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        黑穗醋栗果實(shí)超聲波降解多糖的結(jié)構(gòu)及抗糖基化活性

        2017-06-05 15:00:27徐雅琴郭瑩瑩陳宏超王麗波

        徐雅琴,劉 菲,郭瑩瑩,陳宏超,王麗波,楊 昱

        黑穗醋栗果實(shí)超聲波降解多糖的結(jié)構(gòu)及抗糖基化活性

        徐雅琴,劉 菲,郭瑩瑩,陳宏超,王麗波,楊 昱※

        (東北農(nóng)業(yè)大學(xué)理學(xué)院,哈爾濱 150030)

        為了充分利用黑穗醋栗果實(shí)中的多糖資源,該文對水提醇沉,大孔樹脂純化制得黑穗醋栗果實(shí)多糖進(jìn)行超聲波降解,并對分離純化后得到的低分子量多糖的理化性質(zhì)、結(jié)構(gòu)特征和抗糖基化反應(yīng)活性進(jìn)行了研究。利用葡聚糖凝膠Sephadex G-100對降解多糖進(jìn)行分離純化,高效液相色譜法測定分子量,氣相色譜法測定單糖組成,紅外光譜、紫外光譜、剛果紅試驗(yàn)和電鏡掃描初步表征多糖結(jié)構(gòu)。結(jié)果表明:黑穗醋栗果實(shí)降解多糖(BCP I)純度為83.88% ± 0.76%;重均分子量為235 955 Da;BCP I為酸性雜多糖,單糖組成及物質(zhì)量比為:半乳糖醛酸:鼠李糖:阿拉伯糖:甘露糖:葡萄糖:半乳糖=2.31 : 1.11 : 3.14 : 0.34 : 0.36 : 1.00。BCP I具有多糖的特征吸收峰,不含多酚、蛋白質(zhì)和核酸;不具有三股螺旋結(jié)構(gòu),呈現(xiàn)片狀不規(guī)則的形態(tài)??固腔钚詼y定結(jié)果表明BCP I對糖基化反應(yīng)3個(gè)階段(Amadori產(chǎn)物形成階段、二羰基化合物形成階段和糖基化終產(chǎn)物形成階段)產(chǎn)物的形成均表現(xiàn)出良好的抑制作用,抑制率隨濃度與時(shí)間的增加而增大。最大抑制率分別為49.55% ± 0.79%,41.82% ± 0.72%和42.01% ± 0.13%,均高于對照氨基胍。研究結(jié)果可為后續(xù)深入探討黑穗醋栗果實(shí)多糖結(jié)構(gòu)與降血糖活性之間的構(gòu)效關(guān)系提供理論基礎(chǔ)。

        純化;超聲波;降解;黑穗醋栗果實(shí);多糖;結(jié)構(gòu);抗糖基化

        0 引 言

        多糖,又稱多聚糖,是自然界含量最豐富的生物聚合物,廣泛存在于高等植物、藻類、菌類及動(dòng)物體內(nèi)。研究表明多糖具有多種生理活性,如增強(qiáng)免疫調(diào)節(jié)、抗腫瘤、抗氧化、降血糖、降血脂、抑菌等功效[1-3]。然而大多數(shù)天然提取的多糖由于其分子量較大,水溶性差,不利于生物體有效吸收并發(fā)揮生物學(xué)功能[4]。研究發(fā)現(xiàn)通過適當(dāng)?shù)姆椒ń到舛嗵?,把大分子斷裂成在一定分子量范圍?nèi)的較小片段,可提高其生物活性[5]。目前,有關(guān)多糖降解的研究主要集中在酶法降解、化學(xué)方法降解和物理方法降解等[6]。酶法降解總體成本較高且專一性酶不易獲得[7];化學(xué)方法費(fèi)時(shí)、難以控制且對環(huán)境不友好[8];物理降解法是一種綠色高效的降解方法,操作簡單,可控性好,常用的方法有微波法[9]、輻射法[10]和超聲波法[11]。與微波法和輻射法相比,超聲波降解在獲取所需性質(zhì)的多糖的同時(shí)不會破壞多糖的單元結(jié)構(gòu)[12],從而保證了多糖的生物活性,在食品體系中發(fā)揮著重要作用。

        黑穗醋栗(Ribes nigrum L.)又稱黑加侖,因果實(shí)中含有豐富的活性成分[13-14],與藍(lán)莓、樹莓和沙棘等一起被稱為“第三代”新型水果,受到消費(fèi)者的廣泛關(guān)注。近年來,有關(guān)黑穗醋栗果實(shí)中花色苷、黃酮類、多酚類等活性物質(zhì)的研究較多[14-15],而對于多糖的研究僅有少量報(bào)道。目前,黑穗醋栗果實(shí)多糖因其顯著的抗氧化、抗腫瘤、降血糖、增強(qiáng)免疫力等多種功效[16-17],正逐漸引起植物學(xué)家和醫(yī)藥學(xué)家的關(guān)注,成為研究熱點(diǎn)。課題組前期已從黑穗醋栗果實(shí)中提取出具有體外抗氧化活性的天然多糖。但是這些多糖因較大的分子量和較低的溶解度,一定程度上限制了其生物活性的發(fā)揮[17-18]。此外,前期研究主要將超聲波技術(shù)應(yīng)用于黑穗醋栗多糖的輔助提取,提高多糖提取率[18-19],而有關(guān)超聲波降解對黑穗醋栗果實(shí)多糖結(jié)構(gòu)和活性的影響研究未見報(bào)道。因此,本文通過超聲波法降解黑穗醋栗果實(shí)多糖,初步探討降解多糖的結(jié)構(gòu)特征和抗糖基化反應(yīng)活性,為進(jìn)一步開發(fā)利用黑穗醋栗果實(shí)多糖資源提供科學(xué)依據(jù)。

        1 材料與方法

        1.1 材料與試劑

        黑穗醋栗(黑豐)成熟果實(shí),2015年7月采于黑龍江省農(nóng)科院牡丹江農(nóng)科所,洗滌后于-20 ℃儲藏備用。

        大孔吸附樹脂D4006,南開大學(xué)化工廠;SephadexG-100,瑞典Pharmacia公司進(jìn)口分裝;半乳糖醛酸,上海源葉生物科技有限公司;葡聚糖T-10、T-40、T-70、T-110、T-2000,北京拜爾迪生物公司;D-葡萄糖、D-半乳糖、D-鼠李糖、L-阿拉伯糖、D-甘露糖、D-木糖,美國Sigma公司;其他藥品均為分析純。

        1.2 儀器與設(shè)備

        JY92-2D超聲波細(xì)胞粉碎機(jī)(寧波新芝生物科技股份有限公司);TU-1901雙光束紫外可見分光光度計(jì)(北京普析通用儀器有限責(zé)任公司);FE-20K酸度計(jì)(上海精密儀器儀表有限公司);R-205旋轉(zhuǎn)蒸發(fā)儀(上海申勝生物技術(shù)有限公司);FTS135型傅立葉變換紅外光譜儀(美國BID-BAD公司);LC-10AVP高效液相色譜儀(日本島津公司);GC-2010氣相色譜儀(日本島津公司);S-3400N型顯微鏡(日本HITACHI公司)。

        1.3 方法

        1.3.1 黑穗醋栗果實(shí)多糖的制備

        參考課題組前期研究方法[19],稍加修改。稱取一定量黑穗醋栗果實(shí)勻漿,按料液比1:20 g/mL加入去離子水,溫度80 ℃,電動(dòng)攪拌,轉(zhuǎn)速200 r/min,提取時(shí)間2 h。提取液抽濾、濃縮(50 ℃,真空度< 0.09 MPa),體積分?jǐn)?shù)40%乙醇溶液醇沉,4 ℃冰箱靜置過夜,所得沉淀微孔濾膜抽濾(0.45 μm),凍干(-50 ℃,真空度< 15 Pa,干燥24 h),得到粉紅色的黑穗醋栗果實(shí)粗多糖。

        使用D4006型大孔樹脂(2.0 cm × 30 cm)對黑穗醋栗果實(shí)粗多糖進(jìn)行純化,純化條件:溫度25 ℃,上樣液質(zhì)量濃度4.00 mg/mL,洗脫劑為去離子水,洗脫流速1.00 mL/min。將洗脫后的黑穗醋栗果實(shí)多糖溶液濃縮(50 ℃,真空度< 0.09 MPa),凍干(-50 ℃,真空度<15 Pa,干燥24 h),命名為BCP,苯酚-硫酸法檢測其含量[20]。

        1.3.2 黑穗醋栗果實(shí)降解多糖的制備

        稱取2.400 g黑穗醋栗果實(shí)多糖(BCP),加入400 mL去離子水使BCP質(zhì)量濃度為6 mg/mL,充分溶解后,利用超聲波細(xì)胞粉碎機(jī)進(jìn)行超聲降解。超聲條件:超聲時(shí)間30 min,超聲溫度25 ℃,超聲功率600 W。降解后多糖溶液經(jīng)過透析(3 500 Da,72 h)、濃縮,凍干,得到降解多糖。

        將得到的降解多糖配制成15 mg/mL溶液,利用葡聚糖凝膠Sephadex G-100(1.8 cm × 40 cm)分離純化[21],上樣量為1.00 mL,洗脫劑為去離子水,洗脫流速1.0 mL/min,每1.00 mL為一管,收集洗脫液,苯酚-硫酸法跟蹤檢測至無糖檢出。根據(jù)洗脫曲線收集多糖組分,濃縮,凍干,得到黑穗醋栗果實(shí)降解多糖(BCP I),苯酚-硫酸法測其含量。

        1.3.3 黑穗醋栗果實(shí)降解多糖理化性質(zhì)的測定

        將BCP I溶解在去離子水、乙醇、乙醚、乙酸乙酯、丙酮、氯仿等溶劑,觀察其溶解性。利用酸度計(jì)、茚三酮試驗(yàn)、碘-碘化鉀試驗(yàn)、斐林試劑反應(yīng)、三氯化鐵反應(yīng)測定BCP I的化學(xué)性質(zhì)。

        1.3.4 黑穗醋栗果實(shí)降解多糖分子量和單糖組成測定

        分子量測定:液相色譜條件:日本Shimadzu公司高效液相色譜儀;Waters Ultrahydroge 2000色譜柱(7.8 mm × 300 mm);示差折光檢測器RID-10A;Shimadzu CLASSVp數(shù)據(jù)處理工作站;進(jìn)樣量:10 μL;壓力:2.3 MPa;洗脫劑:Na2SO4溶液(0.05 mol/L),流速:1.0 mL/min。

        將BCP I與標(biāo)準(zhǔn)品(T-10、T-40、T-70、T-110、T-2000)精確配制成2.0 mg/mL溶液,0.45 μm微孔濾膜過濾后進(jìn)樣,測定色譜峰保留時(shí)間,GPC分析軟件計(jì)算得到多糖分子量。

        單糖組成的測定:按照聶永心等[22]的方法,將BCP I進(jìn)行酸水解和衍生化后進(jìn)樣,各單糖標(biāo)準(zhǔn)品也進(jìn)行衍生化后混合進(jìn)樣,氣相色譜儀進(jìn)行分析檢測(肌醇作為內(nèi)標(biāo)),計(jì)算BCP I的單糖組成。

        氣相色譜條件:RTX-1701石英毛細(xì)管色譜柱(0.25 μm × 30.0 m);氫火焰離子化的檢測器(FID);程序升溫:180(5 ℃/min)-220 ℃(5 min),220(10 ℃/min)-280 ℃(20 min);汽化和檢測器的溫度為280 ℃;載氣:高純氮?dú)狻?/p>

        1.3.5 黑穗醋栗果實(shí)降解多糖的紅外光譜和紫外光譜

        采用溴化鉀壓片法,在4 000~400 cm-1范圍內(nèi)進(jìn)行紅外光譜掃描;采用雙光束紫外可見分光光度計(jì)在波長190~630 nm范圍內(nèi)進(jìn)行紫外光譜掃描。

        1.3.6 黑穗醋栗果實(shí)降解多糖的剛果紅試驗(yàn)和掃描電鏡測定

        參照文獻(xiàn)[21]的方法,將BCP I和剛果紅試劑配制成不同濃度NaOH溶液,紫外-可見光譜掃描,測量樣品溶液的最大吸收波長。將樣品BCP I放入離子濺射鍍膜儀,樣品表面鍍一層100 nm左右的金膜,然后利用掃描電鏡觀察BCP I的形貌特征。

        1.3.7 黑穗醋栗果實(shí)降解多糖的抗糖基化反應(yīng)活性測定

        [23]的方法,建立牛血清蛋白-葡萄糖糖基化反應(yīng)體系,以氨基胍作為陽性對照,測定不同質(zhì)量濃度(0.05、0.20、0.40 mg/mL)BCP I溶液對Amadori產(chǎn)物、二羰基化合物、末期糖基化終產(chǎn)物(advanced glycation end products, AGEs)的抑制作用。

        1.4 數(shù)據(jù)處理

        所有試驗(yàn)數(shù)據(jù)均以3次試驗(yàn)結(jié)果的平均數(shù)±標(biāo)準(zhǔn)誤差(mean ± SD)表示,SPSS軟件進(jìn)行差異顯著性分析,以P<0.05作為顯著性差異標(biāo)準(zhǔn)。

        2 結(jié)果與分析

        2.1 黑穗醋栗果實(shí)降解多糖的制備

        BCP經(jīng)超聲波降解,葡聚糖凝膠Sephadex G-100分離純化后,得到主要組分BCP I,洗脫曲線見圖1。如圖1所示,洗脫峰為單一吸收峰,峰形對稱,且無拖尾現(xiàn)象,說明BCP I為均一組分。苯酚-硫酸法測得多糖回收率為88.04% ± 0.51%,純度為83.88% ± 0.76%。

        圖1 BCP I的葡聚糖凝膠SephadexG-100洗脫曲線Fig.1 Elution curve of BCP I by Sephadex G-100

        2.2 黑穗醋栗果實(shí)降解多糖理化性質(zhì)

        BCP I為白色疏松狀(棉花糖狀)固體,易溶于水,難溶于乙醇、乙醚、乙酸乙酯等有機(jī)溶劑。BCP I水溶液pH值為3.35±0.47,表明它是酸性多糖。BCP I與茚三酮反應(yīng)呈陰性,表明BCP I中不含有氨基酸、蛋白質(zhì);BCP I與三氯化鐵反應(yīng)無顏色變化,表明該多糖不含多酚類物質(zhì);與斐林試劑進(jìn)行反應(yīng)后,無磚紅色沉淀氧化亞銅生成,表明BCP I中不含有游離還原糖。

        2.3 黑穗醋栗果實(shí)降解多糖分子量和單糖組成

        BCP I高效液相色譜圖見圖2a。根據(jù)BCP I保留時(shí)間(TR=13.669 min),GPC軟件計(jì)算得到多糖BCP I重均分子量Mw為235 955 Da,數(shù)均分子量Mn為34 205 Da,黏均分子量Mz為1 105 803 Da。未降解多糖BCP重均分子量Mw為441 320 Da,數(shù)均分子量Mn為58 492 Da,黏均分子量Mz為1 547 684 Da。由此可見,BCP經(jīng)過超聲波處理后,分子量降低,多糖發(fā)生降解。

        各標(biāo)準(zhǔn)品和BCP I的氣相色譜圖見圖2b,2c。在相同色譜條件下,保留時(shí)間可作為定性分析的依據(jù),通過與標(biāo)準(zhǔn)單糖保留時(shí)間相比較,可確定多糖樣品中的單糖組分。氣相色譜分析結(jié)果表明,BCP I是由6種單糖組成的酸性雜多糖,單糖組成及物質(zhì)的量比為:半乳糖醛酸:鼠李糖:阿拉伯糖:甘露糖:葡萄糖:半乳糖=2.31 : 1.11 : 3.14 : 0.34 : 0.36 : 1.00。

        圖2 BCP I液相色譜和氣相色譜圖Fig.2 HPLC and GC chromatograms of BCP I

        2.4 黑穗醋栗果實(shí)降解多糖紅外光譜和紫外光譜

        由BCP I紅外光譜圖(圖3a)可知,BCP I在4 000~500 cm-1范圍內(nèi)具有明顯的多糖特征吸收峰。3 428.29 cm-1處和2 930.71 cm-1處分別為O-H的伸縮振動(dòng)峰和C-H伸縮振動(dòng)峰[24],1 744.23和1 618.44 cm-1處分別為酯化羰基C=O和酯化羧基COO-的伸縮振動(dòng)峰[25]。此外,吸收峰出現(xiàn)在1 441.08 cm-1處,表明多糖BCP I中含有糖醛酸[26],這與GC測定結(jié)果一致。在1 200~1 000 cm-1處存在吸收峰,表明多糖BCP I為吡喃糖[27]。在919.87 cm-1處和830.12 cm-1處分別出現(xiàn)吸收峰,表明多糖存在α-和β-兩種糖苷鍵[28]。

        紫外光譜掃描結(jié)果如圖3b,BCP I在260、280 nm處無吸收,表明降解多糖中不含蛋白質(zhì)、核酸、花色苷[29]。該測定結(jié)果和2.2的結(jié)果一致。

        圖3 BCP I的紅外光譜和紫外光譜Fig.3 Infrared and ultraviolet spectra of BCP I

        2.5 黑穗醋栗果實(shí)降解多糖剛果紅試驗(yàn)結(jié)果

        研究表明,在堿性條件下,與剛果紅空白對照相比,具有三股螺旋結(jié)構(gòu)的多糖與剛果紅形成的絡(luò)合物的最大吸收波長(λmax)會發(fā)生變化,而如果待測多糖不具有三股螺旋結(jié)構(gòu),其形成的絡(luò)合物將會與空白對照溶液λmax變化趨勢相近[30]。剛果紅、BCP I-剛果紅絡(luò)合物在不同NaOH濃度下最大吸收波長的變化見圖4。由圖4可以看出,多糖BCP I-剛果紅混合溶液與剛果紅對照溶液的λmax變化趨勢相近,表明BCP I不具有三股螺旋結(jié)構(gòu)。

        圖4 不同NaOH濃度下剛果紅,BCP I-剛果紅絡(luò)合物最大吸收波長(λmax)Fig.4 Maximum absorption wave length (λmax) of Congo red and Congo red-BCP I at various NaOH concentrations

        2.6 黑穗醋栗果實(shí)降解多糖掃描電鏡分析

        掃描電鏡是研究多糖形貌特征的重要方法,超聲波降解前后黑穗醋栗多糖的掃描電鏡觀察結(jié)果如圖5所示,為多糖在放大100倍時(shí)的SEM圖像。從圖5可以看出,降解前后黑穗醋栗多糖表面均比較光滑,呈片狀,形態(tài)不規(guī)則。然而,與降解前多糖BCP相比(圖5a),降解后多糖BCP I片狀結(jié)構(gòu)的表面積顯著減小(圖5b),表明超聲波降解黑穗醋栗多糖效果明顯。Yan等采用超聲波法降解桑黃菌絲多糖時(shí)得到同樣的結(jié)論[5]。

        圖5 超聲波降解前后多糖的掃描電鏡圖像(放大100倍)Fig.5 SEM images of ultrasonic polysaccharides before and after degradation(× 100 times)

        2.7 黑穗醋栗果實(shí)降解多糖抗糖基化活性

        在非酶促條件下,蛋白質(zhì)游離氨基與還原糖的羰基經(jīng)過一系列反應(yīng)可以產(chǎn)生穩(wěn)定的末期糖基化終產(chǎn)物(AGEs),該反應(yīng)叫做非酶糖基化反應(yīng)[31]。糖基化反應(yīng)主要3個(gè)階段:初期Amadori產(chǎn)物形成階段、中期二羰基化合物形成階段和反應(yīng)末期終產(chǎn)物AGEs形成階段。AGEs能夠?qū)е略S多慢性疾病,如糖尿病腎臟合并癥、動(dòng)脈粥樣硬化、老年性癡呆癥,嚴(yán)重威脅人類健康[32]。研究表明,抑制劑若能夠抑制上述3個(gè)階段中的任一產(chǎn)物的生成,都可以減少AGEs的形成,有利于治療慢性疾病[33]。

        BCP I抗糖基化活性結(jié)果如圖6所示,不同濃度(0.05,0.20,0.40 mg/mL)BCP I對糖基化反應(yīng)的3個(gè)階段的產(chǎn)物均有一定的抑制作用;且隨著時(shí)間和濃度的增加,多糖的抑制率顯著增加(P<0.05)。

        圖6 BCP I和氨基胍抗糖基化作用Fig.6 Antiglycation effects of BCP I and aminoguanidine

        如圖6a、6b所示,相同濃度下,BCP I的抑制率高于對照氨基胍。第13天時(shí),當(dāng)BCP I質(zhì)量濃度為0.40 mg/mL,BCP I對Amadori產(chǎn)物和二羰基化合物的抑制作用均達(dá)到最大,抑制率分別為49.55%±0.79%,41.82%± 0.72%,高于對照氨基胍44.58%±1.02%,33.01%±0.28%。此外,由圖6c可以看出,第13 天時(shí),不同質(zhì)量濃度(0.40、0.20、0.05 mg/mL)BCP I對AGEs的最大抑制率分別為42.01%±0.13%,36.86%±0.12%,33.05%±0.09%,均顯著高于相應(yīng)濃度下氨基胍的抑制率(30.45%±0.13%,26.59%±0.20%和23.80%±0.49%)。由此可見,在試驗(yàn)濃度范圍內(nèi),BCP I對AGEs的抑制作用均高于對照氨基胍。

        3 討 論

        研究結(jié)果表明超聲波降解對黑穗醋栗果實(shí)多糖分子量和空間結(jié)構(gòu)產(chǎn)生一定影響。多糖分子量從441 320 Da(BCP)降低至235 955 Da(BCP I),減少了46.53%,同時(shí)降解多糖BCP I片狀結(jié)構(gòu)的表面積相對于原多糖BCP明顯減小。Wang等[34]研究發(fā)現(xiàn),低功率超聲條件下,真菌多糖Cs-HKl在3 400和1 064 cm-1處吸收峰減弱,說明超聲波導(dǎo)致維持多糖二級結(jié)構(gòu)的氫鍵發(fā)生斷裂,而在高功率條件下則裂解為大小不同的多糖碎片。Zhu等[12]對冬蟲夏草菌絲多糖進(jìn)行超聲波降解,發(fā)現(xiàn)超聲波處理不改變多糖的特征屬性,單糖殘基的組成和糖苷鍵的類別沒有改變,但是分子量和特性黏度降低,并且超聲處理后的α-螺旋性增強(qiáng),降解多糖的抗腫瘤活性顯著增加。Yu等[35]對紫菜多糖進(jìn)行超聲波降解,得到低分子量、低黏度,高抑制癌細(xì)胞增值活性的降解多糖。Sun等[36]通過超聲波降解,得到系列低分子量海洋石斛降解多糖,降解多糖在清除自由基、抑制脂質(zhì)過氧化以及紅細(xì)胞溶血試驗(yàn)中均比未降解多糖表現(xiàn)出更優(yōu)良的性能。由此可見,超聲波主要導(dǎo)致多糖分子量、單糖組成、分支度以及空間結(jié)構(gòu)發(fā)生變化,進(jìn)而對多糖的溶解度、黏度、結(jié)晶度以及生物活性產(chǎn)生一定的影響。目前,有關(guān)超聲波降解的確切機(jī)制還不明確。有研究者認(rèn)為[5,35],與化學(xué)或熱分解不同,超聲波降解是非隨機(jī)過程,鏈斷裂主要在分子的中心位置發(fā)生。本試驗(yàn)首次對超聲波降解后黑穗醋栗多糖的結(jié)構(gòu)特征與抗糖基化活性進(jìn)行了初步研究,但是有關(guān)超聲波降解黑穗醋栗果實(shí)多糖的作用機(jī)制還有待進(jìn)一步深入探討。

        4 結(jié) 論

        1)黑穗醋栗果實(shí)多糖經(jīng)過超聲波降解,葡聚糖凝膠Sephadex G-100分離純化后,得到均一降解多糖,純度達(dá)到83.88%±0.76%,重均分子量為235 955 Da。降解多糖為酸性雜多糖,單糖組成的物質(zhì)量比為:半乳糖醛酸:鼠李糖:阿拉伯糖:甘露糖:葡萄糖:半乳糖=2.31 : 1.11 : 3.14 : 0.34 : 0.36 : 1.00。

        2)黑穗醋栗果實(shí)降解多糖具有多糖的特征結(jié)構(gòu),不含蛋白、核酸和多酚類物質(zhì)。此外,降解多糖不具有三股螺旋結(jié)構(gòu),表面呈片狀,形態(tài)不規(guī)則。

        3)黑穗醋栗果實(shí)降解多糖對糖基化反應(yīng)3個(gè)階段產(chǎn)物的形成均表現(xiàn)出良好的抑制作用,同一濃度下,抑制率均高于對照氨基胍。因此,黑穗醋栗果實(shí)降解多糖可作為較好的糖基化反應(yīng)抑制劑。

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        Structure and antiglycation activity of polysaccharides after ultrasonic degradation from blackcurrant fruit

        Xu Yaqin, Liu Fei, Guo Yingying, Chen Hongchao, Wang Libo, Yang Yu※
        (College of Science, Northeast Agricultural University, Harbin 150030, China)

        Blackcurrant (Ribes nigrum L.) is a kind of small berry with many health-beneficial substances, such as organic acids, unsaturated fatty acids, vitamins, polysaccharides, flavonoids, and anthocyanins. Recently, polysaccharide from blackcurrant (BCP) has received considerable attention for their prominent benefits to human health, including immunostimulation, antitumor, antimicrobial, antioxidant, and anti-inflammatory activities. Previous work from our laboratory had isolated BCP which showed apparent antioxidant activities in vitro. However, the polysaccharides’ high molecular weight and low solubility in water limit their absorption and utilization in the body. Thus, the degradation of polysaccharides should be carried out to improve the specific and unique properties. Notably, ultrasonic irradiation has been recently viewed as a new technique for the degradation of polysaccharides, mainly due to the fact that the reduction in the molecular weight is simply splitting the most susceptible chemical bonds without causing any major changes in the chemical nature of polysaccharides. At present, there are no reports on preparing degraded BCP with the method of ultrasonic degradation. In this study, the BCP was obtained through water extraction, 40% alcohol precipitation, and purification with D4006 macroporous resin. The BCP was dissolved in water (6 mg/mL) and then was degraded by ultrasonication at 600 W, 25 °C for 30 min. The degraded polysaccharide (BCP I) was obtained through the subsequent separation with Sephadex G-100. Physical and chemical properties, structural characterization and antiglycation activity of BCP I were preliminarily studied. The molecular weight was determined by high performance liquid chromatography, and the monosaccharide composition was determined by gas chromatography. Infrared spectrum, Congo red and electron microscopy were used to characterize the structure of the polysaccharides. The results showed that the purity of BCP I was 83.88%±0.76%, and the weight average molecular weight was 235 955 Da. BCP I was acidic polysaccharide, and consisted of galacturonic acid, rhamnose, arabinose, mannose, dextrose and galactose in a ratio of 2.31 : 1.11 : 3.14 : 0.34 : 0.36 : 1.00. Fourier transform infrared spectrum showed that BCP I had obvious characteristic peaks of polysaccharides, and BCP I was a pyranose form of sugar containing both α-type and β-type glycosidic linkage. Ultraviolet spectrum showed that BCP I did not contain anthocyanins, proteins and nucleic acids. Scanning electron microscope and Congo red test showed that BCP I exhibited sheet structure and had no triple helix structure, and the surface area of BCP I was reduced compared with BCP. The results of antiglycation assay showed that BCP I exhibited significant inhibitory effects on the product formation of 3 stages of glycation reaction, and the inhibitory rate increased with the increase of concentration and time. The maximum inhibitory rates were 49.55%±0.79%, 41.82%±0.72% and 42.01%±0.13%, respectively, which were higher than those of the control aminoguanidine (30.45%±0.13%,26.59%±0.20% and 23.80%±0.49%). Thus, BCP I can be considered as a kind of potential inhibitor of protein glycation. The results can provide a theoretical basis for further study on the structure-activity relationship between structure and hypoglycemic activity of the polysaccharides from blackcurrant.

        purification; ultrasound wave; degradation; blackcurrant fruits; polysaccharides; structure; antiglycation activity

        10.11975/j.issn.1002-6819.2017.05.042

        TS218

        A

        1002-6819(2017)-05-0295-06

        徐雅琴,劉 菲,郭瑩瑩,陳宏超,王麗波,楊 昱. 黑穗醋栗果實(shí)超聲波降解多糖的結(jié)構(gòu)及抗糖基化活性[J]. 農(nóng)業(yè)工程學(xué)報(bào),2017,33(5):295-300.

        10.11975/j.issn.1002-6819.2017.05.042 http://www.tcsae.org

        Xu Yaqin, Liu Fei, Guo Yingying, Chen Hongchao, Wang Libo, Yang Yu. Structure and antiglycation activity of polysaccharides after ultrasonic degradation from blackcurrant fruit[J]. Transactions of the Chinese Society of Agricultural Engineering (Transactions of the CSAE), 2017, 33(5): 295-300. (in Chinese with English abstract)

        doi:10.11975/j.issn.1002-6819.2017.05.042 http://www.tcsae.org

        2016-10-21

        2017-02-03

        國家自然科學(xué)青年基金資助項(xiàng)目(NO. 31600276);黑龍江省自然科學(xué)基金資助項(xiàng)目(NO. C2015004);東北農(nóng)業(yè)大學(xué)SITP計(jì)劃項(xiàng)目(201710224147)

        徐雅琴,女,教授。研究方向:天然產(chǎn)物提取及活性研究。哈爾濱 東北農(nóng)業(yè)大學(xué)理學(xué)院,150030。Email:xuyaqin@neau.edu.cn

        ※通信作者:楊 昱,女,博士,副教授。研究方向:天然產(chǎn)物提取及活性研究。哈爾濱 東北農(nóng)業(yè)大學(xué)理學(xué)院,150030。Email:yangyu_002@163.com

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