An LC/DAD-UV/MS method for the rapid detection of aristolochic acid in plant preparations

A new method using direct on-line coupling between HPLC and UV-DAD/MS was developed for the rapid detection of aristolochic acid I in plant preparations. The use of both UV-DAD and MS detectors enables this method to be very selective. A ng range detection limit was determined for both UV and MS detections. A quantitative determination of aristolochic acid I in plant preparations was achieved using UV and MS detection. A thin layer chromatography assay was also developed for the preliminary detection of aristolochic acid I in complex mixtures.     

Determination of aristolochic acid I and II in North American species of Asarum and Aristolochia

Wild ginger, Asarum canadense, which has folk uses as a medicinal and food plant, has been reported to contain aristolochic acid I. Rhizomes of North American species of Aristolochiaceae were surveyed for the presence of aristolochic acids by HPLC. Aristolochic acid I (1) and aristolochic acid II (2) were present in Aristolochia species and Hexastylis; 1 alone was detected in multiple accessions of A. canadense and Asarum caudatum, though not in Asarum wagneri. Concentrations in A. canadense were highly variable, reaching as much as 0.037 percent of dry weight.     

Detection of aristolochic acid I, tetrandrine and fangchinoline in medicinal plants by high performance liquid chromatography and liquid chromatography/mass spectrometry

Problems with identification and labeling of medicinal plants, as well as substitution/adulteration of non-toxic plants by toxic ones have previously led to cancer, renal failure and even deaths. The non-toxic Stephania tetrandra (Fangji) has been known to be substituted by Aristolochia fangchi (Guang fangji), which contains the nephrotoxic and carcinogenic aristolochic acid (AA). In this study, 10 samples of "Fangji" were bought from local medicinal shops. HPLC-DAD chromatographic fingerprints of each methanol extract were compared with those of A. fangchi and S. tetrandra, using aristolochic acid I (AAI), tetrandrine and fangchinoline as marker compounds. Nine of the samples were found to be similar to A. fangchi. The presence of AAI in the nine samples was confirmed using LC-MS/MS. Neither tetrandrine nor fangchinoline were detected in these samples. The methods developed in this study allow the simultaneous detection of AAI, fangchinoline and tetrandrine. The results suggest possible substitution of S. tetrandra by A. fangchi at wholesale or retail level. This study highlights the importance of greater control of medicinal plants with toxic components as these may still be readily accessible to the public.     

马兜铃酸相关中药材的探讨

目的：对关木通等相关中药材马兜铃酸的含有情况进行检测、分析。方法：采用高效液相色谱法。色谱条件：Platinum C18色谱柱；流动相：0．05mol/L磷酸二氢钠（含磷酸1ml/L）－腈（60：40）；检测波长：400nm。结果：17种相关中药材仅马兜铃属6种可检测出马兜铃酸类成分。细辛属（Asarum）、木通属（Akebia）、铁线莲属（Clematis）、风龙属（Sinomenium）、千金藤属（Stephania）、川木香属（Vladimiric）及云木香属（Aucklandic）相关植物中均未检出相应成分。结论：初步认为仅限于马兜铃属植物含有马兜铃酸类成分。木通的品种问题应进一步深入研究。     

马兜铃酸类成分检测与分析研究进展

马兜铃酸类成分是广泛存在于马兜铃科植物中的硝基菲类化合物,已被证实具有肾毒性、致癌和致基因突变等作用。我国自2003年以来采取一系列风险控制措施,其中马兜铃酸含量高的关木通、广防己和青木香等药材已被禁用。目前,一些马兜铃酸含量低的中药材与中成药仍在使用中,鉴于马兜铃酸成分对人体的严重危害性,有必要进一步加强相关药材与制剂的风险评估。在归纳马兜铃酸类成分结构等基本信息的基础上,对近年来的检测分析方法进展进行了总结,为其风险控制与安全使用提供了科学依据。     

Comparative study of the contents of analogues of aristolochic acid in two kinds of Aristolochiae Fructus by high-performance liquid chromatography

Aristolochiae Fructus (??Madouling??) is derived from the fruits of Aristolochia contorta and A. debilis (Aristolochiaceae). These two species contain potentially nephrotoxic constituents, but are officially used in China. Distinction of constituents and toxicity between these two species remains unclear. A high-performance liquid chromatography method was developed and validated for the simultaneous determination of seven analogues of aristolochic acid (aristolochic acids I, II, IIIa, IVa and VIIa), as well as aristololactams I and II in Aristolochiae Fructus. Chromatographic separation was achieved on a Zorbax SB-C₁₈ column with a gradient mobile phase comprising acetonitrile and 1?% acetic acid?C30?mM triethylamine (20:1, v/v) buffer. Analytes were detected with a diode array detector at 250 and 260?nm. The contents of seven constituents in samples (11 batches of A. contorta fruits, 15 batches of A. debilis fruits and 33 commercial samples of Madouling) were determined. The content of aristolochic acid IVa was higher than that of aristolochic acid VIIa in A. contorta fruits, whereas the opposite was true in A. debilis fruits. This feature can be used to distinguish the two species from each other and identify the resource plant of Madouling. Through a morphological method and a newly found principle based on the ratio AA-IVa/AA-VIIa, we found that the 33 commercial samples collected from 12 provinces in China were all derived from the fruits of A. contorta.     

RP-HPLC法测定14种中药材中马兜铃酸A的含量

目的对14种中药材中的马兜铃酸A进行含量测定。方法采用RP-HPLC法。色谱柱为Kromasil ODS柱(200 mm×4.6 mm,5μm),流动相为甲醇-体积分数为1%的冰醋酸溶液(体积比70∶30),检测波长为319 nm,流速为1.0 mL.min-1,柱温为25℃。结果14种中药材中马兜铃酸A的含量分别为:苕叶细辛0.22 mg.g-1,万源山慈菇0.18 mg.g-1,宜宾防己0.72 mg.g-1,川细辛0.58 mg.g-1,寻骨风0.52 mg.g-1,广西山慈菇0.13 mg.g-1,短尾细辛0.15 mg.g-1,淮通0.94 mg.g-1,关木通0.49 mg.g-1,广元防己3.00 mg.g-1;平昌防己、冕宁防己、穆平马兜铃、北细辛地上及地下部分中均未检出马兜铃酸A。结论应用本实验中所建立的方法对多种中药材中的马兜铃酸A进行检测,为相关药材及中成药应用提供了科学依据。     

6-O-beta-D-glucoside of aristolochic acid IIIa and other components from the roots of Aristolochia baetica

From the root extract of the European Aristolochia baetica L. we isolated aristolochic acid IIIa-6-O-beta-D-glucoside (1) besides magnoflorine chloride, the free aristolochic acids I and II, and five aristolactams. Saccharose and fructose were found as major constituents.     

北马兜铃的化学成分研究——Ⅱ、马兜铃酸E的化学结构

From the root of Aristolochia contorta Bunge six chemical constituents were isolated; one of them is a new phenanthrene compound containing nitro group for which the name aristolochic acid E is suggested. By means of spectral methods combined with chemical analysis the chemical structure of aristolochic acid E was determined to be 7-methoxy-8-hydroxy-aristolochic acid. The other components were identified as allantoin, aristolochic acid A, magnoflorine, β-sitosterol and daucosterol respectively.     

Aristolochic acids in Aristolochia chilensis

The major components of the acid fraction of the leaves and tender stems of Aristolochia chilensis Miers are aristolochic acids I and Ia. Aristolochic acid Ia has been isolated from plant material for the f?rst time, and its PMR spectrum is discussed.     

LC-MS法检测消风止痒颗粒中马兜铃酸A

目的:建立检测消风止痒颗粒中马兜铃酸A的液相色谱-质谱分析方法。方法:采用Venusil mp C18(250 mm×4.6mm,5μm)色谱柱,流动相为乙腈-0.1%甲酸水溶液(40∶60),流速1.0 mL.min-1。采用电喷雾电离源正离子质谱检测。结果:马兜铃酸A在0.005086～0.4069μg范围内线性关系良好(r=1.000),平均回收率(n=7)为97.1%(RSD=2.1%)。87批制剂均未检出马兜铃酸A。结论:本法快速、灵敏、准确,可用于消风止痒颗粒中是否混入关木通的确证检查,以防马兜铃酸A造成的用药安全,为消风止痒颗粒的质量控制提供了科学依据。     

瑶药天钻的质量标准研究

目的:建立瑶药天钻药材的质量标准。方法:分别对天钻药材进行显微鉴别和薄层色谱(TLC)鉴别;按照《中国药典》方法进行水分、总灰分、酸不溶性灰分、浸出物检查;采用高效液相色谱法测定药材中马兜铃酸的含量:色谱柱为Phenomenex Gemini C18(250 mm×4.6 mm,5μm),流动相为乙腈-水(含0.34%三乙胺、0.94%乙酸,梯度洗脱),检测波长为260 nm。结果:天钻药材的显微特征明显;TLC图中能清晰显现与马兜铃酸A对应的点;马兜铃酸A的质量浓度在1.703¹09.000μg/ml范围内与其峰面积积分值呈良好的线性关系(r=0.999 9),平均加样回收率分别为97.54%、101.16%、101.15%,RSD分别为0.22%、0.63%、0.39%(n=3)。结论:所建标准可用于天钻药材的质量控制。产自广西、云南的11批天钻药材在显微、外观等方面质量相对稳定,但马兜铃酸A的含量差异较大。     

广西马兜铃的化学成分研究

广西马兜铃Aristolochia kwangsiensis Chun et How ex CF Liang,民间又名圆叶马兜铃、大百解薯,生长在广西石山地区,有清热解毒、止血止痛作用。我们在1977年曾报道了初步的化学、药理和临床试验,证明其非水溶性总生物碱有解痉及镇痛作用。现从其根的乙醇提出物中,分离得到五种结晶性成分,经鉴定分别为尿囊素、马兜铃酸、β-谷甾醇、3,4-次甲二氧基-6,8-二甲氧基-1-甲酯菲及3,4-次甲二氧基-6,8-二甲氧基-10-硝基-1-甲     

Evaluation of the cytotoxicity and genotoxicity of aristolochic acid I – A component of Aristolochiaceae plant extracts used in homeopathy

The medicinal plants Aristolochia clematitis L. as well as Asarum europaeum L., representatives of the plant family Aristolochiaceae and mentioned in the German Homeopathic Pharmacopeia, contain aristolochic acid. We found that the mother tinctures of A. clematitis and A. europaeum inhibited DNA synthesis in human hepatoma HepG2 cells in a dose-dependent manner. One of the components of the plant extract, aristolochic acid I (AAI), is linked to the development of nephropathy and urothelial cancer in humans. Therefore, we also evaluated the cytotoxicity and genotoxicity of AAI in HepG2 cells. Cell proliferation was inhibited concentration-dependently by AAI using BrdU-ELISA and colony forming assay. AAI formed DNA adducts (measured by ³²P-postlabeling), induced chromosomal aberrations (micronuclei) and DNA strand breaks. DNA damage induced by AAI led to an arrest of cells in the S-phase which was associated with the increased expression of p53 and p21 proteins. The results are discussed under consideration of former studies.     

穆坪马兜铃化学成分的研究

自穆坪马兜铃(Aristolochia moupinensis Franch)根、茎中分得十三个化合物,其中化合物Ⅰ～Ⅻ经鉴定分別为马兜铃酸Ⅰ(aristolochic acid Ⅰ)(Ⅰ),尿囊素(allantoin)(Ⅱ),紫丁香酸(syringic acid)(Ⅲ),香豆酸(P-coumaric acid)(Ⅳ),马兜铃酸Ⅳ(aristolochic acid Ⅳ)(Ⅴ),β-谷甾醇(Ⅵ),木兰碱(magnoflorine)(Ⅶ),马兜铃酸Ⅳ甲醚aristolochic acid Ⅳmethyl ether(Ⅵ),棕榈酸(Ⅸ),马兜铃酸Ⅱ(aristolochic acid Ⅱ)(Ⅹ),N(Phydroxyphenethy1)P-coumaramide(Ⅺ),马兜铃酸Ⅳ甲醚甲酯(aristolochic acid Ⅳmethyl ether methyl ester)(Ⅻ),化合物ⅩⅢ为新化合物,经光谱分析和化学合成等方法证明其结构为N(P-hydroxyphenethyl)-ferulamide,命名为穆坪马兜铃酰胺(moupinamide)。初步药理试验表明穆坪马兜铃酰胺体外有抑制血小板聚集和影响血小板内前列腺素合成的作用。     

减肥类保健食品中非法添加盐酸西布曲明和酚酞的定性定量检测

目的建立准确检测减肥类保健食品中非法添加盐酸西布曲明和酚酞的定性定量方法。方法采用超高效液相色谱-串联质谱(UPLC-MS/MS)法,以0.1%甲酸的甲醇溶液和水溶液为流动相,通过分子离子峰、二级质谱的碎片离子、多反应检测模式的色谱峰等信息,对盐酸西布曲明和酚酞进行定性鉴别和定量测定。结果 4批保健食品中均检出盐酸西布曲明和酚酞。结论该方法准确可靠、专属性强、灵敏度高,可作为检测减肥类保健食品中非法添加盐酸西布曲明和酚酞的有效方法。     

NMR regulatory analysis: determination and characterization of Chinese-herb aristolochic acids

1H NMR methodology for the simultaneous determination and characterization of the nephrotoxic components of Aristolochia plants aristolochic acid I (AA-I) and aristolochic acid II (AA-II) was developed utilizing a 400 MHz spectrometer without the need of reference standards. The developed methodology is able to differentiate and assess chemical structures of these toxic injurious compounds. The quantity of each was calculated on the basis of the integrals for the signals of the H-7 and H-8 of the phenanthrene ring of AA-I and AA-II at delta7.38 and delta8.31, respectively, and the vinylic protons of the internal standard maleic acid at delta6.06. The accuracy of the method was established through the analysis of synthetic mixtures containing the internal standard maleic acid, with purified AA-I or combined AA-I and AA-II sodium salts. Excellent agreements were verified between the assay results and the quantities in the mixtures. The mean +/- SD recovery values for purified AA-I and combined AA-I and AA-II from two sets of 10 synthetic mixtures were 99.8 +/- 0.6% and 99.6 +/- 0.8%, respectively. The assay of 4 lots of commercial aristolochic acid by 1H NMR spectroscopy indicated AA-I and AA-II contents in the ranges 45.3-97.1% and 0-15.4%, respectively.     

Interaction of phospholipase A2 from Vipera russelli venom with aristolochic acid: a circular dichroism study

The interaction of aristolochic acid, an alkaloid from Aristolochia species, with phospholipase A2 (PLA2) from Vipera russelli venom was followed by circular dichroism measurements. Aristolochic acid is a non-competitive inhibitor of PLA2. The binding of aristolochic acid to PLA2 induces an extrinsic CD band at 320 nm. The association constant was determined by following the intensity of the extrinsic CD band. Aristolochic acid forms a 1:1 complex with PLA2, with an association constant K, of 5.4 X 10(3) M-1 and a Gibb's free energy change (delta G0) for the reaction of -5.1 kcal/mole. The values of association constant and delta G0 suggest that the interaction is weak. Binding of aristolochic acid causes a change in the secondary structure of the protein which is characterized by an increase in the apparent content of alpha-helix, without any detectable change in the tertiary structure of PLA2.     

Aristolochic acid I metabolism in the isolated perfused rat kidney

Aristolochic acids are natural nitro-compounds found globally in the plant genus Aristolochia that have been implicated in the severe illness in humans termed aristolochic acid nephropathy (AAN). Aristolochic acids undergo nitroreduction, among other metabolic reactions, and active intermediates arise that are carcinogenic. Previous experiments with rats showed that aristolochic acid I (AA-I), after oral administration or injection, is subjected to detoxication reactions to give aristolochic acid Ia, aristolactam Ia, aristolactam I, and their glucuronide and sulfate conjugates that can be found in urine and feces. Results obtained with whole rats do not clearly define the role of liver and kidney in such metabolic transformation. In this study, in order to determine the specific role of the kidney on the renal disposition of AA-I and to study the biotransformations suffered by AA-I in this organ, isolated kidneys of rats were perfused with AA-I. AA-I and metabolite concentrations were determined in perfusates and urine using HPLC procedures. The isolated perfused rat kidney model showed that AA-I distributes rapidly and extensively in kidney tissues by uptake from the peritubular capillaries and the tubules. It was also established that the kidney is able to metabolize AA-I into aristolochic acid Ia, aristolochic acid Ia O-sulfate, aristolactam Ia, aristolactam I, and aristolactam Ia O-glucuronide. Rapid demethylation and sulfation of AA-I in the kidney generate aristolochic acid Ia and its sulfate conjugate that are voided to the urine. Reduction reactions to give the aristolactam metabolites occur to a slower rate. Renal clearances showed that filtered AA-I is reabsorbed at the tubules, whereas the metabolites are secreted. The unconjugated metabolites produced in the renal tissues are transported to both urine and perfusate, whereas the conjugated metabolites are almost exclusively secreted to the urine.     

Aristolochic acid and 'Chinese herbs nephropathy': a review of the evidence to date.

Chinese herbs nephropathy (CHN) is a rapidly progressive interstitial nephropathy reported after the introduction of Chinese herbs in a slimming regimen followed by young Belgian women. It is characterised by early, severe anaemia, mild tubular proteinuria and initially normal arterial blood pressure in half of the patients. Renal histology shows unusual extensive, virtually hypocellular cortical interstitial fibrosis associated with tubular atrophy and global sclerosis of glomeruli decreasing from the outer to the inner cortex. Urothelial malignancy of the upper urinary tract develops subsequently in almost half of the patients. Suspicion that the disease was due to the recent introduction of Chinese herbs in the slimming regimen was reinforced by identification in the slimming pills of the nephrotoxic and carcinogenic aristolochic acid (AA) extracted from species of Aristolochia. This hypothesis was substantiated by the identification of premutagenic AA-DNA adducts in the kidney and ureteric tissues of CHN patients. Finally, induction of the clinical features (interstitial fibrosis and upper urothelial malignancy) typical of CHN in rodents given AA alone removed any doubt on the causal role of this phytotoxin in CHN, now better called aristolochic acid nephropathy (AAN). AAN is not restricted to the Belgian cases. Similar cases have been observed throughout the world, but AA is sometimes incriminated on the basis of the known content of AA in the herbs. The possibility remains that in some individuals in whom AA has not been demonstrated, other phytotoxins might be implicated. Biological and morphological features of AAN are strikingly similar to those reported in another fibrosing interstitial nephropathy of still unknown aetiology, Balkan endemic nephropathy (BEN). Interestingly, AA was incriminated as the cause of BEN many years ago, a hypothesis yet to be fully explored. The intake of AA and the presence of tissular AA-DNA adducts in patients with an unequivocal diagnosis of BEN remains to be demonstrated. The tragic phenomenon of CHN, recognised only 10 years ago, has been at the root of significant research and progress both in nephrology and oncology. It has provided a fascinating opportunity to understand the link between a fibrosing interstitial nephropathy and urothelial carcinoma. It allows the categorisation of interstitial nephritis on the basis of histological findings, of initiating toxic substances and of associated clinical features. Finally, it has led to the withdrawal in several countries of a previously unsuspected carcinogenic and nephrotoxic substance.