Quantitative Determination and Toxicity Evaluation of Aristolochic Acid Analogues in Asarum heterotropoides F. Schmidt (Xixin) and Traditional Chinese Patent Medicines.

Zhong Xian,Yushi Zhang,Fang Wang,Aihua Liang,Jingzhuo Tian,Chenyue Liu,Chen Pan,Dunfang Wang,Yan Yi,Jiayin Han,Shasha Qin,Suyan Liu,Lianmei Wang,Chunying Li,Jing Meng,Yong Zhao

doi:10.3389/fphar.2021.761593

Abstract

Asarum (Xixin), which contains analogues of aristolochic acid (AA), is the only species of the genus Aristolochia included in the Chinese Pharmacopoeia 2020. However, the contents and nephrotoxic effects of AA analogs in Asarum (Xixin) and its formulations have not been clarified. An automatic, effective solid phase extraction process and UPLC-MS/MS method were established for the pretreatment and quantitative detection of AA analogues in commercially available traditional Chinese patent medicines. The cytotoxicity and DNA damage induced by five analogues of AA were evaluated by CCK8 using human kidney cells (HK-2) and comet assays. HPLC was used to detect the analogues of AA in Asarum heterotropoides F. Schmidt (Xixin). The results showed that the contents of AA I, AA II, and AA IIIa were below the detection limit, while AA IVa and AL I presented relatively high contents of Asarum heterotropoides F. Schmidt (Xixin), within the range of 66.50–121.03 μg/g and 19.73–43.75 μg/g, respectively. The levels of AA analogues were in the nanogram-per-gram level in the main traditional Chinese patent medicines. AA I and AL I exhibited relatively high cytotoxicity at 48 h in CCK8 assays, while AA II, AA IIIa, and AA IVa showed weak cytotoxicity even at 800–1,000 μM. AA I induced significant pathological alterations and direct DNA damage at 40 mg/kg and 20 mg/kg, respectively. No distinct nephrotoxicity or hepatotoxicity was observed in mice treated with AA II, AA IIIa, AA IVa, or AL I at 40 mg/kg in this study. Consumption of Asarum heterotropoides F. Schmidt (Xixin) with controlled doses and periods is relatively safe as the contents of AA analogues in Asarum heterotropoides F. Schmidt (Xixin) and its formulations were far below those causing acute toxicity in this study. But, the long-term toxicity of Asarum heterotropoides F. Schmidt (Xixin) still needs further study.

Highlights

Aristolochic acid (AA) exists mainly in plants of the Aristolochiaceae family that have been used in pharmaceutical formulations for hundreds of years to treat various diseases (Heinrich et al, 2009; Li et al, 2017)
There were some regional differences in the contents of AA aristolochic acid IVa (IVa) and AL I, but the changes were within the range of 2 fold
Plants of Aristolochiaceae have been gradually prohibited in the clinical use due to the nephrotoxicity and carcinogenicity of its component AA, with the exception of Asarum (Xixin), which has been used for centuries as an analgesic and antitussive (Wu et al, 2021)

Summary

Introduction

Aristolochic acid (AA) exists mainly in plants of the Aristolochiaceae family that have been used in pharmaceutical formulations for hundreds of years to treat various diseases (Heinrich et al, 2009; Li et al, 2017). Exposure to AA is associated with nephropathy and upper tract urothelial carcinoma (UUC) (Heinrich et al, 2009; Wu and Wang, 2013; Yang et al, 2014). AA is the causative agent of Balkan Endemic Nephropathy (BEN), a chronic, progressive renal disease, which arises from the consumption of bread in which wheat grain is contaminated with the weed of Aristolochia clematitis (Jelaković et al, 2012; Jelaković et al, 2019). The adducts can induce adenine-to-thymine (A>T) transversions, which have been detected both in vitro and in vivo (Chen et al, 2012; Chen et al, 2013)

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Conclusion