細辛之成分分析與基原辨識
Abstract
高效液相層析(HPLC)是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(high-performance liquid chromatography-mass spectrometer, LC-MS)在中草藥的分析上逐漸扮演著重要的角色。本研究旨在開發HPLC與LC-MS方法，分別找尋最適合條件，以分析細辛與丁香藥材之組成成分含量、推測未知成分分子結構以及測定細辛藥材中馬兜鈴酸的含量。同時，利用分析結果，建立細辛化學指紋圖譜辨識基原的方法；用應用感應耦合電漿質譜儀(inductively coupled plasma mass spectrometer, ICP-MS)分析細辛之微量元素含量，以探討其比值與品種之間的關係。
本研究分三個部分，首先開發細辛藥材的分析方法。細辛有發散風寒、溫肺化飲、鎮咳鎮痛的功能，以精油為主要組成成分。本研究以六個可購得標準品化合物(3,4,5-trimethoxytoluene、3,5-dimethoxytoluene、methyleugenol、myristicin、safrole、asarinin)及在細辛藥材中主要的另12個未知成分，共18個化合物為指標，利用高效能液相層析(HPLC)開發細辛藥材成分的分析方法。實驗結果顯示，利用醋酸鈉鹽及氰甲烷/甲醇/水為沖提液組成，在波長240nm偵測下，可在75分鐘內成功地分離這18個成分。本研究進一步利用LC-MS來推測細辛藥材中未知成分的分子結構。
運用該分析方法於市售細辛藥材之基原辨識研究。我們共收集24
件細辛樣品，經顯微鏡確認其中計有遼細辛(Asarum heterotropoides Fr. var mandshuricum (Maxim.) Kitag.)8件、漢城細辛(A. sieboldii Miq.var seoulense Nakai.)8件、杜衡(A. forbesii Maxim.)7件及華細辛(A. sieboldii Miq.)1件。結果發現，華細辛無化合物8(elemicin)、17( (2E,4E,8Z,10E)-N-isobutyl-2,4,8,10-dodecatetraenamide)、18 ( (2E,4E,8Z,10Z)-N-isobutyl-2,4,8,10-dodecatetraenamide)；遼細辛不含成分1(4-(1-methylethyl)benzaldehyde)、10(isoelemicin)、11(myristicin)，但為唯一含有成分4(2,3,5-trimethoxytoluene)；漢城細辛與杜衡兩品種中只有isoelemicin(10)和myristicin(11)有較大差異，8/10的比值：杜衡＜12，且漢城細辛＞90；9/11的比值：漢城細辛＜16，且杜衡＞24。
利用Sep-pak前處理配合LC-MS偵測方法，檢定細辛藥材中馬兜鈴酸含量。馬兜鈴酸為馬兜鈴科屬（Aristolochia）植物重要成分，已有許多報告指出，長期服用有造成腎衰竭的副作用。細辛樣品經前處理，注入以醋酸與氰甲烷組成緩衝溶液的LC-MS系統中，可在20分鐘內分析兩種馬兜鈴酸，再現性良好，而且可大幅降低分析的偵測極限。
我們另用ICP-MS測定細辛不同品種間的水溶性微量元素含量
比例，發現植物因彼此蓄積效果不同，各品種間確實存在不同含量的特徵元素。遼細辛的無機元素總含量為最低，漢城細辛與杜衡之含量則較接近，較明顯有差異在於杜衡的Na、Al、V、Co、Pb含量均大於漢城細辛，漢城細辛中Fe、Mn、Br、Sr、Ga含量則大於杜衡，而可能與冠心病有關的Zn/Cu值，在三種細辛中大小為杜衡>漢城細辛>遼細辛。
最後還開發丁香藥材的分析方法。丁香有驅風止痛、健胃整腸、抗菌、鎮痛等功能，含有精油、有機酸、脂肪酸、黃酮類等成分。本研究以七個可購得標準品化合物(gallic acid、ellagic acid、myricetin、quernetin、kaemnetin、eugenol、acetyl eugenol)及在丁香藥材中主要的另3個未知成分，共10個化合物為指標，利用高效能液相層析(HPLC)開發細辛藥材成分的分析方法。實驗結果顯示，醋酸及氰甲烷/水為沖提液組成，在波長254mm偵測下，可在65分鐘內成功地分析出這10個成分。本研究進一步利用LC-MS來推測丁香藥材中未知成分的分子結構。
At present, high-performance liquid chromatography is the most commonly used quantitative method for analyzing the marker substances of Chinese herb drug materials. Currently high-performance liquid chromatography-mass spectrometer (LC-MS) has gradually played an important role in the analysis of Chinese herb drugs. This study aims at the development of HPLC and LC-MS methods by finding the most optimal conditions for analyzing the constituents of asarum (Asari Herba) and clove (Caryophylli Flos) and for postulating the molecular structures of unknown constituents and determining the aristolochic acid content in asarum. Meantime, the analysis date are used to establish the rules for identifying the botanical sources of the herbs. By the inductively coupled plasma mass spectrometer (ICP-MS), we determine the contents of trace elements whereby we try to investigate the relationship between the content ratios of trace elements and the species of the herbs containing them. This study is divided into three parts. In the first part, we develop the analysis method for asarum which possesses the functions of dispersing wind and cold evils, warming lungs to resolve stagnant water, arresting cough and killing pains, and are attributed to the herb’s essential oil as the main constituent.With 6 purchasable compounds, namely 3,4,5-trimethoxytoluene, 3,5-dimethoxy-toluene, methyleugenol, myristicin, safrole and asarinin, and 12 unknown components from asarum, as the marker substances, high-performance liquid chromatographic (HPLC)method was developed. The experiment results showed that using sodium acetate and acetonitrile/methanol/water as an eluent, the method successfully separated the 18 compounds within 75 min, detected under a wavelength of 240 nm. This study made further efforts to determine the structures of the unknown constituents by LC-MS. For study on source identification for commercial asarum articles by the above mentioned method, we collected 24 samples of asarum articles, which under microscopic examination were identified to include 8 samples belonging to the species of Manchurian asarum (Asarum heterotropoides Fr. var mandshuricum (Maxim.) Kitag.); 8 samples belonging to Seoul asarum (A. sieboldii Miq. var seoulense Nakai); 7 samples belonging to Tu-heng (A. forbesii Maxim.); and 1 sample belonging to Chinese asarum (A. sieboldii Miq.). Test results showed that Manchurian asarum did not contain compounds 8 (elemicin), 17 (2E,4E,8Z,10E)- N-isobutyl-2,4,8,10-dodecatetraenamide), 18 ((2E,4E,8Z,10Z)-N-isobutyle-2,4,8,(myristicin), but was the only sample that contained compound (2,3,5-trimethoxytoluene). The Seoul asarum differed from Tu-heng with respect to their contents of isoelemicin () and myristicin (). The 10-dodecatetraenamide), 10 (isoelemicin) and 11410118/10 was<12 for Tu-heng; >90 for Seoul asarum; and the ratio 9/11 was <16 for Chinese asarum and >24 for Tu-heng. Pretreatment with Sep-pak along with detection by LC-MS was used for determining the asarum articles for their contents of aristolochic acid, which is an important constituent of plants of the Aristolochia genus. Many reports have pointed out that long-term use of herbs containing this compound may lead to the side effect of renal failure. After pretreatment, the asarum samples were injectedinto the LC-MS system with acetic acid and acetonitrile as a buffer, the aristolochic acid content can be analyzed within 20 minutes with good reproducibility and high sensitivity. In addition, we also used ICP-MS in determining the content ratios of water-soluble trace elements in different species of asarum. As a result, we found different species of the herb did contain characteristic elements in different contents. The content of total inorganic elements was lowest in Manchurian asarum, but closely similar in Seoul asarum and Tu-heng. The more noticeable difference lied in the contents of Na, Al, V. Co and Pb in Tu-heng, which were larger than those in Seoul asarum; whereas the Fe, Mn, Br, Sr and Ga contents in Seoul asarum were higher than those in Tu-heng. While the Zn/Cu value which is pertinent to coronary disease appeared in the three species of asarum in the order: Tu-heng > Seoul asarum > Manchurian asarum. Finally, we developed the analysis method for clove. which possesses carminative, analgesic, stomachic, antimicrobial and anodyne effects. This herb contains essential oil, organic acids, fatty acids and flavonoids. This study used 10 compounds including seven purchasable compounds (gallic acid, ellagic acid, myricetin, querntin, kaemnetin, eugenol, acetyl eugenol) and three unknown compounds from clove to work as marker substances and by means of HPLC to develop analysis methods for asarum drug materials. The experiment results showed that under the condition of acetic acid and acetonitrile/water as an eluent and a detection wavelength at 254 nm, the ten compounds could be analyzed successfully within 65 minutes. This study further attempted to elucidate the structures of the unknown constituents in clove drug materials by LC-MS.
At present, high-performance liquid chromatography is the most commonly used quantitative method for analyzing the marker substances of Chinese herb drug materials. Currently high-performance liquid chromatography-mass spectrometer (LC-MS) has gradually played an important role in the analysis of Chinese herb drugs. This study aims at the development of HPLC and LC-MS methods by finding the most optimal conditions for analyzing the constituents of asarum (Asari Herba) and clove (Caryophylli Flos) and for postulating the molecular structures of unknown constituents and determining the aristolochic acid content in asarum. Meantime, the analysis date are used to establish the rules for identifying the botanical sources of the herbs. By the inductively coupled plasma mass spectrometer (ICP-MS), we determine the contents of trace elements whereby we try to investigate the relationship between the content ratios of trace elements and the species of the herbs containing them. This study is divided into three parts. In the first part, we develop the analysis method for asarum which possesses the functions of dispersing wind and cold evils, warming lungs to resolve stagnant water, arresting cough and killing pains, and are attributed to the herb’s essential oil as the main constituent.With 6 purchasable compounds, namely 3,4,5-trimethoxytoluene, 3,5-dimethoxy-toluene, methyleugenol, myristicin, safrole and asarinin, and 12 unknown components from asarum, as the marker substances, high-performance liquid chromatographic (HPLC)method was developed. The experiment results showed that using sodium acetate and acetonitrile/methanol/water as an eluent, the method successfully separated the 18 compounds within 75 min, detected under a wavelength of 240 nm. This study made further efforts to determine the structures of the unknown constituents by LC-MS. For study on source identification for commercial asarum articles by the above mentioned method, we collected 24 samples of asarum articles, which under microscopic examination were identified to include 8 samples belonging to the species of Manchurian asarum (Asarum heterotropoides Fr. var mandshuricum (Maxim.) Kitag.); 8 samples belonging to Seoul asarum (A. sieboldii Miq. var seoulense Nakai); 7 samples belonging to Tu-heng (A. forbesii Maxim.); and 1 sample belonging to Chinese asarum (A. sieboldii Miq.). Test results showed that Manchurian asarum did not contain compounds 8 (elemicin), 17 (2E,4E,8Z,10E)- N-isobutyl-2,4,8,10-dodecatetraenamide), 18 ((2E,4E,8Z,10Z)-N-isobutyle-2,4,8,(myristicin), but was the only sample that contained compound (2,3,5-trimethoxytoluene). The Seoul asarum differed from Tu-heng with respect to their contents of isoelemicin () and myristicin (). The 10-dodecatetraenamide), 10 (isoelemicin) and 11410118/10 was<12 for Tu-heng; >90 for Seoul asarum; and the ratio 9/11 was <16 for Chinese asarum and >24 for Tu-heng. Pretreatment with Sep-pak along with detection by LC-MS was used for determining the asarum articles for their contents of aristolochic acid, which is an important constituent of plants of the Aristolochia genus. Many reports have pointed out that long-term use of herbs containing this compound may lead to the side effect of renal failure. After pretreatment, the asarum samples were injectedinto the LC-MS system with acetic acid and acetonitrile as a buffer, the aristolochic acid content can be analyzed within 20 minutes with good reproducibility and high sensitivity. In addition, we also used ICP-MS in determining the content ratios of water-soluble trace elements in different species of asarum. As a result, we found different species of the herb did contain characteristic elements in different contents. The content of total inorganic elements was lowest in Manchurian asarum, but closely similar in Seoul asarum and Tu-heng. The more noticeable difference lied in the contents of Na, Al, V. Co and Pb in Tu-heng, which were larger than those in Seoul asarum; whereas the Fe, Mn, Br, Sr and Ga contents in Seoul asarum were higher than those in Tu-heng. While the Zn/Cu value which is pertinent to coronary disease appeared in the three species of asarum in the order: Tu-heng > Seoul asarum > Manchurian asarum. Finally, we developed the analysis method for clove. which possesses carminative, analgesic, stomachic, antimicrobial and anodyne effects. This herb contains essential oil, organic acids, fatty acids and flavonoids. This study used 10 compounds including seven purchasable compounds (gallic acid, ellagic acid, myricetin, querntin, kaemnetin, eugenol, acetyl eugenol) and three unknown compounds from clove to work as marker substances and by means of HPLC to develop analysis methods for asarum drug materials. The experiment results showed that under the condition of acetic acid and acetonitrile/water as an eluent and a detection wavelength at 254 nm, the ten compounds could be analyzed successfully within 65 minutes. This study further attempted to elucidate the structures of the unknown constituents in clove drug materials by LC-MS.
Description
Keywords
細辛, 基原辨識