Structural characterization and inhibition on α-d -glucosidase activity of non-starch polysaccharides from Fagopyrum tartaricum

Structural characterization and inhibition on α-d -glucosidase activity of non-starch polysaccharides from Fagopyrum tartaricum

Carbohydrate Polymers 153 (2016) 679–685 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/c...

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Carbohydrate Polymers 153 (2016) 679–685

Contents lists available at ScienceDirect

Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Structural characterization and inhibition on ␣-d-glucosidase activity of non-starch polysaccharides from Fagopyrum tartaricum Xiao-Ting Wang a , Zhen-Yuan Zhu a,b,∗ , Liang Zhao a , Hui-Qing Sun a , Meng Meng a , Jin-Yu Zhang a , Yong-min Zhang c a Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China b Tianjin Food Safety & Low Carbon Manufacturing Collaborative Innovation Center, 300457 Tianjin, PR China c Université Pierre et Marie Curie-Paris 6, Institut Parisien de Chimie Moléculaire, UMR CNRS 8232, 4 Place Jussieu, 75005 Paris, France

a r t i c l e

i n f o

Article history: Received 3 July 2016 Received in revised form 5 August 2016 Accepted 8 August 2016 Available online 9 August 2016 Chemical compounds studied in this article: Ethanol (PubChem CID: 702) Butanol (PubChem CID: 263) Chloroform (PubChem CID: 6212) Sodium hydroxide (PubChem CID:14798) Trifluoroacetic acid(PubChem CID: 6422) Dimethyl sulfoxide (PubChem CID: 679) Acetic acid (PubChem CID: 176) Keywords: Fagopyrum tartaricum Polysaccharide Characterization ␣-d-glucosidase

a b s t r a c t In the present study, the crude polysaccharide was extracted from Fagopyrum tartaricum and purified by Sephadex G-25 and G-75 column to produce a polysaccharide fraction termed TBP-II. Its average molecular weight was 26 kDa. The structural characterization of TBP-II was investigated by gas chromatography, periodate oxidation-Smith degradation, Methylation and NMR. Congo red was applied to explore its advanced structures. The results revealed that chemical composition and structural characteristic of TBP-II was mainly consisted of galactose, arabinose, xylose and glucose with a molar ratio of 0.7:1:6.3:74.2. The backbone of TBP-II was composed of (1 → 4)-linked ␣-d-glucopyranosyl (Glcp), while the branches comprised of (1 → 3)-linked ␣-d-glucopyranosyl (Glcp), (1 → 6)-linked ␣d-galactopyranosyl (Galp) and (1 → 2,4)-linked ␣-d-rhamnopyranosyl (Rhap). The structure of TBP-II was 1,3 and 1,6-branched-galactorhamnoglucan that had a linear backbone of (1 → 4)-linked ␣-dglucopyranose (Glcp). Using Congo red assay showed that it was absent of triple helix structure. The ␣-d-glucosidase inhibitory activity of TBP-II was determined using acarbose as positive control. The result showed that the inhibition rate depended on the concentration of polysaccharides. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Buckwheat (Fagopyrum) belongs to the plant family of Polygonaceae. Fagopyrum esculentum Moench (common buckwheat or sweet buckwheat) and Fagopyrum tartaricum Gaertn (tartary buckwheat or bitter buckwheat) are the two main species (Krkoˇskova´ı & Mra´ızova´ı, 2005). The genus Fagopyrum tartaricum, comprising 15 species in the world, is mainly distributed in the North Temperate Zone. Eight species, including some common crops and medicinal plants occur in China: buckwheat, tartary buckwheat, Fagopyrum urophyllum,etc.

∗ Corresponding author at: Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Science and Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin 300457, PR China. E-mail address: [email protected] (Z.-Y. Zhu). http://dx.doi.org/10.1016/j.carbpol.2016.08.024 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

Fagopyrum tartaricum is an important medicinal and edible herb that is abundant in protein, vitamins, as well as iron, zinc, selenium, and other trace elements. Buckwheat has a high antioxidant activity and is rich in rutin, catechins, and other polyphenols, which have significant dietary value (Oomah & Mazza, 1996; Watanabe, 1998). The main flavonoid of Fagopyrum tartaricum also has a positive effect on reducing capillary vessel brittleness, improving microcirculation, and strengthening body immunity (Tian & Ren, 2007). Fagopyrum tartaricum extracts can effectively reduce blood glucose levels in diabetic rats (Liu et al., 2009). For example, the concentration of rutin, a major polyphenol in buckwheat, can be 81 mg/g in the groats of Fagopyrum tartaricum and 0.2 mg/g in that of common buckwheat(Wijngaard & Arendt, 2006). Moreover, Fagopyrum tartaricum tends to have higher concentrations of certain bioactive phytochemicals (e.g., flavonoids) than common buckwheat. The taste of Fagopyrum tartaricum grains is much bitterer than that of common buckwheat due to the much higher concentrations of flavonoids (Fabjan et al., 2003).

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Although previous chemical studies demonstrated the presence of some phenolics, flavonoid and other compounds in Fagopyrum tartaricum, such as hecogenin, ␤-sitosterol, ferulic acid and shakuchirin(Liu et al., 1983; Yao et al., 1989; Zhang et al., 1994), there are few reports on polysaccharides from Fagopyrum tartaricum and its ␣-glucosidase inhibitory activity. Therefore, the present study aimed to extract, isolate, purify and analyze its structural characterization. In addition, the ␣-Glucosidase inhibitory activity of polysaccharides in vitro was also investigated in order to acquire the most effective higher-performance polysaccharides products and exploit applied potential of polysaccharide in wide pharmacological field. 2. Materials and methods 2.1. Materials and reagents Fagopyrum tartaricum were collected from Zhangjiakou, hebei Province, China. Sephadex G-75 and Sephadex G-25, d-glucose, dxylose, d-galactose, l-rhamnose, d-mannose and d-arabinose were purchased from Sigma Chemical Company. (St. Louis, MO, USA). All other reagents and chemicals used in the study were of analytical grade. 2.2. Extraction, fractionation and purification of polysaccharide The fresh Fagopyrum tartaricum was dried and milled into fine power, passed through an 60 mesh sieve. The dried powder (10 g) was defatted with 95% ethanol at room temperature for 1 h to remove most of the polyphenols and pigments, and the treatment was repeated twice. After filtration, the residues were extracted with 200 mL of distilled water at 80 ◦ C for 2 h, and then repeated for two times. Protease, ␣-amylase and glucoamylase were added into the concentration supernatant to further remove protein and starch to get purified sample. Until the iodine reaction disappear blue, the starch had been removed completely. Afterwards, the supernatant was concentrated via rotary evaporator and precipitated with 4 times volume of 95% (v/v) ethanol at 4 ◦ C overnight. The precipitation was deproteinized with the Sevag reagent (butanol/chloroform, v/v = 1:4) and then lyophilized to produce the substance. The crude polysaccharide was applied to gel filtration on a column of Sephadex G-25, eluted with deionized water at a flow rate of 30 drops/min. The collected eluent was monitored using the phenol-sulfuric acid method. The obtained eluent was pooled, concentrated and lyophilized for further purification. The eluted fractions was subjected to gel filtration on a column of Sephadex G-75, eluted with deionized water at a flow rate of 30 drops/min. Fractions were collected and enriched using the method mentioned above. The total sugar content of the obtained polysaccharide was measured by phenol sulfuric acid method (Lin, Cui et al., 2011; Lin, Dong eta l., 2011). 2.3. UV–vis spectra and optical rotation identification The polysaccharide (1 mg) was dissolved in distilled water, diluted to 1 mg/mL and scanned in the range of 190–400 nm at 25 ◦ C using SP-2102UV spectrophotometer. The sample (10 mg) was dissolved in distilled water (1 mg/mL). Optical rotation was analyzed with a WZZ-2B at 25 ◦ C and calculated as follows: ␣ [␣]tD = I×C ␣ is the optical rotation; t is the temperature; D is the wavelength of light source (589 nm); I is the length of polariscope tube; C is the polysaccharide concentration (g/mL) (Li et al., 2013).

2.4. Color reactions The sample was evaluated for solubility in accordance with the British pharmacopoeia (BP) specification (Kannan, Manivannan, Balasubramaniam, & Kumar, 2010). The nature of polysaccharide was confirmed by sulfuric acid carbazole reaction, coomassie brillian blue, Felhing’s test and iodine- potassium iodide reaction (Kintner & Buren, 1982). 2.5. Determination of molecular weight distribution The molecular weight of polysaccharide was determined by using HPGPC (Agilent-1200), which was performed on TSK gel G4000 PWxl column (7.8 mm × 300 mm, column temperature 30 ◦ C) and detected by differential refraction index detector (RID) at 35 ◦ C. The standard dextrans (T-10, T-40, T-70, T-500 and T-2000) were used for the calibration curve (Wen et al., 2011). 2.6. FT-IR and NMR analyses Mixtures of sample (1 mg) with dry KBr (150 mg) were pressed into a 1 mm thick disk then analyzed with a FT-IR spec-trometer from 4000 to 400 cm−1 (VECTOR-22) (Parikh & Madamwar, 2006). The sample was dissolved in D2 O. The 1 H NMR and 13 C NMR spectra were recorded on a Bruker spectrometer at a probe temperature of 298 K and operated at 400 MHz (Nep & Conway, 2011). 2.7. Monosaccharide composition analysis 2 mL of 2.0 M TFA (Trifluoroacetic acid) was added into the polysaccharide (3 mg) powder and the solution was hydrolyzed at 110 ◦ C for 3 h. After TFA was removed, the acetylated part was analyzed by GC (Yan et al., 2010). d-Glucose, d-galactose, l-rhamnose, d-xylose, d-mannose, and d-arabinose were also derivatized as standards. 2.8. Periodate oxidation and Smith degradation analysis The periodate oxidation and Smith degradation analysis were performed based on the literature methods (Linker, Evans, & Impallomeni, 2001; Dixon & Lipkin, 1954). The sample (20 mg) was dissolved in 25 mL of 30 mM NaIO4 , and then the solution was placed in dark. An aliquot of this solution (0.1 mL) was taken out every 8 h and diluted 250 times with distilled water. The absorbance of the solution was detected at 223 nm by the UV spectrophotometer. When it was stable at 223 nm, 2 mL of reaction liquid was drawn out and titrated with 0.01 M NaOH to measure the production of formic acid. 2.9. Methylation analysis Methylation of polysaccharide was performed according to the method (Needs & Selvendran, 1993) with minor revisions. The sample (5.0 mg) was taken and added into 5 mL DMSO. NaOH (20 mg) was added into the solution and N2 was injected into the flask. It was treated by ultrasonication for 30 min under room temperature to obtain the solution. Later, 1.0 mL of 98% CH3 I was added and placed in dark. Ultrasonic treatment was carried out in 20 ◦ C for 30 min to expel the residual CH3 I. The procedures above were repeated for three times. The reaction was terminated by adding 1 mL water and shocking. Then, it was dialyzed for 48 h with distilled water and lyophilized. 2 mL of 2 M TFA was added and hydrolyzed at 110 ◦ C for 3 h. The product was dissolved in 2 mL distilled water. Afterwards, NaBH4 (25 mg) was added into the solution and shocked at room temperature for 3 h. The solution was neutralized by 25% acetic acid and N2 was injected into the flask.

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The procedures above were repeated for five times. Then GC–MS analysis was carried out. 2.10. Congo-red test The interaction of Congo red with polysaccharide was analyzed with the spectrophotometric method (Rout et al., 2008). The solution of samples (0.5 mg/mL) were mixed with 2 mL, 50 ␮M Congo red and 1 M NaOH to achieve a final NaOH concentration of 0–0.50 M. Meanwhile, mixed solution without polysaccharide was prepared as the control. The UV–vis absorption spectrum was measured in the range from 400 to 600 nm after placing the samples at room temperature for 15 min. 2.11. Scanning electron microscopy (SEM) The sample was fixed onto a copper stub. After sputtering with a layer of gold, the sample was examined on a SU1510 scanning electron microscope (Lai & Yang, 2007). 2.12. In vitro ˛-glucosidase inhibitory activity assay The assay was conducted referring to the glucose-oxidase method (Vinholes et al., 2011). The enzyme and samples were dissolved in 0.1 M sodium phosphate buffer (pH7.0) to obtain the desired concentration. The reaction mixture was described as follows: 0.2 U/mL ␣-glucosidase, 10 ␮L; 30 mM maltose, 5 ␮L; and the samples (0.1-1.0 mg/mL), 5 ␮L. The reaction mixture was incubated at 37 ◦ C for 30 min. The catalytic reaction was stopped by addition of 3% SDS solution (30 ␮L). Then 14 ␮L Tris-glycerol buffer, 2 ␮L glucose oxidase, 2.8 ␮L peroxidase and 1.2 ␮L o-Dianisidine dihydrochloride were added to the mixture and the reaction mixture was incubated for another 20 min at 25 ◦ C. The catalytic reaction was terminated by addition of 40 ␮L of 2.5 M H2 SO4 solution. Acarbose was the positive control (Tran et al., 2014). The reaction system without samples was blank and the system without ␣-glucosidase served as control. Enzyme activity was quantified by measuring its optical density (OD) at 405 nm. All assays were carried out in triplicates. The inhibitory rate of sample on ␣-glucosidase was calculated by the following formula. Inhibitionrate(%) = (ODcontrol − ODsample )/(ODcontrol − ODblank ) × 100%

3. Results and discussion 3.1. Isolation and purification The crude polysaccharide was purified by Sephadex G-25 column to produce the substance. And the elution profile result of HPGPC was showed in Fig. 1(a). It showed that there were two peaks, which indicated that crude polysaccharide mainly contained two kinds of polysaccharides with different molecular weights. And the peak (II) mainly contained reducing sugars via detection. Therefore, the collected eluent (I) was pooled, concentrated, lyophilized and termed TBP-I. Subsequently TBP-I was purified by Sephadex G-75 column, then one homogeneous and purified polysaccharide was obtained. The elution profile and the chromatographic result of HPGPC were showed in Fig. 1(b,c). There was only one peak which indicated that TBP-I mainly contained one kind of fraction, collected designated as TBP-II. The total sugar content of TBP-II was 96.2%.

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3.2. Physicochemical properties of TBP-II TBP-II had no absorption at 260 and 280 nm in the UV spectrum, which indicated that it was absent of nucleic acids and proteins. The specific optical rotation was +137.5◦ . TBP-II was soluble in water but practically insoluble in acetone, ethyl acetate, butyl alcohol. TBP-II exhibited negative Fehling’s reagent and iodinepotassium iodide reaction which indicated that it did not contain reducing sugar and starch type ploysaccharide (Xu et al., 2009). The absence of amino acid and protein in TBP-II was confirmed by sulfuric acid carbazole reaction and coomassie brillian blue test. And TBP-II was neutral sugar. The calibration curve of polysaccharide molecular weight was established with T- Dextran series as standard: y = −0.4415x + 9.4164, R2 = 0.9494 (where y represents the loyarithm of molecular weight, while x represents retention time). As shown in Fig. 1(d), the average molecular weight of TBPII was estimated to 26 kDa with the retention time of 11.334 min based on the regression equation. 3.3. Structural characterization 3.3.1. Monosaccharide identification of TBP-II Growing evidence showed that the bioactivity of the polysaccharide was associated with its structure such as monosaccharide composition, glycosidic linkage types and the conformation (J. Wang et al., 2013; K.P. Wang et al., 2013). TBP-II was hydrolyzed with TFA into individual monosaccharides tagged with GC analysis. GC analysis revealed that TBP-II was composed of arabinose (Ara), rhamnose (Rha), galactose (Gal), xylose (Xyl) and glucose (Glc) with amolar ratio of 1:0.5:0.7:6.3:74.2 (Fig. 2a,b). 3.3.2. Linkage analysis of TBP-II polymer chain The periodate oxidation of TBP-II consumed 0.919 mol periodic acid and produced 0.081 mol formic acid by each mole of anhydroglucose unit. The fact that the consumption of formic acid suggested that 1 → 6 or 1 → linkage was present. The fact that the consumption of periodate was more than two times the production of formic acid indicated that there might present 1 → 2, 1 → 4, 1 → 2,6 or 1 → 4,6 linkages, which only consumed periodic acid but not produced formic acid. After Smith degradation of the oxidized product of TBP-II, compared with, glycerol, erythritol and rhamnose were found in the degradation products. The molar ratios of glycerol, erythritol and rhamnose was 2.29:15.66:1.00 in TBP-II. The presence of rhamnoses indicated the existence of 1 → 3, (1 → 2,3), (1 → 2,4), (1 → 3,6) or (1 → 2,3,4) linkages in TBP-II which could hardly be oxidized (Zhang et al., 2010). The relatively high content of erythritol exhibited 1 → 4 linkage might be consisted in the main chain or branch. The relatively low content of glycerol suggested a small amount of 1 → 6 or 1 → linkage. The results from periodate oxidation-Smith degradation showed that 1 → 4, 1 → 3, 1 → 2 and 1 → 6 linkages or non-reducing terminal might exist in TBP-II. 3.3.3. Methylation analysis Methylation is an essential method to analyze linkages, and it forms methoxyl groups on the free hydroxyl groups of polysaccharides. The methylated polysaccharides were acid hydrolyzed and acetylated for the GC–MS analysis. As summarized in Table 1, TBP-II showed four glycosidic linkages, namely 2,3,4-Tri-O-Me-Gal, 2,3,4,6-Tri-O-Me-Glc, 3,5,-di-O-Me-Rha and 2,3,6-Tri -O-Me-Glc. These data demonstrated that the main chain of TBP-II was composed of different types of linkages presented above and maximum amount of monosaccharide of TBP-II was (1 → 4)-linked-glucose. Residues of branch structure might be (1 → 6)-linked-galactose, (1 → 3)-linked-glucose and (1 → 2,4)-linked-rhamnose, which was

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Fig. 1. Elution profiles of (a) crude polysaccharide in Sephadex G-25 column chromatography and (b) TBP-I in Sephadex G-75 column chromatography. HPGPC distribution of (c) TBP-I.

Table 1 Results of the methylation analysis of TBP-II. Methylation sugar residues

Linkages types

Major mass fragments (m/z)

2,3,4-Tri-O-Me-Gal 2,3,4,6-Tri-O-Me-Glc 3,5,-di-O-Me-Rha 2,3, 6-Tri-O-Me-Glc

→ 6) Gal(1 → → 3)Glc(1 → → 2,4) Rha(1 → → 4) Glc(1 →

43,117,129 43,45,87,117 43,87 71,87,131

Fig. 3. FT-IR spectra of TBP-II.

consistent with the overall monosaccharide composition described in Section 3.3.1. 3.4. FT-IR spectra analysis of TBP-II

Fig. 2. GC profile of (a) standard monosaccharides and (b) TBP-II Peaks of (a): (1) Rhamnose, (2) Arabinose, (3) Xylose, (4) Mannose, (5) Glucose, (6) Galactose.

To characterize polysaccharide fractions, the characteristic absorption of polysaccharides was performed in the range of 4000–400 cm−1 by FT-IR (Dou et al., 2015). The FI-IR spectrum of TBP-II showed in Fig. 3. The wide and strong band around 3046 cm−1 exhibited the presence of hydroxyl stretching vibration (Qiu et al., 2013). A weak peak at 2927 cm−1 was assigned to C H ( CHOH, CH2 and CH3 ) asymmetric stretching vibration (Zou et al., 2010). The peak at 1417 cm−1 was for C H deforma-

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Fig. 4.

1

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H NMR spectra of (a) TBP-IIand 13 C NMR spectra of (b) TBP-II.

tion vibration. In addition, three stretching peaks at 1150 cm−1 , 1078 cm−1 and 1030 cm−1 indicated the presence of pyranoside, and the absorption band at 848 cm−1 was attributed to the presence of ␣-type glycosidic linkage (Luo et al., 2010). Therefore, TBP-II was mainly composed of ␣-glycosidic bond-linked glucopyranose. 3.5. Structural characteristics of TBP-II derived from NMR The structural features of TBP-II were further elucidated by NMR spectral analysis. The chemical shifts at ı 4.98–ı 5.5 ppm in the 1 H NMR spectrum (Fig. 4a) could be assigned to the typical signals of anomeric protons of ␣-terminal-d-Glcp (A). Moreover, three main anomeric protons at ı 4.98–ı 5.5 ppm indicated that TBP-II was mainly composed of three types of sugars. The peak of TBP-II at around ı 4.787 ppm was attributed to the chemical shifts of D2 O. A weak triple peak of TBP-II at around ı 4.6 ppm showed that TBP-II contained a small amount of ␤-glycosidic bond. In addition, The 13 C NMR spectrum of TBP-II showed in Fig. 4b. The spectrum had no signal at low field from ı 160–ı 180 ppm, which illustrated TBP-II did not contain uronic acid. In the 13 C NMR spectrogram, ı 90–ı 110 ppm was the anomeric carbon area. TBP-II

contained several types of anomeric carbon signals which indicated that TBP-II was composed of variety of sugars. The signal peak at ı 98.74 ppm was assigned to the anomeric carbon of ␣-d-Glcp (Popov et al., 2011). The signal peaks at ı 102.46 ppm was medium, and it was resulted from anomeric carbon of ␣-d-Xylp (J. Wang et al., 2013; K.P. Wang et al., 2013; Patra et al., 2013). The signal peak at ı 94.73 ppm was lower and it was probably caused by anomeric carbon of ␣-d-Arap (Sarkar et al., 2012). 3.6. Congo red test The conformational transition of the polysaccharide was determined by employing CR–polysaccharide complexes spectrophotometric method at various alkali concentrations. As shown in Fig. 5, it showed the changes in maximum absorption wavelength of complex with alkali concentrations ranging from 0 to 0.5 M. Compared with pure Congo red, the maximum absorption ␭max of the TBP-II complexes red-shifted to longer wavelengths. The red shifts of ␭max of the samples were not observed at all concentrations. Thus, it could be concluded that TBP-II did not exist triple-helical conformation in solution.

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Fig. 5. The maximum absorption wavelength of Congo red and TBP-II at various concentrations of sodium hydroxide solution.

3.7. SEM analysis It was a qualitative tool to analyze the surface morphology of polysaccharides and identify whether polysaccharides were starch by scanning electron microscopy (SEM) (Wang et al., 2015). The microstructure of TBP-II at ×400 times and ×1000 times were showed in Fig. 6. The images showed that the surface present sticks, accumulation, intertwined, and lamellar structure. It indicated that TBP-II was mainly of amorphous structure, and a part of the polysaccharide was scattered. Images demonstrated that TBPII was different from starch in Fagopyrum tartaricum (Wang et al., 2011). Therefore, TBP-II was a non-starch polysaccharide. 3.8. ˛-d-Glucosidase inhibitory activity As shown in Fig. 7, TBP-II with different concentrations exhibited inhibitory activity on ␣-d-glucosidase and was in a dose-dependent manner. The percentage inhibition depended on the concentration of polysaccharides. The higher concentration was, the stronger the inhibition was. When the concentration reached 0.8 mg/mL, inhibition rate of growth was slower. And the higher the purity was, the higher inhibition rate of ␣-d-glucosidase was, showed that the polysaccharides played a leading role in ␣-d-glucosidase reaction. 4. Conclusion In conclusion, the polysaccharide from Fagopyrum tartaricum was purified by Sephadex G-25 chromatography and Sephadex G-75 chromatography successively to obtain a neutral polysaccharide TBP-II, containing about 96.2% of total polysaccharide content. The TBP-II was a polysaccharide with molecular weight of 26 kDa

Fig. 7. Effect of different purity polysaccharides on ␣-d-glucosidase.

and few branches. Structural characterization showed that TBPII was a novel polysaccharide which was mainly composed of galactose, arabinose, xylose and glucose with a molar ratio of 0.7:1:6.3:74.2. Characteristic absorption peaks were found in FTIR spectroscopy. Moreover, the backbone of TBP-II was composed of (1 → 4)-linked ␣-d-Glcp whereas the branches of (1 → 3)-linked ␣-d-Glcp, (1 → 6)-linked ␣-d-Galp and (1 → 2,4)-linked ␣-d-Rhap. It had amorphous surface and was a non-starch polysaccharide. The ␣-d-glucosidase inhibitory activities of polysaccharides were determined using acarbose as positive control. TBP-II exhibited an excellent inhibitory activity on ␣-d-glucosidase, which was superior to TBP-I as well as crude polysaccharide and the percentage inhibition depended on the concentration of polysaccharides. These experimental results offered chemical and bioactive fundamental for the benefits of tartary buckwheat toward exploring the advanced structure, and recommended more researches on hypoglycaemic activity of TBP-II in vitro and vivo. Further studies are worthy to investigate the relative pharmacological mechanism(s) of TBP-II. Acknowledgments This work was financially supported by the key program of the Natural Science Foundation of Tianjin in China (16JCZDJC34100), the National Spark Key Program of China (2015GA610001) and the International Science and Technology Cooperation Program of China (2013DFA31160). References Dixon, J. S., & Lipkin, D. (1954). Spectrophotometric determination of vicinal glycols. Analytical Chemistry, 26, 1092–1093.

Fig. 6. SEM images of TBP-II (A: × 400, B: × 1000).

X.-T. Wang et al. / Carbohydrate Polymers 153 (2016) 679–685 Dou, J., Meng, Y. H., Liu, L., Li, J., Ren, D. Y., & Guo, Y. R. (2015). Purification, characterization and antioxidant activities of polysaccharides from thinned-young apple. International Journal of Biological Macromolecules, 72, 31–40. Fabjan, N., Rode, J., Kosir, I. J., Wang, Z., Zhang, Z., & Kreft, I. (2003). Tartary buckwheat (Fagopyrum tataricum Gaertn.) as a source of dietary rutin and quercitrin. Journal of Agricultural and Food Chemistry, 51, 6452–6455. Wang, J., Jin, W., Zhang, W., Hou, Y., Zhang, H., & Zhang, Q. (2013). Hypoglycemic property of acidic polysaccharide extracted from Saccharina japonica and its potential mechanism. Carbohydrater Polymers, 95, 143–147. Wang, K. P., Zhang, Q. L., Liu, Y., Wang, J., Cheng, Y., & Zhang, Y. (2013). Structure and inducing tumor cell apoptosis activity of polysaccharides isolated from Lentinus edodes. Journal of Agricultural and Food Chemistry, 61, 9849–9858. Kannan, S., Manivannan, R., Balasubramaniam, A., & Kumar, N. S. (2010). Formulation and evaluation of aspirin delayed release tablet. International Journal of Comprehensive Pharmacy, 4, 1–3. Kintner, P. K., & Buren, J. P. (1982). Carbohydrate interference and its correction in pectin analysis using m-hydroxydiphenyl method. Journal of Food Science, 47, 756–759. Krkoˇskova´ı, B., & Mra´ızova´ı, Z. (2005). Prophylactic components of buckwheat. Food Research International, 38(5), 561–568. Lai, L. S., & Yang, D. H. (2007). Rheological properties of the hot-water extracted polysaccharides in Ling-Zhi (Ganoderma lucidum). Food Hydrocolloids, 21, 739–746. Li, N., Yan, C., Hua, D., & Zhang, D. (2013). Isolation, purification, and structural characterization of a novel polysaccharide from Ganoderma capense. International Journal of Biological Macromolecules, 57, 285–290. Lin, L. Z., Cui, C., Wen, L. R., Yang, B., Luo, W., & Zhao, M. M. (2011). Assessment of in vitro antioxidant capacity of stem and leaf extracts of Rabdosia serra (MAXIM.) HARA and identification of the major compound. Food Chemistry, 126, 54–59. Lin, L. Z., Dong, Y., Zhao, H. F., Wen, L. R., Yang, B., & Zhao, M. M. (2011). Comparative evaluation of rosmarinic acid, methyl rosmarinate and pedalitin isolated from Rabdosia serra (MAXIM.) HARA as inhibitors of tyrosinase and aglucosidase. Food Chemistry, 129, 884–889. Linker, A., Evans, L. R., & Impallomeni, G. (2001). The structure of a polysaccharide from infectious strains of Burkholderia cepacia. Carbohydrate Research, 335, 45–54. Liu, Y. L., Fang, Q. N., Zhang, X. C., Feng, X. X., Zhang, L. F., & He, X. W. (1983). Study on the active constituents from Fagopyrum dibotrys. Acta Pharmaceutica Sinica, 18, 545–547. Liu, Y., Liu, C., Ryuichiro, K., & Eri, M. (2009). Effect of buckwheats on blood glucose non-enzymatic protein glycated reaction in STZ-induced diabetic rats. Journal of Liao Ning University of TCM, 05, 195–197. Luo, A., He, X., Zhou, S., Fan, Y., & Chun, Z. (2010). Purification, composition analysis and antioxidant activity of the polysaccharides from Dendrobium nobile Lindl. Carbohydrater Polymers, 79, 1014–1019. Needs, P. W., & Selvendran, R. R. (1993). Avoiding oxidative degradation during sodium hydroxide, methyl iodide-mediated carbohydrate methylation in dimethyl sulfoxide. Carbohydrate Research, 245, 1–10. Nep, E. I., & Conway, B. R. (2011). Physicochemical characterization of grewia polysaccharide gum: Effect of drying method. Carbohydrater Polymers, 84, 446–453. Oomah, B. D., & Mazza, G. (1996). Flavonoid and antioxidative activities in buckwheat. Journal of Agricultural and Food Chemistry, 44(7), 1746–1750. Parikh, A., & Madamwar, D. (2006). Partial characterization of extracellular polysaccharide from cyanobacteria. Bioresource Technology, 97, 1822–1827.

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Patra, S., Patra, P., Maity, K. K., Mandal, S., Bhunia, S. K., Dey, B., et al. (2013). A heteroglycan from the mycelia of Pleurotus ostreatus: Structure determination and study of antioxidant properties. Carbohydrate Research, 368, 16–21. Popov, S. V., Ovodova, R. G., Golovchenko, V. V., Popova, G. Y., Viatyasev, F. V., Shashkov, A. S., et al. (2011). Chemical composition and anti-inflammatory activity of a pectic polysaccharide isolated from sweet pepper using a simulated gastric medium. Food Chemistry, 124, 309–315. Qiu, T., Ma, X., Ye, M., Yuan, R., & Wu, Y. (2013). Purification, structure, lipid lowering and liver protecting effects of polysaccharide from Lachnum YM281. Carbohydrater Polymers, 98, 922–930. Rout, D., Mondal, S., Chakraborty, I., & Islam, S. S. (2008). The structure and conformation of a water-insoluble (1 → 3)-, (1 → 6)-␣-d-glucan from the fruiting bodies of Pleurotus florida. Carbohydrate Research, 343, 982–987. Sarkar, R., Nandan, C. K., Bhunia, S. K., Maiti, S., Maiti, T. K., Sikdar, S. R., et al. (2012). Glucans from alkaline extract of a hybrid mushroom (backcross mating between PfloVv12 and Volvariella volvacea): Structural characterization and study of immunoenhancing and antioxidant properties. Carbohydrate Research, 347, 107–113. Tian, X., & Ren, T. (2007). Nutritional health function and utilization of tartary buckwheat. Food and Nutrition in China (China), 10, 44–46. Tran, H. H., Nguyen, M. C., Le, H. T., Nguyen, T. L., Pham, T. B., Chau, V. M., et al. (2014). Inhibitors of ␣-glucosidase and ␣-amylase from Cyperus rotundus. Pharmaceutical Biology, 52, 74–77. Vinholes, J., Grosso, C., Andrade, P. B., Gil-Izquierdo, A., Valentão, P., De Pinho, P. G., et al. (2011). In vitro studies to assess the antidiabetic, anti-cholinesterase and antioxidant potential of Spergularia rubra. Food Chemistry, 129, 454–462. Wang, S. Q., Wang, L. L., Fan, W. H., Cao, H., & Cao, B. S. (2011). Morphological analysis of common edible starch granules by scanning electron microscopy. Food Science, 32, 74–79. Wang, Y. F., Li, Y. F., Liu, Y. Y., Chen, X. Q., & Wei, X. L. (2015). Extraction, characterization and antioxidant activities of Se-enriched tea polysaccharides. International Journal of Biological Macromolecules, 77, 76–84. Watanabe, M. (1998). Catechins as antioxidants from buckwheat (Fagopyrum esculentum Moench) groats. Journal of Agricultural and Food Chemistry, 46(3), 839–845. Wen, L. R., Lin, L. Z., You, L. J., Yang, B., Jiang, G. X., & Zhao, M. M. (2011). Ultrasoundassited extraction and structural identification of polysaccharides from Isodon lophanthoides var Gerardianus (Bentham) H. Hara. Carbohydrate Polymers, 85, 541–547. Wijngaard, H. H., & Arendt, E. K. (2006). Buckwheat. Cereal Chemistry, 83, 391–401. Xu, H. S., Wu, Y. W., Xu, S. F., Sun, H. X., Chen, F. Y., & Yao, L. (2009). Antitumor and immunomodulatory activity of polysaccharides from the roots of Actinidia eriantha. Journal of Ethnopharmacology, 125, 310–317. Yan, J. K., Li, L., Wang, Z. M., & Wu, J. Y. (2010). Structural elucidation of an exopolysaccharide from mycelial fermentation of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis. Carbohydrate Polymers, 79, 125–130. Yao, R. C., Huang, M. F., Wu, Y. R., & Yang, C. R. (1989). Active constituents of anti-tumor from Fagopyrum cymosum. Acta Botanica Yunnanica, 11, 215–218. Zhang, W. J., Li, X. C., Liu, Y. Q., Yao, R. C., Nonaka, G., & Yang, C. R. (1994). Phenolic constituents from Fagopyrum dibotrys. Acta Botanica Yunnanica, 16, 354–356. Zhang, Y., Ming, G., Wang, K. P., Chen, Z. X., Dai, L. Q., Liu, J. Y., et al. (2010). Structure chain conformation and antitumor activity of a novel polysaccharide from Lentinus edodes. Fitoterapia, 81, 1163–1170. Zou, S., Zhang, X., Yao, W., Niu, Y., & Gao, X. (2010). Structure characterization and hypoglycemic activity of a polysaccharide isolated from the fruit of Lycium barbarum L. Carbohydrater Polymers, 80, 1161–1167.