CN115477604B - 马卡萃取物及其用途 - Google Patents
马卡萃取物及其用途 Download PDFInfo
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- CN115477604B CN115477604B CN202210581564.3A CN202210581564A CN115477604B CN 115477604 B CN115477604 B CN 115477604B CN 202210581564 A CN202210581564 A CN 202210581564A CN 115477604 B CN115477604 B CN 115477604B
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
- C07D207/32—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
- C07D207/33—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms with substituted hydrocarbon radicals, directly attached to ring carbon atoms
- C07D207/333—Radicals substituted by oxygen or sulfur atoms
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
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Abstract
本发明提供一种马卡萃取物及其用途，所述马卡萃取物使用极性溶剂萃取的部分具有抗血栓生成的活性，使用中低极性溶剂萃取的部分则具有抗中性粒细胞炎症以及抗过敏的活性，而使用低极性溶剂萃取的部分也具有抗中性粒细胞炎症的活性，且也具有促血管生成的活性。
Description
技术领域
本发明涉及一种马卡萃取物及其用途，特别是一种使用特定溶剂所萃取的马卡萃取物而使其作为抗血栓生成、抗中性粒细胞炎症、抗过敏以及促血管生成等用途。
背景技术
马卡，学名为Lepidium meyeniiWalp.，又名秘鲁人参，属于十字花科植物，为秘鲁安地斯山脉的一种药用植物，其块根或块茎部分被秘鲁当地人作为功能性食品及民用药物，用以提升性功能及生育能力。
近年来，多种药理学研究指出，马卡萃取物具有多种活性，举例而言，如抗疲劳、壮阳、抗骨质疏松、抗发炎、抗病毒以及抗肿瘤等活性。
此外，也有研究指出，马卡萃取物具有免疫功能调节以及护肝的活性，并对停经后综合征(postmenopausal syndrome)与良性摄护腺肥大症(benign prostatehyperplasia)也有治疗效果。
马卡可食用部分的块茎含有大量碳水化合物、蛋白质及脂质作为初级代谢物(primary metabolites)，以及各种次级代谢物(secondary metabolites)如生物碱(alkaloids)、黄酮木脂素(flavonolignans)、三萜类化合物(triterpenoids)和硫代葡萄糖苷(glucosinolates)等。
其中，生物碱衍生物被认为是生物活性成分，其代表了大多数已被研究的马卡次级代谢物，包括生物碱酰胺(macamides)、乙内酰脲衍生物(如macahydantoins、meyeniihydantoins、macathiohydantoins等)、六氢咪唑噻唑衍生物(如meyeniins)、咪唑生物碱(如lepidiline)、吡啶衍生物(macaridine)以及吡咯衍生物(macapyrrolins)。
过去三十年里，马卡已成为一种需求量大的天然保健材料及膳食补充剂，在全球的各类产品中具有很高的市场价值，因此导致多数相关学者对马卡进行广泛的研究探索，以找出其对于人体有益的成分及实验证据。
尽管马卡在天然保健品市场及作为研究目标中广受欢迎，但马卡中仍有未被发现的植物化学物质及生物活性。
发明内容
有鉴于上述问题，本发明的创作者通过不同溶剂进行萃取分离，并针对各种不同萃取成分进行试验，以寻找马卡中尚未被人们发现的活性化合物以及生物活性。
将马卡块茎使用乙醇水溶液萃取后，经减压浓缩获得一马卡粗萃物，将马卡粗萃物利用乙酸乙酯及水进行分相萃取，获得一乙酸乙酯层以及一第一水层；第一水层再以正丁醇水溶液进行分相萃取，获得一第二水层以及一正丁醇层；而乙酸乙酯层则再以甲醇及正己烷的混合溶液进行分相萃取，获得一甲醇层及正己烷层。
接着，使用管柱层析进一步对正己烷层以及甲醇层进行洗脱(elute)纯化，获得如图1所示的8种马卡萃取生物碱衍生化合物；其中化合物1、5～8为已知的生物碱衍生化合物，而化合物2～4则为首次分离出的新生物碱衍生化合物。其中化合物1为(5S)-乙酰基-1-苄基吡咯烷-2-酮(马卡吡咯烷酮A)，化合物2为(5S)-乙酰基-1-(间-甲氧基苄基)-吡咯烷-2-酮(马卡吡咯烷酮B)，化合物3为5-甲氧基甲基-1-(间-甲氧基苄基)-2-醛基吡咯(马卡吡咯啉D)，化合物4为5-羟甲基-1-(间甲氧基苄基)-2-醛基吡咯，化合物5为5-甲氧基甲基-1-(苄基)-2-醛基吡咯，化合物6为5-羟甲基-1-(苄基)-2-醛基吡咯，化合物7为3-(苄基)-1-(羟基)-4-醛基吡啶，化合物8为3-羟基-1-氰甲基苯；后续为方便阅读，均以化合物1～8代称各化合物。
而经生物活性测试后，发现马卡萃取物使用极性溶剂萃取的部分(即第二水层与正丁醇层部分)具有抗血栓生成的活性，使用中低极性溶剂萃取的部分(即甲醇层部分)则具有抗中性粒细胞炎症以及抗过敏的活性，而使用低极性溶剂萃取的部分(即正己烷层部分)除了具有抗中性粒细胞炎症的活性，且也具有促血管生成的活性。
以下将以具体的实施例配合所附的附图详加说明本发明的技术特征，以使所属技术领域具有通常知识者可易于了解本发明的目的、技术特征及其优点。
附图简单说明
以下搭配附图，对于本发明进行进一步说明：
图1为本发明所萃取出的马卡萃取生物碱衍生物的化合物结构示意图；
图2为本发明对马卡进行液相萃取的简略流程示意图；
图3为本发明对马卡进行液相萃取后，对甲醇层进行管柱层析的简略流程示意图；
图4为正己烷层及富含马卡酰胺的馏分对人类内皮祖细胞管形成的影响，
其中图4A为正己烷层，图4B为富含马卡酰胺的馏分。
实施方式
以下内容将搭配附图，通过特定的具体实施例说明本发明的技术内容，熟悉此技术的人士可由本说明书所揭示的内容轻易地了解本发明的其他优点与功效。本发明也可通过其他不同的具体实施例加以施行或应用。本说明书中的各项细节也可基于不同观点与应用，在不背离本发明的精神下，进行各种修饰与变更。
除非另有定义，本发明所使用的所有术语(包括技术和科学术语)具有与本发明所属技术领域的普通技术人员通常理解的相同含义。将进一步理解的是，诸如在通常使用的字典中定义的那些术语应当被解释为具有与它们在相关技术和本发明的上下文中的含义一致的定义，并且将不被解释为理想化或过度正式的意义，除非本文中明确地这样定义。
材料
马卡(Lepidium meyeniiWalp.)的干燥块茎切片植物材料凭证标本(No.Lepidium-1)已存放于台湾高雄市高雄医学大学天然药物研究所。
实施例1
萃取及分离方法
请参照图2，图2为本发明对马卡进行液相萃取的简略流程示意图。将马卡的干燥块茎切片(1.5kg)在室温下以95％的乙醇水溶液进行萃取，每次以4L的乙醇水溶液萃取，连续萃取4次，其后减压浓缩合并所得萃取物，获得马卡粗萃物(400.3g)。将马卡粗萃物以乙酸乙酯及水(体积比1：1)进行分相萃取，获得第一水层萃取物(370.1g)及乙酸乙酯层萃取物(27.7g)；第一水层以正丁醇(两者体积比为1：1)再次进行分相萃取，获得第二水层萃取物(351.6g)以及正丁醇层萃取物(15.0g)；而乙酸乙酯层萃取物以75％的甲醇水溶液及正己烷(体积比1：1)再次进行分相萃取，获得甲醇层萃取物(6.7g)以及正己烷层萃取物(3.32g)。
正己烷层萃取物使用Sephadex LH-20开放管柱层析(150cm×4.0cm i.d.)进行洗脱(elute)，洗脱液为二氯甲烷/甲醇(体积比1：1)，经洗脱后获得富含生物碱酰胺(macamides，或称马卡酰胺)的馏分(fraction)。
再参照图3，图3为本发明对马卡进行液相萃取后，对甲醇层进行管柱层析的简略流程示意图。甲醇层萃取物使用硅胶层析管柱(30cm×5.5cm i.d.)进行洗脱，洗脱液为正己烷/丙酮(共500mL)，两者体积比依序以4：1、3：1、2：1、1：1及0：1(即仅有丙酮)进行洗脱，其后收集馏分合并后，将合并后的馏分以层析法分为7个主要馏分(F-1至F-7)，取F-2馏分(461.01mg)使用硅胶层析管柱(30cm×3.0cm i.d.)进行洗脱，洗脱液为正己烷/二氯甲烷/甲醇(共200mL)，三者体积比依序以50：10：0、40：10：1、20：10：1、10：10：1、5：10：1及0：10：1进行洗脱，获得7个馏分(F-2-1至F-2-7)；取F-2-2馏分(348.40mg)及F-2-3馏分(65.84mg)合并后，使用ODS(OctaDecylSilane，十八烷基硅烷，即碳18)层析管柱(6.5cm×4.0cmi.d.)进行洗脱，洗脱液为甲醇/水(共160mL)，即甲醇水溶液，依序以体积比10％、30％、50％、60％、70％、80％、90％及100％进行洗脱，获得7个馏分(F-2-2-1至F-2-2-7)；取F-2-2-2馏分(115.50mg)以硅胶层析管柱(20cm×2.5cm i.d.)进行洗脱，洗脱液为二氯甲烷/乙酸乙酯(共100mL)，两者体积比依序为20：1、10：1、5：1、1：1及0：1进行洗脱，获得6个馏分(F-2-2-2-1至F-2-2-2-6)；取F-2-2-2-4馏分(58.14mg)以硅胶层析管柱(14cm×2.5cm i.d.)进行洗脱，洗脱液为正己烷/乙酸乙酯(共80mL)，两者体积比依序以5：1、3：1、1：1及0：1进行洗脱，获得化合物7(23.62mg)及化合物8(8.44mg)；取F-2-2-3馏分(23.95mg)以Luna硅胶层析管柱(正己烷/乙酸乙酯体积比3：1，流速2mL/min)，通过高效能液相层析仪(HPLC)分离获得化合物5(6.41mg)。
另取F-3馏分(728.16mg)以ODS层析管柱(6.5cm×4.0cm i.d.)进行洗脱，洗脱液为甲醇/水(共200mL)，即甲醇水溶液，依序以体积比10％、30％、50％、60％、70％、75％、80％、85％、90％、95％及100％进行洗脱，获得8个馏分(F-3-1至F-3-8)；取F-3-3(203.68mg)以硅胶层析管柱(20cm×2.5cm i.d.)进行洗脱，洗脱液为二氯甲烷/乙酸乙酯(共100mL)，两者体积比依序为9：1、4：1、2：1、1：1及0：1进行洗脱，获得7个馏分(F-3-3-1至F-3-3-7)；取F-3-3-3馏分(8.86mg)以Luna硅胶层析管柱(正己烷/乙酸乙酯体积比3：2，流速2mL/min)，通过高效能液相层析仪(HPLC)分离获得化合物4(2.04mg)及化合物6(2.22mg)；取F-3-3-4馏分(94.00mg)以硅胶层析管柱(18cm×2.5cm i.d.)进行洗脱，洗脱液为正己烷/乙酸乙酯(共100mL)，两者体积比依序以3：1、2：1、1：1及0：1进行洗脱，获得4个馏分(F-3-3-4-1至F-3-3-4-4)；取F-3-3-4-3馏分(51.53mg)以CN层析管柱(正己烷/乙酸乙酯体积比1：1，流速2mL/min)，通过高效能液相层析仪(HPLC)分离获得化合物1(9.10mg)及化合物2(0.78mg)；取F-3-4馏分(95.45mg)以硅胶层析管柱(20cm×2.5cm i.d.)进行洗脱，洗脱液为正己烷/乙酸乙酯(共100mL)，两者体积比依序为5：1、3：1、1：1及0：1进行洗脱，获得6个馏分(F-3-4-1至F-3-4-6)；取F-3-4-2馏分(1.66mg)以Luna硅胶层析管柱(正己烷/乙酸乙酯体积比3：1，流速2mL/min)，通过高效能液相层析仪(HPLC)分离获得化合物3(0.42mg)。
实施例2
促血管生成试验
内皮细胞培养：人类CD34-阳性内皮祖细胞(endothelial progenitor cells,EPCs)的分离及培养使用公知方法(Chen et al.,2018)所进行；人类脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells,HUVECs)购自PromoCell(Heidelberg,Germany)；并根据公知方案(Chung et al.,2013)将EPC及HUVEC维持于MV2培养基中。
细胞生长测定：将EPC及HUVEC在96孔板中以每孔5×103个细胞的密度培养过夜，接着在测试样品(即马卡萃取物)存在，以MV2基础培养基更换原培养基并培养48小时，EPC细胞生长的测定法如Yang et al.,2019所述。
毛细血管样管形成测定：将EPC以每孔1.25×104个细胞的密度接种于基质胶(matrigel)涂布的96孔板中，并在MV2基础培养基及测试馏分中培养24小时，并根据Yanget al.,2019所述方案，检查EPC分化与毛细血管样管形成。
在各细胞培养后于10μg/mL的各液相层、5μg/mL的富含马卡酰胺馏分(即经洗脱后的正己烷层)以及50μM的各分离化合物进行试验，结果如下表1所示，其结果为平均值±SEM(试验皆进行三重复)，与对照组(DSMO)相较之下，*表示p<0.05、**表示p<0.01、***表示p<0.001；-表示未经测试。
表1
血管内皮生长因子(VEGF)为血管生成的重要介质，将其作为阳性对照组，由表1中可看出正己烷层萃取物显著增加了EPC及HUVEC的细胞生长；此外，源自正己烷层的富含马卡酰胺馏分则进一步将细胞生长率提升至128±6％，为了评估马卡萃取物的促血管生成活性，进一步比较了正己烷层以及富含马卡酰胺馏分。
请参考图4，图4为正己烷层及富含马卡酰胺的馏分对人类内皮祖细胞管形成的影响，其中图4A为正己烷层，图4B为富含马卡酰胺的馏分。由图中可知，正己烷层以及富含马卡酰胺的馏分均为浓度依赖性，即随其浓度增加，EPC的毛细管状管的形成长度增加。因此，由上述实验结果表明正己烷层的萃取物及其富含马卡酰胺的馏分皆有促血管生成的活性，可改善组织缺血及血管再生。
实施例3
抗血栓试验
体外血栓形成测定：使用Kao et al.,2021所述的方法进行体外血栓形成测定，将载有荧光染料DiOC6(3)的人类全血通过胶原蛋白图层流动腔室(μ-Slide VI 0.1)，以1500s-1的壁剪切速率灌注2分钟，其后以磷酸盐缓冲液洗涤后，用CCD相机记录每个腔室的三个随机视野，并使用ImageJ软件分析血栓覆盖的区域。
将浓度50μg/mL的马卡粗萃物以及各液相萃取的分层(即正己烷层、第二水层、甲醇层以即正丁醇层)处理全血，以测定全血流动条件下的血栓形成，其中甲醇层与正己醇层未进行测试，因其在高浓度使用下会自行引起血小板聚集。结果如下表2所示，其结果为平均值±SEM(试验皆进行三重复)，*表示p<0.05、**表示p<0.01、***表示p<0.001；N/A表示样品加入培养基后形成菌丝体，因此结果无法证明。
表2
由表2中可看出，经马卡粗萃物处理的全血样品，其血栓面积减少了27.8±4.2％，而正丁醇层及第二水层表现出更佳的抗血栓活性，其血栓面积分别减少了59.9±6.1％以及50.5±12.1％。因此，由上述实验结果表明马卡以极性溶剂萃取的部分具有抗血栓生成的活性。
实施例4
抗中性粒细胞炎症试验
人类中性粒细胞分离：根据葡聚糖沉降的标准方法，从外周血中获得中性粒细胞，接着在Ficoll-Hypaque梯度中离心并进行红细胞低渗裂解以纯化出中性粒细胞；其后使用台盼蓝(trypan blue)排除测试以证实纯化出的中性粒细胞含有大于98％的活细胞，进行试验前将纯化出的中性粒细胞悬浮在4℃的无钙HBSS中(Yang et al.,2013)。
超氧阴离子生成测定及弹性蛋白酶释放抑制测定：通过中性粒细胞活化确定超氧阴离子生成水平是基于Yang et al.,2013所述的铁细胞色素c还原法进行测定，即，将人类中性粒细胞(6×105个细胞/mL)与0.6mg/mL的铁细胞色素c及1mmol/L的CaCl2在37℃下混合，其后使用不同浓度的测试样品(即马卡萃取物)或DMSO(0.1％作为对照组)处理细胞5分钟；接着用100nM的fMLF活化10分钟之前，将细胞与细胞松弛素B(1μg/mL)一起培养3分钟，使用分光亮度计在不断搅拌下连续监测500nm波长处随铁细胞色素c减少的吸亮度变化，并计算基于有无SOD(超氧化物歧化酶，100U/mL)的反应中，除以减少的铁细胞色素c(21.1mM- 1cm-1)还原的消光系数，以获得其差异值。
使用弹性蛋白酶(elastase)的释放以测定活化的中性粒细胞上嗜天青颗粒(azurophilic granule)的脱颗粒(Yang et al.,2013)；使用甲氧基琥珀酰-Ala-Ala-Pro-Val-p-硝基苯胺作为弹性蛋白酶基质进行测定。在37℃下，将中性粒细胞(6×105个细胞/mL)添加到基质(100μmol/L)及CaCl2(1mmol/L)的混合物中，使用不同浓度的测试样品即马卡萃取物)或DMSO(0.1％作为对照组)处理5分钟后，使用fMLF(100nM)/CB(0.5μg/mL)刺激细胞，并连续监测405nm波长处吸亮度的变化，以测定弹性蛋白酶的释放，并以染料木黄酮(Genistein)及LY294002作为阳性对照组。试验结果如下表3所示，其结果为平均值±SEM(试验进行三重复或四重复)，*表示p<0.05、**表示p<0.01、***表示p<0.001；-表示未经测试。
表3
由表3中可看出，甲醇层及正己烷层的抗中性粒细胞炎症活性均非常显著，通过完全消除fMLF活化的中性粒细胞功能(分别为101.12±0.26％和110.88±0.24％)，可见甲醇层(10μg/mL)在所有液相萃取层中对超氧阴离子生成及弹性蛋白酶释放表现出最强的抑制作用；此外，正己烷层(10μg/mL)对超氧阴离子生成(81.46±5.56％)和弹性蛋白酶释放(96.39±5.89％)也表现出有效的抑制作用，而正丁醇层仅对超氧阴离子生成(75.96±2.89％)有显著抑制作用。
在此试验中，仅有化合物5(10μM)对fMLF/CB所诱导的中性粒细胞中超氧阴离子生成有轻微的抑制作用(21.59±5.35％)；与甲醇层及正己烷层的结果相比，分离出的化合物对于抗中性粒细胞炎症的活性均不显著。因此，由上述实验结果表明马卡以中低极性溶剂萃取以及低极性溶剂萃取的部分具有抗中性粒细胞炎症的活性。
实施例5
抗过敏试验
细胞培养：使用Korinek et al.,2017所述的方法培养黏膜肥大细胞(mucosalmast cell)衍生的大鼠嗜碱性白血病(RBL-2H3)。使用10％FBS补充的DMEM培养基以及额外的100U/mL青霉素与100μg/mL链霉素培养细胞，细胞在10cm的培养皿中于37℃的5％CO2培养箱内培养，细胞在80％融合度的胰蛋白酶中继代培养，接着以2×105个细胞/mL的密度于培养板接种用于分泌测定。
细胞存活测定：将浓度为2×104个细胞/孔的RBL-2H3细胞接种在96孔板中隔夜，接着使用不同浓度的样品(即马卡萃取物，溶解于DMSO中)或未处理的对照组(1％DMSO于Tyrode缓冲液中；135mM NaCl、5mM KCl、1.8mM CaCl2、1.0mM MgCl2、5.6mM葡萄糖、20mMHEPES以及1mg/ml BSA，pH 7.4)处理细胞，在37℃、5％CO2中培养1小时后，除去每个孔上的培养基，将MTT溶液(5mg/mL)原液在培养基中按1:10稀释，然后加入孔中(每孔100μL)；其后，将细胞在37℃、5％CO2中培养1小时，除去培养基，将形成的甲臜晶体(正常健康细胞中方可形成)溶解在100μL DMSO中，使用酶标仪(microplate reader)在574nm波长处测量吸亮度之前轻轻摇动板后测量。每个样品的细胞活力值计算以对照组(未处理细胞)的百分比(％)表示。
A23187与抗原诱导的RBL-2H3细胞中的β-胺基己糖苷酶释放抑制测定：将RBL-2H3细胞以2×104个细胞/孔的密度分配在96孔板中，而抗原诱导实验的细胞在以3×104个细胞/孔的密度接种于48孔板后，用抗DNP IgE(0.05μg/ml)致敏，接着在37℃、5％CO2中培养隔夜，使细胞完全黏附在孔的底部；其后将不同浓度的样品(即马卡萃取物，溶解于DMSO中)或Tyrode缓冲液(1％DMSO，作为未处理的对照组)添加至每个孔(100μL)中，并于37℃、5％CO2中培育30分钟；后续去除上清液并通过钙离子载体A23187(0.5μM，A23187诱导测定)或交联抗原DNP-BSA(100ng/ml，抗原诱导测定)刺激细胞，于37℃、5％CO2中培育30分钟，而未经刺激的细胞使用0.5％Triton X-100溶液裂解以释放β-己糖胺酶，抑或不处理以自发释放β-胺基己糖苷酶。将对照孔及实验孔的等分上清液(50μL)与在0.1M柠檬酸盐缓冲液(pH4.5)中制备的等体积(50μL)的1μM p-NAG一起培养，该缓冲液成为释放的β-胺基己糖苷酶的基质；在37℃下培养1小时后，加入100μL终止缓冲液(0.1M Na2/NaHCO3，pH 10.0)使反应终止，接着在酶标仪上测量405nm波长处的吸亮度。RBL-2H3细胞释放β-胺基己糖苷酶的抑制百分比计算为对照组(未处理的刺激细胞)的百分比(％)，氮卓斯汀(Azelastine，20μM)作为阳性对照组。试验结果如下表4至表6所示，其结果为平均值±SEM(试验进行三重复)，*表示p<0.05、**表示p<0.01、***表示p<0.001；-表示未经测试；N/A表示样品加入培养基后形成菌丝体，因此结果无法证明。
表4
表5
表6
由表4中可看出，甲醇层在抗过敏试验中具有显著的效果，在A23187(IC50 97.1μg/mL)和抗原(IC50 102.7μg/mL)诱导的RBL-2H3细胞中，抑制了β-胺基己糖苷酶的释放。
由表5中可看出，在甲基噻唑基四唑(MTT)测定中显示>90％的活性，而通过抑制A23187和抗原诱导的RBL-2H3细胞脱粒中的β-胺基己糖苷酶释放来评估化合物的抗过敏活性，甲醇层的抑制效果依然显著。
由表4及表6中可看出，化合物5(IC50 96.1μM)对A23187诱导的RBL-2H3细胞脱粒表现出最强的抑制作用，且化合物5的β-胺基己糖苷酶释放抑制活性在10～500μM之间以剂量依赖性方式增加；此外，化合物1(500μM)对A23187诱导的β-胺基己糖苷酶释放活性表现出轻度抑制(38.0±3.4％)。然而，在抗原诱导的β-胺基己糖苷酶释放试验中，没有一种化合物表现出显著的抑制作用，故这些结果表明化合物5系通过不依赖IgE的过敏基至而发挥作用，特别是与抑制肥大细胞中的钙流入有关。
因此，由上述实验结果表明马卡以中低极性溶剂萃取的部分具有抗中性粒细胞炎症的活性。
综上各实施例所得的试验结果，马卡萃取物使用极性溶剂萃取的部分具有抗血栓生成的活性，使用中低极性溶剂萃取的部分则具有抗中性粒细胞炎症以及抗过敏的活性，而使用低极性溶剂萃取的部分也具有抗中性粒细胞炎症的活性，且也具有促血管生成的活性；此外，于萃取马卡的过程中，也首次分离出3种的新生物碱衍生化合物。
本发明所属技术领域技术人员能够自前述内容理解，本发明可通过其他具体形式例式的而不改变本揭露内容的技术概念或本质特征。就此而言，本文中揭露的例示性态样仅用于例示性说明之用，且不应解释为限制本揭露内容的范畴。反之，本揭露内容倾向于不仅涵盖该等例示性态样，也涵盖可包括于如后所附申请专利范围定义者的本发明内容的精神及范畴内的多种变更、修饰、均等物、及其他态样。
Claims (2)
1.一种马卡甲醇层萃取物，其萃取流程如下：
将马卡块茎在室温下以95％的乙醇水溶液进行萃取获得马卡粗萃物；
将所述马卡粗萃物以体积比1：1的乙酸乙酯及水进行分相萃取，获得乙酸乙酯层萃取物；以及
将所述乙酸乙酯层萃取物以体积比1：1的75％的甲醇水溶液及正己烷进行分相萃取，获得所述马卡甲醇层萃取物。
2.一种马卡甲醇层萃取物的用途，其用于制备抗中性粒细胞炎症及/或抗过敏的药物的用途，所述马卡甲醇层萃取物的萃取流程如下：
将马卡块茎在室温下以95％的乙醇水溶液进行萃取获得马卡粗萃物；
将所述马卡粗萃物以体积比1：1的乙酸乙酯及水进行分相萃取，获得乙酸乙酯层萃取物；以及
将所述乙酸乙酯层萃取物以体积比1：1的75％的甲醇水溶液及正己烷进行分相萃取，获得所述马卡甲醇层萃取物。
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| US20220387536A1 (en) | 2022-12-08 |
| US11771729B2 (en) | 2023-10-03 |
| TW202300165A (zh) | 2023-01-01 |
| JP2022184813A (ja) | 2022-12-13 |
| JP7446003B2 (ja) | 2024-03-08 |
| CN115477604A (zh) | 2022-12-16 |
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