CN105294628B - 一种从野菊花中分离制备黄酮类成分的方法 - Google Patents
一种从野菊花中分离制备黄酮类成分的方法 Download PDFInfo
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- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
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Abstract
本发明公开了一种从野菊花中分离制备黄酮类成分的方法。工艺步骤为:(1)野菊花原料粉碎,加入5‑10倍量工业乙醇溶液超声提取2‑3次,提取液浓缩得到浸膏;(2)浸膏以水分散,分别用有机溶剂正己烷,乙酸乙酯,正丁醇萃取,浓缩乙酸乙酯萃取液得到粗提物;(3)再用高速逆流色谱仪分离纯化粗提物中的黄酮类成分(金合欢素/刺槐素,芹菜素,木犀草素,蒙花苷),其两相溶剂系统为正己烷‑氯仿‑甲醇‑水,上相为固定相,下相为流动相,紫外检测器在线监测,收集各流份真空浓缩,结晶,干燥既得。本发明操作简单,污染少。
Description
技术领域
本发明属于天然药物提取分离技术领域,特别涉及一种从野菊花中分离制备高纯黄酮类成分的方法。
背景技术
野菊花为菊科植物野菊(Chrysanthemum indicum L.)的干燥头状花序,味苦、辛,性凉,清热解毒,疏风平肝,用于疔疮、痈肿、丹毒,风热感冒、咽喉肿痛、高血压眩晕、头痛等病症。野菊花主要有效成分是黄酮类化合物,含量较高的有金合欢素/刺槐素,芹菜素,木犀草素和蒙花苷,其中蒙花苷含量最高,作为我国2010版药典规定的野菊花药材的质控指标。
现在研究表明,金合欢素/刺槐素,芹菜素,木犀草素和蒙花苷具有抗缺氧、抗不良刺激、抗疲劳、双向调节机体、抑制血糖升高和影响学习记忆等多种作用。由此可见,目标成分的开发具有广泛的商业价值。结构式如下:
金合欢素/刺槐素(Acacetin);芹菜素(Apigenin);木犀草素(Luteolin);蒙花苷(Linarin)
发明内容
本发明的目的是提供一种从野菊花中分离制备高纯度黄酮类成分的方法。该方法克服传统分离技术的缺陷,采用高速逆流色谱仪(HSCCC)从野菊花中同时制备金合欢素/刺槐素,芹菜素,木犀草素和蒙花苷的专属方法,该方法操作简便,回收率高,分离量大。
本发明技术方案为:
一种从野菊花中分离制备高纯度黄酮类成分的方法,包括如下步骤:
(1)将粉碎后的野菊花原料,以固液质量比1:5-10加入体积分数为30-90%的乙醇,超声提取,分离液过滤,分离出滤渣和滤液;
(2)将滤渣按步骤(1)所属分离方法重复分离2次,合并滤液,减压浓缩至无醇味,得浸膏;
(3)将1质量份的步骤(2)中分离物加入100质量份水溶解,并依次用正己烷,乙酸乙酯,正丁醇萃取,乙酸乙酯萃取液减压浓缩得到粗提物;
(4)将所述步骤(3)中的粗提物采用高速逆流色谱法分离得到四个黄酮类化合物(金合欢素/刺槐素,芹菜素,木犀草素,蒙花苷),其中高速逆流色谱中的溶剂系统由正己烷,氯仿,甲醇,水按体积0.5-3:4:2-4:2组成,该溶剂系统的上相为固定相,转动主机,泵入下相做流动相,流动相溶解粗提物,由进样阀进样,紫外检测器在线检测,收集目标成分,真空浓缩,低温干燥即得四个黄酮类化合物。同时,所用溶剂可以回收再利用。
所述步骤(4)中的高速逆流色谱分离过程中,主机转速750-1000rpm/min,流动相为2.5-15.0ml/min,水浴温度为10-35℃,紫外检测波长为254nm。
目前,野菊花中黄酮类化合物的提取分离主要采用柱色谱法,分离周期长,消耗溶剂多,操作复杂。高速逆流色谱(High-speed countercurrent chromatography,HSCCC)作为一种新型分离纯化技术,操作方便,快捷,分离度较高,克服了由固相载体带来的样品结合、失活、污染等缺点。已被广泛应用于天然产物活性成分的分离制备和总草药的分析鉴定。
运用高速逆流色谱技术分离纯化制备野菊花中黄酮类成分,通过文献检索,国内外尚未见报道,而且本发明通过多次摸索溶剂体系,可以用一个溶剂体系同时分离出极性跨度比较大的四个黄酮类物质,更加高效也更易于放大生产操作。
相比于现有技术,本发明具有以下有益效果:
本发明分离方法简便,快捷,分离周期短,可以短时间内同时分离制备四个黄酮类化合物。传统的柱色谱方法操作繁琐,需要反复的进行柱层析分离,分离周期长,目标成分死吸附严重。采用本方法,大大缩短了时间,提高了效率。
本发明单体制备量大,根据所需四个黄酮类化合物的质量,可以调节被分离样品的进样量,采用制备型高速逆流色谱仪,工艺重现性好,产品纯度高。
采用本发明制备金合欢素/刺槐素,芹菜素,木犀草素,蒙花苷,产品纯度高,品质好,适用于各种型号高速逆流色谱仪制备野菊花中的金合欢素/刺槐素,芹菜素,木犀草素,蒙花苷,易于产业化放大。
附图说明
图1为本发明实施例1中分离得到金合欢素/刺槐素,芹菜素,木犀草素,蒙花苷的高速逆流色谱色谱图
图2为本发明实施例1中分离到的金合欢素/刺槐素,芹菜素,木犀草素,蒙花苷的高效液相色谱检测图
附图中数字标记:1金合欢素/刺槐素;2芹菜素;3木犀草素;4蒙花苷
具体实施方式
下面结合具体实施方案,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外,应理解,在阅读本发明讲授的内容后,本领域技术人员可以对发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。实施例1:
取经干燥的野菊花原料粉碎,称1000g,每次加入10L 80%乙醇溶液超声提取60分钟,提取3次,提取液减压浓缩得到浸膏253g。按1质量份浸膏加入100质量份水溶解加正己烷,乙酸乙酯,正丁醇依次萃取,每种溶剂萃取3次,合并萃取液回收乙酸乙酯得粗提物56g,用于高速逆流分离。
在分液漏斗里配制正己烷、氯仿、甲醇、水两相溶剂体系,按1:4:1:2比例混合,振摇后静置分层,超声脱气30min。取上相注满高速逆流色谱仪作为固定相,转动主机,转速800rpm,同时以5ml/min泵入下相做流动相,建立动态平衡后,用流动相溶解粗提物,由进样阀进样,紫外检测器在254nm波长下在线监测,收集各目标成分,连续制备,相应合并回收试剂,浓缩后甲醇结晶,从500mg乙酸乙酯粗提物中分离得到高纯度的金合欢素/刺槐素10mg,芹菜素10.5mg,木犀草素27.1mg,蒙花苷37.4mg。经HPLC检测,含量均大于97%,经UV、IR、MS、HNMR、CNMR等表征其物理性状的数据与现有技术相一致。
图1为本发明实施例1中分离得到金合欢素/刺槐素,芹菜素,木犀草素,蒙花苷的高速逆流色谱色谱图,可以看出采用高速逆流色谱仪结合本发明采用的溶剂体系可以短时间内分离制备出四个黄酮类物质,简便高效。
图2为本发明实施例1中分离到的金合欢素/刺槐素,芹菜素,木犀草素,蒙花苷的高效液相色谱检测图,可以看出采用高速逆流色谱仪结合本发明采用的溶剂体系分离制备得到的四个黄酮类物质,纯度均在97%以上。
实施例2:
取经干燥的野菊花原料粉碎,称500g,每次加入8L 70%乙醇溶液超声提取60分钟,提取2次,提取液减压浓缩得到浸膏330g。按1质量份浸膏加入100质量份水溶解加正己烷,乙酸乙酯,正丁醇依次萃取,每种溶剂萃取3次,合并萃取液回收乙酸乙酯得粗提物64g,用于高速逆流分离。
在分液漏斗里配制正己烷、氯仿、甲醇、水两相溶剂体系,按2:4:2:2比例混合,振摇后静置分层,超声脱气30min。取上相注满高速逆流色谱仪作为固定相,转动主机,转速900rpm,同时以15ml/min泵入下相做流动相,建立动态平衡后,用流动相溶解粗提物,由进样阀进样,紫外检测器在254nm波长下在线监测,收集各目标成分,连续制备,相应合并回收试剂,浓缩后甲醇结晶,从乙酸乙酯粗提物中分离得到高纯度的金合欢素/刺槐素,芹菜素,木犀草素,蒙花苷。经HPLC检测,含量均大于97%,经UV、IR、MS、HNMR、CNMR等表征其物理性状的数据与现有技术相一致。
实施例3:
取经干燥的野菊花原料粉碎,称500g,每次加入8L 60%乙醇溶液超声提取40分钟,提取3次,提取液减压浓缩得到浸膏。按1质量份浸膏加入100质量份水溶解加正己烷,乙酸乙酯,正丁醇依次萃取,每种溶剂萃取3次,合并萃取液回收乙酸乙酯得粗提物,用于高速逆流分离。
在分液漏斗里配制正己烷、氯仿、甲醇、水两相溶剂体系,按0.5:4:1.5:2比例混合,振摇后静置分层,超声脱气30min。取上相注满高速逆流色谱仪作为固定相,转动主机,转速9850rpm,同时以10ml/min泵入下相做流动相,建立动态平衡后,用流动相溶解粗提物,由进样阀进样,紫外检测器在254nm波长下在线监测,收集各目标成分,连续制备,相应合并回收试剂,浓缩后甲醇结晶,从乙酸乙酯粗提物中分离得到高纯度的金合欢素/刺槐素,芹菜素,木犀草素,蒙花苷。经HPLC检测,含量均大于97%,经UV、IR、MS、HNMR、CNMR等表征其物理性状的数据与现有技术相一致。
Claims (2)
1.一种从野菊花中分离制备高纯黄酮类成分的方法,其特征在于,包括以下步骤:
(1)将粉碎后的野菊花原料,以固液质量比1:5‐10加入体积分数为30‐90%的乙醇溶液,超声提取,提取液过滤,分离出滤渣和滤液;
(2)将滤渣按步骤(1)提取分离方法重复提取分离2‐3次,合并滤液,减压浓缩至无醇味,得浸膏;
(3)将1质量份步骤(2)中的浸膏加入100质量份水溶解,并依次用正己烷,乙酸乙酯,正丁醇萃取,乙酸乙酯萃取液减压浓缩得到粗提物;
(4)将所述步骤(3)中的粗提物采用高速逆流色谱法分离得到四种黄酮类化合物,所述的四种黄酮类化合物为金合欢素,芹菜素,木犀草素,蒙花苷,其中高速逆流色谱中的溶剂系统由正己烷,氯仿,甲醇,水组成,它们的体积比为1:4:1:2、2:4:2:2或0.5:4:1.5:2,该溶剂系统的上相为固定相,下相为流动相;
所述步骤(4)中的高速逆流色谱分离过程中,主机转速750‐1000rpm/min,流动相为2.5‐15.0ml/min,水浴温度为10‐35℃。
2.根据权利要求1所述的黄酮类成分的制备方法,其特征在于,紫外检测器在线监测,收集流份减压浓缩,结晶,干燥,得到高纯度的金合欢素,芹菜素,木犀草素,蒙花苷。
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