| Aristolochic acid is an important content in the toxicity research of traditional Chinese medicine in recent years.It is mainly distributed in plants of Aristolochia and Asarum.Aristolochic acid has many pharmacological effects such as anti-infection,anti-early pregnancy and enhancing phagocytic cell activity.But it is also nephrotoxic and carcinogenic.In order to explore and clarify the type,quantity and content of aristolochic acids in medicinal materials containing aristolochic acid toxic substances,with a view to rationally controlling aristolochic acids in such medicinal materials and related patent medicines to ensure that people use medicines Safety.In this study,nine Chinese medicinal materials containing toxic substances like aristolochic acid were selected from the tuber of Aristolochia cinnabarina(ZSL),the herbs of Asarum forbesii(DH),the stems of Aristolochia manshuriensis.(GMT),the fruits of Aristolochia debilis(MDL),the roots of Aristolochia debilis(QMX),the stems and leaf of Aristolochia debili(TXT)s,the herbs of Aristolochia mollissima(XGF),the roots of Aristolochia fangchi(GFJ),and the roots of Asarum heterotropoides var.mandshuricum(XX).The qualitative and quantitative analysis of the ingredients of toulchic acid has been carried out,and the type,quantity and content of the aristolochic acid of this kind of medicinal materials have been clarified.It will be of great significance for the safety control of this kind of medicinal materials and their preparations.The reasonable formulation of control items of aristolochic acid in the quality standard lays the foundation.In this paper,high performance liquid chromatography-diode array detection is used to detect Aristolochic acid I,Aristolochic acid Ⅱ,Aristolochic acid Ⅲa,Aristolochic acid IVa,Aristolochic acid Ⅶa,Aristololactam I,Aristololactam Ⅱ 7 kinds of reference substance single reference substance solution and 9 kinds of medicinal material samples of the test substance solution each 20 μL,injected into the high performance liquid chromatograph,according to 0～100 min,5%～90%(B)chromatographic conditions gradient wash The UV spectrum of the single peak in the chromatogram of the reference substance and the UV spectrum of each chromatographic peak in the chromatogram of the test substance are obtained.Aristolochic acid components were qualitatively analyzed by high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(HPLC-Q-TOF-MS)method for the suspected aristolochic acid components selected in the chromatogram of the test product,Confirmed the chromatographic peak of aristolochic acid compounds.Through continuous optimization of chromatographic separation conditions,the baseline separation of 7 reference substances including Aristolochic acid Ⅰ,Aristolochic acid Ⅱ,Aristolochic acid Ⅲa,Aristolochic acid Ⅳa,Aristolochic acidⅦa,Aristololactam Ⅰ,Aristololactam Ⅱ,and other aristolochic acids in 9 kinds of medicinal materials were achieved within 1 hour,and the optimal chromatographic conditions were obtained:the mobile phase was 0.2%acetic acid aqueous solution(A)-acetonitrile(B);gradient elution:0～20min,18%～28%B;20～38min,28%～35%B;38～41min,35%～42%B;41～45min,42%～43%B;45～60min,43%～60%B.The HPLC-Q-TOF-MS was used to qualitatively analyze the aristolochic acid components in 9 kinds of medicinal materials.The mass spectrometer uses the ESI positive ion mode,and the data is collected in the positive ion mode.The mass scan range is 50～1000 m/z.Analyze the mass spectrum ionization law of 7 reference substances of aristolochic acid Ⅰ,Ⅱ,Ⅲa,Ⅳ a,Vila,and aristolochactam Ⅰ,Ⅱ,and use this law to screen the ultraviolet spectra and 7 of 9 medicinal materials samples.The structural analysis of components with similar spectra of a reference substance was carried out,and a total of 22 aristolochic acid compounds including 7 reference substances were presumably identified.Among the 22 compounds identified,11 aristolochic acid and 11 aristolochic acid were identified as Aristolochic acid Ⅲa-N-β-D-glucoside(1)and Aristolochic acid Ⅲa-O-β-D-glucoside(2),Aristolochic acid Ⅰ a-N-β-D-glucoside(3),Aristolochic acid Ⅳ a-O-β-D-glucoside(4),Aristolochactam Ⅱ-N-β-D-glucoside(5),Aristolochia lactam A Ⅲ a(6),Aristolochactam Ⅰ-N-β-D-glucoside(7),Aristolochactam Ⅲ(8),Aristolochic acidⅢa(9),Aristolochic acid Ⅲa(10),Aristolochic acid Ⅱ(11),Aristolochic acid Ⅶa(12),Aristolochic acid Ⅳa(13),Aristolochic acid Ⅳa(14),Aristolochic acid Ⅱ(15),Aristolochic acid Ⅱ(16),Aristolochic acid Ⅲ(17),Aristolochic acid Ⅲ(18),Aristolochic acid Ⅵ(19),Aristolochic acid Ⅰ(20),Denitroaristolochic acid Ⅰ(21),Aristolochic acid Ⅳ(22).The presence of aristolochic acid and aristolochic acid in the 9 medicinal materials in sequence is as follows:the tuber of Aristolochia cinnabarina,and the roots of Aristolochia debilis have 9 and 6 respectively;the stems of Aristolochia manshuriensis.has 9 and 5;the fruits of Aristolochia debilis has 8 and 6;5 and 4 of the stems and leaf of Aristolochia debilis;2 and 6 of the roots of Aristolochia fangchi;only 6 aristolochic acid in the herbs of Aristolochia mollissima,but aristolochic acid was not detected;2 and 3 in the roots of Asarum heterotropoides var.mandshuricum.;the herbs of Asarum forbesii only has 1 each.There are many types of aristolochic acids in the fruits of Aristolochia debilis,the tuber of Aristolochia cinnabarinay the stems of Aristolochia manshuriensis.and the root of Aristolochia debilis,all of which are more than ten.The types of aristolochic acids are the most in the tuber of Aristolochia cinnabarina and the root of Aristolochia debilis There are 15 species,followed by the fruits of Aristolochia debilis and the stems of Aristolochia manshuriensis.with 14 species;the herbs of Asarum forbesii has the least aristolochic acids,with only two.This paper established a method for determining the content of 22 aristolochic acid compounds in 9 kinds of medicinal materials,and classified the aristolochic acid components screened out according to the similarity of the ultraviolet spectrum,and divided them into six categories.The content of the component is directly determined by the reference substance,and the reference substance with the most similar component to the spectrum of the component without the reference substance is selected as the reference substance for content determination,and the content is calculated by the correction factor.The aristolochic acid components identified in 52 batches of medicinal materials were quantitatively analyzed by high performance liquid chromatography with ultraviolet detection(HPLC-UV)using external standard method,the chromatographic column was C18;the mobile phase was 0.2%acetic acid aqueous solution(A)-Acetonitrile(B),gradient elution;detection wavelength 254nm;results:no interference in specificity test negative;aristolochic acid Ⅰ,aristolochic acid Ⅱ,aristolochic acid Ⅲa,aristolochic acid Ⅳa,aristolochic acid The average recovery rates of lingoic acid Ⅶa,aristolochid Ⅰ,and aristolochid Ⅱwere 97.40%,95.46%,91.34%,89.77%,93.19%,97.06%,95.11%,and the RSD(n=6)was 0.44,respectively%,0.86%,0.99%,1.26%,0.23%,0.40%,0.93%;the average content of the repeatability test is 0.072%,0.007%,0.037%,0.047%,0.041%,0.064%,0.006%,the content of RSD(n=6)were 0.63%,1.12%,0.45%,1.58%,0.51%,0.49%,1.12%,respectively.Quantitative results found that all 9 kinds of medicinal materials have aristolochic acid Ⅰ,but d the roots of Asarum heterotropoides var.mandshuricum.is used as the root of the medicine,and its aristolochic acid Ⅰ content is very small.The types and contents of aristolochic acids in different medicinal materials are quite different.From the 52 batches of medicinal materials collected,the qualitative results of aristolochic acids in different batches of the same medicinal materials The difference is not big,and the quantitative results are somewhat different,but generally the relative level of the content of each component tends to be the same between batches.On the whole,the content of aristolochic acid Ⅰ and aristolochic acid Ⅱ is generally higher than other aristolochic acid substances,among which the tuber of Aristolochia cinnabarina,the herbs of Asarum forbesii,the stems of Aristolochia manshuriensis.,Aristolochic acid,the herbs of Aristolochia mollissima Aristolochic acid The sum of the content of acid Ⅰ and aristolochic acid Ⅱ accounted for more than 30%of the total,and the highest content in the herbs of Aristolochia mollissima was 78%.The total amount of aristolochic acid in the 9 kinds of medicinal materials is between 0.04 mg g-1～13.40 mg g-1,and the total amount of medicinal materials is ZSL≈GMT>QMX>MDL>GFJ>TXT>XGF>XX>DH;Among them,the total amount of aristolochic acid is 0.02mg g-1～12.63 mg g-1,and the content of medicinal materials is GMT≈ZSL>QMX>MDL>GFJ>TXT>XGF>XX>DH;The total amount of aristololactam was not detected～1.36 mg g-1,and the content of medicinal materials was ZSL>MDL>QMX>XX>GMT>GFJ>DH≈TXT>XGF.Considering that traditional Chinese medicines are mostly used in decoctions,this article also conducted a qualitative and quantitative analysis of the aristolochic acids in the decoctions of nine kinds of medicinal materials,and found that 22 aristolochic acids were also detected in the decoctions of 9 kinds of medicinal materials.Acidic compounds,the total amount of aristolochic acid is about 30%to 60%extracted by 70%ultrasonic methanol,indicating that the decoction also contains a lot of aristolochic acid,which is also toxic.Not all aristolochic acids are toxic,but aristolochic acid Ⅰ is not the only one.The literature reported that aristolochic acid Ⅰ was the most toxic and the main toxic component.Later,it was carried out based on zebrafish.To evaluate the toxicity of the fruits of Aristolochia debilis and Aristolochic acid I,compare the LD50,the maximum non-lethal concentration and the maximum lethal concentration of the fruits of Aristolochia debilis and Aristolochic acid Ⅰ.The results showed that the decoction of the fruits of Aristolochia debilis and Aristolochic acid Ⅰ obviously showed better than that of aristolochic acids.aristolochic acid Ⅰ is more toxic,which means that it is imperfect to judge the toxicity of drugs containing aristolochic acid-like toxic substances only by using aristolochic acid Ⅰ as a marker component. |
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