP-U: C. morifolium polysaccharides ultrasonic assisted extraction. obtained by ultrasonic assisted extraction. 3.4. Molecular Weight Distribution of CMPs 3.4. Molecular Weight Distribution of CMPs The molecular weights and monosaccharide composition of CMP samples were determined by HPGPCThe molecular weights and HPLC. As is shown an in d monosa Table 4 and ccha Figur ride compos e 4, both it CMP-H ion of CMP sampl and CMP-Ueshowed s were det thre ee rmi peaks ned by HPGPC and HPLC. As is shown in Table 4 and Figure 4, both CMP-H and CMP-U showed three in size exclusion chromatography. Moreover, the low molecular weight fractions in CMP-U (73.72%) wer peaks e higher in si than ze exclu those sion ch of CMP-H romato (62.56%). graphy. Moreove This could r, thbe e low mo attributed lecuto lar we the degradation ight fractionsof in C CMPs MP-U (73.72%) were higher than those of CMP-H (62.56%). This could be attributed to the degradation of caused by ultrasonic power, which was correspondent with previous literature [24,38]. CMPs caused by ultrasonic power, which was correspondent with previous literature [24,38]. Antioxidants 2020, 9, 59 11 of 17 Antioxidants 2020, 9, x FOR PEER REVIEW 11 of 18 (a) (b) Figure 4. Gel permeation chromatography (GPC) spectrums of C. morifolium polysaccharides. (a) Figure 4. Gel permeation chromatography (GPC) spectrums of C. morifolium polysaccharides. CMP-H; (b) CMP-U. CMP-H: C. morifolium polysaccharides obtained by hot reflux extraction; CMP- (a) CMP-H; (b) CMP-U. CMP-H: C. morifolium polysaccharides obtained by hot reflux extraction; U: C. morifolium polysaccharides obtained by ultrasonic assisted extraction. CMP-U: C. morifolium polysaccharides obtained by ultrasonic assisted extraction. Table Table 4. 4. Molecular Molecular weight di weight distribution stribution of C of CMP-U MP-U and and CM CMP-H. P-H. Molecular Weight Distribution Molecular Weight Distribution Polysaccharides Retention Time (min) Mw (Da) Mn (Da) Area % Polysaccharides Retention Time (min) Mw (Da) Mn (Da) Area % 13.987 669,143 153,373 26.27 13.987 669,143 153,373 26.27 CMP-U 18.492 2115 1231 36.21 18.492 2115 1231 36.21 CMP-U 20.205 214 179 37.51 20.205 214 179 37.51 14.350 521,905 108,418 37.44 CMP-H 14.35018.557 2628 521,905 108,4181464 28.74 37.44 CMP-H 18.557 2628 1464 28.74 20.058 321 225 33.82 20.058 321 225 33.82 CMP-H: C. morifolium polysaccharides obtained by hot reflux extraction; CMP-U: C. morifolium CMP-H: C. morifolium polysaccharides obtained by hot reflux extraction; CMP-U: C. morifolium polysaccharides polysaccharides obtained by ultrasonic assisted extraction. obtained by ultrasonic assisted extraction. 3.5. FT-IR Analysis 3.5. FT-IR Analysis The FT-IR spectra of CMP-U and CMP-H are shown in Figure 5. No significant difference was −1 −1 observed between the spectra of the two CMPs. The peaks at ~3400 cm and 2930 cm were the The FT-IR spectra of CMP-U and CMP-H are shown in Figure 5. No significant di erence was 1 1 stretching vibration of the hydroxyl group and C–H bond, respectively [39]. The absorption band observed between the spectra of the two CMPs. The peaks at ~3400 cm and 2930 cm were the −1 −1 near 1740 cm was the stretching vibration of C=O [40]. The absorption peaks near 1600–1622 cm stretching vibration of the hydroxyl group and C–H bond, respectively [39]. The absorption band was the stretching vibration of the carboxylate anion, indicating the presence of uronic acids [24]. The 1 1 near 1740 cm was the stretching vibration of C=O [40]. The absorption peaks near 1600–1622 cm −1 absorption in the range 1244–1000 cm could be attributed to the stretching vibration of C–O–C or was the stretching vibration of the carboxylate anion, indicating the presence of uronic acids [24]. C–OH bonds of a pyranose ring, which comprises the characteristic absorbance of polysaccharides Antioxidants 2020, 9, x FOR PEER REVIEW 12 of 18 The absorption in the range 1244–1000 cm could be attributed to the stretching vibration of C–O–C or [31]. C–OH bonds of a pyranose ring, which comprises the characteristic absorbance of polysaccharides [31]. Figure 5. Fourier transform infrared (FT-IR) spectra of CMP-H and CMP-U. CMP-H: C. morifolium Figure 5. Fourier transform infrared (FT-IR) spectra of CMP-H and CMP-U. CMP-H: C. morifolium polysaccharides obtained by hot reflux extraction; CMP-U: C. morifolium polysaccharides obtained by polysaccharides obtained by hot reflux extraction; CMP-U: C. morifolium polysaccharides obtained by ultrasonic assisted extraction. ultrasonic assisted extraction. 3.6. Antioxidant Activity Assay of CMPs by Different Extraction Methods 3.6.1. Scavenging Activity of DPPH Radicals DPPH free radical scavenging activity has been widely used as an important index to evaluate the antioxidant activity of natural products [41]. As shown in Figure 6a, both CMP-U and CMP-H exhibited scavenging activity of the DPPH free radical in a dose-dependent manner. Moreover, the DPPH scavenging ability of CMP-U was higher than that of CMP-H at different concentrations. It was suggested that UAE may be an effective method to obtain CMPs with excellent antioxidant activity. Furthermore, the IC50 values of DPPH free radical scavenging activity for CMP-U and CMP- H were 1.850 mg/mL and 3.587 mg/mL, which were higher and hence worse than that of BHA (0.047 mg/mL). (a) (b) Antioxidants 2020, 9, x FOR PEER REVIEW 12 of 18 Figure 5. Fourier transform infrared (FT-IR) spectra of CMP-H and CMP-U. CMP-H: C. morifolium polysaccharides obtained by hot reflux extraction; CMP-U: C. morifolium polysaccharides obtained by ultrasonic assisted extraction. Antioxidants 2020, 9, 59 12 of 17 3.6. Antioxidant Activity Assay of CMPs by Different Extraction Methods 3.6. Antioxidant Activity Assay of CMPs by Di erent Extraction Methods 3.6.1. Scavenging Activity of DPPH Radicals DPPH free radical scavenging activity has been widely used as an important index to evaluate 3.6.1. Scavenging Activity of DPPH Radicals the antioxidant activity of natural products [41]. As shown in Figure 6a, both CMP-U and CMP-H DPPH free radical scavenging activity has been widely used as an important index to evaluate exhibited scavenging activity of the DPPH free radical in a dose-dependent manner. Moreover, the the antioxidant activity of natural products [41]. As shown in Figure 6a, both CMP-U and CMP-H DPPH scavenging ability of CMP-U was higher than that of CMP-H at different concentrations. It exhibited scavenging activity of the DPPH free radical in a dose-dependent manner. Moreover, the was suggested that UAE may be an effective method to obtain CMPs with excellent antioxidant DPPH scavenging ability of CMP-U was higher than that of CMP-H at di erent concentrations. It was activity. Furthermore, the IC50 values of DPPH free radical scavenging activity for CMP-U and CMP- suggested that UAE may be an e ective method to obtain CMPs with excellent antioxidant activity. H were 1.850 mg/mL and 3.587 mg/mL, which were higher and hence worse than that of BHA (0.047 Furthermore, the IC values of DPPH free radical scavenging activity for CMP-U and CMP-H were mg/mL). 1.850 mg/mL and 3.587 mg/mL, which were higher and hence worse than that of BHA (0.047 mg/mL). Antioxidants 2020, 9, x FOR PEER REVIEW 13 of 18 (a) (b) (c) (d) Figure 6. Antioxidant activities of CMP-H and CMP-U. (a) DPPH free radical scavenging activity; (b) Figure 6. Antioxidant activities of CMP-H and CMP-U. (a) DPPH free radical scavenging activity; (b) hydroxyl radical scavenging activity; (c) ferrous chelating activity, and (d) total reducing power. CMP-H: hydroxyl radical scavenging activity; (c) ferrous chelating activity, and (d) total reducing power. C. morifolium polysaccharides obtained by hot reflux extraction; CMP-U: C. morifolium polysaccharides CMP-H: C. morifolium polysaccharides obtained by hot reflux extraction; CMP-U: C. morifolium obtained by ultrasonic assisted extraction. polysaccharides obtained by ultrasonic assisted extraction. 3.6.2. Scavenging of Hydroxyl Radicals 3.6.2. Scavenging of Hydroxyl Radicals The scavenging activities of hydroxyl radical play a significant role in protecting living cells The scavenging activities of hydroxyl radical play a significant role in protecting living cells due due to the bad e ects of the free radicals such as causing cancer, and damaging DNA or proteins [3]. to the bad effects of the free radicals such as causing cancer, and damaging DNA or proteins [3]. The The scavenging activities of CMP-U and CMP-H are shown in Figure 6b. Similar to DPPH scavenging scavenging activities of CMP-U and CMP-H are shown in Figure 6b. Similar to DPPH scavenging activity, the hydroxyl radical scavenging ability of CMPs also exhibited a good concentration dependent activity, the hydroxyl radical scavenging ability of CMPs also exhibited a good concentration manner. It was obvious that CMP-U possessed higher hydroxyl radical scavenging activity than dependent manner. It was obvious that CMP-U possessed higher hydroxyl radical scavenging CMP-H. Moreover, the hydroxyl radical scavenging activity of CMP-U and CMP-H reached a peak activity than CMP-H. Moreover, the hydroxyl radical scavenging activity of CMP-U and CMP-H value of 66.77% and 54.46% at 5.0 mg/mL. The IC values of hydroxyl radical scavenging activity reached a peak value of 66.77% and 54.46% at 5.0 mg/mL. The IC50 values of hydroxyl radical for CMP-U and CMP-H were 2.612 mg/mL and 4.236 mg/mL, respectively. It was proposed that the scavenging activity for CMP-U and CMP-H were 2.612 mg/mL and 4.236 mg/mL, respectively. It was hydrogen or electron abstraction mechanism might be attributed to the hydroxyl radical inhibition of proposed that the hydrogen or electron abstraction mechanism might be attributed to the hydroxyl radical inhibition of polysaccharides [42]. According to Fan et al. [43], stronger hydroxyl radical inhibition activity might be related to higher contents of uronic acids. Based on this report, a higher content of uronic acids in CMP-U contributed to its higher hydroxyl radical scavenging activity. 3.6.3. Ferrous Chelating Activity The ferrous chelating activity of CMPs is presented in Figure 6c. Both polysaccharides displayed ferrous chelating activity in a concentration dependent manner, and CMP-U showed greater ferrous chelating activity. The IC50 values of ferrous chelating activity for CMP-U and CMP-H were 2.523 mg/mL and 3.982 mg/mL. At the concentration of 5.0 mg/mL, the ferrous chelating activity of CMP- U and CMP-H reached the peak of 63.47% and 52.29%, respectively. It was reported that the observed chelating activity of polysaccharides was likely related to the content of galactose in the polysaccharide. It is interesting to note that CMP-U possessed higher galactose content than CMP-H, and this observation was consistent with previous reports [18,44]. 3.6.4. Total Reducing Power The antioxidant can reduce the reactive groups to more stable species by donating electrons to them [3]. The total reducing power of an antioxidant compound essentially indicates whether it is a good electron donor and is related to its antioxidant activity [45]. In this study, greater ultraviolet absorption was indicative of better reducing power [46]. As can be seen from Figure 6d, the total reducing power increased in a dose dependent manner according to CMP concentration. Moreover, the reducing power of CMP-U was higher than that of CMP-H. Reducing power was reported to be associated with reductones, which were proposed to react with precursors of peroxide [47]. 3.7. Alpha-Glucosidase Inhibitory Activities Antioxidants 2020, 9, 59 13 of 17 polysaccharides [42]. According to Fan et al. [43], stronger hydroxyl radical inhibition activity might be related to higher contents of uronic acids. Based on this report, a higher content of uronic acids in CMP-U contributed to its higher hydroxyl radical scavenging activity. 3.6.3. Ferrous Chelating Activity The ferrous chelating activity of CMPs is presented in Figure 6c. Both polysaccharides displayed ferrous chelating activity in a concentration dependent manner, and CMP-U showed greater ferrous chelating activity. The IC values of ferrous chelating activity for CMP-U and CMP-H were 2.523 mg/mL and 3.982 mg/mL. At the concentration of 5.0 mg/mL, the ferrous chelating activity of CMP-U and CMP-H reached the peak of 63.47% and 52.29%, respectively. It was reported that the observed chelating activity of polysaccharides was likely related to the content of galactose in the polysaccharide. It is interesting to note that CMP-U possessed higher galactose content than CMP-H, and this observation was consistent with previous reports [18,44]. 3.6.4. Total Reducing Power The antioxidant can reduce the reactive groups to more stable species by donating electrons to them [3]. The total reducing power of an antioxidant compound essentially indicates whether it is a good electron donor and is related to its antioxidant activity [45]. In this study, greater ultraviolet absorption was indicative of better reducing power [46]. As can be seen from Figure 6d, the total reducing power increased in a dose dependent manner according to CMP concentration. Moreover, the reducing power of CMP-U was higher than that of CMP-H. Reducing power was reported to be associated with reductones, which were proposed to react with precursors of peroxide [47]. 3.7. Alpha-Glucosidase Inhibitory Activities Alpha-glucosidase is a key enzyme associated with the digestion of carbohydrates in the small intestine, and the inhibition of -glucosidase can delay the breakdown of starch, keeping the blood glucose at low levels [48]. Therefore, -glucosidase inhibitors are key factors for the treatment of type II diabetes. As shown in Figure 7, both CMP-U and CMP-H exhibited -glucosidase inhibitory activity in a concentration dependent manner. The IC values of CMP-U and CMP-H were 3.606 and 4.854 mg/mL, higher than that of acarbose (0.01 mg/mL). It was suggested that CMP-U had better -glucosidase inhibition activity than CMP-H when the concentration was higher than 1.0 mg/mL (p < 0.05), showing a similar result to those from antioxidant assays. The observation corresponded well with previous studies in which positive correlations were found between antioxidant activities of polysaccharides from oolong tea and their -glucosidase inhibition activities [31,49]. The discovery of natural anti-diabetic agents is becoming more and more popular due to the side e ects of synthetic anti-diabetic drugs. Polysaccharides extracted from several plants have been reported to exhibit -glucosidase or -amylase inhibition activity [18,31,48]. However, there has been no report on -glucosidase inhibition activity of C. morifolium polysaccharides to the best of our knowledge. Therefore, the findings in our study indicate that UAE is an ecient way to obtain CMPs exhibiting inhibitory potential against -glucosidase. In addition, CMPs may be used as functional food additives that are beneficial for diabetic patients. Antioxidants 2020, 9, x FOR PEER REVIEW 14 of 18 Alpha-glucosidase is a key enzyme associated with the digestion of carbohydrates in the small intestine, and the inhibition of α-glucosidase can delay the breakdown of starch, keeping the blood glucose at low levels [48]. Therefore, α-glucosidase inhibitors are key factors for the treatment of type II diabetes. As shown in Figure 7, both CMP-U and CMP-H exhibited α-glucosidase inhibitory activity in a concentration dependent manner. The IC50 values of CMP-U and CMP-H were 3.606 and 4.854 mg/mL, higher than that of acarbose (0.01 mg/mL). It was suggested that CMP-U had better α- glucosidase inhibition activity than CMP-H when the concentration was higher than 1.0 mg/mL (p < 0.05), showing a similar result to those from antioxidant assays. The observation corresponded well with previous studies in which positive correlations were found between antioxidant activities of polysaccharides from oolong tea and their α-glucosidase inhibition activities [31,49]. The discovery of natural anti-diabetic agents is becoming more and more popular due to the side effects of synthetic anti-diabetic drugs. Polysaccharides extracted from several plants have been reported to exhibit α- glucosidase or α-amylase inhibition activity [18,31,48]. However, there has been no report on α- glucosidase inhibition activity of C. morifolium polysaccharides to the best of our knowledge. Therefore, the findings in our study indicate that UAE is an efficient way to obtain CMPs exhibiting inhibitory potential against α-glucosidase. In addition, CMPs may be used as functional food Antioxidants 2020, 9, 59 14 of 17 additives that are beneficial for diabetic patients. Figure 7. -glucosidase inhibitory e ect of CMP-U and CMP-H. CMP-H: C. morifolium polysaccharides Figure 7. α-glucosidase inhibitory effect of CMP-U and CMP-H. CMP-H: C. morifolium obtained by hot reflux extraction; CMP-U: C. morifolium polysaccharides obtained by ultrasonic polysaccharides obtained by hot reflux extraction; CMP-U: C. morifolium polysaccharides obtained by assisted extraction. ultrasonic assisted extraction. 3.8. Relationship between Physicochemical Properties of CMPs and Their Antioxidant Activity or 3.8. Relationship between Physicochemical Properties of CMPs and Their Antioxidant Activity or α- -Glucosidase Inhibition Activities Glucosidase Inhibition Activities According to previous literature, the antioxidant activity of polysaccharides was a ected by a According to previous literature, the antioxidant activity of polysaccharides was affected by a combination of many factors, including molecular weight, structure, monosaccharide composition, combination of many factors, including molecular weight, structure, monosaccharide composition, and conformation [50]. In our present work, it was suggested that CMP-U with lower protein content and conformation [50]. In our present work, it was suggested that CMP-U with lower protein content and higher uronic acid content exhibited stronger scavenging activity of DPPH radicals and hydroxyl and higher uronic acid content exhibited stronger scavenging activity of DPPH radicals and hydroxyl radicals, ferrous chelating activity, reducing power, and -glucosidase inhibitory activity. The result radicals, ferrous chelating activity, reducing power, and α-glucosidase inhibitory activity. The result was consistent with previous reports. Uronic acids were considered to play a significant role in the was consistent with previous reports. Uronic acids were considered to play a significant role in the antioxidant activity of polysaccharides; a higher content of uronic acids in polysaccharides tended to antioxidant activity of polysaccharides; a higher content of uronic acids in polysaccharides tended to exhibit stronger antioxidant activity [36]. Moreover, the number of low molecular weight fractions exhibit stronger antioxidant activity [36]. Moreover, the number of low molecular weight fractions of of CMP-U was higher than that of CMP-H, contributing to the stronger antioxidant activity and CMP-U was higher than that of CMP-H, contributing to the stronger antioxidant activity and α- -glucosidase inhibitory activity of CMP-U. Dong et al. [51] reported that the higher the number of low glucosidase inhibitory activity of CMP-U. Dong et al. [51] reported that the higher the number of low molecular weight fractions, the higher the antioxidant activity of polysaccharides. However, the exact molecular weight fractions, the higher the antioxidant activity of polysaccharides. However, the exact mechanism by which these physicochemical properties a ect the antioxidant activity or -glucosidase inhibition activity is unclear, and it will be deeply investigated in our further work. 4. Conclusions In our present work, ultrasonic technology was used to extract polysaccharides from C. morifolium. HRE was employed as a control to evaluate the eciency of UAE. The best conditions of UAE optimized by response surface methodology were ultrasonic power 501 W, extraction time 19 min, and ratio of liquid-to-raw material 41 mL/g. Under these conditions, the yield of CMP-U was 8.29 0.18%. Compared to HRE, the extraction yield of UAE was increased, the extraction time was greatly shortened, and the power consumption was lower. Investigation of physicochemical properties indicated that polysaccharides extracted by UAE had lower content of protein and higher content of low molecular weight fractions and uronic acids. Meanwhile, CMPs by the two methods showed similar monosaccharide composition and Fourier transform infrared (FT-IR) spectra. Moreover, polysaccharides extracted by UAE exhibited higher antioxidant and -glucosidase inhibition activity. All these results indicated that UAE was an ecient way to obtain C. morifolium polysaccharides with high antioxidant and -glucosidase inhibitory activity. In addition, CMPs could be used as natural food additives with antioxidant and -glucosidase inhibition activities in the food industry. Antioxidants 2020, 9, 59 15 of 17 Author Contributions: Data curation, G.J. and M.L.; formal analysis, X.H. (Xia Huang), J.L., Q.L., and M.Y.; investigation, X.L.; project administration, Z.Z.; supervision, G.S. and A.C.; writing, original draft, X.H. (Xiaoyan Hou); writing, review and editing, S.L. and H.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Scientific Fund of the Application Fundamental Project (2016JY0118) and the Key Research & Development Project (8ZDYF1175). Conflicts of Interest: The authors declare no conflict of interest. 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Journal
Antioxidants – Multidisciplinary Digital Publishing Institute
Published: Jan 9, 2020