Food Chemistry 199 (2016) 694–701

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Food Chemistry

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Purification, characterization and antiglycation activity of a novelpolysaccharide from black currant

http://dx.doi.org/10.1016/j.foodchem.2015.12.0780308-8146/� 2015 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: yangyu_002@163.com (Y. Yang).

Yaqin Xu a, Gaijie Liu a, Zeyuan Yu b, Xiumei Song a, Xingguo Li b, Yu Yang a,⇑, Libo Wang a, Lu Liu b,Jing Dai b

aCollege of Science, Northeast Agricultural University, Harbin 150030, ChinabCollege of Horticulture, Northeast Agricultural University, Harbin 150030, China

a r t i c l e i n f o

Article history:Received 16 September 2015Received in revised form 15 December 2015Accepted 17 December 2015Available online 18 December 2015

Chemical compounds studied in this article:D-Glucose (PubChem CID: 5793)D-Galacturonic acid (PubChem CID: 439215)D-Galactose (PubChem CID: 6036)D-Rhamnose (PubChem CID: 439548)D-Mannose (PubChem CID: 18950)D-Arabinose (PubChem CID: 66308)D-Fucose (PubChem CID: 94270)D-Fructose (PubChem CID: 5984)Carbazole (PubChem CID: 6854)Trifluoroacetic acid (PubChem CID: 6422)Girard-T (PubChem CID: 67156)

Keywords:Black currantPolysaccharidesPurificationCharacterizationAntiglycation activity

a b s t r a c t

A novel polysaccharide fraction (BCP-1) was extracted from the black currant fruit by ultrasound-assistedcompound enzyme and purified by chromatography on macroporous resin D4006, anion-exchangeQ-Sepharose FF and Sephadex G-100 columns. BCP-1 consisted of galacturonic acid, xylose, mannose,glucose and galactose in a ratio of 1.00:3.14:1.83:17.90:1.98 and its molecular weight was 14,050 Da.The preliminary structure features of BCP-1 were investigated by FT-IR and NMR. SEM and Congo red testshowed that BCP-1 had honeycomb-like structure, but no triple helix structure. BCP-1 exhibitedsignificant inhibitory abilities on protein glycation. Especially, BCP-1 showed obvious inhibitory effectson the formation of dicarbonyl compounds and AGEs (% inhibition of 66.95 ± 0.33% and 67.15 ± 0.40%respectively), but weaker inhibition Amadori compound production (max. 37.15 ± 0.36% inhibition).This suggested that the inhibitory action of BCP-1 on protein glycation was more effective on the laterphases of dicarbonyl compounds and AGEs formation.

� 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Non-enzymatic glycation is a complicated reaction processbetween the aldehydic group in reducing sugars and amino groupsin proteins react to produce advanced glycation end products(AGEs) (Li et al., 2014). AGEs is one of the major risk factors forthe development of chronic complications of diabetes, such as dia-betic atherosclerosis (Wu, Hsieh, Wang, & Chen, 2009), peripheralneuropathy (Wada & Yagihashi, 2005) and diabetic cataract(Lapolla, Traldi, & Fedele, 2005). Non-enzymatic glycation reactionof protein proceeds in 3 phases. In the early phase, the reducing

sugar reacts with the free amino groups of proteins to form Schiffbase, and then Amadori products are produced. In the intermediatephase, the Amadori products are converted to series of dicarbonylcompounds such as glycolaldehyde, glyoxal and methylglyoxalthrough oxidation and dehydration. In the last phase, the dicar-bonyl compounds continue to react with amino groups to produceAGEs (Wang, Zhang, & Dong, 2012). Thus, inhibitors that can inhi-bit any process of the three phases can alleviate the formation ofAGEs and be beneficial to treat chronic complications of diabetes.Some compounds or agents, including aminoguanidine, benfoti-amine and pyridoxamine (Kim, Kim, Jung, & Kim, 2010; Peyroux& Sternberg, 2006), have been used to treat diabetic complications.However, these drugs were reported to have adverse effects (Singh,Barden, Mori, & Beilin, 2001). Therefore, searching for more

Y. Xu et al. / Food Chemistry 199 (2016) 694–701 695

effective and safer antiglycation drugs has received considerableattention.

Polysaccharides are natural polymers and widely distributed inplants and microorganisms. They are highly stable, safe, non-toxic,non-carcinogenic and possess a variety of bioactivities, such asantitumor, anticoagulant, antiviral, anti-inflammatory, anti-thrombotic, hypoglycemic and antiglycation activities (Song, Li,Hu, Ni, & Li, 2011). In recent years, some bioactive polysaccharides,such as the polysaccharides from Dendrobium huoshanense andPolygonum multiflorum Thunb, were found to have antiglycationactivities (Li et al., 2014; Lv, Cheng, Zheng, Li, & Zhai, 2014; Qianet al., 2014). So polysaccharides may be a promising drugcandidate for diabetic complications.

Black currant (Ribes nigrum L.), which originates from NorthernAsia and Europe, is a shrubby tree with many health-beneficialsubstances, such as ascorbic acid, flavone, anthocyanin, polyphenoland polysaccharide (Khoo, Clausen, Pedersen, & Larsen, 2012; Liu,Kallio, & Yang, 2014; McDougall, Gordon, Brennan, & Stewart,2005; Takata, Yamamoto, Yanai, Konno, & Okubo, 2005). Black cur-rant fruits and leaves have been used in both Asian and Europeantraditional medicine for treatment of a variety of diseases(Bishayee et al., 2011). Black currant berry has evoked tremendousinterest due to its diverse health benefits such as the inhibition ofhypertension, neurodegenerative disease, ocular disease and anti-carcinogenesis (Gopalan et al., 2012; Tabart, Kevers, Evers, &Dommes, 2011; Yamamoto et al., 2014). Recently, the polysaccha-rides from black currant were reported to be one of the main fac-tors responsible for the health benefits, exhibiting antioxidant,antitumor, anti-inflammatory activities (Tabart et al., 2012;Takata et al., 2005). In our previous research, the crude polysaccha-rides isolated from black currant fruit were found to exhibit goodantioxidation and antiglycation activities in vitro (Yu, Ren, Xu, &Li, 2012), but there exist no reports on the structure and antiglyca-tion activity of the purified polysaccharides.

In this study, the polysaccharide (BCP-1) was obtained bypurification using macroporous resins D4006, anion-exchangeQ-Sepharose FF and Sephadex G-100. Then the structure of BCP-1was characterized by Fourier-transform infrared (FT-IR) spec-troscopy and nuclear magnetic resonance (NMR). Moreover,the antiglycation activity of BCP-1 was also evaluated using thenon-enzymatic glycation of protein test in vitro.

2. Materials and methods

2.1. Materials

The fruits of black currant (Heifeng) were provided by Mudan-jiang Institute of Agricultural Science in Heilongjiang Province(Mudanjiang, China). The fruits at the fully mature stage werewashed and stored at �20 �C until use. Before extraction process,the fruits were gently defrosted and then homogenized using aJJ-2 homogenizer (Changzhou Guohua Electric Appliance Co., Ltd.,Jiangsu Province, China).

Papain (23.74 U/mg) was purchased from Beijing Obo StarBiotechnology Co., Ltd (Beijing, China). Pectinase (28.48 U/mg)was obtained from Shanghai Lanji Biotechnology Co., Ltd (Shanghai,China). D4006 Macroporous Resin was purchased from NanKaiUniversity Chemical Plant (Tianjin, China). Anion-exchangeQ-Sepharose FF was obtained from Shanghai Yuanye BiotechnologyCo., Ltd (Shanghai, China). Sephadex G-100 and standard monosac-charides (D-galacturonic acid, D-glucuronic acid, D-glucose,

D-galactose, D-rhamnose, D-mannose, D-arabinose, D-fucose and

D-fructose) were purchased from Sigma–Aldrich Co. (St, Louis, Mo,USA). Dextrans of different molecular weights (T-10, T-40, T-70,

T-110) were provided from Baierdi Biotechnology Co. (Beijing,China). All other chemicals were of analytical grade.

2.2. Extraction and purification

The black currant fruit homogenate (20.0 g) with 2% enzymemix (papain: pectinase = 2:1) were put into a 500 mL beaker, thena buffer solution (pH = 5.3) was added at 20:1 mL/g. The polysac-charides were extracted at 40 �C in the ultrasonic cell disintegrator(JY92-2D, Xinzhi Bio-Sciences Co. Ltd, Ningbo, China) for 45 min,and the ultrasonic power was 600W. The extract was centrifugedat 3500 rpm for 20 min and the supernatant was filtrated, concen-trated and precipitated with ethanol to a final concentration of 60%(v/v) at 4 �C overnight. The formed precipitate was gathered bymembrane filtration (0.45 lm, Millipore, USA) under vacuum andlyophilized. Then the crude polysaccharides (BCP) were obtained.

The polysaccharide solution (4.00 mg/mL) was loaded on aD4006 macroporous resin column (2.0 cm � 30 cm) and elutedwith deionized water at a flow rate of 1.00 mL/min. The obtainedelute (1 mL/tube) was collected automatically and monitored byphenol–sulfuric acid method at 490 nm using D-glucose as astandard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Thecollected fraction was lyophilized and dissolved in deionizedwater to a concentration of 20 mg/mL. Then, the sample wasfractionated by anion-exchange chromatography of Q-SepharoseFF (2.0 cm � 30 cm) and eluted with deionized water at a flow rateof 2.00 mL/min. The obtained elute was pooled, concentrated andlyophilized. The eluted fraction (15 mg/mL) was further loadedon a Sephadex G-100 gel-permeation chromatography column(1.8 cm � 40 cm) and eluted with deionized water at a flow rateof 0.60 mL/min. Fractions were collected and enriched using themethod mentioned above. The main fraction BCP-1 was obtainedand lyophilized for further structure characterization and bioactiv-ity assays.

2.3. Physicochemical properties and structural analysis of BCP-1

2.3.1. Physicochemical properties of BCP-1BCP-1 was evaluated for solubility in water, ethanol, diethyl

ether, ethyl acetate, acetone and chloroform in accordance withthe British pharmacopoeia (BP) specification (Kannan,Manivannan, Balasubramaniam, & Kumar, 2010). The nature ofBCP-1 was confirmed by Ninhydrin test, Iodine test, Felhing’s testand Ferric chloride reaction. Uronic acid content was determinedby the carbazole-sulfuric acid method using D-galacturonic acidas standard (Kintner & Buren, 1982).

2.3.2. Determination of molecular weightThe molecular weight of BCP-1 was determined by high perfor-

mance liquid chromatography (HPLC, LC-10AVP, Shimadzu Corpo-ration, Japan), which was performed onWaters Ultrahydrogel 2000column (7.8 mm � 300 mm) and detected by differential refractionindex detector (RID-10A) at 35 �C. The pressure was constantlykept at 1.4 MPa. BCP-1 was dissolved in deionized water andpassed through 0.45 lm filter, applied to gel-filtration chromato-graphic column. Various standard dextrans of known molecularweight (T-10, T-40, T-70, T-110) were passed through the column.A standard curve was plotted according to the retention time andthe logarithm of their respective molecular weights. The molecularweight of BCP-1 was estimated by reference to the calibrationcurve made above.

2.3.3. Analysis of monosaccharide compositionGas chromatography (GC, GC-2010, Shimadzu Corporation,

Japan) was used for identification and quantification of themonosaccharide compositions of BCP-1. Briefly, the polysaccharide

696 Y. Xu et al. / Food Chemistry 199 (2016) 694–701

sample (30 mg) was hydrolyzed with 2.0 mL of 2 mol/L trifluo-roacetic acid (TFA) solution at 100 �C for 6 h. The hydrolyzatewas co-concentrated with ethanol to dryness under reduced pres-sure and this procedure was repeated for a few times to fullyremove TFA. The hydrolyzed sample was acetylated by the addi-tion of a mixture of hydroxylamine hydrochloride, pyridine andacetic anhydride (Qiao et al., 2009). The nine standard sugars(D-galacturonic acid, D-glucuronic acid, D-xylose, D-galactose,

D-mannose, D-glucose, D-fucose, D-arabinose and D-rhamnose) wereacetylated in a similar manner. All the derivatives were then ana-lyzed by GC equipped with flame ionization detector (FID) and aRTX-1701 silica capillary column (30.0 m � 0.25 mm � 0.25 lm).The column temperature was increased from 180 �C to 220 �C ata rate of 5 �C/min and held at 220 �C for 5 min, then elevated to280 �C at a rate of 10 �C/min and finally held at 280 �C for20 min. The temperature of detector and injector were set at280 �C. High purity nitrogen gas (N2) was used as the carrier gas.The injection volume was 0.4 lL for each run.

2.3.4. FT-IR and UV spectroscopy analysisThe FT-IR spectra of BCP-1 were recorded with a Fourier trans-

formed infrared spectrophotometer (FTS135, BID-BAD Company,USA), and the samples were analyzed as KBr pellets. Briefly, sam-ples were ground with KBr powder and then pressed into pelletsfor FT-IR spectral measurement within 4000–400 cm�1. The UVspectra of BCP-1 were measured using a double beam UV–visiblespectrophotometer (TU-1901, Beijing Purkinje General InstrumentCo., Ltd., Beijing, China) in the range of 190–450 nm.

2.3.5. Nuclear magnetic resonance spectroscopy analysisBCP-1 (20 mg) was dissolved in 2.0 mL D2O and transferred into

a 5 mm NMR-tube. NMR spectra (1H NMR and 13C NMR) wererecorded at 300 K by Bruker AVANCE III NMR Spectrometer (BrukerCo., Billerica, MA, USA) at 400 MHz.

2.3.6. Scanning electron microscope analysisThe morphological features of BCP-1 were studied with

JSM-6480A Scanning electron microscope (JEOL, Japan). The driedsample was mounted on a metal stub and sputtered with gold ina vacuum sputter coater.

2.3.7. Congo red testThe Congo red test was performed according to reported meth-

ods with some modifications (Rout, Mondal, Chakraborty, & Islam,2008). Briefly, 5 mg BCP-1 was mixed with 2 mL Congo red(80 lmol/L) and 2 mL distilled water. Different volumes of1.00 mol/L NaOH solution (0–2.00 mL) were added to the mixture(final concentration of NaOH, 0–0.50 mol/L). Meanwhile, 2 mLCongo red (80 lmol/L) was mixed with different volumes of1.00 mol/L NaOH solution (0–2.00 mL) and 2 mL distilled wateras the control. Maximum absorption wavelength (kmax) was mea-sured using the TU-1901 double beam UV–visible spectropho-tometer in the range from 200 to 800 nm after equilibrating for5 min at room temperature.

2.4. Inhibitory effects of polysaccharides on non-enzymatic glycationof proteins

2.4.1. In vitro glycation of bovine serum albumin (BSA)The BSA/glucose model was employed to evaluate the ability of

polysaccharide antiglycation and performed according to thereported method (Wu et al., 2009) with some modification. Briefly,all reagents were filtrated through 0.22 lm microporous mem-brane (Shanghai Xiyan Co., Ltd., Shanghai, China) to removemicroorganisms. The total 20 mL of glycated mixture contained

bovine serum albumin (BSA, 20 mg/mL), glucose (500 mmol/L),1% sodium azide phosphate buffer (200 mmol/L, pH 7.4), andBCP-1 with different concentrations (0.025, 0.05, 0.10, 0.15 and0.20 mg/mL). All the mixtures were placed in the incubator at37 ± 0.5 �C for 15 days and the glycated solution of 1.5 mL wastaken from the whole mixture every 2 days for the followingexperiments.

2.4.2. Analysis of Amadori productsThe Amadori products were measured using the method of

nitro blue tetrazolium (NBT) reductive assay. The procedure ofNBT reductive assay followed the reported method with minormodification (Zhang, Wang, & Dong, 2011). Briefly, the glycatedmaterials of 0.5 mL was mixed with 2.0 mL NBT reagent(0.3 mmol/L) in sodium carbonate buffer (100 mmol/L, pH 10.35),incubated at room temperature for 20 min, and the absorbancewas read at 530 nm. The inhibitory activity was calculated asfollow:

IR ¼ 1� A� Ac � Ad

Aa � Ab

� �� 100% ð1Þ

where A is the absorbance of complete glycosylation system con-taining the polysaccharide solution; Aa is the absorbance of theblank which contains all the reagents except the polysaccharide;Ac, Ad, Ab are defined as the absorbance of glycated solution (withoutBSA), glycated solution (without glucose) and glycated solution(without polysaccharide and glucose), respectively.

2.4.3. Analysis of dicarbonyl compoundsThe content of dicarbonyl compounds was tested spectrophoto-

metrically by Girard-T assay (Wells-Knecht, Zyzak, Litchfield,Thorpe, & Baynes, 1995). Briefly, 0.4 mL glycated material wasmixed with 0.2 mL Girard-T stock solution (500 mmol/L) and3.4 mL sodium formate (500 mmol/L, pH 2.9), followed by a incu-bation at room temperature for 1 h. The absorbance was measuredat 294 nm. A calibration curve was prepared using glyoxal as astandard which was treated in a similar manner. The content ofdicarbonyl compounds was estimated by the calibration curvemade above and the inhibitory activity was calculated accordingto Eq. (1).

2.4.4. Analysis of AGEsThe levels of AGEs were determined as described previously

with slight modification (Wang et al., 2012). The glycated material(0.5 mL) was diluted to 10 mL with phosphate buffer (pH 7.4). Thenthe fluorescence of the material was quantified using a LS45 fluo-rescence spectrofluorometer (Perkin Elmer, USA) at an excitation/emission wavelength of 370/440 nm, which is characteristic ofAGEs. The inhibitory activity was calculated as follow:

IR ¼ 1� F � Fc � Fd

Fa � Fb

� �� 100% ð2Þ

where F is the fluorescence of complete glycosylation system con-taining the polysaccharide solution; Fa is the fluorescence of theblank which contains all the reagents except the polysaccharide;Fc, Fd, Fb are defined as the fluorescence of glycated solution (with-out BSA), glycated solution (without glucose) and glycated solution(without polysaccharide and glucose), respectively.

2.5. Statistical analysis

Data were expressed as mean ± standard deviations (SD) ofthree replicated determinations. All statistical analysis was carriedout by using SPSS 18.0 for Windows. P < 0.05 was consideredstatistically significant.

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3. Results and discussion

3.1. Isolation and purification of BCP

The crude polysaccharide (BCP) was isolated from black currantfruit. After purification by macroporous resin D4006, anion-exchange Q-Sepharose FF and Sephadex G-100 column, one homo-geneous and purified polysaccharide (BCP-1) was obtained and theelution profile was showed in Fig. 1A. The UV spectra (not shownhere) showed that BCP-1 only had an absorption peak at 204 nm,which was the characteristic UV absorption peak for a polysaccha-ride. BCP-1 had no absorption at 260 and 280 nm, indicating theabsence of protein and nucleic acid. There was no absorption at520 nm in the region of 400–600 nm (data not shown), indicatingthe absence of anthocyanin in BCP-1.

3.2. Physicochemical properties of BCP-1

BCP-1 was soluble in water and practically insoluble in ethanol,diethyl ether, ethyl acetate, acetone and chloroform. BCP-1 showednegative Fehling’s reagent and iodine-potassium iodide reactions,indicating that it did not contain reducing sugar and starch type

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 10 20 30

Tube num

Abs

orba

nce

B

A

C

Fig. 1. Physicochemical analysis of BCP-1. (A) Elution curve of BCP-1 on Sephade

polysaccharide (Xu et al., 2009). The absence of amino acid andprotein in BCP-1 was confirmed by Ninhydrin test. Ferric chloridereaction showed the absence of polyphenol in BCP-1. Moreover,the uronic acid content in BCP-1 was found to be 3.89%.

3.3. Molecular weight and monosaccharide composition of BCP-1

The HPLC system was used to determine the molecular weightof BCP-1. The equation of the standard curve was:lgMw = �0.5459t + 12.403 (where Mw represents the molecularweight, while t represents retention time) with a correlation coef-ficient of 0.9907. As shown in Fig. 1B, the single, narrow and sharppeak on HPLC revealed that BCP-1 was a homogeneous fraction.The average molecular weight of BCP-1was calculated as14050 Da with the retention time of 16.144 min based on theregression equation.

For the monosaccharide composition analysis, the results fromGC spectrum (Fig. 1C) indicated that BCP-1 was composed of galac-turonic acid, xylose, mannose, glucose and galactose with molarratio of 1.00:3.14:1.83:17.90:1.98. These results indicated thatBCP-1 was a heteropolysaccharide and glucose is the main sugarunit.

40 50 60

ber

x G-100 column; (B) HPLC elution profile of BCP-1; (C) GC profile of BCP-1.

Fig. 2. Infrared spectra of BCP-1.

Fig. 3. NMR spectra of BCP-1. (A) 1H NMR; (B) 13C NMR.

698 Y. Xu et al. / Food Chemistry 199 (2016) 694–701

Fig. 4. Photomicrographs as recorded by SEM of BCP-1. ((A) magnification 100�, scale bar 100 lm. (B) Magnification 200�, scale bar 100 lm. (C and D) magnification500�, scale bar 50 lm.)

475

480

485

490

495

500

0 0.1 0.2 0.3 0.4 0.5

NaOH concentration(mol/L)

λmax

/nm

CR+BCP-1CR

Fig. 5. Maximum absorption (kmax) of Congo red and Congo red + BCP-1 at variousNaOH concentrations.

Y. Xu et al. / Food Chemistry 199 (2016) 694–701 699

3.4. Structural analysis of BCP-1

3.4.1. FT-IR analysisThe FT-IR spectrum of BCP-1 showed typical absorption peaks

of polysaccharides. As shown in Fig. 2, the band 3419.93 cm�1 rep-resented the stretching of the OAH and the band at around2974.26 cm�1 was due to the CAH stretching and bendingvibrations (Chen et al., 2011). The bands at 1743.50 cm�1,1632.22 cm�1 were caused by the stretching vibrations of C@Oand COO�, respectively (Gnanasambandam & Proctor, 2000). Inaddition, the band at around 1446.15 cm�1 indicated the presenceof uronic acids, which was consistent with the result of carbazole-sulfuric acid reaction (Wang, Pei, Ma, Cai, & Yan, 2014). Theabsorption band at 1050.07 cm�1 in the range of 1200–1000 cm�1 indicated a pyranose form of sugar (Ying, Han, & Li,2011), and a characteristic band at 882.00 cm�1 was due tob-type glycosidic linkage (Pan et al., 2012).

3.4.2. NMR analysisThe structural features of BCP-1 were further analyzed using

NMR spectroscopy. As shown in Fig. 3A, the 1H NMR spectrumwas mostly crowded in a narrow region ranging from 3 to 5 ppmwhich was typical of polysaccharides (Gao et al., 2015). The chem-ical shifts of anomeric protons were higher or lower than 4.95 ppm,indicating the existence of both a- and b-configurations in BCP-1.In 13C-NMR spectrum (Fig. 3B), the signals from the anomeric car-bons appeared between 92.07 and 104.42 ppm, which indicatedthat there were both a (d95–101 ppm) and b (d101–105 ppm)anomeric configurations existing in BCP-1 (Huang, Li, Li, & Wang,2011). This result was in agreement with that of the 1H NMR

spectrum. Moreover, in the anomeric region of Fig. 3A, thesignal at 5.15 ppm was assigned to an anomeric proton ofa-glucopyranose units in the main chain and the signal at98.04 ppm in 13C NMR spectra supported this deduction (Miaoet al., 2014). The anomeric proton chemical shift at 4.44 and4.57 ppm (Fig. 3A) and the 13C chemical shift at 101.48 and104.42 ppm (Fig. 3B) could be assigned to b-galactopyranoseand b-galacturonic acid, respectively (Du et al., 2015; Zhu et al.,2013). These results were also confirmed by GC analysis. The signalat 173.34 ppm was attributed to C-6 of galacturonic acid, whichwas consistent with the results of carbazole-sulfuric acid reactionand FT-IR spectrum (Zhu et al., 2013).

A

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B

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1 3 5 7 9 11 13 15Incubation time(day)

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C

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Incubation time(day)

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0.025mg/ml 0.05mg/ml0.1mg/ml 0.15mg/ml0.2mg/ml

Fig. 6. Inhibitory effects of BCP-1 on antiglycated activities. (A) Inhibit Amadoriproduct formation; (B) inhibit dicarbonyl compound formation; (C) inhibit AGEsformation.

700 Y. Xu et al. / Food Chemistry 199 (2016) 694–701

3.4.3. SEM analysisAs shown in Fig. 4, the scanning electron micrographs (SEM) of

BCP-1 provide the surface morphology of the polysaccharide. TheSEM images showed that BCP-1 existed in the state of aggregation,and its surface showed the honeycomb structure.

3.4.4. Congo red testCongo red can form complexes with polysaccharides in a helical

conformation. Compared with pure Congo red, the maximumabsorption kmax of the complex will undergo a red shift (Wanget al., 2014). Fig. 5 showed the changes in maximum absorptionwavelength of complex with alkali concentrations ranging from 0to 0.5 mol/L. The red shifts of kmax of the samples were notobserved at all concentrations. Thus, it could be concluded thatBCP-1 did not exist in a triple-helical conformation in solution.

3.5. Antiglycation analysis of BCP-1

In the early phase of non-enzymatic glycation of proteins, Schiffbase and Amadori products were formed. NBT could be reduced byAmadori products and then yielded a colored reactant with a max-imum absorption at 530 nm (Zhang et al., 2011). As shown inFig. 6A, BCP-1 with different concentrations (0.025, 0.05, 0.10,0.15 and 0.20 mg/mL) exhibited inhibitory activity on the forma-tion of Amadori products and was in a dose-dependent manner.The inhibitory effects of BCP-1 on Amadori products increasedfrom 1 to 13 days and then decreased to different level. Duringthe culture time of 13 days, the difference of inhibitory effects ofBCP-1 at different concentrations on Amadori products were sig-nificant (P < 0.05). BCP-1 at 0.2 mg/mL concentration showed themaximal inhibitory activity of 37.15 ± 0.36% and IC50 was1.91 mg/mL.

In the intermediate phase, after oxidation and dehydration, thedicarbonyl compounds were generated. As shown in Fig. 6B, theinhibitory effects of BCP-1 (0.025, 0.05, 0.10, 0.15 and 0.20 mg/mL) on production of dicarbonyl compounds significantlyincreased from 1 to 15 days and showed a dose–response relation-ship. Meanwhile, after 15 days of incubation, the maximal inhibi-tory rate (66.95 ± 0.33%) of BCP-1 was observed at 0.2 mg/mLand IC50 was 0.023 mg/mL. In addition, during the culture time of15 days, significant difference between the inhibitory effects ofBCP-1 at different concentrations on the formation of dicarbonylcompounds was observed (P < 0.05).

In the last phase, AGEs were produced. As shown in Fig. 6C, alltest samples (BCP-1, 0.025, 0.05, 0.10, 0.15 and 0.20 mg/mL) exhib-ited strong response against AGEs formation in a dose-dependentmanner. Moreover, the capability of inhibiting AGEs formation ofBCP-1 increased from 1 to 13 days and then levelled off. At the cul-ture time of 13th day, BCP-1 performed the highest inhibitioneffects (67.15 ± 0.40%) with an IC50 value of 0.042 mg/mL at0.2 mg/mL. There was a significant difference between the inhibi-tory effects of BCP-1 with different concentrations on AGEs(P < 0.05).

The inhibitory effects of BCP-1 with different concentrations onnon-enzymatic glycation of proteins increased with the increasingconcentration. In addition, the ability of inhibiting the formation ofdicarbonyl compounds and AGEs of BCP-1 was stronger than thatof Amadori products. Thus, the inhibitory action of BCP-1 on pro-tein non-enzymatic glycation occurred primarily during the lasttwo phases. Results of these activity tests in vitro showed thatBCP-1 would possibly possess a remarkable antiglycation effect,which might due to its structures, such as molecular weight,monosaccharide composition and type of glycosyl linkage.Recently, it had been reported that some polysaccharides exhibitedantiglycation activity, which were correlated with their antioxi-dant properties (Zha et al., 2013). In our previous work, thepolysaccharides of black currant had been confirmed to be goodpotential for scavenging free radicals in vitro, including oxygen freeradicals, superoxide anion and DPPH (Yu et al., 2012). Therefore,the inhibitory effects of BCP-1 on protein nonenzymatic glycationmight be attributed to their antioxidant activities. However, toour best knowledge, the specific mechanism of antiglycation activ-ity of BCP-1 is unclear, and further studies will be carried out.

4. Conclusion

This study illustrated that BCP-1 was a heteropolysaccharideconsisting of galacturonic acid, xylose, mannose, glucose and galac-tose with molar ratio of 1.00:3.14:1.83:17.90:1.98, and a-Glc wasthe major components of the backbone structure. The inhibitoryaction of BCP-1 on protein glycation was more effective on the later

Y. Xu et al. / Food Chemistry 199 (2016) 694–701 701

phases of dicarbonyl compounds and AGEs formation. However,the hypoglycemic activity of BCP-1 in vivo need be further investi-gated in our later work.

Acknowledgements

This work was financially supported by the Scientific andTechnological Research Project Foundation of HeilongjiangProvince of China (C2015004).

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