CN115144491B - 一种提纯并检测霍山石斛鲜茎中6-羟基石斛次碱的方法 - Google Patents
一种提纯并检测霍山石斛鲜茎中6-羟基石斛次碱的方法 Download PDFInfo
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Abstract
本发明涉及一种高效提纯并检测霍山石斛鲜茎中6‑羟基石斛次碱的方法。该方法以GC‑MS法检测霍山石斛中的6‑羟基石斛次碱,先自霍山石斛鲜茎中提取含6‑羟基石斛次碱的待测液用于检测,提取方法为:将霍山石斛鲜茎冻干粉碎过筛得到石斛干粉,加水超声处理后,加入复合酶酶解,得到酶解液;酶解液中加入酸性醇,超高压下提取1min后,取滤液;滤液真空浓缩后,使用MCX萃取柱纯化,甲醇‑乙腈溶液洗脱,收集洗脱液,氮吹至无水分后,再用甲醇溶解过滤,得到待测液。本发明为霍山石斛中的特征生物碱6‑羟基石斛次碱的提取纯化和分离鉴定提供了一套高效且完整的方法,对6‑羟基石斛次碱的提取具有重要意义。
Description
技术领域
本发明属于检测技术领域,具体涉及一种高效提纯并检测霍山石斛鲜茎中6-羟基石斛次碱的方法。
技术背景
石斛中生物碱具有清热养胃、明目清音、退热、止痛作用,还可用于降低血压、心率,减慢呼吸,并可解巴比妥中毒。
生物碱在植物体内的存在形式,少数碱性极弱的以游离状态存在,多数都以成盐的形式存在于植物细胞内。目前,在生物碱提取方面的研究表明:游离的生物碱溶于亲脂性有机溶剂或者醇类溶剂而不溶于水,而成盐的生物碱则较易溶于水。按照提取溶剂的不同对其进行分类,可分为:亲脂性有机溶剂提取法、醇提法和酸水提取法。在石斛生物碱的提取研究方面,主要使用亲脂性有机溶剂提取法和醇提法。
亲脂性有机溶剂提取法多以三氯甲烷为提取溶剂,但具有毒性大等缺点;醇提法主要以甲醇或者乙醇为提取溶剂,但醇提法得到的石斛提取液中常含有较多脂溶性杂质;若以乙醇-水为提取溶剂,石斛提取液还会存在一些水溶性杂质,如多糖等,不利于纯化。
另外,石斛生物碱中对特定生物碱的分离难度较大,目前从石斛属植物中已经发现约41种生物碱。按照其结构骨架分类,主要有五种不同结构骨架的生物碱,分别是倍半萜类、吲哚里西啶类、吡咯烷类、苯酞类和咪唑类,同类间的石斛碱分离更易受到影响。
总生物碱含量、成分种类在不同种属之间差异较大。局限分布于于安徽霍山的霍山石斛(Dendrobium huoshanense,D.huoshanense)被誉为“中华九大仙草之首”。近年来,霍山石斛生物碱还证实具有抗菌作用,并可能有抗癌作用。其中的6-羟基石斛次碱是存在于霍山石斛中的一类含氮的具有复杂的环状结构的小分子有机化合物。但因其含量低,提取困难,目前对6-羟基石斛次碱的研究内容较少。另外石斛中生物碱受到干燥、存储、提取工艺等因素的影响,会存在不同程度的流失,6-羟基石斛次碱天然含量本就少,受提取工艺影响大,目前大多数的生物碱提取方法甚至无法检测出6-羟基石斛次碱。现阶段,对该物质的成功高效提取以及快速准备的分析确认,是阻碍6-羟基石斛次碱研究的一大障碍。
论文“崔楠楠.霍山石斛生物碱提取纯化及真空冷冻干燥加工工艺研究[D].安徽农业大学,2013.”研究了霍山石斛的总生物提取工艺,但是没有对6-羟基石斛次碱的特定提取进行研究。论文“陈婧超.石斛中生物碱分离纯化及神经保护作用研究[D].合肥工业大学,2019.”研究了霍山石斛的总生物提取和成分分析,但是没有对6-羟基石斛次碱的特定提取进行研究。
发明内容
为了解决上述问题,本发明提供一种高效提纯并检测霍山石斛鲜茎中6-羟基石斛次碱的方法。
本发明采用了以下技术方案:
一种高效提纯并检测霍山石斛鲜茎中6-羟基石斛次碱的方法,所述6-羟基石斛次碱采用GC-MS法检测,自霍山石斛鲜茎中提取含6-羟基石斛次碱的待测液用于检测,提取方法包括以下步骤:
S1.将霍山石斛鲜茎通过冻干法脱水,粉碎过筛得到石斛干粉,备用;
S2.取S1中石斛干粉加入水中,超声处理20~30min后,加入占石斛干粉质量1.5%的复合酶,酶解1.5h,得到酶解液,所述石斛干粉和水的使用比为1g:6mL;
S3.在酶解液中加入设定量的酸性醇,室温放置18~24h后,在真空且超高压100MPa环境下处理1min,过滤残渣,取滤液;
S4.将所述滤液真空浓缩至5~8mL后,使用MCX萃取柱纯化,甲醇-乙腈溶液洗脱,收集洗脱液,氮吹至无水分后,再用甲醇溶解过滤,得到含有6-羟基石斛次碱的待测液。
优选的,所述步骤S1中,冻干的温度为-50℃,冻干时间40~48h,粉碎细度为80目。
优选的,所述步骤S2中,复合酶由纤维素酶和果胶酶以10000U/g等活力配制。
优选的,所述步骤S3中,以酶解液:酸性醇体积比1:40添加所述酸性醇,所述酸性醇为体积浓度70%的乙醇水溶液,pH值为3~3.5。
优选的,所述步骤S4中,MCX萃取柱纯化的具体操作为:
所述步骤S4中,MCX萃取柱纯化的具体操作为:
浓缩后的滤液使用质量浓度2~5%H3PO4水溶液溶解,过0.22μm水系滤膜,得到上样液体;MCX萃取柱依次使用5~8ml甲醇和5~8ml水活化,将上样液体按照0.5~1.0ml/min流速上样,上样结束后,使用3~5mL质量浓度2-5%甲酸水溶液进行淋洗;淋洗后用含有占溶液体积比3~5%氨水的甲醇/乙腈溶液进行洗脱,得到洗脱液。
优选的,所述GC-MS法检测包括气相色谱和质谱,其中气相色谱的具体操作为:色谱柱初始柱温70℃,并保持2℃/min上升,直至150℃后维持1min,再以3℃/min上升至180℃后维持1min,再以20℃/min上升至240℃后维持3min;期间,载气为高纯度He,以1.0ml/min流速进气;分流比设定为20:1;
质谱的具体操作为:EI离子源;离子能量设置为70eV;温度设定为200℃;全扫描测定方式的扫描范围m/z 40-500。
与现有技术相比,本发明的有益效果在于:
总生物碱含量在不同石斛种属之间差异较大,在霍山石斛中仅占到0.043%,而在其它品种中,如金钗石斛,可以达到0.548%(干重)。霍山石斛中生物碱种类较多,生物碱的提取和分离难度大。
6-羟基石斛次碱(6-hydroxynobiline),是一类含氮杂环化合物,属于倍半萜类生物碱,又叫石斛型生物碱,其结构特点是含氮吡咯环与倍半萜部分组成紧密的四环体系,在石斛中含量相对其它碱含量尤其少,其化学结构式如下,:
目前,石斛多采用传统烘干技术干燥,有效成分流失严重,从而影响到药材的品质和疗效。真空冷冻干燥是近代快速发展的一种干燥技术,能够较好保证药材的外观、色泽,最大限度地保存药材有效成分活性,干燥彻底,易于保存。本发明首先将石斛鲜茎置于冷冻干燥机中脱水干燥,相较于传统干燥技术,可大大减少石斛中有效物质的损失,避免因温度过高而造成生物碱分解减少。
超声波具有细胞破碎性强、提取效率高及降低时间成本的特点。所使用的纤维素酶和果胶酶来源广泛且价格低廉,反应条件温和,易于操作。通过超声波与酶的结合,可以更大程度上使霍山石斛中的生物碱溶出,再加入酸性醇浸提18~24h后在超高压的作用下,使生物碱充分被浸提,增大生物碱提取率。
6-羟基石斛次碱是纯碱性的一类含氮杂环有机小分子化合物,MCX是一种高分子聚合物,是一种阳离子交换吸附剂,能直接吸附碱性化学物质,因此采用本申请MCX纯化是一种简单有效的分离方法。
本发明以霍山石斛为原料,提取液制备步骤少且提取纯化效率高,基于超声波/超高压提取,结合酶提、冻干等技术,为霍山石斛中的特征生物碱6-羟基石斛次碱的提取纯化和分离鉴定提供了一套高效且完整的方法,对6-羟基石斛次碱的提取具有重要意义。本发明为霍山石斛的有效利用和资源开发提供科学依据,也为其它种类石斛中的6-羟基石斛次碱探索与鉴定提供的技术支持,还为其他活性物质及其他植物的生物碱提取纯化提供了新思路。
附图说明
图1为6-羟基石斛次碱在主谱库中的标准质谱图;
图2为实施例1制备的样品的GC-MS检测结果,图中右上角为圆圈部分的放大图,为6-羟基石斛次碱特征峰,表明检出;
图3为对比例1制备的样品的GC-MS检测结果,未检出6-羟基石斛次碱;
图4为对比例2制备的样品的GC-MS检测结果,未检出6-羟基石斛次碱;
图5为对比例3制备的样品的GC-MS检测结果,图中圈出位置54.841min为6-羟基石斛次碱。
具体实施方式
下面结合实施例对本发明的技术方案做出更为具体的说明,除非另有说明,本文中所使用的术语均具有本领域技术人员常规理解的含义。
实施例1
采用本发明方法提取和鉴定6-羟基石斛次碱,步骤如下:
S1.将霍山石斛鲜茎通过冻干法脱水,粉碎过80目筛得到石斛干粉;冻干的温度为-50℃,冻干时间40~48h。
S2.取5g霍山石斛干粉加入约30mL水后,置于100W超声波功率下超声处理20~30min,再加入0.075g复合酶(纤维素酶与果胶酶以10000U/g等质量配置),酶解1.5h,得到酶解液。
S3.在酶解液中以酶解液:酸性醇体积比1:40加入设定量的酸性醇,室温放置18h后,在真空且超高压100MPa环境下处理1min,过滤残渣,取滤液;酸性醇为体积浓度70%的乙醇水溶液,pH值为3~3.5。
S4.将所述滤液真空浓缩至5mL后,使用MCX萃取柱纯化,甲醇-乙腈溶液洗脱,收集洗脱液。纯化的具体操作为:
浓缩后的滤液使用H3PO4 2~5wt%水溶液溶解,涡旋2~3min使样品充分溶解,样品上柱前过0.22μm水系滤膜,得到上样液体;
对MCX萃取柱进行活化,首先使用5~8ml甲醇活化固相萃取小柱,再使用5~8ml水平衡小柱。柱子平衡后,将上样液体按照0.5~1.0ml/min流速上样,上样结束后,使用3~5mL质量浓度为2-5%的甲酸水溶液进行淋洗,随后将固相萃取小柱放置在真空泵中,将柱内剩余溶液抽干。淋洗后,用含有3-5%体积比氨水的甲醇/乙腈(1:1体积比)溶液进行洗脱,得到洗脱液。
洗脱液氮在氮吹仪上吹干,得到纯化后含6-羟基石斛次碱样品的待测样品。
将待测样品用甲醇溶解过滤,得到含有6-羟基石斛次碱的待测液,利用GC-MS法检测样品,包括气相色谱和质谱检测。
其中气相色谱的具体操作为:色谱柱为Agilent DB-5MS(30m×0.25μm×0.25mm);载气选择高纯度He,以1.0mL/min的流速进气。柱温:设定初始温度70℃,温度的上升速度为2℃/min,直至上升到150℃,维持稳定1min;再以3℃/min的温速上升至180℃,且维持1min稳定状态;再以20℃/min的温速上升至240℃,维持稳定3min。分流比设定为20:1,待测液的总进样量10μL。质谱的具体操作为:EI离子源;离子能量设置为70eV;温度设定为200℃;全扫描测定方式的扫描范围m/z 40-500。检测结果如图2所示。
对比例1
以实施例1中的霍山石斛干粉为试验材料,参考本课题组前期研究“崔楠楠.霍山石斛生物碱提取纯化及真空冷冻干燥加工工艺研究[D].安徽农业大学,2013.”中提供的方法。取1g霍山石斛干粉加入0.1%纤维素酶,酶解2h,温度40℃提取并通过AB-8型大孔树脂纯化霍山石斛中的生物碱。
通过GC-MS检测,色谱柱为Agilent DB-5MS(30m×0.25μm×0.25mm);载气:高纯氦气,流速1.0mL/min;柱温:初始温度60℃,以3℃/min上升到150℃,保持1min;再以3℃/min上升至200℃,保持1min;再以20℃/min上升至280℃,保持3min。进样方式:分流进样,分流比为30:1,进样量10μL。检测结果如图3所示。
对比例2
以实施例1中的霍山石斛干粉为试验材料。取1g霍山石斛干粉加入酸性乙醇,于100w超声波功率下工作20min提取霍山石斛中的生物碱。
通过GC-MS检测,色谱条件:DB-5毛细管柱(0.25μm×0.25mm×30m),载气为高纯氦气,进样口温度250℃,检测器温度250℃,进样量10μL,分流比1:10,升温程序为初始温度80℃,以10℃/min的速率升温至250℃,保持13min。检测结果如图4所示。
对比例3
以实施例1中的霍山石斛干粉为试验材料。参考论文“陈婧超.石斛中生物碱分离纯化及神经保护作用研究[D].合肥工业大学,2019.”的提取方法,取霍山石斛鲜条800g,切段,置于80℃冰箱中冷冻24h后,真空冷冻干燥24h,使用中药粉碎机粉碎,过100目筛,得霍山石斛冻干粉。按照1:25料液比加入提取溶剂酸性乙醇(70%Et OH,pH=3,),80℃水浴浸提3次,每次2h,合并滤液,得到乙醇提取液,经减压浓缩后,加入2%HCl水溶液溶解,使用等体积二氯甲烷萃取1次后,使用等体积2%HCl水溶液萃取二氯甲烷相两次,合并2%HCl水溶液层得酸水相,氨水调节酸水相pH至10.0,使用等体积三氯甲烷萃取5次,合并三氯甲烷相。三氯甲烷相减压浓缩得到样品,样品经SPE纯化后得到样品。进行GC-MS检测。
利用GC-MS检测,气相色谱条件:色谱柱为Agilent DB-5MS(30m×0.25μm×0.25mm);载气选择高纯度He,以1.0mL/min的流速进气;柱温:设定初始温度70℃,温度的上升速度为2℃/min,直至上升到150℃,维持稳定1min;再以3℃/min的温速上升至180℃,且维持1min稳定状态;再以20℃/min的温速上升至240℃,维持稳定3min。分流比设定为20:1,进样量10μL。质谱条件:EI离子源;离子能量设置为70eV;温度设定为200℃;全扫描测定方式的扫描范围m/z 40-500。检测结果如图5所示。
实验结果
实施例1和对比例1-3的检测结果如下表1所示,其中6-羟基石斛次碱参考附图1所示标准质谱。
表1 6-羟基石斛次碱结果对比
上述对比例分别参考了现有文献公开的几种霍山石斛生物碱提取方法,并利用GC-MS进行检测,GC-MS灵敏度高,样品用量少,可检测出ng/g级的物质,有利于只能获得微量样品的检测。
通过GC-MS监测结果,可以看出,采用本发明提供的方法提取霍山石斛中的生物碱,总生物碱提取量较高,且6-羟基石斛次碱占总生物碱含量高,损失最小,可以明显检出,远高于现有的其它检测方法。
以上实施方式仅用以说明本发明的技术方案,而并非对本发明的限制;尽管参照前述实施方式对本发明进行了详细的说明,本领域的普通技术人员应当理解:凡在本发明创造的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明创造的保护范围之内。
Claims (4)
1.一种提纯并检测霍山石斛鲜茎中6-羟基石斛次碱的方法,其特征在于,所述6-羟基石斛次碱采用GC-MS法检测,自霍山石斛鲜茎中提取含6-羟基石斛次碱的待测液用于检测,提取方法包括以下步骤:
S1. 将霍山石斛鲜茎通过冻干法脱水,粉碎过筛得到石斛干粉,备用;
S2. 取S1中石斛干粉加入水中,超声处理20~30 min后,加入占石斛干粉质量1.5%的复合酶,酶解1.5 h,得到酶解液,所述石斛干粉和水的使用比为1 g : 6 mL;所述复合酶由纤维素酶和果胶酶以10000U/g等活力配制;
S3.在酶解液中加入设定量的酸性醇,室温放置18~24 h后,在真空且超高压100MPa环境下处理1min,过滤残渣,取滤液;所述酸性醇为体积浓度70%的乙醇水溶液,pH值为3~3.5;
S4. 将所述滤液真空浓缩至5 ~ 8 mL后,使用MCX萃取柱纯化,甲醇-乙腈溶液洗脱,收集洗脱液,氮吹至无水分后,再用甲醇溶解过滤,得到含有6-羟基石斛次碱的待测液;
所述GC-MS法检测包括气相色谱和质谱,其中气相色谱的具体操作为:色谱柱为Agilent DB-5MS色谱柱,初始柱温70℃,并保持2℃/min上升,直至150℃后维持1 min,再以3℃/min上升至180℃后维持1 min,再以20℃/min上升至240℃后维持3 min;期间,载气为高纯度He,以1.0ml/min 流速进气;分流比设定为20:1;
质谱的具体操作为:EI离子源;离子能量设置为70 eV;温度设定为200℃;全扫描测定方式的扫描范围m/z 40-500。
2.如权利要求1所述的一种提纯并检测霍山石斛鲜茎中6-羟基石斛次碱的方法,其特征在于,所述步骤S1中,冻干的温度为-50℃,冻干时间40~48 h,粉碎细度为80目。
3.如权利要求1所述的一种提纯并检测霍山石斛鲜茎中6-羟基石斛次碱的方法,其特征在于,所述步骤S3中,以酶解液:酸性醇体积比1:40添加所述酸性醇。
4.如权利要求1所述的一种提纯并检测霍山石斛鲜茎中6-羟基石斛次碱的方法,其特征在于,所述步骤S4中,MCX萃取柱纯化的具体操作为:
浓缩后的滤液使用质量浓度2~5% H3PO4 水溶液溶解,过0.22μm水系滤膜,得到上样液体;MCX萃取柱依次使用5~8 ml甲醇和5~8 ml水活化,将上样液体按照0.5~1.0 ml/min流速上样,上样结束后,使用3~5 mL 质量浓度2-5% 甲酸水溶液进行淋洗;淋洗后用含有占溶液体积比3~5%氨水的甲醇/乙腈溶液进行洗脱,得到洗脱液。
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