CN105816702B - 一种甘蔗多酚提取物及其制备方法和应用 - Google Patents
一种甘蔗多酚提取物及其制备方法和应用 Download PDFInfo
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- CN105816702B CN105816702B CN201610196161.1A CN201610196161A CN105816702B CN 105816702 B CN105816702 B CN 105816702B CN 201610196161 A CN201610196161 A CN 201610196161A CN 105816702 B CN105816702 B CN 105816702B
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- ethanol
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Abstract
本发明属于保健食品技术领域,公开了一种甘蔗多酚提取物及其制备方法和应用。所述制备方法为:取甘蔗或蔗渣,粉碎后过筛得粗粉,然后将粗粉用乙醇溶液提取,过滤除去滤渣,滤液减压浓缩至浸膏;所得的浸膏加水分散,采用石油醚进行萃取,得到石油醚相和水相,用乙酸乙酯对水相进行萃取,收集乙酸乙酯相,减压浓缩至浸膏;然后通过聚酰胺柱层析分离,乙醇梯度洗脱,收集30%乙醇体积浓度的洗脱物,浓缩干燥得到甘蔗多酚提取物。本发明来源于大宗农作物甘蔗,通过多级萃取及柱层析梯度洗脱分离纯化,产品具有更强的降血糖活性,可用于食品添加剂和功能食品。
Description
技术领域
本发明属于保健食品技术领域,具体涉及一种甘蔗多酚提取物及其制备方法和应用。
背景技术
糖尿病是一种多因素引起的内分泌失调综合征,包括糖类、蛋白质以及脂肪的代谢紊乱。糖尿病患者分布在全世界各国家,患病人数也在逐年上升。糖尿病以及并发症不仅严重影响了患者的生活质量,也给个人和社会带来了沉重的经济负担。在所有的糖尿病确诊病例中,Ⅱ型糖尿病约占其中的90-95%。Ⅱ型糖尿病的发生,在很大程度上是由于生活习惯以及饮食方式的不合理,摄入的糖类过量,从而导致胰岛素及其相关受体的功能丧失。当前的降血糖药物在Ⅱ型糖尿病的治疗中起着重要的作用,但是,长期服用这些药物会对人体健康产生潜在的威胁,如诱发癌症、心肌梗塞以及骨折等。临床研究发现,二糖水解酶在糖尿病患者的小肠绒毛中异常活跃。蔗糖酶和麦芽糖酶是将淀粉水解物分解成游离的葡萄糖的两个关键酶。因此抑制蔗糖酶和麦芽糖酶的活性是治疗Ⅱ型糖尿病的关键所在;在降低多糖分解水平的同时,促进机体相关组织和器官对游离葡萄糖的摄取和转化,对于提高糖尿病治疗的效果具有重要的作用。因此,探寻对人体损伤小,抑制二糖水解酶的同时,能提高人体对葡萄糖的吸收的药物是一项具有意义的研究。
甘蔗广泛种植于全球热带地区和亚热带地区,是世界生产蔗糖的重要原料。甘蔗的年产量超过1亿吨。自古以来,甘蔗广泛用于中医治疗中,《本草纲目》有“蔗,脾之果也,其浆甘寒,能泻火热”的记载。经研究发现,甘蔗中含有丰富的天然多酚类物质,如槲皮素、阿魏酸、芹菜素等。甘蔗提取物在抗氧化、抗肿瘤、抑菌、DNA保护以及抗突变等方面有着潜在的生物活性。在甘蔗加工和食用的过程中,甘蔗往往会被当做废弃物,其生物价值被低估,因此探寻甘蔗的生物活性,尤其是甘蔗的降血糖生物活性具有重要的意义。
发明内容
为了解决现有技术的缺点和不足之处,本发明的首要目的在于提供一种甘蔗多酚提取物及其制备方法。
本发明的另一目的在于提供一种通过上述方法制备得到的甘蔗多酚提取物。
本发明的再一目的在于提供上述甘蔗多酚提取物在制备预防和治疗糖尿病的功能食品和药物中的应用。
本发明目的通过以下技术方案实现:
一种甘蔗多酚提取物的制备方法,包括以下步骤:
(1)取甘蔗或蔗渣,粉碎后过筛得粗粉,然后将粗粉用乙醇溶液提取,过滤除去滤渣,滤液减压浓缩至浸膏;
(2)将步骤(1)所得的浸膏加水分散,采用石油醚进行萃取,得到石油醚相和水相,用乙酸乙酯对水相进行萃取,收集乙酸乙酯相,减压浓缩至浸膏;
(3)将步骤(2)中获得的浸膏通过聚酰胺柱层析分离,乙醇梯度洗脱,收集30%乙醇浓度(体积比)的洗脱物,浓缩干燥得到甘蔗多酚提取物。
需要说明的是,上述提取物的制备方法中,还可直接对甘蔗或蔗渣用乙酸乙酯进行萃取后,采用聚酰胺柱层析分离,同样可以得到含量较低的甘蔗多酚提取物。
优选地,步骤(1)中所述粗粉与乙醇溶液的固液质量比为1:(5~15),所述的乙醇溶液是指体积分数为60%~95%的乙醇水溶液。
优选地,步骤(1)中所述的提取是指在静态或动态下,进行浸渍提取或加热提取或回流提取或超声辅助提取。更优选超声辅助提取,超声辅助提取温度为50~70℃,提取时间为60~90min,超声功率为350W。
优选地,步骤(2)中所述的石油醚进行萃取是指用1~2倍体积的石油醚萃取,所述的用乙酸乙酯对水相进行萃取是指用1~2倍体积的乙酸乙酯萃取。
优选地,步骤(3)中所述聚酰胺柱层析分离是指采用30~60目聚酰胺柱层析分离;所述的乙醇梯度洗脱是指依次用体积比为30%、50%、70%和95%乙醇水溶液进行梯度洗脱,得到4个不同的极性部位,分别进行减压蒸发浓缩,得到30%、50%、70%和95%(30%E、50%E、70%E和95%E)共四个乙醇提取物,其中30%乙醇提取物即为具有高降血糖活性的甘蔗多酚提取物。
一种甘蔗多酚提取物,通过上述方法制备得到,所述甘蔗多酚提取物中总酚的质量百分含量为20%~98%,所述总酚包括二甲花翠素-4-乙烯基愈创木酚-3-葡萄糖苷(malvidin 3-glucoside-4-vinylguaiacol,MGV)、槲皮素-3-甲基醚(quercetin 3-methylether,Q3M)、苜蓿素(tricin)、苜蓿素-4-O-愈创木基丙三酯-7-O-吡喃葡萄糖苷(tricin4-O-(threo or erythro)guaiacylglyceryl ether-7-O-glucopyranoside,TGG)、p-香豆酸(p-coumaric acid,PCA)和染料木苷(genistin)这6种多酚的混合物,其中上述6种多酚占总酚质量的15%~85%。
上述6种多酚占总酚的质量百分含量为:1%~12%的MGV、5%~7%的Q3M、5%~20%的tricin、2%~35%的TGG、1%~5%的PCA、1%~6%的genistin。上述多酚的结构式分别如下所示:
TGG:R1=R3=R5=H,R2=OH,R4=OGlc,R6=R8=OCH3,R7=guaiacylglyceryl(threo or erythro);
tricin:R1=R3=R5=H,R2=R4=R7=OH,R6=R8=OCH3;
Q3M:R1=OCH3,R2=R4=R6=R7=OH,R3=R5=R8=H。
上述甘蔗多酚提取物在制备预防和治疗糖尿病的功能食品和药物中的应用。
相对于现有技术,本发明具有如下优点及有益效果:
本发明来源于大宗农作物甘蔗,通过不同极性的有机溶剂对甘蔗多酚进行萃取,以纯化降血糖活性组分,减少或去除与降血糖活性无关的成分,纯化后的产品具有更强的降血糖活性,可用于食品添加剂和功能食品,具有安全、高效、生产工艺简单、成本低、原料易得的优点,具有广阔的市场开发前景。
附图说明
图1为本发明实施例1和2的甘蔗多酚提取物的提取技术路线图;
图2为本发明实施例所得产物甘蔗多酚提取物的葡萄糖吸收实验结果图;
图3为本发明实施例所得产物甘蔗多酚提取物的MS总粒子流图;
图4为本发明实施例所得产物甘蔗多酚提取物的MS/MS总离子流图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1
本实施例的一种甘蔗多酚提取物的制备方法,其提取技术路线图如图1所示,具体提取步骤如下:
(1)选购新鲜的原料蔗,剥皮后将甘蔗粉碎,40℃烘干。过40目筛后得到粗粉,于-20℃保存。取300g上述甘蔗粗粉,用3000mL体积比为60%的乙醇水溶液,水浴回流温度60℃,提取90min,过滤,提取3次,真空抽滤后,合并滤液,减压浓缩至浸膏A1;
(2)将步骤(1)所得浸膏A1加水100mL分散,用石油醚萃取3次,每次加入石油醚100mL,分别收集石油醚萃取液和水相,将水相用100mL的乙酸乙酯萃取3次,收集乙酸乙酯相,减压浓缩至浸膏,制得浸膏A2;
(3)将浓缩浸膏A2通过30目聚酰胺柱层析,依次用体积比为30%、50%、70%和95%乙醇水溶液进行梯度洗脱,得到4个不同的极性部位,收集30%乙醇提取物组分,进行减压蒸发浓缩,冷冻干燥,得到产物甘蔗多酚提取物A3。
实施例2
本实施例的一种甘蔗多酚提取物的制备方法,其提取技术路线图如图1所示,具体提取步骤如下:
(1)取干燥蔗渣300g,粉碎后过40目筛得到粗粉,用1500mL体积比为95%的乙醇水溶液,在50℃超声辅助提取90min,超声功率为350w,提取3次。真空抽滤后,合并滤液,减压浓缩至浸膏B1;
(2)将步骤(1)所得浸膏B1加水100mL分散,用石油醚萃取3次,每次加入石油醚100mL,分别收集石油醚萃取液和水相,将水相用100mL的乙酸乙酯萃取3次,收集乙酸乙酯相,减压浓缩至浸膏,制得浸膏B2;
(3)将浓缩浸膏B2通过60目聚酰胺柱层析,依次用体积比为30%、50%、70%和95%乙醇水溶液进行梯度洗脱,得到4个不同的极性部位,收集30%乙醇提取物组分,进行减压蒸发浓缩,冷冻干燥,得到产物甘蔗多酚提取物B3。
实施例所得甘蔗多酚提取物理化性质测定
(1)总多酚含量的测定:
采用福林酚法。没食子酸标准曲线的制作:准确称量没食子酸标准品25.00mg,用超纯水溶解配制成1mg/mL的溶液,避光4℃保存备用。用超纯水稀释上述溶液得到0.1mg/mL的没食子酸标准溶液。将0.1mg/mL的没食子酸标准品配制成浓度依次为:0、20、60、100、150、200、300、400、500、600(μg/mL)的标准液。在试管中添加100μL的标准品,400μL的超纯水和100μL Folin-Ciocalteu试剂混合后静置6min。静置后加入7%Na2CO3溶液1mL和纯水0.8mL混合均匀。静置1.4h后在760nm波长下测定样品的吸光度值。至少重复三组平行实验。以没食子酸浓度(X)为横坐标,以吸光度(Y)为纵坐标绘制出没食子酸的标准曲线。
得到回归方程:Y=0.0036X+0.0249,R2=0.9995(式中Y为吸光度值,X为没食子酸浓度,0<X<600μg/mL)。
准确吸取化合物溶液100μL加入400μL的超纯水于试管中,采用福林酚法在760nm波长下测定样品的吸光度值,重复三组平行实验。根据上述回归方程计算出样品中多酚含量。多酚含量如下式计算。
多酚含量%=(Y-0.0249)×100%/(0.0036×C×1000)
Y-吸光值;C-样品浓度(mg/mL)
(2)黄酮含量测定:
黄酮含量的测定采用硼氢化钠/四氯代苯对醌法(SBC)。称量29.0mg儿茶素用四氢呋喃:乙醇(1:1)溶解配制成10.0mmol/L的溶液。以四氢呋喃:乙醇(1:1)为溶剂将上述儿茶素溶液配置成浓度为0.3,0.5,1.0,2.0,4.0,5.0,6.0,8.0,10.0mmol/L的儿茶素标准品。在试管中加入1mL的儿茶素标准品,并做三个平行。加0.5mL NaBH4溶液和0.5mL AlCl3·6H2O溶液,混合均匀,在振荡器上震荡30min。加入0.5m LNaBH4溶液,再震荡30min。每个试管中加入2mL冰乙酸,再震荡15min(避光)。向每个试管加入1mL四氯代苯对醌溶液,将试管置于95℃的油浴锅中,进行1h的油浴。自然冷却,用甲醇定容至4mL。向每个试管加入1mL的16%香草醛甲醇溶液,再加入2mL12M的HCl,在室温下避光放置15min。后将试管中溶液转移至离心管,在2500rpm转速下,离心3min。之后在490nm波长下测其吸光度。以儿茶素浓度(X)为横坐标,以吸光度(Y)为纵坐标绘制出儿茶素的标准曲线。得到回归方程:Y=0.1429X+0.0179,R2=0.9923(式中X为儿茶素浓度,Y为吸光度值,0<X<10mmol/L)。
准确吸取化合物溶液100μL到试管中用氮吹仪干燥,用四氢呋喃:乙醇(1:1)定容到1mL。在490nm波长下测其吸光度,重复三组平行实验。根据上述回归方程计算出黄酮含量。黄酮含量按下式计算。
黄酮含量%=(Y-0.0179)×290.27×100%/(0.1429×C×1000);
Y-吸光值;C-样品浓度(mg/mL)。
经检测各提取阶段提取物中多酚和黄酮的含量如表1所示:
表1甘蔗提取物多酚和黄酮含量
(3)抗氧化活性(ORAC)测定:
用pH为7.4的75mM NaH2P04-Na2HPO4缓冲液配制浓度为500μM的荧光素钠储备液,配制完成后在-80℃下避光保存。用磷酸缓冲溶液稀释上述荧光素钠储备液,即得到使用液(50μM)。取4℃下保存的ABAP,用磷酸缓冲溶液配制成119.4μM,每次使用的ABAP均为新鲜配制。精确称量Trolox,溶于磷酸缓冲溶液,配成50μM溶液,并用磷酸缓冲溶液稀释成不同浓度,以磷酸缓冲溶液稀释样品溶液。
将酶标仪预温至37℃,设定激发波长为485nm,发射波长为538nm。在96孔板中,每孔加入20μL样品溶液或Trolox溶液,或磷酸缓冲溶液(空白对照),然后加入200μL荧光素钠使用液,将孔板放入酶标仪中,37℃孵育20分钟。加入20μL ABAP后,开始计时反应并读数,共读35次(共计反应155分钟),将每次读数连成曲线。每个样品设置3个复孔。AUC表示曲线下的面积。Trolox浓度与其Net AUC是成正比的,将样品Net AUC代入所得标准曲线,换算得到ORAC值,即得每毫克粗提物(或浸膏)相当于Torlox的量(μmolTE/g)。实施例1所得A3,抗氧化活性结果如表2所示。
表2实施例1所得甘蔗多酚提取物A3抗氧化活性
(4)体外降血糖实验
将实施例2中所取得的样品用乙醇溶解,用磷酸盐缓冲液稀释成不同浓度的待测液,储存于-20℃冰箱备用,阿卡波糖(AB)为阳性对照。取1g鼠小肠丙酮粉用10mL 0.9%NaCl溶解,依次进行超声(1min×3),离心(3000rpm×30min)处理,取上清液用水稀释4倍作为酶溶液备用。蔗糖、麦芽糖和样品均用100mM磷酸盐缓冲溶液(pH6.9)配成所需浓度。取底物(0.2mL的3.5mM麦芽糖溶液或0.4mL的56mM蔗糖溶液)于10mL比色管中,加入50μL待测液,于37℃的水浴中孵育5min。然后,添加0.2mL酶溶液,37℃继续反应20min。最后,将比色管放入沸水浴,5min进行灭酶。冷却后用葡萄糖检测试剂盒检测样品吸光度。以阿卡波糖为阳性对照,同时设定空白组(缓冲液+酶液+底物),空白对照组(缓冲液+缓冲液+底物),样品测定组(样品+酶液+底物),样品对照组(样品+缓冲液+底物)。
蔗糖酶/麦芽糖酶抑制率%=(1–(A1–A0)/(A3–A2))×100%
式中A0,A1,A2,A3分别为505nm处空白对照组、空白组、样品对照组和样品测定组的吸光值。其结果如表3,表4所示。
表3各组分不同浓度提取物蔗糖酶抑制率
表4各组分不同浓度提取物麦芽糖酶抑制率
(5)葡萄糖吸收实验
细胞培养:人肝癌HepG2细胞用高糖Dulbecco minimum essential medium(DMEM)培养基添加10%胎牛血清培养于含有5%CO2,37℃的培养箱中。
葡萄糖吸收:将用高糖DMEM培养基(含10%胎牛血清)培养的人肝癌HepG2细胞以1.5×104个/孔的密度种植于黑色96孔板上,24h之后将实施例3中所制备的30%乙醇洗脱相(30%E)溶解于DMSO中,用高糖培养基(无血清,含有50μM葡萄糖类似物2-NBDG,1μM胰岛素)稀释至一定的浓度,阿卡波糖(10μM)和罗格列酮(10μM)为阳性对照。药物作用24h之后,去除培养基,用冷的磷酸盐缓冲液冲洗细胞3次,将96孔板至于荧光酶标仪下读数。激发波长设定于485nm,发射波长设定于535nm。结果如图2所示。
(6)HPLC-MS/MS测定甘蔗多酚成分
用HPLC-MS/MS对实施例所得产物甘蔗多酚提取物进行成分分析。取10mg甘蔗多酚提取物A3或B3溶于10mL甲醇溶液中,离心(12,000g),用0.45μm的滤膜过滤后,用HPLC-MS/MS系统分析。
色谱条件:进样体积为20μL,流动相A:含有0.05%乙酸的超纯水,流动相B:色谱甲醇;梯度洗脱条件:0-12min,70A/30B;12-13min,30A/70B;13-15min,100B。
质谱条件:离子源:ESI;离子化模式:正离子模式;毛细管电压:2000V;干燥气:N2;雾化气流量:4L/min;雾化器压力:1bar;干燥器温度:180℃。液质分析结果如表5所示。
表5实施例所得产物甘蔗多酚提取物的HPLC-MS/MS分析结果
a为MS或者MS/MS离子片段。其MS总粒子流图和MS/MS总离子流图分别如图3和图4所示。
由上述结果可以看出,本发明的甘蔗多酚提取物(30%乙醇提取物)具有很好的体外降血糖效果。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其它的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (8)
1.一种甘蔗多酚提取物的制备方法,其特征在于包括以下步骤:
(1)取甘蔗或蔗渣,粉碎后过筛得粗粉,然后将粗粉用乙醇溶液提取,过滤除去滤渣,滤液减压浓缩至浸膏;
(2)将步骤(1)所得的浸膏加水分散,采用石油醚进行萃取,得到石油醚相和水相,用乙酸乙酯对水相进行萃取,收集乙酸乙酯相,减压浓缩至浸膏;
(3)将步骤(2)中获得的浸膏通过聚酰胺柱层析分离,乙醇梯度洗脱,收集30%乙醇体积浓度的洗脱物,浓缩干燥得到甘蔗多酚提取物;
所述甘蔗多酚提取物中总酚的质量百分含量为20%~98%,所述总酚包括二甲花翠素-4-乙烯基愈创木酚-3-葡萄糖苷、槲皮素-3-甲基醚、苜蓿素、苜蓿素-4-O-愈创木基丙三酯-7-O-吡喃葡萄糖苷、p-香豆酸和染料木苷这6种多酚的混合物,其中上述6种多酚占总酚质量的15%~85%。
2.根据权利要求1所述的一种甘蔗多酚提取物的制备方法,其特征在于:步骤(1)中所述粗粉与乙醇溶液的固液质量比为1:(5~15)g/mL,所述的乙醇溶液是指体积分数为60%~95%的乙醇水溶液。
3.根据权利要求1所述的一种甘蔗多酚提取物的制备方法,其特征在于:步骤(1)中所述的提取是指在静态或动态下,进行浸渍提取或加热提取或回流提取或超声辅助提取。
4.根据权利要求3所述的一种甘蔗多酚提取物的制备方法,其特征在于:所述超声辅助提取温度为50~70℃,提取时间为60~90min,超声功率为350W。
5.根据权利要求1所述的一种甘蔗多酚提取物的制备方法,其特征在于:步骤(2)中所述的石油醚进行萃取是指用1~2倍体积的石油醚萃取,所述的用乙酸乙酯对水相进行萃取是指用1~2倍体积的乙酸乙酯萃取。
6.根据权利要求1所述的一种甘蔗多酚提取物的制备方法,其特征在于:步骤(3)中所述聚酰胺柱层析分离是指采用30~60目聚酰胺柱层析分离;所述的乙醇梯度洗脱是指依次用体积比为30%、50%、70%和95%乙醇水溶液进行梯度洗脱,分别进行减压蒸发浓缩,得到30%、50%、70%和95%共四个乙醇提取物,其中30%乙醇提取物即为最终产物。
7.根据权利要求1所述的一种甘蔗多酚提取物的制备方法,其特征在于:直接对甘蔗或蔗渣用乙酸乙酯进行萃取后,收集乙酸乙酯相,减压浓缩至浸膏;然后将获得的浸膏通过聚酰胺柱层析分离,乙醇梯度洗脱,收集30%乙醇体积浓度的洗脱物,浓缩干燥得到甘蔗多酚提取物。
8.根据权利要求1所述的一种甘蔗多酚提取物的制备方法,其特征在于:上述6种多酚占总酚的质量百分含量为:1%~12%的二甲花翠素-4-乙烯基愈创木酚-3-葡萄糖苷、5%~7%的槲皮素-3-甲基醚、5%~20%的苜蓿素、2%~35%的苜蓿素-4-O-愈创木基丙三酯-7-O-吡喃葡萄糖苷、1%~5%的p-香豆酸、1%~6%的染料木苷。
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