厚朴、防己之成分與基原分析研究

蘇政宏(Su Cheng-hung)

指導教授 ： 許順吉

國立臺灣師範大學/理學院/化學系/碩士(2001年)

摘要

高效液相層析（HPLC）及毛細管電泳（CE）同屬液相分離技術，具有多種分離模式，為目前最常用於定量中藥材指標成分的分析方法；而近年來高效液相層析質譜儀(LC-MS)在中草藥的分析上逐漸扮演著重要的角色，本研究應用這三種儀器於厚朴、防己藥材的分析。LC-UV及CE-UV的偵測極限有一定的範圍，對藥材微量成分的分析，常有困難，本文開發毛細管電泳掃集法，成功地測定廣防己藥材中馬兜鈴酸的含量。
本研究分三個部分，第一部分為開發厚朴藥材的分析方法。厚朴為一芳香化濕藥，主要由酚類、精油及多種微量生物鹼組成。市售藥材主要有厚朴及和厚朴(Magnolia obovata THUNB.)兩種。其中厚朴又可分為川厚朴(M. officinalis REHD.et WILS.)及凹葉厚朴[M. officinalis ssp. Biloba(REHD.et WILS.)Law]兩種。本研究以七個生物鹼成份(1,(-)-magnocurarine，2,(+)-magnoflorine，3,(+)-laurifoline，4,(+)-oblongine，5, (+)-menisperine，6(+)-xanthoplanine，7(+)-N-methylglaucine)及兩個酚類成分(8,Honokiol，9,Magolol)，共九個化合物為指標，利用高效能液相層析(HPLC)開發厚朴藥材成分的分析方法。實驗結果顯示，厚朴藥材經酸鹼處理後，利用過氯酸鹽及氰甲烷水溶液為衝提液，在波長280mm偵測下，可在60分鐘內成功地分析出這九個成分。本研究進一步利用LC-MS來確認厚朴藥材中指標成分存在的真實性。
第二部分為運用該分析方法於市售厚朴藥材之基原辨識及炮製、安定性之研究。市售厚朴藥材28批，用HPLC分析七個生物鹼成份及兩個酚類成分之含量。研究顯示，以M/H之比值，可以初步區分正品、偽品之厚朴，再利用七個生物鹼成分之間含量比值的差異，加強化學辨識的真實性。炮製方面，醋炒及薑炒對各成分的影響最大，而且在時間上也較為經濟。安定性研究方面，結果顯示，加熱溫度越高，對藥材的保存越不利。
第三部分為開發防己藥材之HPLC分析方法。市售防己藥材主要有防己科的日防己、粉防己、木防己外，還有馬兜鈴科的廣防己，防己藥材具利水消腫、祛風止痛的功效。本研究利用醋酸銨、醋酸和氰甲烷混合液為沖提液，在60分鐘內分析出四種防己的十五個成分，該條件亦適用於LC-MS系統。此外，利用此分析方法對四種防己之基原加以區分。以馬兜鈴酸來區分馬兜鈴科的廣防己；以sinomenine來區分日防己；以tetrandrine來區分粉防己；以magnoflorine來區分木防己。
最後，本文運用兩種毛細管電泳技術來分析廣防己藥材中馬兜鈴酸的含量。在rev. EOF CZE方面，利用陽離子界面活性劑(CTAB)和有機修飾劑之緩衝溶液，可在15分鐘內分析兩種馬兜鈴酸；在Sweeping方面，以70 mM SDS之50 mM 磷酸溶液，及氰甲烷為緩衝溶液( 75：25,v/v )，在30分鐘內能完全分離出二成分峰，再現性良好，而且可大幅降低分析的偵測極限。

關鍵字

厚朴 ； 防己 ； 基原 ； 高效能液相層析 ； 毛細管電泳 ； 高效能液相層析質譜儀

並列摘要

Both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) belong to the liquid-chromatography technology. They possess a number of separation modes and are currently the methods most commonly used .in assaying the marker substances of Chinese herb drugs. In current years, liquid chromatograph-mass spectrograph (LC-MS) gradually plays an important role. In this study we use these three instruments in analyzing the herb drugs of the magnolia and fangchi items. LC-UV and CE-UV have limited detection scopes and it is usually difficult for them to analyze trace elements. This study has successfully used CE-Sweeping method in detecting the content of aristolochic acid in Aristolochiae Fangchi Radix. .
The present study includes three parts. The first part is on the development of analysis methods for analyzing Magnoliae Cortex that is an aromatic dehumidifying Chinese herb drug containing chiefly phenols, essential oil and trace amounts of alkaloids. Commercial samples include Magnoliae Cortex and Magnoliae Obovatae Cortex. The former is also divided into Suchuanese magnolia (Magnoliae Officinales Radix) and concave-leaf magnolia (Magnoliae Officinales Ssp. Bilobae Radix). The present study takes nine compounds as the drug’s marker substances, which include seven alkaloids (1. (-)-magnocurarine; 2. (+)-magnoflorine; 3. (+)-laurifoline; 4. (+)-oblongine; 5. (+)-menispermine; 6. (+)-xanthoplanine; 7. (+)-N-methylglaucine) and two phenols (8. honokiol; 9. magnolol). High-performance liquid chromatography is utilized in developing the methods for analyzing the constituents of Magnoliae Cortex. Experimental results show that after acid and alkaline processing, and using perchlorate salt and acetonitrile-water solution as an eluent, the nine constituents can be analyzed at 280 mm within 60 min. In this study, LC-MS is also used to further confirm the reality of existence of the marker substances in Magnoliae Cortex.
The second part uses this method for identifying the origin and for the study on the processing and stability of the herb drugs. Twenty-eight batches of the commercial articles of Magnoliae Cortex are assayed for the contents of seven alkaloids and two phenols. Results show that by means of the M/H ratio, we are able to discriminate the orthodox from the counterfeit samples. Then by comparing the differences of ratios between the contents of the seven alkaloids, the validity of chemical identification can be augmented. In terms of stability study, the results show that the higher temperature the herb is subjected to during processing, the worse its preservation quality will be.
The third part deals with the development of methods for analyzing the herbs of the fangchi item by HPLC. Commercial samples of the fangchi item chiefly include those derived from the Menispermaceous Sinomenium acutum, Stephania tetrandra and Cocculus trilobus, and those from the Artistolochiaceous Aristolochia fangchi and Aristolochia heterophylla. Herb drugs of the fangchi item possess diuretic, detumescent, anemofugic and analgesic effects. This study uses a mixture of ammonium acetate, acetic acid and acetonitrile as an eluent to analyze 15 constituents from four samples of fanchi within 60 minutes. The condition is also applicable to the LC-MS system. Besides, This method can also be used for identifying the four fangchi samples, using aristolochic acid to identify Aristolochiae Fangchi Radix, sinomenine to identify Sinomenii Acuti Radix, tetrandrine to identify Stephaniae Tetrandrae Radix and magnoflorine to identify Cocculi Triobi Radix.
Finally, this study uses two CE techniques in analyzing the contents of aristolochic acid in samples of Aristolochiae Fangchi Radix. In the regard of rev. EOF CZE, with the use of anionic surfactant (CTAB) and organic modifier as a buffer, two kinds of aristolochic acid can be analyzed within 15 min. In the aspect of the Sweeping method, using a buffer made of 70 mM SDS in 50 mM phosphoric acid and acetonitrile (75 : 25, v/v), we can have two constituent peaks eluted out within 30 min. with good reproducibility, thereby lowering the detection limits of analyses to a very great extent.

並列關鍵字

Magnoliae Cortex ； fangchi ； resource ； HPLC ； CE ； LC-MS