【作者】 齐凤琴；

【导师】 李廷利；

【作者基本信息】 黑龙江中医药大学 ， 中药学， 2011， 博士

【摘要】 目的：利用中药血清药物化学的方法,确认酸枣仁汤改善睡眠作用的药效物质基础。方法：1、酸枣仁汤冻干粉中芒果苷和甘草苷的含量测定：以芒果苷和甘草苷为对照品,采用HPLC-UV色谱法,分别在257nm和276nm波长下对酸枣仁汤冻干粉中芒果苷和甘草苷的含量进行测定。2、酸枣仁汤冻干粉指纹图谱的建立：本实验遵照国家药品监督管理局《中药注射剂指纹图谱研究的技术要求(暂行)》的技术规定,采用HPLC-UV色谱法,确定酸枣仁汤的HPLC-UV色谱条件,测定十批样品,建立酸枣仁汤HPLC-UV指纹图谱。3、酸枣仁汤冻干粉给药后大鼠血中移行成分的分析：选择Wistar大鼠,灌胃给予酸枣仁汤冻干粉,连续给药7天,以甲醇法除蛋白制备含药血清,在酸枣仁汤冻干粉指纹图谱色谱条件下,进行给药后血中移行成分的确认及药物来源的认定。4、酸枣仁汤冻干粉给药后大鼠脑脊液中移行成分的分析：选择Wistar大鼠,灌胃给予酸枣仁汤冻干粉,连续给药7天,以甲醇法除蛋白制备含药脑脊液。在酸枣仁汤指纹图谱色谱条件下,进行给药后脑脊液中移行成分的分析。5、酸枣仁汤改善睡眠作用药效物质基础的确认：在酸枣仁汤冻干粉指纹图谱色谱条件下,对体外色谱图、给药后大鼠血中移行成分色谱图、脑脊液中移行成分色谱图进行对比分析,初步确认该方改善睡眠作用的药效物质基础。6、脑脊液中内源性物质的初步认定：在酸枣仁汤冻干粉指纹图谱色谱条件下,记录神经递质的HPLC色谱图,通过对比分析,确认脑脊液中内源性物质。结果：1、酸枣仁汤冻干粉中芒果苷和甘草苷的含量测定：芒果苷含量为4.8091±0.1806mg/g,甘草苷含量为2.1259±0.0623mg/g。2、酸枣仁汤冻干粉指纹图谱的建立：确定了酸枣仁汤冻干粉指纹图谱色谱条件—色谱柱：Waters公司Symmetry C18色谱柱；洗脱系统：乙腈-0.1%磷酸水梯度洗脱；流速：1ml/min:柱温：30℃；检测波长：280nm；进样量10μl；对10批样品进行了指纹图谱的测定,经HPLC-UV比对确定了11个共有峰的来源。3、酸枣仁汤冻干粉给药后大鼠血中移行成分的分析：在酸枣仁汤冻干粉色谱条件下,给药七天后大鼠血中共检出6个移行成分,均为酸枣仁汤入血后产生的新生代谢产物。其中,血中移行成分1、2由酸枣仁、甘草所含成分入血而产生的代谢产物；血中移行成分3、4、5为甘草所含成分入血而产生的代谢产物；血中移行成分6为酸枣仁和知母所含成分入血而产生的代谢产物；血中移行成分4在单味药血清色谱图中未见体现,推测该移行成分不是由方中任何一个单味药产生,可能是复方煎煮过程中产生的新的化学成分入血后产生的新生代谢产物。4、酸枣仁汤冻干粉给药后脑脊液中移行成分的分析：酸枣仁汤冻干粉给药后大鼠脑脊液中出现了某种内源性物质的升高,从峰面积看,给药组是空白组峰面积的近4倍。5、酸枣仁汤改善睡眠作用药效物质基础的确认：酸枣仁汤冻干粉给药后大鼠血清中出现的6个移行成分有可能成为酸枣仁汤改善睡眠作用的药效物质基础。6、脑脊液中内源性物质的初步认定：通过与神经递质的比对,发现该内源性物质不是5-羟色胺、5-羟吲哚乙酸、去甲肾上腺素、多巴胺、腺苷及尿苷中的任何一种,至于该成分为何种神经递质有待进一步研究。结论：1、酸枣仁汤冻干粉中芒果苷和甘草苷的含量测定：酸枣仁汤冻干粉中芒果苷的含量应不低于4.8091±0.1806 mg/g,甘草苷的含量应不低于2.1259±0.0623mg/g。2、酸枣仁汤冻干粉指纹图谱的建立建立了酸枣仁汤冻干粉指纹图谱,在280nm下共确定了11个共有峰。1号峰来源于知母；2号峰来源酸枣仁；3号峰来源于知母；4号来源于酸枣仁；5、6号来源于甘草；7号峰来源于川芎；8号峰来源于酸枣仁和川芎；9号峰来源于川芎、甘草；10号峰来源于川芎；11号峰来源于甘草。3、酸枣仁汤冻干粉给药后大鼠血中移行成分的分析：在酸枣仁汤冻干粉色谱条件下,给药后大鼠血中共检出6个移行成分,均为酸枣仁汤入血后产生的新生代谢产物。其中,血中移行成分1、2由酸枣仁、甘草所含成分入血而产生的代谢产物；血中移行成分3、4、5为甘草所含成分入血而产生的代谢产物；血中移行成分6为生药酸枣仁和知母所含成分入血而产生的代谢产物；血中移行成分4在单味药血中移行成分色谱图中未见体现,推测该移行成分不是由方中任何一个单味药产生,可能是复方煎煮过程中产生的新的化学成分入血后产生的新生代谢产物。4、酸枣仁汤给药后大鼠脑脊液中移行成分的分析：给药后脑脊液中,并未见体外成分的检出,而是出现了某种内源性物质的增高。推测酸枣仁汤改善睡眠作用机制可能是通过升高该内源性物质来实现的。5、酸枣仁汤改善睡眠作用药效物质基础的初步确认：酸枣仁汤给药后大鼠血中出现的6个移行成分有可能成为酸枣仁汤改善睡眠作用的药效物质基础。6、脑脊液中内源性物质的初步认定：通过与神经递质的比对,发现该内源性物质不是5-羟色胺、5-羟吲哚乙酸、去甲肾上腺素、多巴胺、腺苷及尿苷中的任何一种,至于该成分为何种物质有待进一步研究。上述研究结果将为酸枣仁汤作用机制的阐明及深层次的开发研究奠定实验基础。更多还原

【Abstract】 Objective:Efficacy of improvement sleep function material base of Suanzaoren decoction was determined preliminarily by using the method of traditional Chinese medicine serum pharmaceutical chemistry.Method:1.The content determination of the mangiferin and the Licorice glucoside in the Suanzaoren decoction:The qualification of the Suanzaoren decotion and ensue standardization of the experimental sample was determined by the contents of the mangiferin and the Licorice glucoside in the 257nm and 276nm by HPLC-UV with the mangiferin and the Licorice glucoside as the reference substance.2.To establish.the fringerprint of Suanzaorendecotion:The experiment complied with the technical regulations of The National Drug Supervision And Administration’The requirement of the research techniques of Traditional Chinese Medicine Injection fingerprint (temporary)’, The chromatographic conditions of ten batch sample of Suanzaoren decotion was determined by HPLC-UV, and the fingerprint of HPLC-UV of the Suanzaorentang decotion was established.3.To confirm the Suanzaoren decotion freeze-dried the analysis and source of the mobiling composition in the blood of the rats:the wistar rats were irrigated for seven days with methanol removed the protein and prepared the cerebrospinal fluid. The mobiling composition of the cerebrospinal fluid was ensured and the source after the rats given medicine was affirmed in the the fingerprint of the Suanzaoren decotion.4.To analyse the Suanzaoren decotion freeze-dried the analysis and source of the mobiling composition in the blood of the rats:The wistar rats were irrigated for seven days, with methanol removed the protein and prepared the cerebrospinal fluid. The mobiling composition of the cerebrospinal fluid after the rats given the medicine was analysed by the fingerprint of the Suanzaorentang decotion condition.5.To preliminary confirm the efficacy material base of the improvement of the Suanzaorendecotion:in the condition of the fingerprint of the Suanzaoren freeze-dried, to analyse and contrast the external fingerprint, the mobiling composition fingerprint of the rats after giving medicine, the mobiling composition fingerprint in the cerebrospinal fluid preliminary confirmed the efficacy of material base of this prescription in the function of the improvement sleep6.To preminilary confirm endogenous substances in the cerebrospinal fluid:in the condition of the fingerprint chromatographic of Suanzaoren decotion reezed-dried, recording the HPLC of the neurotransmitter confirmed the endogenous substances in the cerebrosphinal.Result:1.In the Suanzaoren decoction the content determination of the mangiferin and the Licorice glucoside:the content determination of the mangiferin is 4.8091±0.1806mg/g,the content determination of the Licorice glucoside is 2.1059±0.0623mg/g.2.To establish the fringerprint of Suanzaoren decotion:Confirmed the fingerprint chromatographic conditions of Suanzaoren decotion freezed-dried-chromatographic column:waters company Symmetry C18 chromatographic column; elution system:acetonitrile-0.1% phosphoric acid water gradient elution; column temperature:30℃;detected wavelength:280nm; injection volume:10μl. The fingerprint for 10 batch samples was detemined, and confirmed 10 common peaks.3.To confirm the Suanzaoren decotion freeze-dried the analysis and source of the mobiling composition in the blood of the rats:in the chromatographic of the Suanzaoren decotion condition after giving seven days in the rats blood, it detected 6 mobiling contents, they are all metabolism products of producing by Suanzaoren decotion in the blood. Among them, mobiling contents 1,2 in the blood are all the metabolism products of the content of the Ziziphi spinosae, licorice in the blood;in the blood mobiling contents 3,4,5 are the metabolism products of the content of the licorice in the blood;the mobiling contents6 is the metabolism products of the contents of the Ziziphi spinosae and licorice in the blood;the mobiling content 4 don’t appear in the serum chromatograms of the serum, it speculates that this mobiling contents don’t product by any single herb, it is probable the new metabolism products of the new contents in the blood of the compound in the decoction process.4.To analyse the Suanzaoren decotion freeze-dried the analysis and source of the mobiling composition in the blood of the rats.After giving the medicine in the cerebrospinal fluid of the rats it appears that some endogenous material rises.5.To preliminary confirm the efficacy material base of the improvement of the Suanzaoren decotion:6 mobiling contents are probably the efficacy material base of the improvement of the Suanzaoren decotion in the serum after giving the medicine.6.To preminilary confirm endogenous substances in the cerebrospinal fluid:by contrast to the neurotransmitter,it detects that the endogenous substances are not anyone of the serotonin,5-hydroxyl ethyl indoles, norepinephrine, dopamine adenosine and urine glucoside what the composition is reminded to further research.Conclusion:1.The content of the mangiferin in the Suanzaoren decotion should be no less than 4.8091±0.1806mg/g, the content of the Licorice glucoside should be no less than 2.1259±0.0623mg/g.2.Establish the fringerprint of Suanzaoren decotion in the first, at 280nm, it detects 10 common peaks. They can fully reflect the existing state of the external components of the Suanzaorentang decotion.3.To confirm the Suanzaoren decotion freeze-dried the analysis and source of the mobiling composition in the blood of the rats:in the chromatographic of the Suanzaoren decotion condition after giving seven days in the rats blood, it detected 6 mobiling contents, they are all metabolism products of producing by Suanzaoren decotion in the blood. Among them, mobiling contentsl,2 in the blood are all the metabolism products of the content of the Ziziphi spinosae, licorice in the blood;in the blood mobiling contents 3,4,5 are the metabolism products of the content of the licorice in the blood;the mobiling contents6 is the metabolism products of the contents of the Ziziphi spinosae and licorice in the blood;the mobiling content 4 don’t appear in the serum chromatograms of the serum, it speculates that this mobiling contents don’t product by any single herb, it is probable the new metabolism products of the new contents in the blood of the compound in the decoction process.4.To analyse the Suanzaoren decotion freeze-dried the analysis and source of the mobiling composition in the blood of the rats.After giving the medicine in the cerebrospinal fluid of the rats it appears that some endogenous material rises.5.To preliminary confirm the efficacy material base of the improvement of the Suanzaoren decotion:6 mobiling contents are probably the efficacy material base of the improvement of the Suanzaoren decotion in the serum after giving the medicine.6.To preminilary confirm endogenous substances in the cerebrospinal fluid:by contrast to the neurotransmitter,it detects that the endogenous substances are not anyone of the serotonin,5-hydroxyl ethyl indoles, norepinephrine, dopamine adenosine and urine glucoside what the composition is reminded to further research.更多还原