中草藥品質管制 鍾娟碧

中草藥之標準化 中草藥品質控管所面對的問題 中草藥化學成分分析方法建立 中草藥潛在的污染源 中草藥標準化之參考規範 2019/1/2

中草藥品質控管所面對的問題 中草藥由許多的化學成分所組成 活性成分很難被真正的確認 有效的分析方法可能尚未開發 對照標準品可能尚未商業化及標準化 植物本身的化學差異 自然環境對化學成分的影響 品種差異及產地的影響 採收、乾燥及儲存條件的影響 2019/1/2

影響中草藥品質的因素 採收季節、產地、炮製： 北方藥農--------春採茵陳夏採蒿 知母黃芩全年刨 九月中旬摘菊花 十月上山採連翹 南方夏季早------正月茵陳二月蒿 三月蒿子當柴燒 2019/1/2

中草藥化學成分分析方法建立 化學指紋圖譜 (Chemical Fingerprint) 有機化合物 GC GC/MS 水抽提物 醇抽提物 TLC HPLC LC/MS 無機元素 總重金屬(比色法) AA ICP-MS 2019/1/2

中草藥化學成分分析方法建立 指標成分含量測定 (Chemical Assay) 一般中草藥指標成分選擇原則  市售標準品  符合原藥材功效成分(ex. 大黃-瀉下藥, 指標成分-Sennosides)  具其他生理活性成分  特異性成分(ex. 甘草-Glycyrrhizic acid)  主要含量成分(ex. 桔梗, 指標成分-Inulin)  熱/光安定性(安定性試驗考量)  UV/Vis吸收成分(依不同實驗室設備而有不同需求) 2019/1/2

中草藥潛在的污染源 他種植物夾雜 草麻黃中混有中麻黃 微生物 黴菌、病原菌(大腸桿菌、沙門式桿菌、金黃葡萄球菌) 微生物衍生毒素 黃麴毒素(Aflatoxin) 農藥 有機氯農藥(BHC、DDT) 、有機磷農藥 煙燻試藥 硫磺 金屬 鎘、汞、銅、鉛 2019/1/2

麻黃素(ephedrine, E) ：興奮交感神經及中樞神經作用。 草麻黃 Ephedra sinica (E  PE) (E  PE) 中麻黃 Ephedra intermedia 麻黃素(ephedrine, E) ：興奮交感神經及中樞神經作用。 偽麻黃素(pseudoephedrine, PE) ：抑制交感神經及中樞神經作用。 2019/1/2 8 8

中草藥潛在的污染源 他種植物夾雜 草麻黃中混有中麻黃 微生物 黴菌、病原菌(大腸桿菌、沙門式桿菌、金黃葡萄球菌) 微生物衍生毒素 黃麴毒素(Aflatoxin) 農藥 有機氯農藥(BHC、DDT) 、有機磷農藥 煙燻試藥 硫磺 重金屬 鎘、汞、銅、鉛 2019/1/2

中草藥的標準化之參考規範 WHO: World Health Organization(世界衛生組織) ABC: The American Botanical Council (美國植物委員會 ) AHP: The American Herbal Pharmacopoeia (美國草藥藥典) ESCOP: The European Scientific Cooperative on Phytotherapy (歐洲植物療法科學合作組織) USP: The United States Pharmacopoeia (美國藥典) JP: The Japan Pharmacopoeia(日本藥典) 中華人民共和國藥典 台灣藥典 2019/1/2

中草藥品質管制 正確性 (Authenticity) 安全性 (Safety) 有效性 (Efficacy) 均一性 (Consistency) 安定性 (Stability) 2019/1/2

正確性－藥材基原鑑定 顯微鏡檢 鑑定藥材基原 2019/1/2

化學指紋圖譜 HPLC-DAD Scutellariae Radix (黃芩) HPLC-MSD BG 280 nm WG B OG W O 2019/1/2

正確性－化學指紋圖譜 Marker substance of Herbs Herbal Product (IS) 正確性－化學指紋圖譜 (IS) Herbal Product (IS) (IS) Radar fingerprint for product consistency 2019/1/2

安全性 微生物 黃麴毒素 農藥殘留 煙燻試藥－硫磺 重金屬 馬兜鈴酸 Next, the Discriminative Use of Drug Materials. Viewed by the external appearance, the ephedra drug materials can be differentiated by the number of stipules into ephedra intermedia and Ephedra sinica of which the former has three stipules and the latter, 2 stipules. However, because most commercial articles contain only the internodal portions and the nodes have been removed, it is difficult to identify by external appearance. It is then necessary to resort to internal histological identification. 2019/1/2

微生物檢驗 Microbiological Analysis 需氧微生物總數 / Total Aerobic Microbial Count 黴菌與酵母菌 / Yeast & Mold 大腸桿菌 / Escherichia coli 沙門氏桿菌 / Salmonella 大腸桿菌群 / Coliform 金黃色葡萄球菌 / Staphylococcus aureus 綠膿桿菌 / Pseudomonas aeruginosa Sun Ten is equipped with 26 HPLC with various detectors to analyze the following inspections: 1. Diode array detector: batch-to-batch biological marker 2. Inductively coupled plasma mass spectrometry: heavy metal elements 3. Fluorescent detector: aflatoxin 4. Liquid chromatography tandem mass spectrometry: aristolochic acid 5. Gas chromatography coupled mass spectroscopy: pesticide residue In-house Microbiology Laboratory 2019/1/2

黃麴毒素檢測 Aflatoxin Analysis 高效能液相層析/螢光偵測 (LC-FD) HPLC Coupled Fluorescent Detector (FD) 美國FDA規定人類食品的黃麴毒素限量為20 ppb 台灣衛生署針對較可能被污染黃麴毒素之中藥材訂定限量為15 ppb 台灣衛生署公告必須檢驗黃麴毒素之中藥材: HPLC Coupled Fluorescent Detector (FD) Sun Ten is equipped with 26 HPLC with various detectors to analyze the following inspections: 1. Diode array detector: batch-to-batch biological marker 2. Inductively coupled plasma mass spectrometry: heavy metal elements 3. Fluorescent detector: aflatoxin 4. Liquid chromatography tandem mass spectrometry: aristolochic acid 5. Gas chromatography coupled mass spectroscopy: pesticide residue 黃耆, 薏苡仁 , 延胡索 , 蓮子 , 紅棗 , 大腹皮, 女貞子 , 小茴香, 山茱萸 ,山楂, 枸杞子. 2019/1/2

藥材中殘留農藥的主要來源及種類 有機氯農藥 有機磷農藥 生長期使用 氨基甲酸鹽類 農藥 其他 貯存時使用 有機氯: BHC, DDT…. 生長期使用的農藥大多屬於殺蟲劑或除草劑，貯放時使用的農藥則是為了延長藥材保存期限為主要目的 針對某些藥材而言，通常含有適量的重金屬含量 有機氯: BHC, DDT…. 有機磷: 達馬松,巴拉松.. 氨基甲酸鹽: 得滅克,加保利… 其它 2019/1/2

農藥殘留檢驗 氣相層析質譜儀(GC-MS) 靈敏度高 定性準確 多殘留同時檢測 現有GC-MS 1台。與傳統GC的價差數倍以上。 曾有台灣某廠所製人參粉末輸出至香港，被檢出農藥殘留超量，以致全批銷毀。 2019/1/2 19

藥材中重金屬的主要來源及種類 道地藥材(天然) 土壤 土壤污染 重金屬來源 切削器械 加工 粉碎研磨設備 生長期使用的農藥大多屬於殺蟲劑或除草劑，貯放時使用的農藥則是為了延長藥材保存期限為主要目的 針對某些藥材而言，通常含有適量的重金屬含量 鉛(Pb) 、汞(Hg) 、鎘(Cd) 、 砷(As) 、銅(Cu) 2019/1/2

重金屬檢驗 離子偶合電漿質譜儀(ICP/MS) 多元素同步檢測 具有分析大量樣品的能力 靈敏度高 為原子分析光譜儀的數十倍以上 而傳統使用AA之分析能力無法滿足現有生產需求。 2019/1/2 21

安全性－馬兜鈴酸分析 1.長期服用易造成腎衰竭副作用 2.馬兜鈴科藥材多數含有馬兜鈴酸 例如廣防己、關木通、靑木香、天仙藤及馬兜鈴等 3.含防己、木通、木香等藥材處方需檢測馬兜鈴酸成分 2019/1/2

馬兜鈴酸LC/UV and LC/MS分析 (390 nm) 抽提液樣品的HPLC-UV 圖 m/z 359 m/z 324 m/z 298 AA-I (390 nm) AA-I AA-II 抽提液樣品的HPLC-UV 圖 圖 2. AA-I的選定離子圖 (m/z 359, m/z 324, m/z 298 and m/z 296) 2019/1/2

馬兜鈴酸檢驗極限比較 紫外光譜液相層析儀(LC-UV) 液相層析質譜儀(LC-MS) 二次液相層析質譜儀(LC-MS-MS) 1～2 ppm 生技LC-MS-MS 1台， 必安 LC-MS 1 台。 檢驗能力是法規規定的10倍以上。 馬兜鈴酸1～2ppm長期服用即有腎臟衰的風險。龍膽瀉肝湯即為實例。 2019/1/2 24

Clematis montana Buch.-Ham. <植物外觀明顯不同> 白木通 Akebia trifoliata (Thunb.) Koidz. var. australis (Diels) Rehd. 關木通 Aristolochia manshuriensis Kom. 五葉木通 Akebia quinata Decne. 小木通 Clematis armandii Franch. 繡球藤 Clematis montana Buch.-Ham. 2019/1/2

木 通 <正品> 關木通類 馬兜鈴科 Aristolochiaceae 關木通 Aristolochia manshuriensis Kom. 川木通類 毛茛科 Ranunculaceae 繡球藤 Clematis montana Buch.-Ham. 小木通 Clematis armandii Franch. The careful selection of drug materials. We know the Chinese herb fangchi or stephania is of many varieties, including Aristolochia fangchi and stephania tetrandra as the two main sources. The Aristochia fanchi variety contains aristochic acid which is a compound capable of antitumor, immuno-stimulation --- and so on. However, although there are reports in literature that it may cause renal failure, many Chinese herbal physicans have been using Aristochia fanchi safely all the time. Hence, we believe that the herb is not totally unusable. It is just a matter of dosage whereof we must use the herb carefully. 關木通 Aristolochia manshuriensis Kom. 川木通 Clematis montana Buch.-Ham. 2019/1/2

防 己 <正品> 廣防己 Aristolochia fangchi Wu 防 己 <正品> 廣防己 Aristolochia fangchi Wu 粉防己 Stephania tetrandra S. Moore. 日防己 Sinomenium acutumn Rehd. et Wils. <代用品及偽品> 鐵牛入石 Cocculus sarmentosus Diels 玉栚葛藤 Stephania cephalantha Hayata 小果微花藤 Iodes ovalis Bl. var. vitiginea (Hance) Gagnep. 瘤枝微花藤 Iodes seguinii (Hevl.) Rehder The careful selection of drug materials. We know the Chinese herb fangchi or stephania is of many varieties, including Aristolochia fangchi and stephania tetrandra as the two main sources. The Aristochia fanchi variety contains aristochic acid which is a compound capable of antitumor, immuno-stimulation --- and so on. However, although there are reports in literature that it may cause renal failure, many Chinese herbal physicans have been using Aristochia fanchi safely all the time. Hence, we believe that the herb is not totally unusable. It is just a matter of dosage whereof we must use the herb carefully. 2019/1/2

<植物外觀明顯不同> 粉防己 (防己科) Stephania tetrandra S. Moore. 漢中防己(馬兜鈴科) Aristolochia heterophylla Hemsl. 小果微花藤 (茶茱萸科) Iodes ovalis Bl. var. vitiginea (Hance) Gagnep. 廣防己(馬兜鈴科) Aristolochia fangchi Wu 2019/1/2

Stephania tetrandra S. Moore. <藥用部位不易辨別> 小果微花藤 Iodes ovalis Bl. var. vitiginea (Hance) Gagnep. 瘤枝微花藤 Iodes seguinii (Hevl.) Rehder 粉防己 (防己科) Stephania tetrandra S. Moore. 2019/1/2

組織切片 (Histological slides) HPLC chromatogram 組織切片 (Histological slides) Aristolochia fangchi 廣防己 Bogus article 小果微花藤 Stephania tetrandra 粉防己 Aristolochia Fangchi 廣防己 Stephania Tetrandra 粉防己 2019/1/2

馬兜鈴科藥材 HPLC/UV層析圖譜 廣防己 關木通 馬兜鈴 天仙藤 Aristolochic acid I 靑木香 AA-I AA-I 2019/1/2

馬兜鈴酸 Aristolochic Acid Guan Fanji vs Fen Fanji 廣防己 (馬兜鈴科) / Aristolochia fangchi Wu (Aristolochiaceae) 馬兜鈴酸 (AA) 粉防己(防己科) / Stephania tetrandra S. Moore (Menispermaceae) Sun Ten is equipped with 26 HPLC with various detectors to analyze the following inspections: 1. Diode array detector: batch-to-batch biological marker 2. Inductively coupled plasma mass spectrometry: heavy metal elements 3. Fluorescent detector: aflatoxin 4. Liquid chromatography tandem mass spectrometry: aristolochic acid 5. Gas chromatography coupled mass spectroscopy: pesticide residue 2019/1/2

馬兜鈴酸 Aristolochic Acid Guan Mu Tong vs Mu Tong 關木通 (馬兜鈴科 ) / Aristolochia manshuriensis (Aristolochiaceae) 木通 (木通科 ) / Akebia quinata (Lardizabalaceae) Sun Ten is equipped with 26 HPLC with various detectors to analyze the following inspections: 1. Diode array detector: batch-to-batch biological marker 2. Inductively coupled plasma mass spectrometry: heavy metal elements 3. Fluorescent detector: aflatoxin 4. Liquid chromatography tandem mass spectrometry: aristolochic acid 5. Gas chromatography coupled mass spectroscopy: pesticide residue 2019/1/2

有效性 藥理藥效評估 Next, the Discriminative Use of Drug Materials. Viewed by the external appearance, the ephedra drug materials can be differentiated by the number of stipules into ephedra intermedia and Ephedra sinica of which the former has three stipules and the latter, 2 stipules. However, because most commercial articles contain only the internodal portions and the nodes have been removed, it is difficult to identify by external appearance. It is then necessary to resort to internal histological identification. 免疫研究 心血管研究 2019/1/2

均一性 ST Specification (規格值): more than 25 mg/g Ref: JP XIV Next, the Discriminative Use of Drug Materials. Viewed by the external appearance, the ephedra drug materials can be differentiated by the number of stipules into ephedra intermedia and Ephedra sinica of which the former has three stipules and the latter, 2 stipules. However, because most commercial articles contain only the internodal portions and the nodes have been removed, it is difficult to identify by external appearance. It is then necessary to resort to internal histological identification. 2019/1/2

指標含量分析 高效液相層析儀 目前最廣泛使用的定性、定量設備 定性--(化學指紋圖譜建立及比對) 定量--(藥效成分或指標成分含量) 總計有24台全自動高效液相層析儀，為品保最大項目的投資。 2019/1/2 36

葛根湯 HPLC 層析圖 2019/1/2

三批葛根湯統計圖 (原料嚴格控制) Next, the Discriminative Use of Drug Materials. Viewed by the external appearance, the ephedra drug materials can be differentiated by the number of stipules into ephedra intermedia and Ephedra sinica of which the former has three stipules and the latter, 2 stipules. However, because most commercial articles contain only the internodal portions and the nodes have been removed, it is difficult to identify by external appearance. It is then necessary to resort to internal histological identification. 2019/1/2

四批葛根湯統計圖 (原料不嚴格控制) Next, the Discriminative Use of Drug Materials. Viewed by the external appearance, the ephedra drug materials can be differentiated by the number of stipules into ephedra intermedia and Ephedra sinica of which the former has three stipules and the latter, 2 stipules. However, because most commercial articles contain only the internodal portions and the nodes have been removed, it is difficult to identify by external appearance. It is then necessary to resort to internal histological identification. 2019/1/2

安定性試驗 ICH Guideline (New) (2~8ºC, 25ºC/60%RH, 30ºC/65%RH, 40ºC/75%RH) 長期試驗及加速試驗 長期試驗時程為產品預期有效期限再加一年 加速試驗時程為六個月 外觀性狀/五大項/HPLC指紋圖譜/指標成分含量 規格制定要求 安定性試驗報告 指標成分標準品安定性 2019/1/2

成品安定性試驗規劃 範例 1. 風險評估 產品內容(賦型劑比例), 產品包裝 1. 風險評估 產品內容(賦型劑比例), 產品包裝 2. 執行品項 不同劑型, 不同包裝, 不同批次, 安慰劑(Placebo) 3. 品管項目 Full items as T0 or Partially items 4. 規格制定 Appearance, Water content, Marker Content, Fingerprint 範例 ICH Guidelines 3 months / 1st year 6 months / 2nd year T0 T1 T2 T3 T6 T9 T12 T18 T24 T36 12 months / 3rd year 40ºC/75% RH ● ● ● ● ● 加速試驗 30ºC/65% RH ● ● ● ● ● 長期試驗 25ºC/60% RH ● ● ● ● ● ● ● ● ● ● 2~8ºC ● ● ● ● ● ● ● ● ● ● 2019/1/2

藥材品質控管 藥材來源證明 裁切規格 藥材包裝/儲存要求 採樣標準作業 藥典依據 基原鑑定(外觀性狀/顯微鏡檢) 外物摻雜(Foreign Organic Matter) 一般性理化檢驗 (乾燥減重、水抽提物、稀醇抽提物、總灰份、酸不溶性灰份) 化學指紋鑑定 (薄層液相層析TLC、高效液相層析HPLC) 指標成分含量測定 農藥殘留/重金屬檢測 馬兜鈴酸(Aristolochic Acid) 黃麴毒素 微生物 2019/1/2

甘 草 [藥材來源] 1. 基原：Glycyrrhiza uralensis Fisher 2. 典藉記載的產地：新疆、內蒙古、甘肅、陜西、 甘 草 [藥材來源] 1. 基原：Glycyrrhiza uralensis Fisher 2. 典藉記載的產地：新疆、內蒙古、甘肅、陜西、 山西、遼寧、吉林、黑龍江、 河北、青海、寧夏。 3. 購買地點：內蒙古。（A） 4. 代用品或偽品：土甘草、苦甘草(苦豆子)、黃甘草。 5. 裁切大小：長度0.5 cm。 A 級：產地農民供應(產地明確) B 級：自屬專員與產地二道販結合(產地明確) C 級：自屬專員定位進行大區域採購 (產地為2~3省份) D 級：一般大型市場購入(產地較難掌控) 2019/1/2

【組織切片)】 【 (一般檢驗項目規格) 】 Article (品名) Item (項目) Licorice(甘草) (MH117) Specification (規格) Reference (出處) 1 Loss on drying 乾燥減重 （％） 11.34 Less than 12.0% JP (日局方) 2 Water extractives 水抽提物 （％） 25.49 Less than 20.0% ST Factory specified (廠規) 3 Dilute alcohol extractives 稀醇抽提物 （％） 35.15 More than 25.0 % 4 Total ash 總灰分 （％） 3.99 Less than 7.0% 5 Acid-insoluble acid酸不溶灰分 （％） 0.51 Less than 2.0% 2019/1/2

【Marker substance and Quantitative Specifications (指標成分及定量值規格)】 【TLC】 【HPLC】 【Marker substance and Quantitative Specifications (指標成分及定量值規格)】 指標成分: glycyrrhizin, liquiritin 日本藥典：glycyrrhizin, not less than 25 mg/g (順天廠規)：1. HPLC指紋圖譜相似. 2. Glycyrrhizin, Not less than 25 mg/g I would like to introduce the “Manufacturing Technology of Chinese Herb Products“. It embodies three parts: Scrupulous selection of drug materials, Strict production control and Analyses of herb preparations. Rf：0.3, Redbrownish spot (紅褐色斑點) 2019/1/2

成品品質控管 採樣標準作業 藥典依據 外觀性狀 一般性理化檢驗 (乾燥減重、水抽提物、稀醇抽提物、總灰份、酸不溶性灰份) 粒度試驗 密度試驗 重容量差異 化學指紋鑑定 (薄層液相層析TLC、高效液相層析HPLC) 指標成分含量測定 農藥殘留/重金屬檢測 馬兜鈴酸(Aristolochic Acid) 微生物 2019/1/2

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THANK YOU VERY MUCH! 藥用牡丹 Mudan for herbs (安徽亳州, Bozhou, Anhui) (2009.4.14) 2019/1/2