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題名 | 以免疫親和管純化及液相層析法檢測中藥材內赭麴毒素A=Determination of Ochratoxin A in Chinese Medicines by Immunoaffinity Column Clean-up and High-Performance Liquid Chromatography |
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作者 | 鄧正賢; | 書刊名 | 中醫藥年報 |
卷期 | 25:4 2007.10[民96.10] |
頁次 | 頁275-306 |
專輯 | 中醫藥品質管理相關研究 |
分類號 | 414.33 |
關鍵詞 | 赭麯毒素A; 免疫親和; 液相層析; Ochratoxin A; Immunoaffinity column; HPLC; |
語文 | 中文(Chinese) |
中文摘要 | 赭麯毒素A(Ochratoxin A)是由麯黴菌和青黴菌所產生的,常分佈汙染了糧食和動物飼料,是一種很強的腎毒性致癌物質並具有免疫特性。在許多國家中赭麯毒素A是引起腎病的原因之一。馬兜鈴酸是一種很強的腎毒性致癌物質已毫無疑慮並已遭禁用,然而在臺灣所發生類似的中草藥腎病卻常無法檢驗出馬兜鈴酸。赭麯毒素引發腎衰竭,卻被誤認為馬兜鈴酸引起,最有名的例子就是發生在巴爾幹半島的流行性腎病,其腎臟病裡特徵與中草藥腎病非常類似,然而後來有足夠的證據證實馬兜鈴酸非兇手而是食物發霉中的赭麯毒素引起的。寶路乾狗糧引起狗隻急性腎衰竭而死亡令飼主驚恐不安,已被證實是位在泰國儲存槽中的玉米發霉而產生的赭麯毒素所引起的。然而在臺灣,有太多的腎衰竭病患不知其病因為何,故對腎毒性物質的調查是必須的,了解致病原因且加以避免,才是保障臺灣民眾健康的最佳方法。故本計畫之目的在於建立中藥材內赭麯毒素A的高效液相色譜檢測方法,並瞭解臺灣地區中藥材內赭麯毒素A的汙染狀況,檢測樣品包括:(1)果實類:枸杞、大棗、酸棗仁、山楂、五味子;(2)麴類:神麴、淡豆豉、綠豆癀;(3)延胡索及橘皮(陳皮);(4)其他易發霉中藥材:人參、牛膝、芍藥、當歸、葛根、地黃、白术、澤瀉、甘草。 本計畫以免疫層析管純化樣品及高效液相層析儀(附螢光檢出器)檢測指標成分赭麴毒素A,其分析方法已建立。分析方法流程為取5g樣品粉碎加入20 mL溶媒(甲醇:1% NaHCO₃),超音波震盪1分鐘,濾紙過濾,入免疫親和力分離管(OchraTest TM affinity column),以10 mL PBS及水洗之,再以1.5 mL甲醇沖提出指標成分。高效液相層析儀分析赭麴毒素指標成分時,移動相為CH₃CN/H₂O/CH₃COOH (45:54:1, v/v),流速為1.0 mL/min,檢測營光波長為333nm、477nm。 利用上述分析技術進行對照標準品赭麴毒素A分析,赭麴毒素A在濃度1-50 ng/mL,得線性迴歸方程式(Y=mX+b)及相關係數(r)為Y=0.5508-0.64834X (r=0.9999),顯示良好線性關係。赭麴毒素A同日內及異日間相對標準偏差0.37-2.23%及1.82-2.55%,顯示再現性可以接受。單位藥材添加回收率為83.5-105.6%,顯示準確性均可以接受。以三倍於指標成分和雜訊之波峰高度比的最小濃度視為儀器的偵測極限,赭麴毒素A的偵測極限為0.01 ng/mL。目前已完成在臺灣地區北、中、南各地市售檢品,包括枸杞(16/47)、大棗(8/41)、酸棗仁(0/13)、山楂(0/15)、五味子(2/15)、神麴(21/25)、淡豆豉(5/13)、綠豆癀(0/10)、延胡索(0/15)、陳皮(1/20)、人參(0/15)、牛膝(5/16)、芍藥(0/0)、當歸(3/16)、葛根(0/15)、地黃(3/16)、白术(2/16)、澤瀉(1/15)、甘草(2/16)、黃耆(1/16)等檢品,含量分別依序0.29-19.8、0.38-16.7、0、0、0.34-7.34、0.15-45.2、0.35-15.8、0、0、0.05`0、0.08-4.3、0、0.28-23.65、0、3.23-17.8、0.45-5.88、0.22-3.66、0.32-5.65、0.16-8.55 µg/kg。 故建議赭麴毒素A受檢藥材為枸杞、大棗、五味子、神麴、淡豆豉、牛膝、當歸、地黃、白术、澤瀉、甘草、黃耆等,並暫時建議規範其限量標準為10 µg/kg,然仍須進一步多點採樣並重複篩選確定。 |
英文摘要 | Ochratoxin A (OTA) is produced by several species of the fungal genera Aspergillus and Penicillium. These fungi are ubiquitous and the poetential for the contamination of food stuffs and animal feed is widespread. OTA has both antibiotic and toxic properties, the most important of which are its nephrotoxic, carcinogenic, and immunotoxic properties. It has been the cause of a nephropathy in many countries. Similarities between Chinese herbs nephropathy and Balkan endemic nephropathy have been described, including the association with urothelial carcinoma. Some evidence suggests that Balkan endemic nephropathy is an environmentally induced disease, related to exposure to fungal such as OTA. Some CHN patients failed to demonstrate the presence of aristolochic acid in the capsules taken by the patients in Taiwan. A correlation between certain questionable batches of Pedigree dog food manufactured in Thailand and numerous cases of kidney failure among dogs in Taiwan had been indirectly confirmed. Further investigations indicated that OTA which formed by Aspergillus ochraceus were detected in Pedigree dry dog food in its Thailand corns. The purpose of this study was to investigate the presence of OTA in Chinese Crude Drugs in Taiwan. We first established the methods of analysis of OTA, then the samples of Chinese crude drugs were collected from traditional markets or retail shops and analyzed for the mycotoxin. Using organic solvents extraction、immunoaffinity column (solid phase extraction) clean up and HPLC to analyze various kinds of Chinese crude drugs. The method used commercial immunoaffinity columns for clean-up and HPLC with fluorescence detection for quantification of the toxin. The samples were extracted with 20 mL solution (methanol: 1% sodium bicarbonate=70:30), filtered and applied to an OchraTest immunoaffinity column. The column was washed with 10 mL PBS buffer solution containing 0.01% Tweeen-20 and followed by water. OTA was eluted with methanol and quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 333 nm, emission wavelength 477 nm) using acetonitrile-water-acetic acid (45:54:1) as mobile phase. The regression equations of OTA was Y=0.5508-0.64834X (r=0.9999). The intraday and interday relative standard deviations of OTA were at the levels of 0.37-2.23% and 1.82-2.55%, respectively. Detection limit was 0.01 ng/mL based on a signal-to-noise ratio of 3:1. The average OTA recoveries from spiked OTA-free crude drugs varied from 83.5-105.6%, and RSD% ranged from 4 to 6%. The method was applied to 47 samples of Fructus Lycii, 41 samples of Fructus Jujubae, 13 samples of Semen Zinziphi Spinosae, 15 samples of Fructus Crataegi, 15 samples of Fructus Schisandrac, 25 samples of Massa Medicata Fermentata, 13 samples of Sojae praeparatum Semen, 10 samples of Praeparatum Mungo, 15 samples of Rhizoma Corydalis, 20 samples of Pericarpium Citri Reticulata, 15 samples of Radix Ginseng, 16 samples of Radix Achyranthis Bidentatac, 0 samples of Rhizoma Paeonia lactiflora, 16 samples of Radix Angelicae Sinensis, 15 samples of Radix Puerariaef, 16 samples of Radix Rehmanniae, 16 samples of Rhizoma Atracylodis Macrocephalac, 15 samples oRhizoma Alismatisf, 16 samples of Radix Glycyrrhizae, 15 samples of Radix Atragali, purchased from retail outlets in Taiwan. OTA was detected in 16, 8, 0, 0, 2, 21, 5, 0, 0, 1, 0, 5, 0, 3, 0, 3, 2, 1, 2, and 1 of samples, measurable at 0.29-19.8, 0.38-16.7, 0, 0, 0.34-7.34, 0.15-45.2, 0.35-15.8, 0, 0, 0.05, 0, 0.08-4.3, 0, 0.28-23.65, 0, 3.23-17.8, 0.45-5.88, 0.22-3.66, 0.32-5.65, 0.16-8.55 µg/kg. In conclusion, this study had shown that the HPLC method could be applied successfully to analyze Ochratoxin A occurred in the traditional Chinese herbal medicines. Therefore, an easy and suitable method for the detection and assay of Ochratoxin A in traditional Chinese medicines was established. |
本系統之摘要資訊系依該期刊論文摘要之資訊為主。