CN111458428A - 百里香挥发油主成分及其检测方法和应用、γ-萜品烯和3-甲基-4-异丙基苯酚的应用 - Google Patents
百里香挥发油主成分及其检测方法和应用、γ-萜品烯和3-甲基-4-异丙基苯酚的应用 Download PDFInfo
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- CN111458428A CN111458428A CN202010272814.6A CN202010272814A CN111458428A CN 111458428 A CN111458428 A CN 111458428A CN 202010272814 A CN202010272814 A CN 202010272814A CN 111458428 A CN111458428 A CN 111458428A
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- thyme
- volatile oil
- terpinene
- methyl
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Abstract
本发明属于中药挥发油技术领域,具体涉及百里香挥发油主成分及其检测方法和应用、γ‑萜品烯和3‑甲基‑4‑异丙基苯酚的应用。本发明通过特定设计的检测方法,包括检测参数,实现对百里香挥发油成分的检测,以有利于确定百里香挥发油主成分并进行针对性应用设计。本发明提供的检测方法检测确认百里香挥发油中含有百里酚、γ‑萜品烯、龙脑、邻伞花烃、反式石竹烯和3‑甲基‑4‑异丙基苯酚。本发明提供的百里香挥发油主成分对大肠杆菌、李斯特菌和金黄色葡萄球菌具有明显抑菌效果;清除DPPH自由基和ABTS自由基效果优异;对胆碱酯酶和α‑葡萄糖苷酶具有显著抑制作用。
Description
技术领域
本发明属于中药挥发油技术领域,具体涉及百里香挥发油主成分及其检测方法和应用、γ-萜品烯和3-甲基-4-异丙基苯酚的应用。
背景技术
百里香(Thymus monoglicus Ronn)属于百里香属,其提取物(如百里香挥发油)广泛用于民间医药和香料,已被FDA批准作为食品添加剂。百里香药理作用广泛,现代药理研究表明,其具有温中散寒、祛风止痛、止咳化痰、利尿痛经、健胃发汗和和胃等作用。
但目前的研究方向主要集中在百里香挥发油的提取提纯以及药理应用,对百里香挥发油的药用机理或其主要成分的研究尚不足。
贾红丽等对百里香(hymus Proximus Serg)挥发油成分进行了分析鉴定,从中分离出73种成分并鉴定了其中的69种,其主要成分有:百里香酚、p-聚伞花素、γ-松油烯、β-甜没药烯、香芹酚等(贾红丽,计巧灵等,新疆拟百里香挥发油的气象色谱-质谱分析[J].质谱学报,2008,29(1):36-41);还对阿勒泰百里香(Thymus altaicus Klok)挥发油成分进行了分析鉴定,从中分离出82种成分并鉴定出其中的72种,结果表明阿勒泰百里香挥发油主要成分有:p-聚伞花素、百里香酚、反-石竹烯、α-松油醇、内冰片等(贾红丽,张丕鸿等.新疆阿勒泰百里香挥发油化学成分GC-MS分析及抗氧化活性测定[J].食品科学,2009,30(4):224-229)。杨敏丽等对宁夏固原百里香(hyms quinquecostatus)挥发油成分进行了分析鉴定,共鉴定出31种成分,其主要成分为:香荆芥酚、1-甲基-3-异丙基苯、百里香酚、1-甲基-4-异丙基-1,4-环己二烯、1-甲氧基-4-甲基2-异丙基苯、桉树脑等(杨敏丽,郝凤霞等.宁夏固原百里香挥发油化学成分的GC-MS研究[J].宁夏大学学报(自然科学版),2004,25(4):353-355);陈丽艳等对百里香(Thymus vulgaris)挥发油成分分析鉴定出9个成分,其主要成分为:百里香酚、对伞花烃、异丙基-2-环己烯-1-醇、香荆芥酚等(陈艳丽等.百里香精油的化学成分分析及其抗菌活性[J].黑龙江医药,2009,22(5):636-637);程霜等对百里香挥发油成分分析鉴定了34个成分,结果表明其主要成分有:麝香草酚、异麝香草酚、龙脑、3,7二甲基-1,6-二烯-3-辛醇等(程霜,马清温等.百里香挥发油化学成分的GC-MS分析[J].香料香精化妆品,2002,5:1-3);张继等对百里香(Thymus sarpyllum L.)精油成分进行了分析鉴定,分离出81个化合物并鉴定了其中的68个,其主要成分为:百里酚、1-甲基-3-(1-甲基乙基)苯、7,11-二甲基-1,6,10-十二三烯、2-甲基-5-(1-甲基乙基)苯酚、桉油醇等(张继,刘阿萍等.百里香挥发油成分的研究[J].四川大学学报(自然科学版),2004,41(4):890-892);张知侠等对采自甘肃平凉的百里香(Thymus monoglicus Ronn)挥发油成分进行了鉴定,从中分离出53个化合物并鉴定了其中的40种,结果表明其主要成分为:百里香酚、p-聚伞花素、1-甲乙基-4-甲烯基环已烯、香芹酚、1,8-桉叶素等(张知侠.百里香芳香油化学成分的研究[J].西北农业学报,2004,13(3):151-153);杨丽莉等也对百里香(Thymusmonoglicus Ronn)挥发油进行了鉴定,其结果表明主要成分为:百里香酚、香芹酚、冰片等(杨丽莉,韩基明等.百里香精油提取及化学成分鉴定[J].中国野生植物资源,17(2):36,45);马莉等对普通百里香(Thymus vulgaris)和葡萄百里香(Thymus sarpy1lm)精油成分进行了比较,普通百里香(Thymus vulgaris)主要成分为:百里香酚、对伞花烃、δ-松油烯、芳樟醇等,而葡萄百里香(Thymus serpyllum)的主要成分为:百里香酚、龙脑、红没药烯、对伞花烃、4-松油醇等(马莉,姚雷等.2种百里香的植物学性状和精油成分的比较[J].上海交通大学学报(农业科学版),2008,26(2):150-152)。
以上文献就百里香挥发油的成分进行了研究,但仍不完备,对百里香挥发油的主成分进行检测研究并针对检测得到的主成分进行应用设计,对提高百里香的应用具有重要意义。
发明内容
有鉴于此,本发明的目的在于提供百里香挥发油主成分及检测方法、γ-萜品烯和3-甲基-4-异丙基苯酚的应用,通过本发明提供的检测方法明确了百里香挥发油的几种主成分,有利于针对性提出百里香挥发油的应用;本发明还提供了百里香挥发油主成分的应用。
为了实现上述发明的目的,本发明提供以下技术方案:
本发明提供了一种百里香挥发油主成分的检测方法,包括以下步骤:
提供百里香地上部分挥发油的稀释液;
使用气相色谱-质谱法对所述百里香挥发油的稀释液进行检测;
所述气相色谱-质谱法的气相色谱条件为:使用SE-54色谱柱;载气为氦气;载气流速为1mL/min;升温程序为:初始温度50℃,以5℃/min的升温速率升温至200℃,再以1℃/min的升温速率升温至220℃;所述气相色谱-质谱法的质谱条件为:离子化方式为EI,70eV;离子源温度为230℃;扫描速率为0.2scan/s;质量扫描范围m/z为50~500;
检测结果为:含有双环[3.1.0]己-2-烯、4-甲基-1-(1-甲基乙基)、α-蒎烯、莰烯、1-辛烯-3-醇、月桂烯、α-萜品烯、邻伞花烃、γ-萜品烯、龙脑、4-萜烯醇、百里酚、3-甲基-4-异丙基酚、反式石竹烯、葎草烯、大根香叶烯D、(3Z,6E)-α-法呢烯、1-异丙基-4,7-二甲基-1,2,3,5,6,8a-六氢萘、T-杜松醇和α-杜松醇。
优选的,所述百里香挥发油的稀释液中稀释剂为正己烷、石油醚或乙醚。
优选的,所述百里香挥发油的稀释液中百里香挥发油和稀释剂的体积比为1:(5~20)。
本发明还提供了上述技术方案所述检测方法检测得到的百里香挥发油主成分,包括以下质量百分含量的组分:百里酚32.3%、γ-萜品烯22.1%、龙脑12.0%、邻伞花烃11.6%、反式石竹烯3.8%和3-甲基-4-异丙基苯酚3.7%。
本发明还提供了上述技术方案所述百里香挥发油主成分在抑菌剂、抗氧化剂或酶抑制剂中的应用。
优选的,所述抑菌剂抑制的菌种为革兰氏阴性细菌-大肠杆菌、革兰氏阳性细菌-李斯特菌和革兰氏阳性细菌-金黄色葡萄球菌中的一种或多种。
优选的,所述酶抑制剂抑制的酶为胆碱酯酶和/或α-葡萄糖苷酶。
本发明还提供了γ-萜品烯在抑菌剂、抗氧化剂或酶抑制剂中的应用。
本发明还提供了3-甲基-4-异丙基苯酚在抑菌剂、抗氧化剂或酶抑制剂中的应用。
本发明提供了一种百里香挥发油主成分的检测方法,包括以下步骤:提供百里香地上部分挥发油的稀释液;使用气相色谱-质谱法对所述百里香挥发油的稀释液进行检测;所述气相色谱-质谱法的气相色谱条件为:使用SE-54色谱柱;载气为氦气;载气流速为1mL/min;升温程序为:初始温度50℃,以5℃/min的升温速率升温至200℃,再以1℃/min的升温速率升温至220℃;所述气相色谱-质谱法的质谱条件为:离子化方式为EI,70eV;离子源温度为230℃;扫描速率为0.2scan/s;质量扫描范围m/z为50~500;检测结果为含有双环[3.1.0]己-2-烯、4-甲基-1-(1-甲基乙基)、α-蒎烯、莰烯、1-辛烯-3-醇、月桂烯、α-萜品烯、邻伞花烃、γ-萜品烯、龙脑、4-萜烯醇、百里酚、3-甲基-4-异丙基酚、反式石竹烯、葎草烯、大根香叶烯D、(3Z,6E)-α-法呢烯、1-异丙基-4,7-二甲基-1,2,3,5,6,8a-六氢萘、T-杜松醇和α-杜松醇。本发明通过特定设计的检测方法,包括检测参数,实现对百里香挥发油主成分的检测,以有利于确定百里香挥发油主成分并进行针对性应用设计。
实验数据表明,本发明提供的检测方法检测确认百里香挥发油中含有百里酚、γ-萜品烯、反式石竹烯和3-甲基-4-异丙基苯酚。
本发明还对百里香挥发油主成分针对性地进行了抑菌、抗氧化和酶抑制测试,实验数据表明,本发明提供的百里香挥发油主成分对大肠杆菌、李斯特菌和金黄色葡萄球菌具有明显抑菌效果;清除DPPH自由基和ABTS自由基效果优异;对胆碱酯酶和α-葡萄糖苷酶具有显著抑制作用。
具体实施方式
本发明提供了一种百里香挥发油主成分的检测方法,包括以下步骤:
提供百里香地上部分挥发油的稀释液;
使用气相色谱-质谱法对所述百里香挥发油的稀释液进行检测;
所述气相色谱-质谱法中气相色谱条件为:使用SE-54色谱柱;载气为氦气;载气流速为1mL/min;升温程序为初始温度为50℃,以5℃/min的升温速率升温至200℃,再以1℃/min的升温速率升温至220℃;所述气相色谱-质谱法中质谱条件为:离子化方式为EI,70eV;离子源温度为230℃;0.2scan/s;质量扫描范围m/z为50~500。
本发明提供百里香挥发油的稀释液。
在本发明中,所述百里香挥发油优选经提取纯化制得,包括以下步骤:
提供百里香地上部分的碎片;
将所述百里香地上部分的碎片进行水蒸气蒸馏法,得到含水挥发油;
将所述含水挥发油依次进行萃取、脱水和过滤,得到所述百里香挥发油。
本发明提供百里香地上部分的碎片。在本发明中,所述百里香地上部分的碎片优选为新鲜百里香地上部分的碎片;本发明对所述碎片的大小没有特殊限定,以所述碎片能够进行水蒸气蒸馏法为准。在本发明的实施例中,所述百里香优选采自吉林省龙井市。
得到百里香地上部分的碎片后,本发明将所述百里香地上部分的碎片进行水蒸气蒸馏法,得到含水挥发油。在本发明中,所述水蒸气蒸馏法中百里香碎片和水的质量比优选为1:(2~10),更优选为1:(4~8),最优选为1:5。在本发明中,所述水蒸气蒸馏法的时间优选为3~6h,更优选为3.5~5.5h;温度优选为120℃。本发明对所述水蒸气蒸馏法没有特殊限定,采用本领域技术人员熟知的水蒸气蒸馏法即可。
得到含水挥发油后,本发明将所述含水挥发油依次进行萃取、脱水和过滤,得到所述百里香挥发油。
在本发明中,所述萃取用溶剂优选为有机溶剂,所述有机溶剂优选为乙酸乙酯、丙烯酸乙酯、正己烷和乙醚中的一种或多种。在本发明中,所述萃取后体系会发生油水分离;本发明优选弃取水层,向有机层中加入干燥剂进行脱水。在本发明中,所述干燥剂优选为无水硫酸钠或无水硫酸镁。本发明对所述过滤没有特殊限定,采用本领域技术人员熟知的过滤即可;所述过滤后得到的滤液,即为所述百里香挥发油。
得到百里香挥发油后,本发明对所述百里香挥发油进行稀释,得到百里香挥发油的稀释液。在本发明中,所述百里香挥发油的稀释液中稀释剂优选为正己烷、石油醚或乙醚。在本发明中,所述百里香挥发油的稀释液中百里香挥发油和稀释剂的体积比优选为1:(5~20),更优选为1:(7~18),再优选为1:(10~15)。本发明对所述稀释的方式没有特殊限定,采用本领域技术人员熟知的稀释方式即可。
得到百里香挥发油的稀释液后,本发明使用气相色谱-质谱法对所述百里香挥发油的稀释液进行检测。在本发明中,所述气相色谱-质谱法的气相色谱条件为:使用SE-54色谱柱;载气为氦气;载气流速为1mL/min;升温程序为:初始温度50℃,以5℃/min的升温速率升温至200℃,再以1℃/min的升温速率升温至220℃;所述气相色谱-质谱法的质谱条件为:离子化方式为EI,70eV;离子源温度为230℃;扫描速率为0.2scan/s;质量扫描范围m/z为50~500。
本发明优选基于质谱鉴定化合物,化合物所占浓度百分比表示为每个峰面积相对于总面积的相对百分比。
在本发明中,所述气相色谱-质谱法的设备优选为Thermo Fisher Trace 1300-ISQ GC/MS。
检测结果见表1。
表1气相色谱-质谱法检测结果
本发明还提供了上述技术方案所述检测方法检测得到的百里香挥发油主成分,包括以下质量百分含量的组分:百里酚32.3%、γ-萜品烯22.1%、龙脑12.0%、邻伞花烃11.6%、反式石竹烯3.8%和3-甲基-4-异丙基苯酚3.7%。
本发明还提供了上述技术方案所述百里香挥发油主成分在抑菌剂、抗氧化剂或酶抑制剂中的应用。在本发明中,所述抑菌剂抑制的菌种优选为革兰氏阴性细菌-大肠杆菌、革兰氏阳性细菌-李斯特菌和革兰氏阳性细菌-金黄色葡萄球菌中的一种或多种;所述抗氧化剂清除的自由基优选为DPPH自由基和/或ABTS自由基;所述酶抑制剂抑制的酶为胆碱酯酶和/或α-葡萄糖苷酶,更优选为α-葡萄糖苷酶、乙酰胆碱酯酶和丁酰胆碱酯酶中的一种或多种。
本发明还提供了一种γ-萜品烯在抑菌剂、抗氧化剂或酶抑制剂中的应用。在本发明中,所述抑菌剂抑制的菌种优选为革兰氏阴性细菌-大肠杆菌、革兰氏阳性细菌-李斯特菌和革兰氏阳性细菌-金黄色葡萄球菌中的一种或多种;所述抗氧化剂清除的自由基优选为DPPH自由基和/或ABTS自由基;所述酶抑制剂抑制的酶为胆碱酯酶和/或α-葡萄糖苷酶,更优选为α-葡萄糖苷酶、乙酰胆碱酯酶和丁酰胆碱酯酶中的一种或多种。
本发明还提供了一种3-甲基-4-异丙基苯酚在抑菌剂、抗氧化剂或酶抑制剂中的应用。在本发明中,所述抑菌剂抑制的菌种优选为革兰氏阴性细菌-大肠杆菌、革兰氏阳性细菌-李斯特菌和革兰氏阳性细菌-金黄色葡萄球菌中的一种或多种;所述抗氧化剂清除的自由基优选为DPPH自由基和/或ABTS自由基;所述酶抑制剂抑制的酶为胆碱酯酶和/或α-葡萄糖苷酶,更优选为α-葡萄糖苷酶、乙酰胆碱酯酶和丁酰胆碱酯酶中的一种或多种。
为了进一步说明本发明,下面结合实施例对本发明提供的百里香挥发油主成分及其检测方法和应用、γ-萜品烯和3-甲基-4-异丙基苯酚的应用进行详细地描述,但不能将它们理解为对本发明保护范围的限定。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
在吉林省龙井市收集百里香地上部分,并根据植物学特征鉴定为百里香(Thymusmonoglicus Ronn);刀切碎新鲜材料,材料质量和水体积之间的关系为1/5,浴温为120℃,在水蒸气蒸馏装置中提取挥发油4小时;分离挥发油层,用无水硫酸钠干燥,并储存在4℃的琥珀色玻璃瓶中备用;
用Thermo Fisher Trace 1300-ISQ GC/MS分析EO样品,其配备有SE-54石英毛细管柱,载气为500kPa的恒定压力的氦气,将初始GC设定在50℃,温度以5℃/min升高至200℃后,以1℃/min升高至220℃,在炉温250℃下以分流模式(1:10)注射1.0μL挥发油的稀释液;MS转移线温度设定为280℃,离子源温度设定为230℃,化合物的质谱为70eV,0.2scan/s,m/z50-500。
基于质谱鉴定化合物,浓度表示为每个峰面积相对于总面积的相对百分比,确定主成分,经测试,百里香挥发油主成分包括百里酚32.3%、γ-萜品烯22.1%、龙脑12.0%、邻伞花烃11.6%、反式石竹烯3.8%和3-甲基-4-异丙基苯酚3.7%,结构式如下:
应用例1
使用三种食源性细菌:革兰氏阴性细菌-大肠杆菌ATCC 25922、革兰氏阳性细菌-李斯特菌ATCC 19111和金黄色葡萄球菌ATCC 29213,通过肉汤微量稀释技术(CLSI,2009)评估百里香挥发油的最低抑菌浓度(MIC)和最低杀菌浓度(MBC),同时,以氯霉素为对比例1,以氨苄西林为对比例2;具体测试方法为:
CLSI.(2009).Methods for dilution antimicrobial susceptibility testsfor bacteria that grow aerobically.8th ed.USA,M07-A;具体为:
最低抑菌浓度
在无菌条件下,样品储备液在96孔板中用MH肉汤培养基采用二倍稀释法稀释(浓度从1024μg/mL稀释至1μg/mL,稀释后的DMSO含量应小于1%)。制备了1×105CFU/mL的细菌溶液。使用移液枪将100μL细菌溶液添加到1至11孔中。将100μL不含样品的MH肉汤培养基添加到第11孔作为阳性对照孔,将200μL MH肉汤培养基添加到第12孔作为空白对照孔。接种后将96孔微孔板置于微型振动器上震荡5min,将药物溶液和细菌溶液充分混合,并在37℃的培养箱中培养24h。用肉眼从低浓度开始观察孔是混浊还是澄清,第一个澄清孔的浓度即为最低抑菌浓度。
最低杀菌浓度
根据MIC评估结果,从无菌生长孔中吸取100μL培养基,均匀地涂在MH固体培养基上,在37℃放置12h后观察到细菌的生长。根据一般规定,在培养皿培养基中,计数不到5个菌落作为样品的MBC。
实验结果见表2。
表2百里香挥发油及其主成分抗菌能力测试结果(mg/L)
由表2可见,本发明所述百里香挥发油主成分均对革兰氏阴性细菌-大肠杆菌ATCC25922、革兰氏阳性细菌-李斯特菌ATCC 19111和金黄色葡萄球菌ATCC 29213有明显抑制作用,说明本发明所述百里香挥发油及其主成分均对食源性细菌具有抑制作用。
应用例2
通过清除DPPH自由基和ABTS自由基评估挥发油的抗氧化能力,同时,以抗坏血酸为对比例3,以Trolox为对比例4;实验方法为:Dong,L.M.,Jia,X.C.,Luo,Q.W.,Zhang,Q.,Luo,B.,Liu,W.B.,Zhang,X.,Xu,Q.L.,&Tan,J.W.(2017).Phenolics from Mikaniamicrantha and their antioxidant activity.Molecules,22(7),E1140;具体为:
1.DPPH
将100μL的挥发油甲醇溶液和100μL的DPPH甲醇溶液(50μM)在微孔板中混合,然后在室温下避光放置20min,在492nm处记录样品的吸光度;抗坏血酸和trolox用作阳性参考;计算半数最大抑制浓度(IC50)值(清除测试溶液中存在的DPPH自由基的50%所需的浓度),并表示为平均值±标准偏差。
2.ABTS
将1mL的2.6mM过硫酸钾加入1mL的7mM的ABTS中,混合后室温下避光保存12~16小时方可使用;使用甲醇稀释ABTS溶液,使其在734nm下吸光度为0.70±0.02;将190μL稀释的ABTS+溶液和10μL挥发油DMSO溶液在微孔板中混合,并在黑暗中孵育20min,以trolox(0-1mg/mL)为标准绘制了校准曲线;在734nm处记录样品的吸光度;抗坏血酸和trolox用作阳性参考;计算IC50值。
实验结果见表3。
表3百里香挥发油及其主成分的抗氧化能力测试结果
由表3可见,本发明所述百里香挥发油主成分均对DPPH自由基和ABTS自由基有明显清除作用,抗氧化效果明显,可以应用作为抗氧化剂中的抗氧化活性成分。
应用例3
通过抑制胆碱酯酶评估挥发油的治疗阿尔茨海默病的能力,测试方法为:Ozturk,M.,Kolak,U.,Topcu,G.,Oksuz,S.,&Choudhary,M.I.(2011).Antioxidant andanticholinesterase active constituents from Micromeria cilicica by radical-scavenging activity-guided fractionation.Food Chemistry,126(1),31–38;具体为:
胆碱酯酶抑制活性评价
①乙酰胆碱酯酶(AChE)抑制活性
不同浓度样品10%DMSO溶液20μL加入到120μL磷酸盐缓冲液(pH8.0,0.1M)和20μLAChE溶液(pH8.0,0.8U/mL,0.1M磷酸盐缓冲液)中;在25℃下孵化15min,然后分别加入20μL碘代硫代乙酰胆碱(ATCI)溶液(pH8.0,1.78mM,0.1M磷酸盐缓冲液)和20μL5,5'-二硫代双-(2-硝基苯甲酸)(DTNB)溶液(pH8.0,1.25mM,0.1M磷酸盐缓冲液),在25℃下孵化5min;使用酶标仪在405nm下测量孵育前后的吸光度;使用多奈哌齐作为阳性对照;AChE抑制活性表示为%抑制,计算公式如下:
②丁酰胆碱酯酶(BChE)抑制活性
不同浓度样品10%DMSO溶液20μL加入到120μL磷酸盐缓冲液(pH8.0,0.1M)和20μLBChE溶液(pH8.0,0.8U/mL,0.1M磷酸盐缓冲液)中。在25℃下孵化15min,然后分别加入20μL氯化硫代丁酰胆碱溶液(pH8.0,0.4mM,0.1M磷酸盐缓冲液)和20μLDTNB溶液(pH8.0,1.25mM,0.1M磷酸盐缓冲液),在25℃下孵化5min;使用酶标仪在405nm下测量孵育前后的吸光度;使用多奈哌齐作为阳性对照;BChE抑制活性表示为%抑制,计算公式如下:
实验结果见表4;
通过抑制α-葡萄糖苷酶评估挥发油的治疗糖尿病的能力,同时,以阿卡波糖为对比例5,以多奈哌齐为对比例6;测试方法为:Yuan,T.,Wan,C.,Liu,K.,&Seeram,N.P.(2012).New maplexins F-I and phenolic glycosides from red maple(Acer rubrum)bark.Tetrahedron,68(4),959–964;具体为:
α-葡萄糖苷酶抑制抑制活性
不同浓度样品DMSO溶液20μL加入到100μLα-葡萄糖苷酶溶液(pH6.9,0.1U/mL,0.1M磷酸盐缓冲液)中,在25℃下孵化10min,然后分别加入50μL对硝基苯-α-D-吡喃葡糖苷(pNPG)溶液(pH6.9,5mM,0.1M磷酸盐缓冲液),在25℃下孵化5min,使用酶标仪在405nm下测量孵育前后的吸光度;使用阿卡波糖作为阳性对照;α-葡萄糖苷酶抑制活性表示为%抑制,计算公式如下:
实验结果见表4。
表4百里香挥发油及其主成分的酶抑制能力测试结果
由表4可见,阿卡波糖是一个降血糖的药物,它的降糖能力是39.3mg/L,挥发油的是93.3mg/L,3-甲基-4-异丙基苯酚是64.6mg/L,反式石竹烯是65.8mg/L,均表现了一定的酶抑制(降糖)作用,说明所述百里香挥发油及其主成分有开发为降糖药物的潜力。多奈哌齐是一种胆碱酯酶抑制药物,反式石竹烯对两种胆碱酯酶也都有抑制作用,说明所述百里香挥发油及其主成分有开发为胆碱酯酶抑制剂,治疗阿尔茨海默病的潜力。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (9)
1.一种百里香挥发油主成分的检测方法,其特征在于,包括以下步骤:
提供百里香地上部分挥发油的稀释液;
使用气相色谱-质谱法对所述百里香挥发油的稀释液进行检测;
所述气相色谱-质谱法的气相色谱条件为:使用SE-54色谱柱;载气为氦气;载气流速为1mL/min;升温程序为:初始温度50℃,以5℃/min的升温速率升温至200℃,再以1℃/min的升温速率升温至220℃;所述气相色谱-质谱法的质谱条件为:离子化方式为EI,70eV;离子源温度为230℃;扫描速率为0.2scan/s;质量扫描范围m/z为50~500;
检测结果为:
含有双环[3.1.0]己-2-烯、4-甲基-1-(1-甲基乙基)、α-蒎烯、莰烯、1-辛烯-3-醇、月桂烯、α-萜品烯、邻伞花烃、γ-萜品烯、龙脑、4-萜烯醇、百里酚、3-甲基-4-异丙基酚、反式石竹烯、葎草烯、大根香叶烯D、(3Z,6E)-α-法呢烯、1-异丙基-4,7-二甲基-1,2,3,5,6,8a-六氢萘、T-杜松醇和α-杜松醇。
2.根据权利要求1所述的检测方法,其特征在于,所述百里香挥发油的稀释液中稀释剂为正己烷、石油醚或乙醚。
3.根据权利要求1所述的检测方法,其特征在于,所述百里香挥发油的稀释液中百里香挥发油和稀释剂的体积比为1:(5~20)。
4.权利要求1~3任一项所述检测方法检测得到的百里香挥发油主成分,其特征在于,包括以下质量百分含量的组分:百里酚32.3%、γ-萜品烯22.1%、龙脑12.0%、邻伞花烃11.6%、反式石竹烯3.8%和3-甲基-4-异丙基苯酚3.7%。
5.权利要求4所述百里香挥发油主成分在抑菌剂、抗氧化剂或酶抑制剂中的应用。
6.根据权利要求5所述的应用,其特征在于,所述抑菌剂抑制的菌种为革兰氏阴性细菌-大肠杆菌、革兰氏阳性细菌-李斯特菌和革兰氏阳性细菌-金黄色葡萄球菌中的一种或多种。
7.根据权利要求5所述的应用,其特征在于,所述酶抑制剂抑制的酶为胆碱酯酶和/或α-葡萄糖苷酶。
8.一种γ-萜品烯在抑菌剂、抗氧化剂或酶抑制剂中的应用。
9.一种3-甲基-4-异丙基苯酚在抑菌剂、抗氧化剂或酶抑制剂中的应用。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN115997763A (zh) * | 2022-12-14 | 2023-04-25 | 江西农业大学 | 一种防治蜂螨的组合物 |
CN115997763B (zh) * | 2022-12-14 | 2024-05-31 | 江西农业大学 | 一种防治蜂螨的组合物 |
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