厚朴挥发性成分的HS-SPME-GC指纹图谱建立
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摘要
厚朴(Magnoliae officinalis Cortex)为木兰科植物厚朴(Magnolia officinalis Rehd.et Wils.)及凹叶厚朴(Magnolia officinalis Rehd. et Wils.var. biloba Rehd. et Wils.)的干燥干皮、枝皮和根皮。本课题以厚朴为研究对象,采集和购买了多批不同产地或部位的厚朴药材共15批,采用顶空-固相微萃取-气相色谱法(HS-SPME-GC)对其成分进行分析,并结合顶空-固相微萃取-气相色谱-质谱(HS-SPME-GC-MS)的鉴定结果,建立其中药色谱指纹图谱。通过聚类分析、相似度分析的结果对其品质进行评价。同时为了比较不同方法及不同化学模式之间的异同,采用高效液相色谱法(HPLC)获取反映不同批次厚朴化学本质差异的数量化特征,为厚朴的质量控制提供科学的、系统的评价依据。
1.对HS-SPME-GC法分析厚朴挥发性成分的萃取条件和色谱条件进行优化。通过考察不同条件下出峰个数和总峰面积的变化差异,对不同涂层的SPME商用萃取头、萃取温度、萃取时间和样品用量等影响因素进行优化,结果表明：采用PDMS/DVB萃取头,取厚朴粉末0.2g在70℃恒温条件下萃取60min,可达到较好的萃取效率；同时也对色谱柱、升温程序、流速、分流比等分离因素进行了条件优化,筛选出HP-50毛细管柱(30 m×0.32mm×0.25μm),程序升温：50℃(1min),以4℃/min上升到140℃(2min),以2℃/min上升到180℃,以12℃/min上升到280℃(5 min),流速1.3 ml/min,分流比为15：1的色谱条件较适合建立厚朴药材的中药色谱指纹图谱。
2.按上述优化条件对不同产地或不同部位的15批厚朴药材进行批量分析,共获得42个共有色谱峰,并经HS-SPME-GC-MS鉴定确认了15个组分。以喇叭茶醇为内参照峰,建立指纹图谱。通过聚类分析,15批次药材被分成4类。其中1、2类为推荐品,选取其中10个批次建立共有模式,并计算生成的对照图谱与15个批次之间的相似度,结果表明各批次的相似度均在0.9以上,未发现离群样品,而其中11号批次因保存不当导致挥发性成分流失,其聚类结果在第四类、相似度在15个批次中最低,与实际情况相符,所以本研究所采用的HS-SPME方法适合于中药材GC指纹图谱的建立。
3.对21个批次厚朴的主要药效成分厚朴酚和和厚朴酚进行比较分析,建立其共有模式,以厚朴酚、和厚朴酚为考察指标,用系统聚类分析对其进行化学模式识别研究。21批药材被分为4类,其中1类为推荐品,较好的反映了不同产地的厚朴酚性成分的差异。与厚朴药材GC指纹图谱进行比较,未发现厚朴药材GC指纹图谱的分析结果与其主要药效成分含量的差异性之间的关联。
Cortex Magnoliae officinalis for dessicated skin of the tree trunk, branches and the root of magnoliaceous species, Magnolia officinalis Rehd. Ge et Wils and Magnolia officinalis Rehd.et Wils.var. biloba Rehd.et Wils.This topic's object of study is Cortex Magnoliae officinanlis. Cortex Magnoliae officinalis of 15 different production fields are gathered and bought.Use top empty Solid-phase microextraction-gas chromatography method to determine and analyze compositions of volatile compounds in Cortex Magnoliae officinalis of different origins,furthermore incorporating GC-MS evaluation results,a fingerprint of volatile Chinese medicine is established,through results achieved from cluster analysis and similarity analysis evaluation of the quality of the quality of the herbs and at the same time to look for the difference of volatility and it's relevant relation with presence of phenolic compounds in different herbs of different origins, we adopt HPLC method to find out and compare the phenolic composition of the herbs from different production fields; for scientific control of the quality control of Cortex Magnoliae officinalis we evaluate and judge by a systematic approach.
Firstly, HS-SPME-GC method is established to analyze composition of volatile compounds and it's extraction condition and optimization by chromatographic process. Through investigation of the number of peaks peak under different conditions and total superficial peak area change differences. For commercial extraction of differently coated SPME,extraction temperature,extraction time and sample usage level are influencing factors in optimization process.Result indication-adopt PDMS/DVB extraction at the beginning,take 0.2g of magnolia bark powder and extraction done under conditions of temperature 70℃and for 60min achieves efficient extraction comparatively. Also on chromatographic column, warming procedures, velocity, diversion ratio separation etc, factors for the optimization conditions, Screened HP-50 capillary column (30 m*0.32 mm*0.25μm),temperature programming-initial temperature 50℃(held for 1min),increased by 4℃/min to 140℃(held for 2 min),increased 2℃/min to 180℃, increased 12℃/min to 280℃(held for 5 min),ate of flow 1.3 ml/min, split ratio of 15:1 for chromatographic condition is suitable for analysis of volatile organic compounds magnolia bark.
Secondly, In accordance with the above optimization conditions for batch analytical procedure of bark of Magnoliae officinalis of 15 different origin,altogether obtained 42 common GC peaks,thereby establishing fingerprint,through cluster analysis,those 15 herbs were categorized into 4 different classes,among which classes 1 and 2 are recommended products, choosing 10 Cortex Magnoliae officinalis of different origins,established common mode,furthermore the similarity of comparative illustrations of the calculation produced with the group of 15 herbs. Result indication-similarity even above 0.9, furthermore adopting GC-MS for determining volatile composition in Magnoliae officinalis bark is analyzed; commonly identified 15 constituents among which content ofα-Eucalyptus leaves oleyl alcohol andβ-Eucalyptus leaves oleyl alcohol was highest.
Thirdly, For major efficacy, phenol composition,comparative analysis process of 21 batch herbs of magnolia officinalis,it established that it has a common pattern between them,according to honokiol and honokiol investigated indication,use systematic cluster analysis process for identification research of the chemical pattern,22 batches of medicinal material categorized into 4 classes,among which 1 class is the recommended product sample,better reflection of the fact that different origins of magnolia officinalis have different phenolic composition,be it magnolol or honokiol,furthermore with GC method a fingerprint was established related to the composition of volatile compounds of magnolia officinalis and it's analyzed,researching GC fingerprint and it's analytical results and rather than determining relationship between effective analysis content difference,trials through GC and HPLC, two different chromatographic analytical methods can systematically evaluate quality of the herb.In reality allowing fingerprint to reflect the the inherent quality of Chinese medicine.
1.对HS-SPME-GC法分析厚朴挥发性成分的萃取条件和色谱条件进行优化。通过考察不同条件下出峰个数和总峰面积的变化差异,对不同涂层的SPME商用萃取头、萃取温度、萃取时间和样品用量等影响因素进行优化,结果表明：采用PDMS/DVB萃取头,取厚朴粉末0.2g在70℃恒温条件下萃取60min,可达到较好的萃取效率；同时也对色谱柱、升温程序、流速、分流比等分离因素进行了条件优化,筛选出HP-50毛细管柱(30 m×0.32mm×0.25μm),程序升温：50℃(1min),以4℃/min上升到140℃(2min),以2℃/min上升到180℃,以12℃/min上升到280℃(5 min),流速1.3 ml/min,分流比为15：1的色谱条件较适合建立厚朴药材的中药色谱指纹图谱。
2.按上述优化条件对不同产地或不同部位的15批厚朴药材进行批量分析,共获得42个共有色谱峰,并经HS-SPME-GC-MS鉴定确认了15个组分。以喇叭茶醇为内参照峰,建立指纹图谱。通过聚类分析,15批次药材被分成4类。其中1、2类为推荐品,选取其中10个批次建立共有模式,并计算生成的对照图谱与15个批次之间的相似度,结果表明各批次的相似度均在0.9以上,未发现离群样品,而其中11号批次因保存不当导致挥发性成分流失,其聚类结果在第四类、相似度在15个批次中最低,与实际情况相符,所以本研究所采用的HS-SPME方法适合于中药材GC指纹图谱的建立。
3.对21个批次厚朴的主要药效成分厚朴酚和和厚朴酚进行比较分析,建立其共有模式,以厚朴酚、和厚朴酚为考察指标,用系统聚类分析对其进行化学模式识别研究。21批药材被分为4类,其中1类为推荐品,较好的反映了不同产地的厚朴酚性成分的差异。与厚朴药材GC指纹图谱进行比较,未发现厚朴药材GC指纹图谱的分析结果与其主要药效成分含量的差异性之间的关联。
Cortex Magnoliae officinalis for dessicated skin of the tree trunk, branches and the root of magnoliaceous species, Magnolia officinalis Rehd. Ge et Wils and Magnolia officinalis Rehd.et Wils.var. biloba Rehd.et Wils.This topic's object of study is Cortex Magnoliae officinanlis. Cortex Magnoliae officinalis of 15 different production fields are gathered and bought.Use top empty Solid-phase microextraction-gas chromatography method to determine and analyze compositions of volatile compounds in Cortex Magnoliae officinalis of different origins,furthermore incorporating GC-MS evaluation results,a fingerprint of volatile Chinese medicine is established,through results achieved from cluster analysis and similarity analysis evaluation of the quality of the quality of the herbs and at the same time to look for the difference of volatility and it's relevant relation with presence of phenolic compounds in different herbs of different origins, we adopt HPLC method to find out and compare the phenolic composition of the herbs from different production fields; for scientific control of the quality control of Cortex Magnoliae officinalis we evaluate and judge by a systematic approach.
Firstly, HS-SPME-GC method is established to analyze composition of volatile compounds and it's extraction condition and optimization by chromatographic process. Through investigation of the number of peaks peak under different conditions and total superficial peak area change differences. For commercial extraction of differently coated SPME,extraction temperature,extraction time and sample usage level are influencing factors in optimization process.Result indication-adopt PDMS/DVB extraction at the beginning,take 0.2g of magnolia bark powder and extraction done under conditions of temperature 70℃and for 60min achieves efficient extraction comparatively. Also on chromatographic column, warming procedures, velocity, diversion ratio separation etc, factors for the optimization conditions, Screened HP-50 capillary column (30 m*0.32 mm*0.25μm),temperature programming-initial temperature 50℃(held for 1min),increased by 4℃/min to 140℃(held for 2 min),increased 2℃/min to 180℃, increased 12℃/min to 280℃(held for 5 min),ate of flow 1.3 ml/min, split ratio of 15:1 for chromatographic condition is suitable for analysis of volatile organic compounds magnolia bark.
Secondly, In accordance with the above optimization conditions for batch analytical procedure of bark of Magnoliae officinalis of 15 different origin,altogether obtained 42 common GC peaks,thereby establishing fingerprint,through cluster analysis,those 15 herbs were categorized into 4 different classes,among which classes 1 and 2 are recommended products, choosing 10 Cortex Magnoliae officinalis of different origins,established common mode,furthermore the similarity of comparative illustrations of the calculation produced with the group of 15 herbs. Result indication-similarity even above 0.9, furthermore adopting GC-MS for determining volatile composition in Magnoliae officinalis bark is analyzed; commonly identified 15 constituents among which content ofα-Eucalyptus leaves oleyl alcohol andβ-Eucalyptus leaves oleyl alcohol was highest.
Thirdly, For major efficacy, phenol composition,comparative analysis process of 21 batch herbs of magnolia officinalis,it established that it has a common pattern between them,according to honokiol and honokiol investigated indication,use systematic cluster analysis process for identification research of the chemical pattern,22 batches of medicinal material categorized into 4 classes,among which 1 class is the recommended product sample,better reflection of the fact that different origins of magnolia officinalis have different phenolic composition,be it magnolol or honokiol,furthermore with GC method a fingerprint was established related to the composition of volatile compounds of magnolia officinalis and it's analyzed,researching GC fingerprint and it's analytical results and rather than determining relationship between effective analysis content difference,trials through GC and HPLC, two different chromatographic analytical methods can systematically evaluate quality of the herb.In reality allowing fingerprint to reflect the the inherent quality of Chinese medicine.
引文
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