CN108409703B - 一种模拟移动床分离具有延缓衰老作用的花色苷的方法 - Google Patents
一种模拟移动床分离具有延缓衰老作用的花色苷的方法 Download PDFInfo
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Abstract
本发明公开了一种从浆果果实中提取花色苷的方法。该方法包括下述步骤:(1)将浆果果实打成果浆,加入乙醇水溶液进行提取;将提取液依次经过大孔树脂纯化、旋蒸蒸发回收溶剂、浓缩提取液、真空冷冻干燥,得到花色苷粗提物;(2)采用模拟移动床色谱对所述花色苷粗提物溶液进行分离,得到花色苷提取物。本发明通过大孔树脂及模拟移动床对浆果中花色苷进行分离纯化,分离出杂质少的花色苷提取物,制备成用于抗衰老的花色苷提取物。该方法实现了花色苷提取物的工业化生产,从而提高了花色苷的生产效率。花色苷可以单独或复配在食品或药品中应用,制成任何剂型的保健品或药品,黑果腺肋花楸、桑葚、葡萄等花色苷具有延缓衰老的作用。
Description
技术领域
本发明涉及一种模拟移动床分离具有延缓衰老作用的花色苷的方法。
背景技术
花色苷属于植物多酚的类黄酮,是植物的次生代谢产物,其结构由C6-C3-C6骨架结构的花色素以糖苷键结合葡萄糖、半乳糖等糖苷而成。自然界中已知有20种花色素,食源性花色素主要有六种形式,多以葡萄糖酯化的形式存在。花色素与葡萄糖、半乳糖、阿拉伯糖、木糖、鼠李糖等,发生酯化形成花色苷;并可与咖啡酸、对香豆酸、芥子酸、对羟基苯甲酸、阿魏酸、丙二酸、苹果酸、琥珀酸和乙酸等发生酰化反应,形成酰化形式花色苷。花色素含有多个酚羟基,具有极强的抗氧化活性,作为自由基清除剂和抗氧化活性物质,因此也极不稳定,但是通过上述一系列的酯化、酰化结构形成的花色苷极好地保护了花色素。摄食后,在人体内酸性或酶的作用下会缓慢的水解,释放出花色素,表现出强烈的抗氧化活性。花色苷具有制癌细胞增殖、抗炎、抑制肥胖,糖尿病和心血管疾病等作用。
在自然界中花色苷广泛存在于浆果类水果如桑葚、黑莓、树莓、覆盆子、黑醋栗、接骨木梅、蓝莓、黑加仑等,其中桑葚主要含有花青素-3-O-葡萄糖苷和花青素-3-O-芸香糖苷,蓝莓主要含有花翠素-3-O-半乳糖苷,葡萄果实中主要含有3,5-二甲基花翠素-3-O-葡萄糖苷等。
黑果腺肋花楸属于蔷薇科植物,主要种植在北美、欧洲及俄罗斯等地方,我国八十年代引进在辽宁等地种植,用于园林观赏植物。黑果腺肋花楸果实含有丰富的多酚类物质,其中矢车菊3-O-半乳糖苷、矢车菊3-O-阿拉伯糖苷、矢车菊3-O-木糖苷和矢车菊3-O-葡萄糖苷为主要花色苷,主要类黄酮包括槲皮素。但是目前黑果腺肋花楸开发利用度低,花色苷价值还未被完全开发,多酚类物质主要集中在降血脂、降血糖、抗氧化等方面,并且采用模拟移动床分离黑果腺肋花楸花色苷的工艺未见报道。
模拟移动床色谱(Simulated moving bed chromatography,SMB)是连续色谱的一种,由色谱柱或类似色谱柱的固定床层串联起来的分离系统。目前,模拟移动床一类是由美国UOP公司开发的Sorbax系统,一类是采用旋转木马式,通过旋转阀与一系列柱串联而成。旋转阀由两个不锈钢盘构成,下部钢盘静止而上部钢盘每隔一定时间逆时针旋转一定角度,由此模拟固定相的逆流移动。系统中洗脱液、进样液的进口以及萃取液和残余液的出口位置均是固定的。
模拟移动床色谱系统具有精细分离,常温操作,无热损伤,质耗低,能耗低、效率高等优点,适用于分离手性药物、热敏性物质,在食品、生物医药、化妆品及精细化工产品中广泛应用。
发明内容
本发明的一个目的是提供一种从浆果果实中提取花色苷的方法。
本发明所提供的从浆果果实中提取花色苷的方法,包括下述步骤:
(1)将浆果果实和/或皮渣打成果浆,加入乙醇水溶液进行提取,收集提取液;将所述提取液依次经过大孔树脂纯化、旋转蒸发回收乙醇,收集浓缩的提取液,真空冷冻干燥,得到花色苷粗提物;
(2)将所述花色苷粗提物以体积分数为25%~30%的乙醇水溶液加0.1%~1%盐酸或柠檬酸为溶剂,配制成花色苷粗提物溶液;采用模拟移动床色谱对所述花色苷粗提物溶液进行分离,得到花色苷提取物;
其中,所述模拟移动床色谱中的模拟移动床为IV带结构,每带由1-2根C18色谱柱串联组成;采用的流动相是体积分数为20%~50%的乙醇水溶液,其中加入质量分数1%~5%的盐酸或柠檬酸;进样液流速为0.4~1mL/min,洗脱液流速为4~15mL/min(优选为4~10mL/min),萃取液流速为1~10mL/min(优选为1~5mL/min),萃余液流速为4~10mL/min(优选为6~10mL/min),阀切换时间为100~150s(优选为130~150s),色谱操作的温度为20~32℃。
上述方法步骤(1)中,所述乙醇水溶液中乙醇的体积分数为40%-90%;所述超声提取至少进行1次,优选进行1-5次。
所述提取中,所述果浆与所述乙醇水溶液的质量比(即料液比)为1:2~1:10。
所述提取为超声辅助提取,所述提取的时间为15-30min,所采用的超声频率为200~400W。
所述大孔树脂具体可选自下述任意一种:XAD-7HP、AB-8、D101和HPD。
上述方法步骤(1)中,在提取前还向所述果浆中加入柠檬酸护色,所述柠檬酸的加入量为所述果浆和乙醇水溶液总质量的1%-5%。
所述浆果包括但不限于下述物质:黑果腺肋花楸、葡萄、桑葚、蓝莓、黑加仑、黑莓、树莓、覆盆子、黑醋栗、接骨木梅、草莓、黑加仑、越橘、樱桃、黑李子、石榴和火龙果。
当所述浆果为桑葚、黑果腺肋花楸、蓝莓、黑莓、树莓、覆盆子、黑醋栗和接骨木梅、草莓、黑加仑、越橘、樱桃等时,可选用其果实打浆;当所述浆果为葡萄、蓝莓、黑果腺肋花楸、黑李子、石榴、火龙果等时,可以选用皮渣,如酿酒后的葡萄皮渣。
上述方法制备得到的花色苷提取物也属于本发明的保护范围。
本发明的再一个目的是提供上述制备的花色苷提取物的应用。
本发明所提供的花色苷提取物的应用包括下述方面:1)在制备延缓衰老产品中的应用;2)在制备预防和/或治疗阿尔茨海默病的产品中的应用;3)在制备改善学习记忆功能的产品中的应用;4)在制备对PC12细胞损伤保护用的产品中的应用。
其中,所述PC12细胞损伤是由Aβ(β淀粉样蛋白)诱导引起的损伤。
所述产品包括食品、药品或保健品。
本发明提供的花色苷可以单独或复配在食品或药品中应用,制成任何剂型的保健品或药品,黑果腺肋花楸、桑葚、葡萄等花色苷具有延缓衰老的作用。
本发明以模拟移动床分离技术为手段,通过大孔树脂及模拟移动床对浆果(黑果腺肋花楸)中花色苷进行分离纯化,分离出杂质少的花色苷提取物,制备成用于抗衰老的花色苷提取物。模拟移动床分离方法纯化效率高,可适用于连续化生产,纯度满足于食品或保健品的添加需求,该方法实现了花色苷提取物的工业化生产,从而提高了花色苷的生产效率。
本发明采用浆果花色苷提取物为原料,利用Aβ诱导的PC12细胞凋亡,通过体外细胞增殖实验得出,花色苷提取物可以抑制Aβ对PC12细胞的凋亡作用,保护细胞免于Aβ的伤害。
本发明采用黑果腺肋花楸花色苷提取物为原料,以快速老化小鼠SAMP8为模型,通过水迷宫实验得出,花色苷提取物提高小鼠行为认知能力,提升小鼠体力,减缓衰老。由此可见,浆果(如黑果腺肋花楸)花色苷提取物具有延缓衰老的作用,是开发天然、抗衰老保健品的优质原料。
附图说明
图1为实施例1中经大孔树脂纯化后得到的黑果腺肋花楸花色苷粗提物的液相色谱图。
图2为实施例1中黑果腺肋花楸花色苷粗提物经模拟移动床纯化后萃取物的液相色谱图。
图3为实施例1中黑果腺肋花楸花色苷粗提物经模拟移动床纯化后萃余物的液相色谱图。
图4为黑果腺肋花楸花色苷提取物冷冻干燥产品图。
图5为实施例5中不同种类的浆果花色苷提取物对PC12细胞保护作用。
图6为实施例5中黑果腺肋花楸花色苷提取物对快速老化小鼠SAMP8的学习记忆能力的影响。
具体实施方式
下面通过具体实施例对本发明的方法进行说明,但本发明并不局限于此,凡在本发明的精神和原则之内所做的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中所使用的模拟移动床色谱购自北京优联宏远生物技术有限公司,规格型号为HYSMB6-500;
RPMI Medium Modified 1640,withL-Glutamine(HyClone,SH30809.01)。
实施例1、制备花色苷提取物
(1)将25g黑果腺肋花楸果实打浆,按照1:4料液比,加入体积分数为的60%乙醇水溶剂提取,加入1%柠檬酸护色,超声辅助提取30min(超声功率320w),连续提取3次,合并提取液,经大孔树脂XAD-7HP纯化,经旋转蒸发回收溶剂,浓缩提取液,经真空冷冻干燥,得到黑果腺肋花楸粗提物,其纯度以矢车菊素半乳糖苷计为35%。
(2)将所述黑果腺肋花楸粗提物用体积分数为25%的乙醇水溶液溶液加1%盐酸为溶剂,配制成花色苷粗提物溶液;采用模拟移动床色谱分离技术分离黑果腺肋花楸花色苷,模拟移动床为IV带结构,有8根C18色谱柱组成,每带由2根色谱柱串联组成,流动相为乙醇:水=25:75(v/v),加入质量分数1%的盐酸,进样液流速为0.42mL/min,洗脱液流速为4.5mL/min,萃取液流速为1.5mL/min,萃余液流速为6.5mL/min,切换时间为141s,色谱操作温度为26℃。高效液相色谱法检测,采用Intertsil ODS-SPC 18柱,A相为酸化去离子水(冰醋酸/磷酸/去离子水=10:1:89),B相为乙腈,A:B=80:20,流速:1mL/min,PDA检测器在波长278nm分析。得到黑果腺肋花楸花色苷经液相色谱分析(面积归一化法)纯度为80%。
实施例2、制备花色苷提取物
(1)将25g黑果腺肋花楸果实打浆,按照1:4料液比,加入体积分数为的50%乙醇水溶剂提取,再加入1%柠檬酸护色,超声辅助提取20min(超声功率240w),连续提取5次,经大孔树脂AB-8纯化,经旋蒸蒸发回收溶剂,浓缩提取液,经真空冷冻干燥,得到黑果腺肋花楸粗提物,其纯度以矢车菊素半乳糖苷计为35%。
(2)将所述黑果腺肋花楸粗提物用体积分数为25%的乙醇水溶液溶液加1%盐酸为溶剂,配制成花色苷粗提物溶液;采用模拟移动床色谱分离技术分离黑果腺肋花楸花色苷,模拟移动床为IV带结构,有6根C18色谱柱组成,每带由1-2根色谱柱串联组成,流动相为乙醇:水=25:75(v/v),加入质量分数1%的盐酸,进样液流速为0.5mL/min,洗脱液流速为4.5mL/min,萃取液流速为1.5mL/min,萃余液流速为8mL/min,切换时间为131s,色谱操作温度为25℃。得到黑果腺肋花楸花色苷经液相色谱分析(面积归一化法)纯度为68%。
实施例3、制备花色苷提取物
(1)将25g黑果腺肋花楸果实打浆,按照1:4料液比,加入90%乙醇水溶剂提取,加入5%柠檬酸护色,超声辅助提取20min(超声功率400w),提取1次,经大孔树脂HPD纯化,经旋转蒸发回收溶剂,浓缩提取液,经真空冷冻干燥,得到黑果腺肋花楸粗提物纯度为30%。
(2)将所述黑果腺肋花楸粗提物用体积分数为30%的乙醇水溶液加1%盐酸为溶剂,配制成花色苷粗提物溶液;采用模拟移动床色谱分离技术分离黑果腺肋花楸花色苷,模拟移动床为IV带结构,有6根C18色谱柱组成,每带由1-2根色谱柱串联组成,流动相为乙醇:水=25:75(v/v),加入质量分数1%的盐酸,进样液流速为1mL/min,洗脱液流速为9.5mL/min,萃取液流速为1.5mL/min,萃余液流速为9mL/min,切换时间为141s,色谱操作温度为30℃。得到黑果腺肋花楸花色苷经液相色谱分析(面积归一化法)纯度为60%。
实施例4、制备花色苷提取物
(1)将25g桑葚果实打浆,按照1:4料液比,加入体积分数为的60%乙醇水溶剂提取,加入1%柠檬酸护色,超声辅助提取30min(超声功率320w),连续提取3次,合并提取液,经大孔树脂XAD-7HP纯化,经旋转蒸发回收溶剂,浓缩提取液,经真空冷冻干燥,得到桑葚粗提物,其纯度以矢车菊素葡萄糖苷计为30%。
(2)将所述桑葚粗提物用体积分数为25%的乙醇水溶液溶液加1%盐酸为溶剂,配制成桑葚粗提物溶液;采用模拟移动床色谱分离技术分离桑葚花色苷,模拟移动床为IV带结构,有8根C18色谱柱组成,每带由2根色谱柱串联组成,流动相为乙醇:水=25:75(v/v),加入质量分数1%的盐酸,进样液流速为0.42mL/min,洗脱液流速为4.5mL/min,萃取液流速为1.5mL/min,萃余液流速为6.5mL/min,切换时间为141s,色谱操作温度为26℃。高效液相色谱法检测,采用IntertsilODS-SPC18柱,A相为酸化去离子水(冰醋酸/磷酸/去离子水=10:1:89),B相为乙腈,A:B=80:20,流速:1mL/min,PDA检测器在波长278nm分析。得到桑葚花色苷经液相色谱分析(面积归一化法)纯度为60%。
实施例5、花色苷提取物的药效试验
(1)PC12细胞增殖活性实验
1.1PC12细胞损伤模型的建立
将PC12细胞均匀接种在96孔板中,接种密度为1.0×105个/mL,每个孔的接种量为100μL。细胞在10%FBS完全培养基RPMI1640中培养24h,更换为2%FBS低血清浓度的培养基RPMI1640,对PC12细胞进行饥饿处理,使得96孔板中的PC12细胞状态均一稳定,并于37℃,5%CO2孵育箱中孵育培养,24h后用于后期药物处理,建立PC12细胞损伤模型用于后期实验。
1.2.MTT法检测细胞存活率
造模24h后,进行药物处理,添加实施例1制备的黑果腺肋花楸花色苷提取物或实施例4制备的桑葚花色苷提取物200μg/mL 24h后,用微型台式真空泵移去上层培养液,加入0.5mg/mL MTT溶液,每孔100μL,放入CO2孵箱,5%CO2,37℃孵育4h。加入DMSO助溶剂150μL/孔,37℃孵育4h,使甲瓒颗粒完全溶解。用酶标仪检测甲瓒的生成量,检测波长为492nm。
存活率(%)=(处理组-空白组)/(对照组-空白组)×100%
结果如图5所示。由图5可知,黑果腺肋花楸花色苷提取物以及桑葚花色苷提取物对于PC12细胞存活率没有显著的细胞毒性。
(2)水迷宫试验
供试样品:
取实施例1制备得到的黑果腺肋花楸花色苷提取物,加水稀释制成所需浓度。
动物:
SAMP8(快速老化模型小鼠)和SAMR1小鼠,SPF级,雄性,4月龄,数量48只(其中,SAMP8小鼠36只,SAMR1小鼠12只)。购自天津中医药第一附属医院实验动物房。
实验动物分组及给药方式:
正常对照组,AD模型阳性对照组,药物组,实施例1制备的黑果腺肋花楸花色苷提取物分高、低组,每组12只,随机分配。正常进食,给水。
灌胃60天。
水迷宫的准备:
采用黑色食用色素将水染黑,水面基本充满摄像头的圆圈视野;控制水温在20~22℃;将平台置于水池第一象限。训练期间平台要低于水面1厘米,保持能够让小鼠触碰平台。在训练的后期以及测试期将平台隐藏至水面以下。
水迷宫训练:
1.定向航行试验:实验开始前,将小鼠在迷宫中适应性游泳一次,让其熟悉环境1分钟,并将动物在平台上停留20s;正式试验时,将小鼠面向池壁分别从各个象限的入水点放入水中,记录小鼠游泳轨迹,每个象限的时间,找到平台时间,平均距离。每只小鼠每次游泳60s,如果60s内未能找到平台,则将其引至平台并停留10s。分别在4个象限训练1次,每次训练间隔20min,连续进行5天。
迷宫水池分东南西北四个象限,应保证动物有机会从每个象限都能入水受训,动物入水时应用手托住,动作缓慢轻柔,以免动物受惊扰,应使动物尾端先入水,切不可强迫或者猛烈将动物头部先投入水中。
2.空间探索试验:在定位航行试验结束后(即第6天,将平台撤离,将小鼠从平台所在象限的对面象限放入水中,自由游泳60s,记录小鼠游泳路线、在平台象限的时间及穿越平台的次数。
结果见图6。由图6可知,花色苷提取物明显提高小鼠空间搜索能力,低剂量组和高剂量组均高于模型组。给予花色苷高低剂量组的小鼠体力更好,在水中更加灵活。
Claims (2)
1.一种从浆果果实中提取花色苷的方法,包括下述步骤:
(1)将浆果果实和/或皮渣打成果浆,加入乙醇水溶液进行提取,收集提取液;将所述提取液依次经过大孔树脂纯化、旋蒸蒸发回收乙醇,浓缩提取液,得到花色苷粗提物;
(2)将所述花色苷粗提物以体积分数为25%~30%乙醇溶液加质量分数0.1%~1%盐酸或柠檬酸为溶剂,配制成花色苷粗提物溶液;采用模拟移动床色谱对所述花色苷粗提物溶液进行分离,得到花色苷提取物;
其中,所述模拟移动床色谱中的模拟移动床为IV带结构,每带由1-2根C18色谱柱串联组成;采用的流动相是体积分数为20%~50%的乙醇水溶液,其中加入质量分数1%~5%的盐酸或柠檬酸;进样液流速为0.4~1mL/min,洗脱液流速为4~15mL/min,萃取液流速为1~10mL/min,萃余液流速为4~6mL/min,阀切换时间为100~150s,色谱操作的温度为20~32℃;
所述方法步骤(1)中,所述乙醇水溶液中乙醇的体积分数为40%~90%;所述提取中,所述果浆与所述乙醇水溶液的质量比为1:4;
所述提取为超声辅助提取,所述提取的时间为15~30min,所采用的超声频率为240~400W;所述提取进行1~5次;
所述浆果选自下述至少一种:黑果腺肋花楸、桑葚;
所述大孔树脂选自下述任意一种:XAD-7HP、D101和HPD;
所述步骤(1)中,在提取前还向所述果浆中加入柠檬酸护色,所述柠檬酸的加入量为所述果浆和乙醇水溶液总质量的1%~5%。
2.根据权利要求1所述的方法,其特征在于:所述大孔树脂为XAD-7HP。
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