CN115385901A - 一种从半夏中同时分离夏佛塔苷和胡芦巴碱的方法 - Google Patents
一种从半夏中同时分离夏佛塔苷和胡芦巴碱的方法 Download PDFInfo
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- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
- C07D407/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing three or more hetero rings
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- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/79—Acids; Esters
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- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
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Abstract
本发明涉及一种从半夏中同时分离夏佛塔苷和胡芦巴碱的方法,属于化合物单体的分离纯化技术领域。本发明是以半夏为原料,经提取、浓缩、AB‑8柱层析、过滤、制备型高效液相色谱仪分离及产品回收,得到夏佛塔苷和胡芦巴碱单体。其中,涉及的分离效率高,生产周期短,工艺稳定,操作简便,成本低廉,可实现夏佛塔苷单体、胡芦巴碱单体的高纯度(面积归一法99%以上)分离。
Description
技术领域
本发明涉及一种从半夏中同时分离夏佛塔苷和胡芦巴碱的方法,属于化合物单体的分离纯化技术领域。
背景技术
半夏,始载于《神农本草经》,《礼记·月令》中记载:“五月半夏生,盖当夏之半也,故名,又名守田、水玉,守田会意,水玉因形”。半夏为天南星科植物半夏Pinelliaternate(Thumb).Breit.的干燥块茎,味辛,性温,有毒,归脾、胃、肺经,具有燥湿化痰、降逆止呕、消痞散结的功效。其于临床上多为复方应用,在张仲景所著的《伤寒论》《金贵要略》书中,含半夏的方剂共42 方(去除重复方),占总方的13.25%。
目前,经对半夏单味药的研究发现,其化学成分包括生物碱类、有机酸类、挥发油类、黄酮类、甾体类和糖类等多种成分;其药理学研究表明,半夏除具有燥湿化痰、降逆止呕、消痞散结等作用外,还具有抗肿瘤、抗菌、抗炎、抗癫痫等药理活性;其毒性主要为刺激性毒性、肝肾毒性、妊娠毒性。近年来,对半夏研究进展的综述文献多侧重于药理作用研究,对其化学成分的总结不直观、针对性不强。
夏佛塔苷(Schaftoside),分子式为C26H28O14,分子量为564.50,结构式如下:
胡芦巴碱(Trigonelline hydrochloride),分子式为C7H8ClNO2,分子量为173.60,结构式如下:
夏佛塔苷、胡芦巴碱来源于天南星科植物半夏Pinelliaternate(Thumb).Breit.的干燥块茎,是半夏药材中的主要活性成分,具有燥湿化痰、降逆止呕、消痞散结等功效。目前,主要采用硅胶柱层析与结晶相结合的方法进行夏佛塔苷、胡芦巴碱单体的精制,但涉及的生产周期长,试剂浪费严重,产品收率低。
现有技术“UPLC测定半夏中胡芦巴碱的含量”中公开:取半夏粉末2g,精密称定,置50mLBP管中,精密加入20ml50%的甲醇溶液,称重,浸泡1h,超声45min,冷却,再次称重,并用50%的甲醇补足失重,摇匀,静置,取上清液1.0mL于离心管中,吹干,用乙腈-甲醇(75:25)液500μL复液,涡旋5min,超声10min,13000r/min离心30min,即得供试品溶液。其重要是基于UPLC,测定半夏中胡芦巴碱的含量。
发明内容
本发明旨在解决现有技术问题,而提出了一种从半夏中同时分离夏佛塔苷和胡芦巴碱的方法。在本技术方案中,以半夏为原料,经提取、浓缩、AB-8柱层析、过滤、制备型高效液相色谱仪分离及产品回收,得到夏佛塔苷和胡芦巴碱单体。其中,涉及的分离效率高,生产周期短,工艺稳定,操作简便,成本低廉,可实现夏佛塔苷单体、胡芦巴碱单体的高纯度(面积归一法99%以上)分离。
为了实现上述技术目的,提出如下的技术方案:
本技术方案提出:一种从半夏中同时分离夏佛塔苷和胡芦巴碱的方法,具体包括如下步骤:
X1.提取:将半夏粉碎为1-4mm的粗粉,以每千克半夏粗粉加入乙醇溶液10-20升计,加入浓度为70%的乙醇溶液,提取3-4次,每次提取2-3h,收集,合并,过滤,得提取液;
X2.浓缩:将所得的提取液在60℃条件下浓缩,得浓缩液;
X3.富集和洗脱:将所得的浓缩液通入至活化的AB-8大孔树脂柱中富集,用纯化水清洗至无色,然后,以浓度为70%的乙醇溶液解析,回收乙醇,剩余水溶液即为层析液;
X4.过滤:将所得的层析液用0.45μm的滤膜过滤,得到包括夏佛塔苷单体和胡芦巴碱单体的过滤液;
X5.分离:将所得的过滤液向制备型高效液相色谱仪中进样,进行夏佛塔苷单体和胡芦巴碱单体的制备,收集夏佛塔苷单体和胡芦巴碱单体的制备液;
其中,制备型高效液相色谱仪中的色谱条件包括:
色谱柱填料:C18填料;
流动相:甲醇-0.1%磷酸水溶液V/V=30︰70;
进样量:1.0-1.5L/针;
检测:紫外光,检测波长为270nm;
X6.产品回收:将所得的夏佛塔苷单体和胡芦巴碱单体的制备液,回收甲醇,剩余水溶液用C18富集和纯甲醇解析,在45-55℃条件下分别浓缩至干,干燥,即得夏佛塔苷单体和胡芦巴碱单体,纯度均为99%以上。
进一步的,在进行制备型高效液相色谱仪分离前,通过液-质联用法确定制备型高效液相色谱仪中夏佛塔苷单体和胡芦巴碱单体各自的峰形,即取经步骤X4所得的过滤液进样,进行夏佛塔苷单体和胡芦巴碱单体的制备分离,根据质谱检测结果,确定夏佛塔苷单体及胡芦巴碱单体在液相色谱中对应的峰形。
本技术方案还提出:一种从半夏中同时分离夏佛塔苷和胡芦巴碱的纯度检测方法,采用分析型高效液相色谱仪;
其中,分析型高效液相色谱仪中的色谱条件包括:
色谱柱填料:十八烷基硅烷键合硅胶,柱长为250mm,内径为4.6mm,粒径5μm;
流动相:甲醇为流动相A,以水为流动相B;
流速:0.9mL/min;
检测波长:270nm;
柱温:30℃,
梯度洗脱如下;
采用本技术方案,带来的有益技术效果为:
1)本发明采用制备型高效液相色谱系统对夏佛塔苷单体、胡芦巴碱单体进行分离纯化,达到了很好的分离效果,且通过在线紫外检测,针对性的收集夏佛塔苷单体、胡芦巴碱单体,目标明确,避免了常规柱层析等造成的资源浪费,而且便于控制产品质量,产品纯度可达99%以上;
2)由于高效液相色谱对样品的色泽、纯度等要求较高,通过简单处理得到的提取液不能直接进样,因此,若在进行高效液相色谱分离前,未对进样溶液进行有效的前处理,这不仅使得分离效果不好,而且还可能对仪器造成严重影响(如:缩短寿命等),进而增加产品的生产成本,以及,影响分离工艺的正常进行;
3)本发明针对半夏药材中存在的各种成分的理化性质,进行了X1-X3步骤的适当顺序搭配,以及适当的参数组合,有效的提取出夏佛塔苷、胡芦巴碱等成分,并除去了大量杂质,由此,获得了可进入制备型高效液相色谱系统的样品溶液,而不至对高效液相色谱系统造成大的影响,尽量延长其使用周期,节约了生产成本;
4)在高效液相色谱分离过程中,各色谱条件的选择及其组合极为重要,其直接影响物质成分的出峰时间、峰形等。其中,色谱条件主要包括色谱柱(包括填料、柱长、柱温等)、流动相(包括成分、流速等)、检测器及检测波长等,本发明通过大量的试验研究和对比分析,确定了特定的色谱条件,从而使物质成分的出峰时间、峰形、分离效果等达到最佳化,实现了夏佛塔苷单体、胡芦巴碱单体的高效分离。
附图说明
图1为实施例3中夏佛塔苷单体产品的HPLC图谱;
图2本实施例3中胡芦巴碱单体产品的HPLC图谱(其中,从下至上依次为:胡芦巴碱盐酸盐对照品溶液、半夏RK20032002、半夏RK21120701、半夏RK21120702、半夏RK20032002(加对照品溶液));
图3本实施例4中半夏的HPLC图谱(其中,峰5(S):鸟苷,峰7:腺苷,峰8:夏佛塔苷;色谱柱:SVEATM C18 Opal)。
具体实施方式
下面通过对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明的一部分实施例,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它实施例,都属于本发明保护的范围。
在下述实施例中,涉及的夏佛塔苷单体和胡芦巴单体的纯度复检,均采用分析型高效液相色谱(HPLC)法;
其中,分析型高效液相色谱仪中的色谱条件包括:
色谱柱填料:十八烷基硅烷键合硅胶,柱长为250mm,内径为4.6mm,粒径5μm;
流动相:甲醇为流动相A,以水为流动相B;
流速:0.9mL/min;
检测波长:270nm;
柱温:30℃,
梯度洗脱如下;
实施例1
一种从半夏中同时分离夏佛塔苷和胡芦巴碱的方法,包括如下工艺步骤:
1)提取
将半夏药材1Kg粉碎成1~4mm的粗粉,加入10L体积百分比浓度为70%乙醇溶液,提取3次,每次2小时,合并,过滤提取液,共30L;
2)浓缩
将步骤1)所得的提取液,60℃浓缩至小体积,所得浓缩液5L;
3)AB-8大孔树脂柱层析
将步骤2)所得的浓缩液用已经活化好的AB-8大孔树脂富集,富集完成后,再用10L水洗、15L体积百分比浓度为70%乙醇解析。解析液回收乙醇后,所得浓缩液共6L;
4)过滤
将步骤3)所得的浓缩液6L用0.45μm滤膜过滤,所得过滤液6.2L;
5)制备型高效液相色谱分离
色谱柱填料为:C18填料;
流动相为:甲醇-0.1%磷酸水溶液V/V=30︰70;
步骤4)所得的过滤液进样,进样量为1L/针,在线紫外检测,检测波长为270nm,分别收集夏佛塔苷制备溶液5L,胡芦巴碱制备溶液6L;
6)产品回收
将步骤5)所得的夏佛塔苷制备溶液5L及胡芦巴碱制备溶液6L,分别回收甲醇,剩余水溶液用C18富集、甲醇解析,45-55℃条件下浓缩至干,分别得夏佛塔苷高纯度的固体和胡芦巴碱高纯度的固体,干燥,即得夏佛塔苷单体0.5g和胡芦巴碱单体0.4g。
利用分析型高效液相色谱(HPLC)法复检产品纯度,测得结果为:夏佛塔苷99.15%、胡芦巴碱99.35%。
实施例2
一种从半夏中同时分离夏佛塔苷和胡芦巴碱的方法,包括如下工艺步骤:
1)提取
将半夏药材10Kg粉碎成1~4mm的粗粉,加入110L体积百分比浓度为70%乙醇溶液,提取3次,每次2小时,合并,过滤提取液,共90L;
2)浓缩
将步骤1)所得的提取液,60℃浓缩至小体积,所得浓缩液10L;
3)AB-8大孔树脂柱层析
将步骤2)所得的浓缩液用已经活化好的AB-8大孔树脂富集,富集完成后,再用80L水洗、120L体积百分比浓度为70%乙醇解析。解析液回收乙醇后,所得浓缩液共18L;
4)过滤
将步骤3)所得的浓缩液18L用0.45μm滤膜过滤,所得过滤液19L;
5)制备型高效液相色谱分离
色谱柱填料为:C18填料;
流动相为:甲醇-0.1%磷酸水溶液V/V=30︰70;
步骤4)所得的过滤液进样,进样量为1L/针,在线紫外监测,检测波长为270nm,分别收集夏佛塔苷制备溶液60L,及胡芦巴碱制备溶液75L;
6)产品回收
将步骤6)所得的夏佛塔苷制备溶液60L、胡芦巴碱制备溶液75L,分别回收甲醇,剩余水溶液用C18富集,甲醇解析,45-55℃条件下浓缩至干,分别得夏佛塔苷高纯度固体和胡芦巴碱高纯度固体,干燥,即得夏佛塔苷5.6g和胡芦巴碱4.3g。
利用分析型高效液相色谱(HPLC)法复检产品纯度,测得结果为:夏佛塔苷单体99.25%,胡芦巴碱单体99.45%。
实施例3
一种从半夏中同时分离夏佛塔苷和胡芦巴碱的方法,包括如下工艺步骤:
1)提取
将半夏药材20Kg粉碎成1~4mm的粗粉,加入200L体积百分比浓度为70%乙醇溶液,提取3次,每次2小时,合并,过滤提取液,共185L;
2)浓缩
将步骤1)所得的提取液,60℃条件下浓缩至小体积,所得浓缩液30L;
3)AB-8大孔树脂柱层析
将步骤2)所得的浓缩液用已经活化好的AB-8大孔树脂富集,富集完成后,再用150L水洗、200L体积百分比浓度为70%乙醇解析。解析液回收乙醇后,所得浓缩液共25L,
4)过滤
将步骤3)所得的浓缩液25L用0.45μm滤膜过滤,所得过滤液26L;
5)制备型高效液相色谱分离
色谱柱填料为:C18填料;
流动相为:甲醇-0.1%磷酸水溶液V/V=30︰70;
步骤4)所得的过滤液进样,进样量为1L/针,在线紫外监测,检测波长为270nm,分别收集夏佛塔苷制备溶液80L,及胡芦巴碱制备溶液95L;
6)产品回收
将步骤5)所得的夏佛塔苷制备溶液80L、胡芦巴碱制备溶液95L,分别回收甲醇,剩余水溶液用C18富集,甲醇解析,45-55℃条件下浓缩至干,分别得夏佛塔苷高纯度固体和胡芦巴碱高纯度固体,干燥,即得夏佛塔苷12g、胡芦巴碱9g。
利用分析型高效液相色谱(HPLC)法复检产品纯度,测得结果为:夏佛塔苷99.45%,胡芦巴碱99.35%;其中,涉及的HPLC图谱如图1-2所示。
实施例4
基于实施例3,本实施例另对半夏进行特征图谱的构建,以用于半夏药材的质量控制,而为从半夏中同时分离夏佛塔苷和胡芦巴碱的方法提供前提保证,为此,进行了方法开发(如:流动相研究、检测波长确定、不同品牌色谱柱及柱温流速考察、供试品溶液制备方法、参照物溶液的制备、色谱条件确定等)、方法学验证(如:专属性、重复性、中间精密度、准确度)、多批次样品检测等研究,然后得出相适应的参照物溶液制备方法、供试品溶液的制备方法和色谱条件等。以对本发明申请中的技术做进一步的说明。
在半夏进行特征图谱的构建中,涉及的方法包括:
1)参照物溶液的制备
取鸟苷对照品适量,精密称定,加50%甲醇分别制成每1mL含鸟苷0.02mg的溶液,即得;
2)供试品溶液的制备
取本品粉末(过四号筛),取约2g,精密称定,置具塞锥形瓶中,精密加入水20ml,密塞,称定重量,超声处理30min(功率360W,频率40kHz),放冷,再称定重量,用水补足减失的重量,摇匀,离心,取上清液滤过,取续滤液,即得;
3)色谱条件及系统适用性试验
以十八烷基硅烷键合硅胶为填充剂(柱长为250mm,内径为4.6mm,粒径5μm);以甲醇为流动相A,以水为流动相B,按下表进行梯度洗脱;柱温为35℃;流速为1.0ml/min;检测波长为272nm。按下表程序进行洗脱。
4)测定法
分别精密吸取参照物溶液与供试品溶液各10μL,注入液相色谱仪,测定,即得。
如图3所示,供试品色谱图中应呈现8个特征峰,与鸟苷参照物相应的峰为S峰,计算各特征峰与S峰的相对保留时间,其相对保留时间应在规定值的±10%之内。规定值为:0.50(峰1)、0.56(峰2)、0.64(峰3)、0.93(峰4)、1.00(峰5)、1.49(峰6)、1.54(峰7)、2.21(峰8)。
实施例5
基于实施例3,本实施例另对半夏中化学成分-胡芦巴碱的含量进行测定,用于半夏药材的质量控制,而为从半夏中同时分离夏佛塔苷和胡芦巴碱的方法提供前提保证,以对本发明申请中的技术做进一步的说明。
在半夏中胡芦巴碱的含量测定中,涉及的方法包括:
1)供试品溶液的制备
取本品粉末(过四号筛)约2g,精密称定,精密加入50%甲醇50mL,密塞,称定重量,放置1h,超声处理(功率300W,频率50kHz)45min,放冷,密塞,再称定重量,用50%甲醇补足减失的重量,摇匀,滤过,取续滤液,即得(每1mL相当于含半夏约0.04g);
2)对照品溶液的制备
取胡芦巴碱对照品适量,精密称定,加50%甲醇制成每1mL含15μg的溶液,即得;
3)色谱条件及系统适用性试验
以甲醇-0.05%十二烷基磺酸钠溶液-冰醋酸(19∶81∶0.1)为流动相;检测波长为265nm,柱温为30℃,流速为0.6ml/min,进样量为20μL;
4)测定法
分别精密吸取参照物溶液与供试品溶液各10μL,注入液相色谱仪,测定,即得。
实施例6
基于实施例3,本实施例另对半夏中化学成分-夏佛塔苷的含量进行测定,用于半夏药材的质量控制,而为从半夏中同时分离夏佛塔苷和胡芦巴碱的方法提供前提保证,为此,进行了色谱条件、供试品溶液制备考察、方法学验证等考察,以对本发明申请中的技术做进一步的说明。
在半夏中夏佛塔苷的含量测定中,涉及的方法包括:
1)供试品溶液的制备
取本品粉末(过二号筛)约4g,精密称定,置具塞锥形瓶中,精密加入80%甲醇100mL,加热回流30min,放冷,摇匀,离心,取上清液,50℃减压浓缩至干,残渣精密加入50%甲醇10mL,密塞,称定重量,超声(功率360W,频率40kHz)处理20min,放冷,再称定重量,用50%甲醇补足减失的重量,摇匀,滤过,取续滤液,即得;
2)对照品溶液的制备
取夏佛塔苷对照品适量,精密称定,加50%甲醇制成每1mL含15μg的溶液,即得;
3)色谱条件及系统适用性试验
以十八烷基硅烷键合硅胶为填充剂;以十八烷基硅烷键合硅胶为填充剂,以甲醇-0.1%甲酸(31∶69)为流动相,柱温为25℃,流速为1.0mL/min,检测波长为270nm;
4)测定法
分别精密吸取对照品溶液和供试品溶液各20µL,注入液相色谱仪,测定,即得。
Claims (4)
1.一种从半夏中同时分离夏佛塔苷和胡芦巴碱的方法,其特征在于,包括如下步骤:
X1.提取:将半夏粉碎为1-4mm的粗粉,以每千克半夏粗粉加入乙醇溶液10-20升计,加入浓度为70%的乙醇溶液,提取3-4次,每次提取2-3h,收集,合并,过滤,得提取液;
X2.浓缩:将所得的提取液在60℃条件下浓缩,得浓缩液;
X3.富集和洗脱:将所得的浓缩液通入至活化的AB-8大孔树脂柱中富集,用纯化水清洗至无色,然后,以浓度为70%的乙醇溶液解析,回收乙醇,剩余水溶液即为层析液;
X4.过滤:将所得的层析液用0.45μm的滤膜过滤,得到包括夏佛塔苷单体和胡芦巴碱单体的过滤液;
X5.分离:将所得的过滤液向制备型高效液相色谱仪中进样,进行夏佛塔苷单体和胡芦巴碱单体的制备,收集夏佛塔苷单体和胡芦巴碱单体的制备液;
其中,制备型高效液相色谱仪中的色谱条件包括:
色谱柱填料:C18填料;
流动相:甲醇-0.1%磷酸水溶液V/V=30︰70;
进样量:1.0-1.5L/针;
检测:紫外光,检测波长为270nm;
X6.产品回收:将所得的夏佛塔苷单体和胡芦巴碱单体的制备液,回收甲醇,剩余水溶液用C18富集和纯甲醇解析,在45-55℃条件下分别浓缩至干,干燥,即得夏佛塔苷单体和胡芦巴碱单体。
2.根据权利要求1所述的从半夏中同时分离夏佛塔苷和胡芦巴碱的方法,其特征在于,在进行制备型高效液相色谱仪分离前,通过液-质联用法确定制备型高效液相色谱仪中夏佛塔苷单体和胡芦巴碱单体各自的峰形,具体包括:
取经步骤X4所得的过滤液进样,进行夏佛塔苷单体和胡芦巴碱单体的制备分离,根据质谱检测结果,确定夏佛塔苷单体及胡芦巴碱单体在液相色谱中对应的峰形。
3.根据权利要求1所述的从半夏中同时分离夏佛塔苷和胡芦巴碱的方法,其特征在于,所述夏佛塔苷单体和胡芦巴碱单体的纯度均为99%以上。
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CN104926719A (zh) * | 2015-07-17 | 2015-09-23 | 湖南杰萃生物技术有限公司 | 一种从火麻仁中提取葫芦巴碱的方法 |
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CN116514916A (zh) * | 2023-06-26 | 2023-08-01 | 江西省药品检验检测研究院 | 一种吲哚生物碱化合物及其制备方法 |
CN116514916B (zh) * | 2023-06-26 | 2023-09-08 | 江西省药品检验检测研究院 | 一种吲哚生物碱化合物及其制备方法 |
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