Chemical structure and inhibition on α-glucosidase of the polysaccharides from Cordyceps militaris with different developmental stages

International Journal of Biological Macromolecules 148 (2020) 722–736

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

Chemical structure and inhibition on α-glucosidase of the polysaccharides from Cordyceps militaris with different developmental stages Lingran Wu a,b,c,1, Huiqing Sun a,b,c,1, Yali Hao a,b,c, Xiaomin Zheng a,b,c, Qiaoying Song a,b,c, Shuhan Dai a,b,c, Zhenyuan Zhu a,b,c,⁎ a b c

State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin 300457, PR China Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, PR China College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China

a r t i c l e

i n f o

Article history: Received 23 November 2019 Received in revised form 24 December 2019 Accepted 19 January 2020 Available online 20 January 2020 Keywords: Cordyceps militaris Polysaccharide Structure analysis

a b s t r a c t The natural form of wild edible fungus is the fruiting body. The cultivation of fruiting bodies from sexual reproduction requires strict conditions and long periods. Some literatures have paid attentions on the mycelia prepared with liquid fermentation to alter fruiting bodies. Cordyceps militaris (C. militaris) is a kind of precious edible fungus. The polysaccharide is an important active ingredient in C. militaris. The manuscript aimed to evaluate the feasibility of alternative of mycelia to fruit bodies with studies of polysaccharides from C. militaris of different developmental stages. The two polysaccharides were separated. The chemical structure and inhibitory activity on α-glucosidase of polysaccharides were explored. The results indicated that the structure and inhibitory activity on α-glucosidase of polysaccharides with different developmental stages had significant differences. The polysaccharides from fruiting bodies had better inhibitory activity on α-glucosidase. It demonstrated that the mycelia of C. militaris from asexual reproduction with liquid fermentation can't be an effective substitute for fruiting bodies from sexual reproduction, from the perspective of polysaccharides. © 2020 Elsevier B.V. All rights reserved.

1. Introduction The wild edible fungus has the functions of antimicrobial, antioxidant properties, anti-tyrosinase and hyperglycaemic inhibitory activities [1,2]. The natural form of wild edible fungus is the fruiting body developed from sexual reproduction. The sexual reproduction of the wild edible fungus needs strict growth conditions, growing season, long development period and special ecological environment. With the utilization, the resources of wild edible fungus have decreased sharply, which can't satisfy the daily needs of human. So the more attentions have been paid on the other form of edible fungus as alternatives of wild edible fungus. In addition to sexual reproduction, asexual reproduction is another mode of reproduction of edible fungi. Taking Cordyceps militaris (C. militaris) as an example, in Fig. 1, asexual reproduction occurred in the generational cycle, the C. militaris produce conidia and the conidia developed to form mycelia. In the stage of sexual

⁎ Corresponding author at: State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin, 300457, PR China. E-mail address: [email protected] (Z. Zhu). 1 The first two authors contributed to the work equally and should be regarded as cofirst authors.

https://doi.org/10.1016/j.ijbiomac.2020.01.178 0141-8130/© 2020 Elsevier B.V. All rights reserved.

reproduction, the mycelium of C. militaris invaded the body of insect, and the tissue and organs in the insect body were used as the material and energy source for growth and development. Therefore some researches have paid attentions on the mycelia prepared with liquid fermentation to alter fruiting bodies [3,4]. C. militaris is a kind of precious edible fungus. C. militaris contains large numbers of biologically active ingredients, such as polysaccharides, amino acids, adenosine, inorganic elements, vitamins and other active substances, and has various biologically activities of hypotension, antitumor, antioxidative, antibacterial, anti-inflammatory, lung protection, kidney benefit, hemostasis, anti-fatigue, anticancer [5–9]. Many studies also showed that the active substances and their pharmacological effects contained in C. militaris were close to or even higher than those of Cordyceps sinensis. The polysaccharide is an important active ingredient in C. militaris. In recent years, there have been more and more reports about the polysaccharide structure and activities of C. militaris. Two polysaccharides, with different molecular weights and composed of D-Man, D-Glc, D-Gal with the molar ratios were purified from C. militaris by Luo et al. [10]. A polysaccharide, mainly composed of LRha, L-Ara, D-Man and D-Gal, was obtained from the C. militaris reported by Dong et al. [11]. Chen et al. extracted the polysaccharides from the fruiting bodies of C. militaris with antioxidant activity to

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Fig. 1. Life-history of the Cordyceps militaris.

scavenge the stable freeradical 1 and DPPH [12]. Wang et al. found that the C. militaris polysaccharides (CMP) had immunomodulatory activity to enhance the spleen lymphocyte activity and macrophage function and [13]. The manuscript aimed to evaluate the feasibility of alternative of mycelia with fruit bodies with studies of polysaccharides from C. militaris with different developmental stages (fruiting bodies from sexual reproduction and mycelia from asexual reproduction with liquid fermentation). In order to get closer to its wild growth environment, the cultivated-type C. militaris grown on silkworm chrysalis medium was selected in manuscript. To increase the accuracy and reliability of the experiment, the manuscript first isolated the strain of C. militaris from fruiting body. The mycelia were prepared with liquid fermentation and then the fruiting body and mycelia were dried and ground into powder. The polysaccharides were separated from fruiting body and mycelia, respectively. The structural properties (in primary structure, advanced structure, physical properties and appearance shape), inhibitory activity on α-glucosidase and Kinetics of α-glucosidase inhibition of polysaccharide were studied by chemical methods and instrumental analysis. 2. Materials and methods 2.1. Experimental materials C. militaris fruiting bodies, cultured on silkworm chrysalis medium, were obtained from the Shanxi Xitamei Health Management Co., Ltd. (Shanxi, China). The Sephadex G-150, Sephadex G-200, p-nitrophenylα-D-glucopyranoside (pNPG), acarbose and standards of monosaccharides, such as D-Glucose (D-Glc), D-Xylose (D-Xyl), D-Galactose (DGal), L-Rhamnose (L-Rha), D-Mannose (D-Man), L-Arabinose (L-Ara),

were bought from Sigma Chemical Co. (USA). The Dextran standards were bought from Pharmacia Biotech (Uppsala, Sweden). The reagents used in HPGPC and GC–MS were chromate-graphically grade. Other reagents used in manuscript were AR. 2.2. Isolation of C. militaris strain, microscopic identification and liquid fermentation 2.2.1 isolation of C. militaris strain The surface of the strain of the C. militaris fruiting body was washed with water, disinfected with 75% alcohol three times, and then scrubbed in sterile water in a UV-sterilized ultra-clean workbench. The fruiting body was cut into two parts and the pieces with 0.5 cm2 were inoculated into PDA medium. The grown mycelia was purified and cultured for regeneration in PDA culture. The culture conditions were 25 °C for 7 days. After rejuvenation, the strain was obtained and the microstructure of the mycelia was observed under a microscope [14]. 2.2.1. Liquid fermentation of C. militaris The mycelia were obtained with liquid submerged fermentation. The experiments were performed according to literature with some modifications [15]. The strain was cultivated in a 250 mL Erlenmeyer flask containing 100 mL of seed culture medium contained (% w/v) glucose 2, peptone 1.0, KH2PO4 0.1, and MgSO4·7H2O 0.1 for 3 days at 25 °C, 150 rpm, nature pH. The harvested seeds were added into 250 mL Erlenmeyer flasks containing 100 mL liquid fermentation medium composed of (% w/v) glucose 4, peptone 1.0, KH2PO4 0.05, and MgSO4·7H2O 0.05. The inoculate rate was 10% (v/v). The mycelia were harvested after incubated at 25 °C on a rotary shaker maintained at 150 rpm for 5 days. The culture broth was filtrated. The mycelia were washed, freezedried and grinded. So do the fruiting bodies.

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2.3. Preparation of polysaccharides from C. militaris 2.3.1. Extraction of crude polysaccharides The fruiting bodies and mycelia were extracted by using distilled water (1:30). The temperature and time were 80 °C and 3 h, respectively. The extraction was performed three times. The above supernatant was combined, evaporated at 60 °C. The extract was precipitated with 80% ethanol [16], deproteinizated with the Sevag and lyophilizated to get the crude polysaccharides [17]. The crude polysaccharides from fruiting bodies and mycelia were named as CBPS-RI and CMPS-RI, respectively. 2.3.2. Purification of polysaccharides The dialysis bag with molecular weight cut off of 3500 D was utilized to remove impurities such as small molecule, oligosaccharides and salts in CBPS-RI and CMPS-RI. Then the CBPS-RI and CMPS-RI were processed by using Sephadex G-150 and Sephadex G-200, respectively. The content of sugar was detected with phenol-sulfuric acid. The tubes of main peaks were collected and freeze-dried to prepare pure polysaccharides. The pure polysaccharides from CBPS-RI and CMPS-RI were named as CBPS-RII and CMPS-RII, respectively. The UV–visible spectralscanning method was applied to detect the purity of the CBPS-RII and CMPS-RII. 2.4. Determination of molecular weight distribution The HPGPC (Agilent-1200) was utilized to detect the molecular weight (Mw) of CBPS-RII and CMPS-RII. The HPGPC was equipped with a TSK gel G4000 PWxl column (7.8 mm × 300 mm, column temperature 30 °C) and Refractive Index Detector (RID, detecting temperature 35 °C). The CBPS-RII and CMPS-RII were accurately weighed, dissolved in distilled water to 2 mg/mL solution, filtered with filter (0.22 mm) and measured according to literature [18]. The T-series Dextrans were used to make standard curve [15]. 2.5. Structural characterization 2.5.1. FT-IR analysis The sample of 1.00 mg and dried KBr of 150.00 mg were accurately weighed and mixed. The FT-IR analysis was performed with FT-IR spectrophotometer (Perkin Elmer Corp., USA) according to literature [19]. 2.5.2. NMR analyses The freeze-dried sample (25 mg) was dissolved with D2O of 500 μL. Put the sample in nuclear magnetic tube. The 1H NMR, 13C NMR and HSQC spectra were recorded on a Bruker spectrometer referred to Yun et al. [20]. 2.5.3. Monosaccharide composition analysis The experiment was referred to literatures [21,22]. The sample was hydrolyzed with TFA. The products were reduced with NaBH4. Then the solution was desalted and the pyridine-Ac2O was used to acetylate the sample. Ultimately, the product was measured by GC–MS method of Ge [23]. The D-Glc, D-Xyl, D-Gal, L-Rha, D-Man, L-Ara were also derived with above method for standards. 2.5.4. Oxidation of periodic acid and smith degradation The oxidation of periodic acid was performed based on the methods of Tang [24]. NaIO4 of 0.03 M was used to oxidize the sample (25 mg). The experiment was performed in the dark at 4 °C. Every 8 h, 0.1 mL of solution was diluted to 25 mL with distilled water and detected by UV spectrophotometer at 223 nm. When the absorbance stabled, the reaction was quenched with ethylene glycol (0.2 mL). The consumption of NaIO4 was calculated with the absorbance. And the production of formic acid was determined by titration with 0.01 M NaOH. The reaction mixture was dialyzed for 36 h and reduced with NaBH4 (30 mg) for 12 h in

dark. Adjust the pH of reaction mixture to 5.0–6.0. The reaction mixture was dialyzed and lyophilized. The sample was hydrolyzed and acetylated according literatures [21,25,26]. The product was measured by GC–MS method of Ge [23] The six monosaccharide standards, erythritol and glycerin were also derived for standards. 2.5.5. Methylation analysis Before methylated, the TAPS-II was reacted with 1-(3Dimethyaminopropyl)-3-ethylcarbodiimide Hydrochloride to reduce the carboxyl accordance with the literature [27]. Then the products were performed with methylation analysis by referring Needs and Selvendran [28]. The degree of methylation was detected by FT-IR [29]. After the methylation, the sample was used to hydrolyzation and acetylation. The experiments were performed with literatures of Sun et al. [30]. Then the product was dissolved in chloroform and analyzed with GC–MS (VARIAN, USA). The search library was NIST05. 2.5.6. Congo red test The Congo-red test analysis of sample was referenced to Liu et al. [31]. 0.5 mg/mL of the polysaccharide samples were mixed with the same volume of 50 mmol/L Congo red solution, and then 1 mol/L NaOH was added to the mixed solutions until the final concentration of NaOH solution was 0–0.4 mol/L. After standing for 10 min, the maximum absorption wavelength was recorded by the UV–visible spectrometer in wavelength range of 400–600 nm. 2.5.7. The optical rotation identification The sample (1 mg/mL) was measured at 25 °C with WZZ-2B and calculation accordance with [32]. 2.5.8. Analysis of differential scanning Calorimetry (DSC) The DSC analyses of sample were referred to literature [33]. 2.5.9. SEM analysis The sample was uniformly attached to the sample stage, placed in an ion sputtering apparatus for vacuum gold plating, then scanned under high vacuum conditions by using scanning electron microscope (SU1510, Hitachi) [34]. 2.6. Inhibition on α-glucosidase in vitro The experiment of inhibition α-glucosidase was performed according to the method of Ren [21]. The enzyme and samples were dissolved in 0.1 M sodium phosphate buffer (pH 7.0) to obtain the desired concentration. The sample of 400 μL and α-glucosidase of 400 μL were mixed. The mixture was incubated at 37 °C for 10 min. The 15 mmol/L PNPG of 100 μL was added. The mixture was incubated at 37 °C for 30 min. The reaction was ended with adding 1.5 mL of 1 mol/L NaOH. The OD value was detected at 410 nm. The acarbose was used as the positive control. The reaction system without samples was blank and the system without α-glucosidase served as control. The inhibitory rate of sample on α-glucosidase was calculated by the following formula. φ ¼ ða−bÞ  100=ða−cÞ where the φ was the inhibitory rate; a, b and c were the OD of control group, sample group, blank group, respectively. 2.7. Kinetics of α-glucosidase inhibition The substrate, pNPG at concentrations of 0.47, 0.94, 1.88, 3.75 mmol/ L, was added into mixtures of polysaccharides solution and αglucosidase, as describe in the procedure for the α-glucosidase inhibition assay [35]. The absorbances were measured at 400 nm as described in previously. The 1/S-1/V double reciprocal curve was plotted

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Fig. 2. Mycelia (a) and microscopic appearance of the mycelia of Cordyceps militaris (b).

Fig. 3. Average molecular weight distribution of CBPS-RI (a) and CMPS-RI (b).

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according to the method of Lineweave-Burk to determine the type of polykinase kinetic reaction.

spore stalk with single, pairs or clusters. The conidia peduncles were solitary or branched. The hyphae had uniform thickness without septum.

2.8. Statistical analysis The data from three independent experiments are presented as means ± SD. The SPSS 19.0 software was utilized to analyze the date. 3. Results and discussion 3.1. Mycelia purification, microscopic identification and liquid fermentation of C. militaris The colonies formed by C. militaris on PDA medium were round or oval, with fluffy surface and raised cotton-like hemispherical shape in Fig. 2(a). The mycelia were white, developed and easy to pick up, with neat edges. The spore morphology of C. militaris under the microscope was shown in Fig. 2(b). As shown in Fig. 2(b), the conidia of C. militaris were round, ovoid or cylindrical. The conidia live on the top of the

3.2. Preparation of the polysaccharides from fruiting bodies and mycelia of C. militaris The crude polysaccharides from fruiting bodies and mycelia of C. militaris were named as CBPS-RI and CMPS-RI, respectively. The total sugar content of CMPS-RI was 8.34% higher than that of CBPSRI which indicated that the crude polysaccharide content of the mycelia was higher than fruiting bodies. The molecular weight distribution of CBPS-RI and CMPS-RI was detected with HPGPC and the results were demonstrated in Fig. 3(a) and (b), respectively. The molecular weight distributions of CBPS-RI and CMPS-RI were completely different, and the spectra contained 3 and 5 peaks which indicated that CBPS-RI and CMPS-RI were mainly composed of three and five kinds of polysaccharides, respectively. It could be seen that both of the polysaccharides with the first peak were the

Fig. 4. HPGPC spectra of CBPS-RII (a) and CMPS-RII (b).

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main component. Then the CBPS-RI and CMPS-RI were purified with Sephadex G-200 and Sephadex G-150 column, respectively, to collect the content of first peak. The elution curves of CBPS-RI and

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CMPS-RI were shown in Fig. S1(a) and (b), respectively. The main peak was concentrated to obtain the pure polysaccharides of fruiting bodies and mycelia which were named as CBPS-RII and CMPS-RII,

Fig. 5. FT-IR spectra of CBPS-RII (a) and CMPS-RII (b).

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respectively. The UV–Vis scanning spectra of CBPS-RII and CMPS-RII were shown in Fig. S2(a) and (b). There were no peaks at 260 nm and 280 nm. It revealed that both the CBPS-RII and CMPS-RII had no nucleic acids and proteins [36].

3.3. Molecular weight analysis of CBPS-RII and CMPS-RII The HPGPC spectra on the purity and Mw of CBPS-RII and CMPSRII were illustrated in the Fig. 4(a) and (b). The spectra were both composed of a single symmetrical peak which revealed that the CBPS-RII and CMPS-RII were both homogeneous polysaccharide. The retention time of CBPS-RII and CMPS-RII were 8.245 min and 8.975 min, respectively. The molecular weight curve of Dextran standards was as follows: y = −0.3433× + 9.4236, R 2 = 0.9916 (y = lgM w , x = R t ). after calculation, the molecular weights of CBPS-RII and CMPS-RII were 3.94 × 10 3 kDa and 2.20 × 10 3 kDa, respectively.

3.4. Structural characterization of CBPS-RII and CMPS-RII 3.4.1. FT-IR analysis The FT-IR spectra of CBPS-RII and CMPS-RII were demonstrated in Fig. 5(a) and (b), respectively. There were wide peaks at 3416 cm−1 (CBPS-RII) and 3396 cm−1 (CMPS-RII) which caused by the stretching vibration of –OH [35,37]. The signals at 2925 cm−1 (CBPS-RII) and 2923 cm−1 (CMPS-RII) were attributed to C\\H stretching vibration [38]. The strong absorption peaks at 1640 cm−1 (CBPS-RII) and 1652 cm−1 (CMPS-RII) were signals of C_O stretching vibration [39,40]. C\\H variable angular vibration was observed at 1413 cm−1 (CMPS-RII) or 1419 cm−1 (CBPS-RII) [41]. The above four points were characteristic absorption peaks of polysaccharides. The characteristic absorption peaks at 1146 cm−1, 1080 cm−1, 1034 cm−1 (CMPS-RII) and 1154 cm−1, 1080 cm−1 and 1023 cm−1 (CBPS-RII), revealed the presence of pyranoside in CBPS-RII and CMPS-RII. The absorption characteristic peaks at 874 cm−1 (CMPS-RII) and 849 cm−1 (CBPS-RII) proved that CBPS-RII and CMPS-RII contained α-type glycosidic bonds [42].

Fig. 6. GC–MS spectra of standards, CBPS-RII and CMPS-RII in monosaccharide compositions analysis.

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Fig. 7. GC–MS spectra of CBPS-RII derivatives (a) and CMPS-RII derivatives (b) in Smith degradation analysis.

Table 1 Results of the methylation analysis of CBPS-RII and CMPS-RII.

CBPS-RII

CMPS-RII

Methylation sugar residues

Linkages types

Relative molar ratio

Major mass fragments (m/z)

2,3,4,6-tetra-O-Me-D-Glc 2,3,6-tri-O-Me-D-Gal 2,3,6-tri-O-Me-D-Glc 2,4,6-tri-O-Me-D-Glc 2,6-di-O-Me-D-Glc 2,3,6-tri-O-Me-D-Man 2,3,4,6-tetra-O-Me-D-Glc 2,3,6-tri-O-Me-D-Gal 2,3,6-tri-O-Me-D-Glc 2,4,6-tri-O-Me-D-Glc 2,3-di-O-Me-Glc 2,3,6-tri-O-Me-D-Man

T→4)Gal(1→ →4)Glc(1→ →3)Glc(1→ →3,4)Glc(1→ →4)Man(1→ T→4)Gal(1→ →4)Glc(1→ →3)Glc(1→ →3,4)Glc(1→ →4)Man(1→

1.00 0.65 4.98 1.30 1.04 1.02 0.83 2.46 3.60 0.35 0.85 1.91

43,75,87,101,117,131,161,205 43,71,87,99,117,113,129,142,161,173,233 43,71,87,99,117,113,129,142,161,173,233 43,59,71,85,101,117,129,161,202,233 43,71,87,99,117,129,161,173,189,233 43,71,87,99,117,113,129,142,161,173,233 43,71,87,101,117,131,161,205 43,71,87,99,117,113,129,142,161,173,233 43,71,87,99,117,113,129,142,161,173,233 43,59,71,85,101,117,129,161,202,233 43,71,87,99,117,129,161,173,189,233 43,71,87,99,117,113,129,142,161,173,233

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Fig. 8. 1H NMR (a), 13C NMR (b) and HSQC (c) spectra of CBPS-RII;

L. Wu et al. / International Journal of Biological Macromolecules 148 (2020) 722–736

3.4.2. Monosaccharide composition analysis The products of degradation of CBPS-RII and CMPS-RII were detected with GC–MS and the spectra were illustrated in Fig. 6. Compared with the standards, the CBPS-RII and CMPS-RII were both composed of DMan, D-Glc and D-Gal with molar ratios of 1.00:9.18:0.71 and 1.00:2.92:1.31, respectively. 3.4.3. Linkage analysis The oxidation of periodic acid analysis, Smith degradation analysis and methylation analysis were performed to analyze the linkages of CBPS-RII and CMPS-RII. The periodate oxidation of CBPS-RII and CMPS-RII produced 0.0074 mmol and 0.036 mmol formic acid, respectively, which indicated that of 1 → 6 linkage [27]. The CBPS-RII and CMPS-RII, both contained saccharides residue of 0.0377 mmol, consumed periodate of 0.0190 mmol and 0.0235 mmol, respectively, which indicated the presence of glycosidic bonds of (1 → 3), (1 → 3, 6), (1 → 2, 3), (1 → 2, 4), (1 → 3, 4) or (1 → 2, 3, 4) in CBPS-RII and CMPS-RII [43]. The consumption of periodic acid is more than twice of the formic acid which revealed the presence of glycosidic bonds of (1 → 2), (1 → 4) and (1 → 4, 6) [27]. The products of oxidation of periodic acid were further treated with Smith degradation and detected with GC–MS. The results were illustrated in the Fig. 7 and Fig. S3. Compared with the standards, the products after Smith degradation of CBPSRII and CMPS-RII contained glycerol, erythritol and D-Glucose with molar ratios of 1.00:3.20:1.10 and 1.00:2.15:0.69, respectively. The presence of glucose indicated the exist 1 → 3, (1 → 2, 3), (1 → 3, 4), (1 → 3, 6) or (1 → 2, 3, 4) linkage [44]. Meanwhile, the glycerol and erythritol indicated that presence of (1 → 2) or (1 → 2, 6), (1 → 4), (1 → 4, 6), (1 → 6) or (1→) glycosidic bonds [31]. The methylation analysis was performed to determine the accurately glycosidic bonds in CBPS-RII and CMPS-RII. The CBPS-RII and CMPS-RII were methylated for several times, and the methylation degree was detected by FT-IR, as shown in infrared spectrum Fig. S4 (a) and (b). The –OH peak at 3000 cm−1-3600 cm−1 changed from wide flat to narrow sharp, and the characteristic absorption peak caused by C\\H stretching vibration in –CH3 around 2900 cm−1 was significantly enhanced which indicated that the methylation was complete and further degradation could be carried out. The products of CBPS-RII and CMPS-RII after methylation were hydrolyzed, acetylated and detected with GC–MS. The methylation results of CBPS-RII and CMPS-RII were analyzed according to the literature of Sims [45]. The results were summarized in Table 1. The results indicated that the CBPS-RII and CMPS-RII were both composed of (1→)-D-Glcp, (1 → 4)-D-Galp, (1 → 4)-D-Glcp, (1 → 3)-D-Glcp, (1 → 3, 4)-D-Glcp and (1 → 3)-DManp with relative molar ratios of 1.00:0.65:4.98:1.30:1.04:1.02 and 0.83:2.46:3.60:0.35:0.85:1.91, respectively. All results revealed that the backbones of the CBPS-RII and CMPS-RII were composed of (1 → 4)-D-Glcp and the branches contained (1 → 4)-D-Galp, (1 → 3)D-Glcp, (1 → 3, 4)-D-Glcp and (1 → 3)-D-Manp. After comparison, in the CBPS-RII and CMPS-RII, There were no difference between the kinds of glycosidic bonds and the backbones, but the number of glycosidic bonds and branches had significant difference. 3.4.4. NMR analysis The 1H NMR spectra, 13C NMR spectra and HSQC spectra of CBPS-RII and CMPS-RII were illustrated in Figs. 8 and 9. Since the solvent peak in the 1H spectrum was too high to affect the nearby signal peak, the 1H NMR spectrum of CBPS-RII (Fig. 8 (a)) was water-suppering spectrum and there was no signal at 4.79 ppm. The chemical shift of the proton at the C-1 position is generally in the lower field (4.2–5.5 ppm) in 1H NMR spectra. In Figs. 8(a) and 9(a), there were signals at 4.2–5.5 ppm which indicated that the CBPS-RII and CMPS-RII both contain αglycosidic bond and β-glycosidic bond [15]. In 13C NMR spectra, signals at 90–100 ppm indicated the presence of α-glycosidic bond, while at 100–103 ppm indicated that the presence of β-glycosidic bond [24]. From the 13C NMR spectra of CBPS-RII (Fig. 8b) and CMPS-RII (Fig. 9b),

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the α-glycosidic bond β-glycosidic bond were both presented in CBPSRII and CMPS-RII which was consistent with results of 1H NMR analysis. The HSQC spectra of CBPS-RII and CMPS-RII were illustrated in Figs. 8c and 9c, respectively. And the regions of H1/C1 were amplified in the Fig. S5(a) and (b), respectively. According to the types and proportions residue from methylation analysis, coupling constants of the peak in hydrogen spectrum and related literatures [46–53], the signals of anomeric carbon and anomeric hydrogen have been attributed. The Fig. 9d, there were six signals at 5.14/91.96 ppm, 4.56/95.86 ppm, 4.88/98.03 ppm, 5.31/99.68 ppm, 5.03/106.16 ppm and 5.08/ 100.87 ppm which present six residues of →3)-α-D-Manp(1→, →3,4)α-D-Glcp(1→, α-D-Glcp(1→, →4)-α-D-Glcp(1→, →3)-α-D-Glcp (1 → and →4)-β-D-Galp(1→, respectively. In Fig. 9h, there were six signals at 5.18/91.97 ppm, 4.60/95.86 ppm, 4.93/98.51 ppm, 5.35/ 99.83 ppm, 5.24/107.12 ppm, 4.99/108.04 ppm and 4.75/101.63 ppm which present six residues of →3)-α-D-Manp(1→, →3,4)-α-D-Glcp (1→, α-D-Glcp(1→, →4)-α-D-Glcp(1→, →4)-β-D-Galp(1→ and →3)β-D-Glcp(1→, respectively. The above results indicated that the residues of CBPS-RII and CMPS-RII have high similarity but the configuration of anomeric carbon in →3)-D-Glcp(1→ were significant different. 3.4.5. Congo red analysis The results of Congo red analysis on CBPS-RII and CMPS-RII were illustrated in Fig. 10 (a). The maximum wavelengths of CMPSRII + Congo red and CBPS-RII + Congo red were changed in different concentrations of NaOH. Compared with the control group, the maximum wavelengths of experimental groups were significant difference and had red shift (from 492 nm to 505 nm) at the NaOH concentration of 0.05 M. With the increase of NaOH concentration, the maximum wavelengths of experimental groups were gradually decreased. When NaOH concentration was 0.15 M, the maximum wavelength of experimental groups were approached to the control group. The results showed that CMPS-RII and CBPS-RII had a triple helix structure in a weakly alkaline solution [44].

3.4.6. Specific rotation analysis The monomer asymmetry and the whole space asymmetry of the macromolecule determine the specific rotation of the macromolecule. The specific rotation of CBPS-RII and CMPS-RII were +120° and +125°, respectively, which revealed that the advanced structure of CBPS-RII and CMPS-RII had discrepancies.

3.4.7. DSC analysis DSC technology can be used to study the advanced structure of complex [25]. The results of DSC analysis on CBPS-RII and CMPS-RII were illustrated in Fig. 10(b) and (c), respectively. It can be seen from the Fig. 10 (b, c) that both CBPS-RII and CMPS-RII had endothermic reactions during heating which indicated that derivatives of CBPS-RII and CMPS-RII were accompanied by disintegration and mutation of homomorphic configuration during heating. Under the condition of nitrogen protection, there was no exothermic peak. There was a large endothermic peak at 75.90 °C and 75.94 °C, respectively, indicating that their solid state configurations were mutated at this temperature. In addition, the CBPS-RII also had endothermic peak at 201.26 °C. It revealed that there are discrepancies in the advanced structure of CBPS-RII and CMPS-RII.

3.4.8. SEM analysis The surface morphology of CBPS-RII and CMPS-RII were observed by scanning electron microscopy (SEM). As shown in Fig. 11(a) and (b), the CBPS-RII and CMPS-RII has similar surface morphology. They had a relatively loose, rough surface and amorphous structure which revealed that there were interactions between polysaccharides molecules.

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Fig. 9. 1H NMR (a), 13C NMR (b) and HSQC (c) spectra of CMPS-RII.

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3.5. α-Glucosidase inhibitory activity The inhibitory activities of polysaccharides were tested against αglucosidase，and the IC50 were summarized. The Fig. 12 (a) showed the inhibitory effect of C. militaris polysaccharides of different purity and concentration of 4 mg/mL on α-glucosidase activity. The inhibition rates of acarbose, CBPS-RI, CMPS-RI, CBPS-RII and CMPS-RII was 90.24 ± 3.15%, 36.65 ± 3.08%, 32.65 ± 2.23%, 68.29 ± 2.67% and 58.06 ± 2.32%, respectively. The inhibition rates of CBPS-RII and CMPS-RII were 31.64% and 25.43% higher than CBPS-RI and CMPS-RI, respectively. The purified polysaccharides CBPS-RII and CMPS-RII were formulated into gradient concentrations to evaluate the inhibitory activity on α-glucosidase, respectively (Fig. 12(b)). As the concentrations of polysaccharide increased, the inhibition rate on α-glucosidase activity increased continuously which indicated that there was a doseeffect relationship. The inhibition rates of CBPS-RII and CMPS-RII on the activity of α-glucosidase reached 68.29 ± 2.67% and 58.06 ± 2.32%, respectively at 4 mg/mL. Then the inhibition rates tended to be gentle. All the results illustrated that the polysaccharide components of C. militaris had inhibitory activity on α-glucosidase. The inhibitory activity of CBPS-RII was higher than that of CMPS-RII in the whole concentration ranging from 0.1 to 4.0 mg/mL. The IC50 values of CMPS-RII and CBPS-RII were 3.270 mg/mL and 1.882 mg/mL, respectively, which revealed that the CBPS-RII had better inhibitory activity on αglucosidase than CMPS-RII.

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and + 125° respectively. The state configurations of CBPS-RII mutated at 75.90 °C and 201.26 °C, while the CBPS-RII at 75.94 °C. The IC50 values of CMPS-RII and CBPS-RII on α-glucosidase were 3.270 mg/mL and 1.882 mg/mL, respectively, which revealed that the CBPS-RII had better inhibitory activity on α-glucosidase than CMPS-RII. The inhibition types of CBPS-RII and CMPS-RII were both Mixed Type inhibition. With comprehensive analysis, the CBPS-RII and CMPS-RII had similarity monosaccharide composition, glycosidic bonds sugar residue and surface morphology, but the molecular weights, proportions of backbones and braches, configuration of glycosidic bonds, and advanced structure had significantly discrepancy. Obviously, the CBPS-RII had better inhibitory activity on α-glucosidase than CMPS-RII. All the results indicated that

3.6. Kinetic of α-glucosidase inhibition As shown in Fig. 12(c) and (d), the concentration of polysaccharides and enzyme was 0, 0.5, 1.0 mg/mL. The concentration of the enzyme was plotted by absorbance (speed) per unit time, and three curves were obtained. With the increasing enzyme concentration, the slope of the curve was decreased under the same inhibitor concentration. All three curves of different inhibitor concentrations passed through the origin. Therefore, the inhibition mechanism of the CBPS-RII and CMPS-RII on the enzyme was reversible. The inhibition kinetics of the active components CBPS-RII and CMPS-RII were studied by Lineweaver-Burk plot analysis. As shown in Fig. 12(e) and (f), the Xaxis represented the reciprocal of the various concentrations of the matrix pNPG, the Y-axis represented the 1/V calculated by absorbance. It can be observed that the double reciprocal curves of inhibitors with different concentrations intersect in the second quadrant, indicating that the maximal velocity (Vmax: reciprocal of y-intercept) decreased, while the reciprocal of Michaelis constant x-intercept) increased after the polysaccharide was added to the reaction system, and the Michaelis constant (Km) was increased mildly, indicating that when the polysaccharide was combined with the enzyme, the affinity of the enzyme for the substrate was reduced. This indicated that the inhibition types of CBPS-RII and CMPS-RII were Mixed Type inhibition. With all results, the CBPS-RII and CMPS-RII were separated from the fruiting bodies and mycelia of C. militaris. The Mw of CBPS-RII and CMPS-RII were 3.94 × 103 kDa and 2.20 × 103 kDa, respectively. The monosaccharides in CBPS-RII and CMPS-RII were D-Man, D-Glc and DGal with molar ratios of 1.00:9.18:0.71 and 1.00:2.92:1.31, respectively. The CBPS-RII and CMPS-RII were both composed of (1→)-D-Glcp, (1 → 4)-D-Galp, (1 → 4)-D-Glcp, (1 → 3)-D-Glcp, (1 → 3, 4)-D-Glcp and (1 → 3)-D-Manp with relative molar ratios of 1.00:0.65:4.98:1.30:1.04:1.02 and 0.83:2.46:3.60:0.35:0.85:1.91,respectively, from linkages analysis. It revealed that the backbones of the CBPS-RII and CMPS-RII were composed of (1 → 4)-D-Glcp and the branches contain (1 → 4)-D-Galp, (1 → 3)-D-Glcp, (1 → 3, 4)-D-Glcp and (1 → 3)-D-Manp. The NMR analysis indicated that the configuration of anomeric carbon in (1 → 3)-D-Glcp of CBPS-RII and CMPS-RII were αtype and β-type, respectively. The results showed that CMPS-RII and CBPS-RII both had a triple helix structure, rough surface and amorphous structure. The specific rotations of CBPS-RII and CMPS-RII were + 120°

Fig. 10. (a) Results of Congo red test; DSC diagram of CBPS-RII (b) and CMPS-RII (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 11. SEM images of CBPS-RII (a) and CMPS-RII (b) (×2000).

the chemical structure and α-glucosidase inhibitory activity of polysaccharides from C. militaris with different developmental stages had significant differences. The literatures have reported that the culture conditions and nutritional conditions have significant influences on the structure and biological activities of polysaccharides. Papinutti found that production of EPS from Ganoderma lucidum can be obviously affected by the nutrients, pH and water potential of cultural medium

[54]. Fraga et al. found that the carbohydrate composition and structural features of the EPS extracted from Ganoderma lucidum were changed significantly according to the different growing conditions (carbon, nitrogen levels and initial pH), especially in the (1 → 3)/(1 → 4)-Glcp ratio and also on the branching degree of EPS [55]. Shen et al. found that the normal, salt stress and mixotrophic culture conditions can significant effect the physicochemical properties and antioxidant activities

Fig. 12. (a) Inhibition rates of CMPS-RII and CBPS-RII with different purities onα-glucosidase activity. (b) Inhibition rates of CMPS-RII and CBPS-RII with different concentrations on αglucosidase activity. (In the same series, different uppercase letters and lowercase letters indicate that the differences between groups are extremely significant (P b .01) and significant (P b .05), respectively. The same lowercase letters indicated that the difference between the groups was not significant (P N .05); Relationship of the α-glucosidase activity with enzyme concentrations at different concentrations of CBPS-RII (c) and CMPS-RII (d); Lineweaver-Burk plots showing inhibition kinetics of α-glucosidase by CBPS-RII (e) and CMPS-RII (f).

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of polysaccharides from Nostoc flagelliforme [56]. This manuscript demonstrated that the mycelia of C. militaris from asexual reproduction with liquid fermentation can't be an effective substitute for fruiting bodies from sexual reproduction, from the perspective of polysaccharides. 4. Conclusions In conclusion, the polysaccharides were separated from the C. militaris with different developmental stages. The chemical structure and inhibitory activity on α-glucosidase of polysaccharides were explored. The results indicated that the chemical structure and αglucosidase inhibitory activity of polysaccharides from C. militaris with different developmental stages had significant differences. The polysaccharides from fruiting bodies had better inhibitory activity on αglucosidase. The studies of the polysaccharides from C. militaris with different developmental stages revealed that the growth conditions and nutritional conditions had obviously effect on the active ingredient and biological activity C. militaris. It demonstrated that the mycelia of C. militaris from asexual reproduction with liquid fermentation can't be an effective substitute for fruiting bodies from sexual reproduction, from the perspective of polysaccharides. The results confirmed the medicinal value of C. militaris fruiting bodies. It also provided the experimental evidence and scientific explanation for the irreplaceability of C. militaris fruiting body. Declaration of competing interest None. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (31871791), Technology Program of Tianjin, China, (18ZYPTJC00020), the Key Program of the Foundation of Tianjin Educational Committee (2018ZD06). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2020.01.178. References [1] S.A. Petropoulos, A. Fernandes, N. Tzortzakis, M. Sokovic, A. Ciric, L. Barros, I. Ferreira, Bioactive compounds content and antimicrobial activities of wild edible Asteraceae species of the Mediterranean flora under commercial cultivation conditions, Food Res. Int. 119 (2019) 859–868. [2] K. Kaewnarin, N. Suwannarach, J. Kumla, S. Lumyong, Phenolic profile of various wild edible mushroom extracts from Thailand and their antioxidant properties, anti-tyrosinase and hyperglycaemic inhibitory activities, J. Funct. Foods 27 (2016) 352–364. [3] W. Liu, H. Wang, X. Pang, W. Yao, X. Gao, Characterization and antioxidant activity of two low-molecular-weight polysaccharides purified from the fruiting bodies of Ganoderma lucidum, Int. J. Biol. Macromol. 46 (4) (2010) 451–457. [4] J. Guan, J. Zhao, K. Feng, D.J. Hu, S.P. Li, Comparison and characterization of polysaccharides from natural and cultured Cordyceps using saccharide mapping, Anal. Bioanal. Chem. 399 (10) (2011) 3465–3474. [5] F.M.N.A. Aida, M. Shuhaimi, M. Yazid, A.G. Maaruf, Mushroom as a potential source of prebiotics: a review, Trends Food Sci. Technol. 20 (11−12) (2009) 567–575. [6] X.N. Liu, B. Zhou, R.S. Lin, L. Jia, P. Deng, K.M. Fan, G.Y. Wang, L. Wang, J.J. Zhang, Extraction and antioxidant activities of intracellular polysaccharide from Pleurotus sp. mycelium, Int. J. Biol. Macromol. 47 (2) (2010) 116–119. [7] C. Zhang, S. Li, J. Zhang, C. Hu, G. Che, M. Zhou, L. Jia, Antioxidant and hepatoprotective activities of intracellular polysaccharide from Pleurotus eryngii SI-04, Int. J. Biol. Macromol. 91 (2016) 568–577. [8] W.W. Deng, X. Cao, Y. Wang, Q.T. Yu, Z.J. Zhang, R. Qu, J.J. Chen, G.B. Shao, X.D. Gao, X.M. Xu, J.N. Yu, Pleurotus eryngii polysaccharide promotes pluripotent reprogramming via facilitating epigenetic modification, J. Agric. Food Chem. 64 (6) (2016) 1264–1273. [9] J. Chen, D. Mao, Y. Yong, J. Li, H. Wei, L. Lu, Hepatoprotective and hypolipidemic effects of water-soluble polysaccharidic extract of Pleurotus eryngii, Food Chem. 130 (3) (2012) 687–694.

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