Preliminary structural characterization and hypoglycemic effects of an acidic polysaccharide SERP1 from the residue of Sarcandra glabra

Accepted Manuscript Title: Preliminary Structural Characterization and Hypoglycemic Effects of an acidic Polysaccharide SERP1 from the Residue of Sarcandra Glabra Authors: Wei Liu, Weisheng Lu, Yin Chai, Yameng Liu, Wenbing Yao, Xiangdong Gao PII: DOI: Reference:

S0144-8617(17)30947-5 http://dx.doi.org/10.1016/j.carbpol.2017.08.071 CARP 12678

To appear in: Received date: Revised date: Accepted date:

10-7-2017 26-7-2017 15-8-2017

Please cite this article as: Liu, Wei., Lu, Weisheng., Chai, Yin., Liu, Yameng., Yao, Wenbing., & Gao, Xiangdong., Preliminary Structural Characterization and Hypoglycemic Effects of an acidic Polysaccharide SERP1 from the Residue of Sarcandra Glabra.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.08.071 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Preliminary Structural Characterization and Hypoglycemic Effects of an acidic Polysaccharide SERP1 from the Residue of Sarcandra Glabra Wei Liu 1, Weisheng Lu1, Yin Chai , Yameng Liu , Wenbing Yao * , Xiangdong Gao ** Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China

* Corresponding author. Tel.: +86 25 83271218; fax: +86 25 83271218. ** Corresponding author. Tel.: +86 25 83271543; fax: +86 25 83271249. E-mail addresses: [email protected] (W. Yao), [email protected] (X. Gao). 1 These two authors contributed equally to this work.

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Highlight  SERP1 has complicated structure due to its composition of nine monosaccharides.  SERP1 shows high hypoglycemic properties in diabetic mice.  SERP1 could be explored as a functional food or supplement from industrial waste.

Abstract An acidic polysaccharide (SERP1) was isolated from the residue of Sarcandra glabra (Thunb.) Nakai by water extraction and purified by decoloration and ion exchange chromatography. The structure of SERP1 was determined by HPLC, HPSECMALLS, FT-IR, and NMR. The results indicated that SERP1 was a homogeneous heteropolysaccharide with the absolute molecular weight of 4.208×104 Da in the aqueous phase. 1,4-linked α-D-galacturonic acid, methyl esterified 1,4-linked α-Dgalacturonic acid, 1,4-linked α-D-glucuronic acid, 1,5-linked α-L-arabinose, 1,3linked β-D-galactose 1,4-linked α-D-glucose, 1,4,6-linked β-D-glucose, 1,6-linked βD-glucose, and 1,2-linked rhamnose existed in SERP1. In vitro α-glucosidase inhibition assay indicated that SERP1 had a low IC50 value of 49.01 µg/mL, which exhibited stronger α-glucosidase enzyme inhibitory activity than acarbose at the same concentration. The treatment of SERP1 to type 2 diabetes mellitus mice alleviated the hyperglycemia, increased glucose utilization of peripheral tissues of the liver and inhibited the liver injury. This study provides a possible exploration to use valuable industrial waste.

Keywords： Acidic polysaccharide, hypoglycemic, residue of Sarcandra glabra, structure characterization

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Introduction Diabetes mellitus (DM) is a chronic metabolic syndrome characterized by elevated levels of blood glucose, which results from a deficiency in the production of insulin, utilization of insulin, or both (Meusel et al., 2014). In diabetes mellitus, the body does not produce (type 1) and/or properly respond (type 2) to insulin, which leads to the entry of glucose from the plasma to cells in energy production (Rother, 2014). Research showed chronic hyperglycemia is associated with dysfunction of bodies and failure of organs, including eyes, kidneys, heart, and blood vessels (Sendrayaperumal, Iyyam Pillai, & Subramanian, 2014). Type 2 diabetes mellitus (T2DM) is the most widespread metabolic disease in the world. It is estimated that 347 million people worldwide are suffered from diabetes, of which 90-95% are T2DM (Danaei et al., 2011). Many hypoglycemic drugs have been synthesized for the treatment of the DMs, but those drugs were limited by side effects and high prices. Therefore, investigators are eager for low-side-effect, cheap, and efficient drugs with wide availability. As active compounds from natural resources, polysaccharides have been investigated in depth in recent years. Numerous studies showed that polysaccharides with anti-hyperglycemic activity were found in plants and medicinal fungi (T. Chen et al., 2016; Li et al., 2015). Sarcandra glabra (Thunb.) Nakai from the family of Chloranthaceae is a natural source of dietary supplement and widely grows in southern China, Japan, and Southeastern Asia (Chau & Wu, 2006; He et al., 2009; Zhou et al., 2013). Many studies showed that S. glabra possessed anti-inflammation (Liu et al., 2016), anticancer(Guo et al., 2013), and antimicrobial (Cao et al., 2012) activities, while sesquiterpenoids, coumarin, flavone, and other small molecules were considered as the main active constituents. As macromolecular components in many functional herb medicines, polysaccharides in S. glabra were not deeply studied. Thus, our group began to study the polysaccharides components in S. glabra . Two polysaccharides from the whole plant of S. glabra with antioxidant (Jin et al., 2012), anticancer (Zhang et al., 2014), and hypoglycemic activities were studied (Liu, Zheng, Zhang, Yao, & Gao, 2014). But the value of S. glabra whole plant is to produce many drugs including Caoshanhu Hanpian, XueKang Koufuye, and Wantong Yankang Pian. CaoshanHu Hanpian is one of the drugs with the sales of more than 1 billion RMB yearly, which is made by the supernatant of ethanol-precipitated S. glabra extract (The State of Pharmacopoeia Commission of People’s Republic of China, 2015). During the preparation procedures of these drugs, tons of ethanol precipitation considered as industrial waste could be produced. Instead of discarding the residue, proper utilization of S. glabra is extremely urgent. In this study, we obtained an acidic polysaccharide from S. glabra extract residue and investigated the physicochemical property and preliminary structure of this polysaccharide. Furthermore, type 2 diabetic mouse model was applied to evaluate the hyperglycemia and insulin resistance ability of this polysaccharide. Materials and methods Materials 3

The concrete residue of S. glabra was a gift from Jiangxi Jiangzhong Pharmaceutical Co., Ltd. (Nanchang, Jiangxi, China). Macroporous adsorption resin D101 was purchased from Tianjin Bohong Resin Technology Co., Ltd. (Tianjin, China). Diethylaminoethylcellulose (DEAE-52) was obtained from Pharmacia Co.,Ltd. (Uppsala, Sweden). p-Nitrophenyl-α-glucopyranoside (pNPG), α-glucosidase from Saccharomyces cerevisiae, metformin (MET), and streptozotocin (STZ) were purchased from Sigma-Aldrich Chemical Co., Ltd. (St. Louis, MO, USA). Acarbose was purchased from Bayer Schering Pharma (Wedding, Berlin, Germany). The kits used in the study for the assay of blood glucose (GLU), insulin (INS), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterin (LDL), high-density lipoprotein cholesterin (HDL), free fatty acids (FFA), the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), total antioxidant capacity (T-AOC), malondialdehyde (MDA), the activities of pyruvate kinase (PK) and hexokinase (HK), the activities of alkaline phosphatase(AKP), glutamicpyruvic transaminase (GPT), and aspartate aminotransferase (AST) were purchased from Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). All other chemicals were of analytical grade. Animals Seventy male C57BL/6J mice (16 ± 2 g, 5-week-old) were provided by the Experimental Animal Center of Yangzhou University (Yangzhou, Jiangsu, China). All mice were housed in plastic cages and maintained under normal conditions (12 h light/dark cycle; ambient temperature 22 ± 2 ºC; 35-60% humidity) with free access to diet and purified water. Regular pellet diet consisting of 5% fat, 23% protein, and 53% carbohydrate with the total caloric value of 25.0 kJ/kg, and high fat diet (HFD) consisting of 22% fat, 20% protein, and 48% carbohydrate with the total caloric value of 44.3 kJ/kg were purchased from Qinglongshan Animal Farm (Nanjing, Jiangsu, China). All animal experiments were conducted with criteria approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University. Separation and purification of polysaccharide from S. glabra extract residue The S. glabra extract residue was dried at 60 ºC for 4 hours by the oven and then ground to powder. The powder was dissolved, a final ethanol concentration of 30% was added to the solution. The precipitation proceeded at 4 ºC for 12 hours. The precipitate was collected by centrifugation, washed successively with ethanol, acetone, and ether, and then dried under vacuum to obtain crude polysaccharides. Crude polysaccharides were decolored by nonpolar macroporous resin D101 at 37 ºC for 30 min. The decolored sample was then dissolved in double-distilled water and the solution was loaded onto a DEAE-52 column, and gradient eluted with 0-2 mol/L NaCl. The main components were collected and enriched according to the result of carbohydrate quantitative determination by phenol-sulfuric acid method (DuBois, Gilles, Hamilton, Rebers, & Smith, 1956). Then the collected fraction was dialyzed against distilled water for 24 hours and followed by lyophilizing. The purified polysaccharide was coded as SERP1. 4

General analysis Neutral carbohydrate contents were determined by the phenol-sulfuric acid method with D-glucose as equivalent (DuBois et al., 1956). Acidic carbohydrate contents were measured by the meta-hydroxydiphenyl method with D-galacturonic acid as equivalent (Blumenkrantz & Asboe-Hansen, 1973). Protein content was performed by Bradford method with bovine serum albumin (BSA) as equivalent (Bradford, 1976). SERP1 was dissolved in distilled water at 1 mg/mL at 20 ºC and measured on PE241MC spectropolarimeter (PerkinElmer, USA) at 589 nm for optical rotation analysis. Elemental analysis of SERP1 was operated by Vario EL III automatic element analyzer (Elementar, German). The ultraviolet spectral analysis of SERP1 was completed by the Multiskan Spectrum (Thermo Fisher, USA). Homogeneity and relative molecular weight determination The homogeneity and relative molecular weight of SERP1 were determined by highperformance size exclusion chromatography (HPSEC). SERP1 was dissolved in double-distilled water and passed through 0.45 µm filter to remove insoluble impurity, then applied to the Agilent 1260 Infinity HPLC (USA) with a gel permeation chromatographic column of Shodex KS-805 (SHOWA DENKO K.K., Japan) at 35 ºC. The samples were eluted with double-distilled water at an isocratic flow rate of 1.0 mL /min and detected with refractive index detector. The column was calibrated with 2.5 mg/mL of 180, 2700, 5250, 9750, 13050, 36800, 64650, 135350, 300600 and 2000000 Da dextrans. HPSEC-MALLS analysis The High-performance size exclusion chromatography coupled with multi-angle laser light scattering (HPSEC-MALLS) system consisted of a pre-column (Shodex OHpak SB-G, Japan), a gel permeation chromatographic column (Shodex OHpak SB-806 HQ, Japan), a separation module (Waters 1100, Milford, USA), and a multi-angle laser light scattering detector (Wyatt DAWN HELEOS-II, Santa Barbara, USA) at 663.6 nm of laser, along with a refractive index monitor (Agilent 1100, Santa Clara, USA). SERP1 was dissolved in double-distilled water at 1 mg/mL, filtered by 0.22 µm filter before injection. 20 µL of the SERP1 solution was injected into the system and eluted with double stilled water at an isocratic flow rate of 0.5 mL/min. Data of MALLS and refractive index was analyzed by ASTRA software. Data of molecular weight and molecular weight distribution were fitted by Debye method. Every experiment was performed in triplicate. Monosaccharide composition analysis High-performance anion-exchange chromatography with pulsed ampere detection method (HPAEC-PAD) was used to determine the monosaccharide composition of SERP1 (Zhenqing Zhang, Khan, Nunez, Chess, & Szabo, 2012). Briefly, the sample was completely hydrolyzed with trifluoroacetic acid (TFA) at 100 ºC for 8 hours. Monosaccharide standard solutions were prepared by mixing each standard 5

monosaccharide with double-distilled water. Sample and monosaccharide standard solutions were passed through 0.45 µm filter to remove insoluble impurity. The hydrolyzed samples were analyzed by HPAEC. HPAEC consisted of a Dionex ICS5000+ system (Thermo Scientific Dionex, Sunnyvale, CA, USA), coupled with a pulsed amperometric detector, and a CarboPacTM PA10 column (4.0 mm × 250 mm; Thermo Scientific Dionex, Sunnyvale, CA, USA). The hydrolyzed samples (25 µL) were injected into the column. The mobile phase contained NaOH (200 mmol/L), CH3COONa (200 mmol/L) and double-distilled water. The elution method was as follows: 0-30 min (13.5% of the 200 mmol/L NaOH and 86.5% of the double-distilled water), 30-60 min (13.5% of the 200 mmol/L NaOH, 75% of the 200 mmol/L CH3COONa, and 11.5% of the double-distilled water). The flow rate was adjusted to an isocratic flow rate of 0.5 mL/min and the column temperature was set at 28 ºC. Fourier transform infrared spectrum analysis The Fourier transform infrared spectroscopy (FT-IR) spectrum of SERP1 (SERP1:KBr =1:200 (w/w)) was recorded by Spectrum 100 instrument (PerkinElmer, Waltham, USA) with the scanning width ranging from 4000 cm-1 to 400 cm-1. Nuclear magnetic resonance (NMR) spectroscopy analysis SERP1 was dissolved in D2O at the concentration of 15 mg/mL. 1H-NMR and 13CNMR spectra were recorded by Bruker spectrometer at 300 MHz at 30 ºC. α-D-glucosidase inhibitory activity and half inhibition concentration (IC50) The α-D-glucosidase inhibitory activity was measured by determining the release of p-nitrophenol (pNP) from pNPG (Chapdelaine, Tremblay, & Dube, 1978). Briefly, 160 µL SERP1 or acarbose range from 15.63 to 1000 μg/mL and 20 µL phosphate buffer (pH 6.8, 0.1 mol/L) with α-D-glucosidase (1 U/mL) were incubated in 96-well plates at 37 ºC for 10 min. The enzymatic reaction was initiated by 5 mmol/L pNPG (20 µL), and terminated by 1 mol/L sodium carbonate (20 µL) after incubating mixture for another 30 min at 37 ºC. Enzyme activity was determined by absorbance of pNP at 405 nm. The inhibitory rate of SERP1 or acarbose on α-glucosidase was calculated by the following equation: Inhibition percentage = [1-(Asample-Ablank)/(Acontrol-Ablank)]×100 % Asample and Acontrol are the absorbances of the sample with SERP1 or acarbose and without SERP1 or acarbose, respectively. Ablank is the absorbance of the sample without α-glucosidase. IC50 values of SERP1 or acarbose were calculated by SPSS (Version 19.0), respectively. Establishment and grouping of hyperglycemic mice model The mice were fed with HFD for three weeks after one-week adaptation for the establishment of type 2 diabetes mellitus. Then the mice were given with a single dose of 50 mg/kg STZ in 0.1 mol/L citrate buffer (pH 4.5) after fasting for 12 h by intraperitoneal administration. After feeding with HFD for another three weeks, all 6

mice with fasting blood glucose over 7.8 mmol/L were chosen for next step study (Ren et al., 2015; Xu, Zhou, Yin, Yao, & Zhang, 2015). The mice in control group were fed with regular diet at the same time. HFD/STZ induced diabetic mice were assigned into 6 groups randomly (n=8), and the mice with regular diet were used as the control. Group 1 (Control, the normal control) was given 0.9% of saline (w/w) by intragastric injection and only kept on the regular diet. For Group 2 (Model, the diabetic control), 0.9% of saline (w/w) by intragastric injection was given and the HFD was fed. Groups 3, 4, and 5 (SERP1-L, SERP1-M, and SERP1-H) were treated with 100, 200, and 400 mg∙kg-1∙d-1 of SERP1 (dissolved in 0.9% of saline (w/w)) respectively by intragastric injection and kept on feeding with the HFD. Group 6 and 7 (Acarbose and Metformin) were treated with 10 mg∙kg1 -1 ∙d of acarbose or 200 mg∙kg-1∙d-1 of metformin (dissolved in 0.9% of saline (w/w)) respectively by intragastric injection and both kept on feeding with the HFD. The fasting blood glucose, body weight, and food consumption were monitored weekly during 28 days of treatment. In the last day of treatment, the mice were fasted for 12 h, serum samples were obtained from orbital sinus and centrifuged at 3500 rpm for 15 min immediately. All mice were killed by cervical dislocation after taking blood and whole pancreas and parts of livers were excised for pathological histology analysis by hematoxylin and eosin (HE) staining. The rest of liver and small intestines tissues were excised from the mice promptly and frozen with liquid nitrogen and stored at − 80 ºC. Insulin sensitivity measurement Oral glucose tolerance test (OGTT), homeostasis model assessment-insulin resistance index (HOMA-IR) and quantitative insulin-sensitivity check index (QUICKI) were calculated to evaluate the sensitivity of insulin. OGTT was performed according to the method reported by Shirwaikar et al. after overnight fasting (Shirwaikar, Rajendran, Kumar, & Bodla, 2004). Glucose (2000 mg/kg) was intragastrically injected and blood samples were collected from the orbital sinus of mice at 0, 15, 30, 60, 90, and 120 min after administration, respectively. The serum obtained by centrifugation was used for measuring serum glucose levels with the glucose oxidase-peroxidase glucose assay kit (Jiancheng, Nanjing, China). The concentrations of fasting serum glucose and fasting serum insulin were measured by glucose assay kit and mouse insulin enzyme-linked immunosorbent assay (ELISA) kits (Jiancheng, Nanjing, China) at the last of treatment, respectively. HOMA-IR and QUICKI were calculated as follows (Motamed et al., 2015; Zhu et al., 2013): HOMA-IR = FBG × FINS /405. QUICKI = 1/log (FBG + FINS). FBG was fasting blood glucose, mg/dL and FINS was fasting food insulin, mU/L Serum lipids Measurement Levels of serum TC, TG, LDL, HDL, and FFA were measured according to the instructions of the each assay kit (Jiancheng, Nanjing, China).

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Oxidative stress measurement of the serum and liver Liver tissues were homogenized in 0.1 g/mL isotonic physiological saline at 0 ºC. The liver suspension was centrifuged at 3000 rpm for 10 min at 4 ºC to achieve liver supernatant. Total protein content was measured following the BCA method. Then, the activity of the SOD, GSH-Px, CAT, T-AOC, and MDA levels of both blood serum and liver supernatant were measured by corresponding assay kits, respectively (Jiancheng, Nanjing, China). Measurement of hexokinase, pyruvate kinase, alkaline phosphatase, alanine transaminase and aspartate aminotransferase activity Liver supernatants were also used for measuring the activity of pyruvate kinase, hexokinase, alkaline phosphatase, alanine transaminase, and aspartate aminotransferase by corresponding assay kits, respectively (Jiancheng, Nanjing, China). Measurement of α-D-glucosidase of small intestines Small intestine tissues were homogenized in 0.1 g/mL isotonic physiological saline at 0 ºC. The small intestine suspension was centrifuged at 3000 rpm for 10 min at 4 ºC to achieve small intestine supernatant. Total protein content was measured following the BCA method. Then, the activity of α-D-glucosidase was measured by the method described in 2.10 (Ademiluyi, Oboh, Boligon, & Athayde, 2014). Statistical analysis Data were shown as a mean ± standard deviation for triplicate determinations of each sample. One-way analysis of variance (ANOVA) was used to evaluate statistical significance by IBM SPSS Statistics (Version 19.0). Probability values <0.05 and <0.01 were considered as statistically significant and highly significant, respectively. Results and Discussion Preparation and basic physicochemical property of SERP1 SERP1 was obtained after ethanol precipitation, macroporous resin D101 absorption, and DEAE-52 column purification with a final yield of 1.30%. The physicochemical properties of SERP1 were elucidated by the following results. Neutral carbohydrate content and acidic carbohydrate content of SERP1 were 40.83 ± 1.31% and 65.48 ± 0.78%, respectively. A negative response to the Bradford test and no absorption at 260 nm or 280 nm in the UV spectrum indicated the absence of protein and nucleic acid in SERP1. The optical rotation value of + 87.5° for SERP1 indicated that both α and β configurations existed in SERP1. Elemental analysis showed SERP1 contained 30.45% of C, 5.63% of H, and 63.92% of O. The high-performance size exclusion chromatography (HPSEC) was employed to determine the homogeneity and relative molecular weight of SERP1. As shown in Fig. 1A, SERP1 eluted as a single and symmetric peak, which indicated that SERP1 was a homogeneous polysaccharide. The average molecular weight of SERP1 was calculated to be 6.35 × 106 Da based on the calibration of the standard curve. 8

Determination of monosaccharide composition was performed by TFA hydrolysis and HPAEC-PAD analysis. As shown in Fig. 1C and 1D, the high peak around 40 min was the gradient elution solvent peak which was caused by the addition of CH3COONa (NaOH 200 mmol/L, CH3COONa 200 mmol/L, and ultrapure water) and SERP1 was consisted of nine monosaccharides, including fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), mannose (Man), xylose (Xyl), galacturonic acid (GalA) and glucuronic acid (GlcA), with the molar ratio of 3.30: 8.75: 11.82: 19.41: 8.77: 1.00: 10.77: 72.61: 27.51. Structure characterization of SERP1 Not only the monosaccharide composition but also the chain conformation of polysaccharide has a large impact on its biological activities. The accuracy of relative molecular weight from HPSEC depends on the similarity between the samples and the standards. While HPSEC-MALLS could be used to determine the macromolecule’s absolute molecular weight in order to eliminate the influence of standard calibration. In the method of HPSEC-MALLS, the dn/dc value is an important index which describes the differences between the refractive index (n) with the concentration (c) (Kisonen et al., 2012). Fig. 1G-1J showed the results of advanced structural analysis of SERP1. The dn/dc of SERP1 was calculated to be 0.1353 (± 0.2415 %) mL/g from Fig. 1G and 1H. As shown in Fig. 1I and 1J, both RI and LS signals of SERP1 were single peaks. But LS signal exhibited as a symmetric peak at the high-molecularweight region while RI signal appeared at the low-molecular-weight region. This was because a specific molecular weight molecule in high concentration was able to exhibit an obvious peak in RI signal due to the concentration-dependent manner of RI. And the polymerization of SERP1 molecules with high-molecular-weight was only able to be observed in LS due to low concentration. As calculated from Fig. 1G-1J, the absolute molecular weight of SERP1 was determined to be 4.208 × 104 (± 5.062 %) Da while Mw/Mn was 2.948 (± 7.874%). The absolute molecular weight of SERP1 determined by HPSEC-MALLS was much lower than that of HPSEC, hydration effect of SERP1 could be one possible reason. Relative molecular weight was a calculation based on standards with different molecular weights. The difference of structure and conformation between SERP1 and standards may also lead to the distinct results compared with these two methods. In Fig. 1B, the IR spectrum of SERP1 showed typical absorption peaks of acidic polysaccharides. In the spectrum, a broadband at 3442.8 cm-1 exhibited the O-H stretch vibration and the peak at 2936.2 cm-1 was assigned to the C-H stretch vibration of CH2. The absorption peaks at 1737.4 cm-1, 1416.3 cm-1, and 1248.2 cm-1 were the characteristic absorptions of C=O stretching vibration, C-O stretching vibration and O-H bending vibration, respectively, which indicated the presence of -COOH in SERP1 (Chen, Zhang, Jiang, Mu, & Miao, 2012). The stretching peaks at 1142.5 cm-1, 1076.3 cm-1, 1047.6 cm-1 , and 1023.6 cm-1 indicated the presence of C-O bonds and furanose ring in the monosaccharides of SERP1 (Jin et al., 2012). These results confirmed that SERP1 was an acidic polysaccharide. The absorption at 1332.7 cm−1 was possibly due to symmetrical and non-symmetrical CH3 bending vibration, which 9

indicated the presence of rhamnose (Zhu et al., 2013). The moderate intense signals ranging from 1350 to 1450 cm−1 corresponded to symmetrical deformations vibration of CH2 and COH groups. The peaks at 896.4 cm-1 and 812.7 cm-1 confirmed the existence of both α- and β-glycosidic bonds and pyranose ring in the monosaccharide blocks of SERP1. Given that the moderate absorbance at 812.7 cm-1 and the weak absorbance at 896.4 cm-1, α-glycosidic bonds was dominant in SERP1, which was in agreement with the result of optical rotation. The structural features of SERP1 were further elucidated by NMR spectral analysis. As shown in Fig. 1E and 1F, signals of 1H-NMR and 13C-NMR spectra of SERP1 were assigned according to monosaccharide composition analysis and literature values (Catoire, Goldberg, Pierron, Morvan, & Hervé Du Penhoat, 1998; Huang, Jin, Zhang, Cheung, & Kennedy, 2007; Silva et al., 2012; Sun, Cui, Tang, & Gu, 2010; Sun, Zhang, Zhang, & Niu, 2010; Yu et al., 2009). Details of the assignment were given in Table 1. Briefly, the anomeric proton signals (δ 4.2 to 5.35 ppm) of SERP1 in the 1HNMR spectrum (Fig. 1E) implied that polysaccharide residues were connected by both α and β-glycosidic bonds. The intense signal at δ 3.86 ppm was attributed to the methyl groups of esterified galacturonic acid. Moreover, typical signals were observed due to the C-6 carboxyl group of galacturonic acid units at δ 177.98 and 173.59 ppm in the 13C-NMR spectrum (Fig. 1F). The two carboxyl signals confirmed the presence of free and esterified carboxyl groups in galacturonic acid. The resonance peak at δ 55.54 ppm was assigned to the methyl ester of galacturonic acid. Signals around δ 98.76-107.67 ppm further confirmed that SERP1 contained both α- and β- anomeric configurations of monosaccharide residues. In general, signals in the 13C-NMR spectrum ranging from δ 67 to 70 ppm confirmed the presence of (1→6) glycosidic linkages in SERP1, while that of δ 80-83 ppm implied the presence of (1→3/4) glycosidic linkages. The NMR results, accompanied with monosaccharide composition and IR results, indicated that SERP1 had a backbone chain mainly composed of 1,4-linked α-D-GalpA, 1,4-linked 6-OMe-α-D-GalpA, 1,5-linked α-LAraf, 1,4-linked α-D-GlcpA, 1,3-linked β-D-Galp, 1,4-linked α-D-Glcp, 1,6-linked βD-Glcp, 1,2-linked α-Rhap, and 1,4,6-linked β-D-Glcp. SERP1 had extremely complicated structure due to its composition of nine monosaccharides. In this part, several typical peaks in spectra of IR, 1H-NMR, and 13 C-NMR confirmed the monosaccharide composition. It was concluded that SERP1 consisted of furan and pyran rings with both α- and β-glycosidic bonds. From the above results, SERP1 was considered as a novel polysaccharide with specific physicochemical properties and structure information from S. glabra extract residue by using the different purification processes with that from the whole S. glabra plant. α-glucosidase inhibitory activity in vitro Mammalian α-glucosidase, located in the brush border of the small intestine, is the key enzyme catalyzing the carbohydrates digestive process (Bisht, Kant, & Kumar, 2013). Hence, α-glucosidase inhibitors are able to delay the release of glucose from dietary complex carbohydrates and retard absorption of glucose, resulting in 10

diminished postprandial plasma blood glucose level and suppression of postprandial hyperglycemia. Thus, oral administration of α-glucosidase inhibitors could control non-insulin-dependent diabetes mellitus (NIDDM) effectively by improving postprandial hyperglycemia (Fujita & Yamagami, 2001). Published reports indicated αglucosidase inhibitors such as acarbose, miglitol, voglibose from microorganisms, nojirimycin and 1-deoxynojirimycin from plants affected blood glucose levels after up taking food (Kim, Jeong, Wang, Lee, & Rhee, 2005). Fig. 2A showed the inhibitory on α-glucosidase of SERP1 and acarbose at different concentrations. Both SERP1 and acarbose exhibited a dose-dependent inhibition on αglucosidase, while SERP1 showed stronger inhibition potential than that of acarbose at all tested concentrations. The α-glucosidase half maximal inhibitory concentration (IC50) of SERP1 and acarbose were 49.01 µg/mL and 148.3 µg/mL, respectively. The results proved that SERP1 had a better inhibitory activity on α-glucosidase than acarbose in vitro. Effect on blood glucose level, body weight and food intake of SERP1 in diabetic mice SERP1 exhibited a great α-glucosidase inhibition activity in vitro in the previous experiment. Thus, the anti-diabetes effect evaluation of SERP1 was performed with HFD/STZ induced diabetic mice. Apart from model group and control group, other two groups of mice were also fed with HFD alone and injection with STZ alone respectively to choose the suitable model of diabetic mice. As shown in

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Table 2, fasting blood glucose (FBG) of the HFD/STZ group was highly significantly raised compared with the control group, STZ alone group, and HFD alone group. Moreover, the HFD/STZ group appeared to show lowest insulin concentrations among all groups, suggesting the formation of insulin resistance in this group. Therefore, mice with fasting blood glucose over 7.80 mmol/L in the HFD/STZ group were chosen as diabetic mice for next step study. Acarbose and metformin were chosen as positive drugs. FBG levels of all groups during the 28-day administration were shown in Table 3. FBG levels of the control group and model group were almost constant and that of the model group remained significantly higher than that of the control group. Acarbose and metformin group showed a significant hypoglycemic effect at day 28 and FBG levels of each group reducing to 6.95 and 6.90 mmol/L respectively, which was significantly lower than that of model group. Moreover, The SERP1 groups exhibited a highly significant reduction in FBG level compared with that of the model group. And FBG levels of SERP1-H group were even lower than that of both the positive drug groups. Simultaneously, the body weight and food intake were also monitored during whole 28-day administration, whereas there were no obvious changes in body weight among all groups after 28 days of treatment (data not shown). On the other hand, the food intake of the model group increased remarkably compared to those of other groups, while the food intake amounts decreased in the SERP1 group, acarbose group, and metformin group to that of model group (data not shown). Effect of SERP1 on the oral glucose tolerance test Oral glucose tolerance test is used for testing whether the experimental subject has regular glucose tolerance or type 2 diabetes mellitus. It is considered as an essential method to evaluate whether the experimental subject has impaired glucose tolerance activity (Bartoli, Fra, & Schianca, 2011). Blood glucose will reach to a high level in 0.5 - 1 h after the administration due to the hyposecretion of insulin, which indicates the impairment of glucose tolerance. Results of all groups were presented in Fig. 2B and the AUC for blood glucose concentrations during the OGTT was shown in Table 3. The model group showed obviously impaired glucose tolerance while SERP1 groups and the positive drug groups presented significant decreases in blood glucose levels of diabetic mice in 0 - 2 h after a single oral uptake of glucose (2 g/kg). The model group exhibited an almost 4-fold increase of blood glucose level compared with that of the control group in 30 min, and the levels of blood glucose from diabetic mice in the model group were unable to be down-regulated to the normal levels in 120 min. While glucose tolerance activities of SERP1 groups were increased, of which SERP1-H group exhibited the strongest effects, suggesting that SERP1 could improve the glucose tolerance activities of diabetic mice. Effects of SERP1 on insulin resistance Insulin resistance is the reason for the generation of many metabolic abnormalities 12

associated with metabolic syndrome (MetS) (Eckel, Grundy, & Zimmet, 2005). Metabolic syndrome includes many factors of cardiovascular risk including obesity, dyslipidemia, hypertension, and hyperglycemia. Hyperinsulinemic-euglycemic clamp technique is regarded as the best way to diagnose insulin resistance. In addition, several low costs and minimally invasive methods have been invented to evaluate insulin resistance with predictive accuracy (Singh & Saxena, 2010). Among these methods, homeostatic model assessment-insulin resistance (HOMA-IR) index and quantitative insulin sensitivity check index (QUICKI) are simply calculated by fasting blood glucose and insulin levels. These indexes are also effective and useful tools to assess insulin resistance in clinical practices (Hrebicek, Janout, Malinčíková, Horáková, & Čížek, 2009). From Fig. 2C, the model group presented higher HOMAIR index than that of normal group. SERP1-H and SERP1-M groups significantly decreased the HOMA-IR index comparing to that of the model group. Fig. 2D showed the QUICKI index of each group, the model group revealed lower QUICKI index than that of normal group. While SERP1 groups significantly improved the QUICKI index. Therefore, decreased values of HOMA-IR and increased values of QUICKI from all groups of SERP1 indicated that SERP1 had the ability to relieve the insulin resistance. Improvement effects of SERP1 on lipid metabolic in serum Diabetes mellitus is one of the most common metabolic diseases. Abnormality of lipid metabolism is generally regarded as the indicator of the development and status of diabetes mellitus (Gupta, Sharma, Bansal, & Prabhu, 2009). In order to investigate the mechanism of SERP1 in the treatment of diabetes mellitus, lipid metabolic parameters were determined in all groups. As shown in Fig. 3A-3E, serum TC, TG, HDL-C, LDL-C, and FFA level of the model group were significantly higher than those of the control group (p < 0.01), while the HDL-C level of the model group decreased compared to that of the control group (p < 0.05). Both acarbose and metformin showed obvious hypolipidemic effects. In addition, SERP1-H group and positive drug groups exhibited significantly reduced levels of TC, TG, LDL-C, and FFA (p < 0.01) and SERP1-H and SERP1-M group presented increased HDL-C levels compared to the model group (p < 0.05). High levels of TG and/or TC concentrations and a low ratio of HDL/LDL are generally considered as characteristics of dyslipidemia (Xiong, Gu, Wang, Sun, & Liu, 2013). These results also indicated that FFA secretion was inhibited in SERP1 groups, especially in SERP1-H group, which was consistent with the result of HOMA-IR and QUICKI determination. In the experiment, concentrations of TC, TG, and LDL-C in diabetic mice were dose-dependently decreased after treatment with SERP1 for 28 days, and concentrations of HDL-C increased in a dosedependent manner. Those results showed that SERP1, effective on hyperglycemia and dyslipidemia, was also able to decrease the risk of atherosclerosis and coronary heart disease. High level of FFA was also able to induce the generation of atherosclerosis by over-production of very-low-density lipoprotein and alteration in glucose metabolism. Therefore, SERP1 could relieve insulin resistance to some extent and have a beneficial effect on dyslipidemia.

13

Improvement effects of SERP1 on activities of antioxidant enzymes and MDA levels in serum and liver Diabetes mellitus is typically correlated with increased formation of free radicals or impaired antioxidant defense mechanism. Growing oxidative stress is considered as one of the participants in the generation and progression of diabetes mellitus and its complications (Maritim, Sanders, & Watkins, 2003). On the other hand, Antioxidant defense system protects the body from the adverse effects of free radical generation under physiological condition. In diabetes mellitus, hyperglycemia and wrong protein glycation may damage the native antioxidant system. The formation of free radicals at excessive rates leads to the consumption of antioxidant defense components, which could lead to the disorder of cellular functions and lipid peroxidation (Preetha, Devi, & Rajamohan, 2012). Thus, the improving activities of antioxidant enzymes including T-AOC, SOD, CAT, and GSH-Px represent the increasing response of antioxidant defense systems (Liu, Sun, Rao, Su, & Yang, 2013). . As shown in Fig. 3F-3O, the liver T-AOC, CAT, and SOD activity could be significantly decreased in model group compared with that of the control group (p < 0.05), which indicated diabetes mellitus was associated with oxidative stress. As the major product of lipid peroxidation, the level of MDA indicates the development of lipid peroxidation. MDA levels in both liver and serum of the model group were obviously increased compared with that of the control group (p < 0.01). On the other hand, the activities of T-AOC, SOD, CAT and GSH-Px in the liver and serum were significantly decreased in the SERP1-H and SERP1-M group compared with that of the model group (p < 0.01). Meanwhile, the SERP1-M and SERP1-H group showed a significant increase in the liver GSH-Px activity compared to that of model group (p < 0.05). In addition, SERP1 could dose-dependently decrease the MDA level in liver and serum. In positive drugs groups, both acarbose and metformin were able to increase the activities of T-AOC, SOD, CAT, and GSH-Px and decrease MDA level in liver and serum. Moreover, metformin exhibited more effective than acarbose, indicating that metformin had better effects on activities of antioxidant enzymes and MDA levels. These results showed that SERP1 could improve the activities of antioxidant enzymes and reduce the MDA levels. α-glucosidase inhibitory activity in vivo The activity of α-glucosidase in the mucous membrane of small intestine was measured to evaluate whether the hypoglycemic effect of SERP1 was associated with the inhibitory activity of α-glucosidase. From Fig. 4A, the α-D-glucosidase activity of model group was significantly increased compared with that of the control group. Both of SERP1-H group and acarbose group exhibited significant decreases of αglucosidase activity. Moreover, SERP1 was able to decrease the activity of α-Dglucosidase significantly in a dose-dependent manner. As a result, inhibition of α-Dglucosidase could be one of the mechanisms for SERP1’s hypoglycemic effect.

14

Improvement effects of SERP1 on activities of pyruvate kinase and hexokinase in liver Previous studies stated that insulin resistance comes along with depressed glucose utilization and decreased activities of hepatic glycolytic enzymes including hexokinase (HK) and pyruvate kinase (PK) (Agius, 2007). HK and PK are key enzymes mainly distributed in the liver and play important roles in glucose metabolism. HK is an important enzyme maintaining glucose homeostasis and is regarded as an indicator in regulating glucose hepatic release/uptake, while PK is a significant rate-limiting enzyme of glucose aerobic oxidation. Therefore, enhancement of glucose utilization by improving the activities of HK and PK is able to alleviate insulin resistance. As shown in Fig. 4B, SERP1-M, SERP1-H, acarbose and metformin groups obviously increase the activity of PK compared with the model group which exhibited a significant decrease level of PK’s activity compared with control group. While in Fig. 4C, only SERP1-M, and SERP1-H showed significant increases of HK’s activity compared with the model group. The results indicated that SERP1 was able to alleviate diabetes mellitus with improving glucose utilization. Improvement effects of SERP1 on activities of alkaline phosphatase, glutamicpyruvic transaminase and aspartate aminotransferase in liver Liver alkaline phosphatase (AKP), glutamic-pyruvic transaminase (GPT) and aspartate aminotransferase (AST) are usually analyzed in the diagnosis of hepatic disease. The increasing activities of these liver transaminases are closely associated with liver fat accumulation, alcoholic liver injury and hepatitis (Tziomalos, Athyros, & Karagiannis, 2012). Non-alcoholic fatty liver disease, liver cirrhosis, liver tumor and bacterial liver abscess are typical liver complications blamed for diabetes mellitus, and subsequent disorder of glycolipid metabolism in complications exacerbate the diabetes mellitus in turn. Moreover, these liver transaminases are significant indicators of hepatic function and will be released into blood manifesting as an increase of enzyme activities in blood in the broken liver. In Fig. 4D-4F, activities of all the transaminases were significantly increased in model group compared with that of the control group, inferring the success of modeling damaged liver. In addition, SERP1 dose-dependently decreased the activities of transaminases, of which SERP1 in high dose presented most significant decrease compared to that of model group. The results showed that SERP1 was able to protect the liver from liver damage complicated with diabetes mellitus. Protection effects of SERP1 on pancreas and liver From Fig. 5, degenerated pathologic morphologies were observed in the pancreas and liver in model groups. Moreover, decreased islet cells and small pancreatic island were presented in the HE-stained pancreas tissue sections of model group. However, the control group indicated no obvious change in pancreas histology after 28 days of treatment. Meanwhile, SERP1 treatment significantly alleviated histopathological changes, and all doses of SERP1, acarbose, and metformin were able to protect pancreas tissues to some extent from injury blamed by diabetes mellitus. For liver 15

tissue sections, hepatic steatosis, characterized as ballooned hepatocytes, and hepatomegaly could be observed clearly in the model group. While the treatments of SERP1, acarbose, and metformin were able to protect and repair liver tissues effectively. Result inferred that SERP1 protected and repaired pancreas and liver tissues from diabetes mellitus. The histopathology sliced images showed evident results that SERP1 as a novel polysaccharide was able to take the control of diabetic complications and organ damage. Conclusion SERP1 was successfully prepared from the residue of S. glabra with the assistance of macroporous resin and DEAE-cellulose-52. The absolute molecular weight of SERP1 in the aqueous phase is 4.208×104 Da detected by HPLC and MALLS. Results of spectropolarimeter and infrared spectroscopy proved that the terminal carbon was mainly α-configuration. SERP1 is composed of fucose, rhamnose, arabinose, galactose, glucose, mannose, xylose, galacturonic acid, and glucuronic acid at a ratio of 3.30: 8.75: 11.82: 19.41: 8.77: 1.00: 10.77: 72.61: 27.51. NMR shows that SERP1 is composed of 1,4-linked α-D-galacturonic acid, methyl-esterified 1,4-linked α-Dgalacturonic acid, 1,4-linked α-D-Glucuronic acid, 1,5-linked α-L-arabinose, 1,3linked β-D-galactose, 1,4-linked α-D-glucose, 1,4,6-linked β-D-glucose, 1,6-linked βD-glucose and 1,2-linked rhamnose. SERP1 exhibited high α-glucosidase inhibitory activity in vitro. While in in vivo assays, SERP1 showed a potent antihyperglycemic effect on HFD/STZ induced diabetic mice for its ability to relieve insulin resistance, reduce postprandial blood glucose levels and ameliorate lipid metabolism, as well as alleviate oxidative stress. SERP1 could be explored as an ingredient of functional foods in the treatment of diabetes and complications. Acknowledgement This study was supported by the National Natural Science Foundation of China (No.81673589, 81473216), Innovative Scientific Research Team Fund and Qing Lan Project of Jiangsu Province, 13th of Six Talent Peak Foundation of Jiangsu Province (SWYY-068), "333 project" of Jiangsu Province in the fourth phase (No. BRA2015322), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Ademiluyi, A. O., Oboh, G., Boligon, A. A., & Athayde, M. L. (2014). Effect of fermented soybean condiment supplemented diet on α-amylase and α-glucosidase activities in Streptozotocin-induced diabetic rats. Journal of Functional Foods, 9, 1–9. Agius, L. (2007). New hepatic targets for glycaemic control in diabetes. Best Practice and Research in Clinical Endocrinology and Metabolism, 21, 587–605. Bartoli, E., Fra, G. P., & Schianca, G. P. C. (2011). The oral glucose tolerance test (OGTT) revisited. European Journal of Internal Medicine, 22, 8–12. Bisht, S., Kant, R., & Kumar, V. (2013). α-D-Glucosidase inhibitory activity of 16

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Journal of Diabetes and Its Complications. doi:10.1016/j.jdiacomp.2015.11.019 Preetha, P. P., Devi, V. G., & Rajamohan, T. (2012). Hypoglycemic and antioxidant potential of coconut water in experimental diabetes. Food & Function, 3, 753–7. Ren, C., Zhang, Y., Cui, W., Lu, G., Wang, Y., Gao, H., et al. (2015). A polysaccharide extract of mulberry leaf ameliorates hepatic glucose metabolism and insulin signaling in rats with type 2 diabetes induced by high fat-diet and streptozotocin. International Journal of Biological Macromolecules, 72, 951–959. Rother, K. (2014). Diabetes Treatment — Bridging the Divide. Journal of Medicine, 356, 1499–1501. Sendrayaperumal, V., Iyyam Pillai, S., & Subramanian, S. (2014). Design, synthesis and characterization of zinc-morin, a metal flavonol complex and evaluation of its antidiabetic potential in HFD-STZ induced type 2 diabetes in rats. ChemicoBiological Interactions, 219, 9–17. Shirwaikar, A., Rajendran, K., Kumar, C. D., & Bodla, R. (2004). Antidiabetic activity of aqueous leaf extract of Annona squamosa in streptozotocin-nicotinamide type 2 diabetic rats. Journal of Ethnopharmacology, 91, 171–175. Silva, D. C., Freitas, A. L. P., Barros, F. C. N., Lins, K. O. A. L., Alves, A. P. N. N., Alencar, N. M. N., et al. (2012). Polysaccharide isolated from Passiflora edulis: Characterization and antitumor properties. Carbohydrate Polymers, 87, 139–145. Singh, B., & Saxena, A. (2010). Surrogate markers of insulin resistance: A review. World Journal of Diabetes, 1, 36–47. Sun, Y., Cui, S. W., Tang, J., & Gu, X. (2010). Structural features of pectic polysaccharide from Angelica sinensis (Oliv.) Diels. Carbohydrate Polymers, 80, 545–551. Sun, Z., Zhang, L., Zhang, B., & Niu, T. (2010). Structural characterisation and antioxidant properties of polysaccharides from the fruiting bodies of Russula virescens. Food Chemistry, 118, 675–680. The State of Pharmacopoeia Commission of People’s Republic of China. (2015). Pharmacopoeia Commission of People’s Republic of China. In Pharmcopeia of People’s Republic of China, 1 (pp. 409–410). Beijing: Chemical Industry Press. Tziomalos, K., Athyros, V. G., & Karagiannis, A. (2012). Non-alcoholic fatty liver disease in type 2 diabetes: Pathogenesis and treatment options. Current Vascular Pharmacology, 10, 162–172. Xiong, W. T., Gu, L., Wang, C., Sun, H. X., & Liu, X. (2013). Anti-hyperglycemic and hypolipidemic effects of Cistanche tubulosa in type 2 diabetic db/db mice. Journal of Ethnopharmacology, 150, 935–945. Xu, W., Zhou, Q., Yin, J., Yao, Y., & Zhang, J. (2015). Anti-diabetic effects of polysaccharides from Talinum triangulare in streptozotocin (STZ)-induced type 2 diabetic male mice. International Journal of Biological Macromolecules, 72, 575– 579. Yu, R., Yin, Y., Yang, W., Ma, W., Yang, L., Chen, X., et al. (2009). Structural elucidation and biological activity of a novel polysaccharide by alkaline extraction from cultured Cordyceps militaris. Carbohydrate Polymers, 75, 166–171. Zhang, Z., Khan, N. M., Nunez, K. M., Chess, E. K., & Szabo, C. M. (2012). 19

Complete monosaccharide analysis by high-performance anion-exchange chromatography with pulsed amperometric detection. Analytical Chemistry, 84, 4104– 4110. Zhang, Z., Zheng, Y., Zhu, R., Zhu, Y., Yao, W., Liu, W., et al. (2014). The ERK/eIF4F/Bcl-XL pathway mediates SGP-2 induced osteosarcoma cells apoptosis in vitro and in vivo. Cancer Letters, 352, 203–213. Zhou, H., Liang, J., Lv, D., Hu, Y., Zhu, Y., Si, J., et al. (2013). Characterization of phenolics of Sarcandra glabra by non-targeted high-performance liquid chromatography fingerprinting and following targeted electrospray ionisation tandem mass spectrometry / time-of-flight mass spectrome- try analyses. Food Chemistry, 138, 2390–2398. Zhu, J., Liu, W., Yu, J., Zou, S., Wang, J., Yao, W., et al. (2013). Characterization and hypoglycemic effect of a polysaccharide extracted from the fruit of Lycium barbarum L. Carbohydrate Polymers, 98, 8–16.

20

Fig. 1 Basic physicochemical properties and structure characterization of SERP1. HPSEC result of SERP1 detected by RID (A). IR spectrum of SERP1 (B). HPAECPAD chromatograms of the mixed standard monosaccharide (C) and monosaccharide composition of SERP1 (D). 1H-NMR spectrum (E) and 13C-NMR spectrum (F) of SERP1. dn/dc determination of SERP1 (G) and (H), HPSEC-RI and later scattering 21

curves of SERP1 (I) and (J).

Fig. 2 Inhibitory on α-glucosidase of SERP1 and acarbose at different concentrations in vitro, n=3. *p < 0.05, **p < 0.01 (A). Oral glucose tolerance test after overnight fasting at the last day of treatment (B). HOMA-IR index (C) and QUICKI index (D) of the diabetic mice in all groups. n=8. #p < 0.01 vs. control group, *p < 0.05 vs. model group, **p < 0.01 vs. model group, ***p < 0.001 vs. model group.

22

23

Fig. 3 Improvement on dyslipidemia and alleviation on oxidative stress in both liver and serum of SERP1. Levels of serum total cholesterol (TC) (A), serum triglyceride (TG) (B), serum high density lipoprotein cholesterin (HDL-C) (C), serum low density lipoprotein cholesterin (LDL-C) (D), free fatty acid (FFA) (E), liver (F) and serum (K) total antioxidant capacity (T-AOC), liver (G) and serum (L) superoxide dismutase (SOD), liver (H) and serum (M) catalase (CAT), liver (I) and serum (N) glutathione peroxidase (GSH-Px), and liver (J) and serum (O) malonaldehyde (MDA) were measured at the end of treatment. n= 8. #p< 0.05 vs. control group, ##p< 0.01 vs. control group, *p< 0.05 vs. model group, **p< 0.01 vs. model group

24

Fig. 4 Inhibitory on α-glucosidase of SERP1 and acarbose in the small intestine (A). Activities of pyruvate kinase (PK) (B), hexokinase (HK) (C), alkaline phosphatase (AKP) (D), glutamic-pyruvic transaminase (GPT) (E), and aspartate aminotransferase (AST) (F) in liver were measured at the last day of treatment. n=8. #p< 0.05 vs. control group, ##p< 0.01 vs. control group, *p< 0.05 vs. model group, **p< 0.01 vs. model group.

25

Fig. 5 Protection effect of SERP1 to the pancreas (100×) and liver (200×) in mice by HE staining. Control group (A and H), Model group (B and I), SERP1-L group (C and J), SERP1-M group (D and K), SERP1-H group (E and L), acarbose group (F and M), 26

and metformin group (G and N) presented different degrees of pancreas and liver injuries.

27

Table 1 Chemical shifts of the signals in the 1H and 13C NMR spectra of SERP1 Chemical shifts, (ppm) Glycosyl residues C-1/ C-2/ C-3/ C-4/ C-5/ C-6/ CH3O H-1 H-2 H-3 H-4 H-5 H-6 →4)-α-D-GalpA-(1→ 101.65 68.91 70.62 79.00 73.17 177.98 5.09 3.86 4.13 4.43 4.94 →4)-6-OMe-α-D-GalpA- 98.76 68.91 70.62 80.54 72.16 173.59 55.54 (1→ 4.94 3.76 4.06 4.42 5.16 3.82 α-D-Glcp-(1→ 98.76 72.00 73.42 70.62 72.96 63.55 4.94 3.51 3.56 3.45 3.82 3.76 β-D-Galp-(1→ 107.67 73.17 73.98 70.76 76.89 63.55 4.42 3.56 3.76 3.91 3.76 3.82 →5)-α-L-Araf -(1→ 109.45 81.02 77.75 81.62 63.84 5.16 4.14 3.91 4.06 3.76 →4,6)-β-D-Glcp-(1→ 105.44 72.96 77.75 79.00 76.89 72.00 4.42 3.37 3.56 3.45 3.56 3.86 →6)-β-D-Glcp-(1→ 106.08 76.22 77.75 72.96 78.23 71.35 4.42 3.37 3.56 3.37 3.56 3.82 →2-α-Rha-(1→ 102.03 79.00 70.62 74.92 70.62 17.92 5.41 3.44 3.91 3.51 3.76 1.30 →4)-α-D-GlcpA-(1→ 103.01 83.90 83.56 81.62 81.33 176.27 5.10 3.45 3.76 3.82 4.27

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Table 2 Characterization of the HFD/STZ mouse model fasting blood glucose (mmol/L)

Serum insulin (mU/L)

Control

6.13±1.96

3.26±0.28

STZ alone

8.55±1.45*

2.38±0.19*

HFD alone

8.13±0.67*

2.68±0.14

HFD/STZ

10.82±1.12**

2.13±0.20**

C57BL/6J mice were given regular diet and administered saline (Control) or STZ (STZ alone) or given HFD and administered saline (HFD alone) or STZ (HFD/STZ). Levels of fasting plasma glucose and serum insulin were determined during feeding. *p < 0.05, **p < 0.01.

29

Table 3 Effect on the fasting blood glucose level and OGTT result of SERP1 OGTT

Fasting blood glucose (mmol/L) Group

AUC 0 day

7 days

14 days

21 days

28 days

(h·mmol/L)

Control

6.13±0.96

6.33±0.74

6.21±1.01

6.47±1.11

6.36±0.95

18.88±2.44

Model

10.14±1.31#

11.31±0.45#

11.46±0.98#

12.29±1.58#

11.81±1.26#

40.54±4.97#

SERP1-L

10.74±0.54#

11.45±1.23

8.21±0.95*

7.56±1.00**

7.66±0.72**

31.55±4.16

SERP1-M

10.67±0.67#

10.98±1.31

8.66±1.88*

7.88±0.33**

7.10±1.44**

25.72±1.50*

SERP1-H

10.53±1.31#

11.39±1.28

8.32±1.29*

7.65±1.12**

6.74±0.44**

26.94±1.73**

Acarbose

10.55±0.98#

9.34±1.23*

8.55±0.81*

7.40±0.77**

6.95±1.99**

26.48±2.26**

Metformin

10.90±1.11#

9.55±0.71*

8.65±1.36*

7.16±0.33**

6.90±2.27**

24.32±1.68**

FBG levels were determined at the specified time. OGTT was performed at the end of the treatment. n=8. #p < 0.01 vs. control group, *p < 0.05 vs. model group, **p < 0.01 vs. model group.

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