Analysis And Testing Methods Of Aristolochic Acid Analogues In Related Traditional Chinese Medicines

Aristolochic acids(AAs)are well known for causing aristolochic acid nephropathy,a renal fibrosis often associated with urothelial carcinoma.AAs are a group of toxins commonly present in the plants of genus Aristolochia and Asarum.In the present study,the detection and quantitative analysis methods of aristolochic acids in Chinese herbal medicines have been investigated.Various chromatographic methods were used for the separation and purification of AA-type compounds from the roots of Aristolochia contorta(A.contorta).As a result,a total of 21 compounds were isolated.On the basis of their spectral data,the structures of these compounds were identified,including 6 aristolochic acids,9 aristolactams,1 aristolochic acid alkyl ester,2 aporphines,2 sodium aristolochates,and 1 alkaloid.Among them,compounds 9 and 10 were charatcerized as new compounds,named sodium 9-hydroxy-10-formyloxyaristolochate I(SHFA I)(9)and sodium 7,9-dihydroxy-10-formyloxy aristolochate I(SDFA I)(10).Then their cytotoxic activity in human proximal tubular cells HK-2 was evaluated by the MTT method,which has been widely used to assess cell viability.Among these molecules,aristolochic acid I and compound 9 were found to have higher cytotoxicity.Furthermore,molecular modeling was used to evaluate,for the first time,the interactions of compound 9 and aristolochic acid I with the target protein organic anionic transporter 1(OAT1)that plays a key role in mediating aristolochic acid nephropathy.The fragmentation rules of aristolochic acid derivatives were systematically approached by using LTQ-Orbitrap mass spectrometer.A few characteristic pathways were summarized,which would be beneficial for distinguishing the compounds and the unknowns with similar structures.Using UHPLC-LTQ-Orbitrap-mass spectrometry,the aristolochic acids and aristolactams displayed rather distinct MS behaviors.In MS1 of aristolochic acids,the characteristic pseudomolecular ions were[M+NH4]+ and[M+H-H2O]+.Ratios of characteristic ions abundance were different,[M+H-NO2]+/[M+H-H2O]+is bigger than I when C-6 position was substituted by methoxy group or hydroxyl group.However,only[M+H]+was found in the MS1 of aristolactams,which was simpler than that of aristolochic acids.[M+H2O+H]+ also occurred in the MS1 of sodium aristolochates,and in-source fragmentation showed that ester bond of aristolochic acid alkyl ester was easily fragmented.UPLC-ESI-MS/MS method was developed for the determination of 13 components including 6 aristolochic acids,5 aristolactams,1 sodium aristolochate and 1 aristolochic acid alkyl ester.The analytes were quantified by a triple quadrupole instrument with dynamic multiple reaction monitoring(DMRM)mode.This assay method was fully validated and applied in analysis of samples of Aristolochia griffithii,Houttuynia cordata Thunb and Clematis hexapetala Pall and different parts of Asarum heterotropoides.The results indicated that aristolochic acid analogues could not be detected in Clematis hexapetala Pall.AL FI could be detected in Houttuynia cordata Thunb.The contents of aristolochic acid analogues in Aristolochia griffithii were reported for the first time,while the contents of aristolochic acid analogues in different parts of Asarum heterotropoides were also tested and discussed.AA I,AA IVa,Aristoxazole and SHFAI were found in collected 5 batches rhizome samples,but both AA I and AA IVa were detected in collected 3 batches leaf samples.However,traces of AA I(2.72—4.99μg/g)were found in all the collected samples that were tested with using LC-MS/MS.Contents of SHFA I(7.61—21.27μg/g)were found in the collected 5 batches rhizome samples while traces of SHFA I were 5.55 and 5.71 μg/g in the collected 2 batches leaf samples,respectively.Due to the complexity of Chinese patent medicines and low concentrations of aristolochic acid in Asarum,a new ultra performance liquid chromatography coupled with photo-diode array fluorescence detector(UPLC-PDA-FLD)method was developed for the sensitive determination of aristolochic acid analogues.The method makes full use of a cysteine-induced denitration reaction that "turns on" the fluorescence of aristolochic acid for fluorometric detection.Our results showed that the combination of cysteine-induced denitration and HPLC-PDA-FLD analysis method allows for the sensitive quantification of AA I and AA II as low as 8.00 and 15.00 ng/ml,respectively.The method has been validated and successfully applied in quantifying aristolochic acids in Chinese patent medicines.To summarize,a comprehensive research has been conducted for the phytochemical analysis of aristolochic acids in related medicinal plants.Quantitative determination methods of aristolochic acids were established and applied to detect aristolochic acid analogues especially in different parts of Asarum heterotropoides,which provided scientific evidence for the safe use of the aristolochic acids-associated herbal drugs like Asarum species and also for the quality control of Asarum-related Chinese patent medicines.