Isolation and quantification of aristolochic acid I from Houttuynia cordata Thunb. by using HPLC-UV

Abstract

Background: Nephropathy caused by Aristolochic acid is a rapidly progressive interstitial renal disease owing to some traditional Chinese herbs containing aristolochic acids (AAs). Houttuynia cordata Thunb. (yuxingcao魚腥草), is used to detoxify, reduce inflammation, and as a diuretic drug in traditional Chinese medicine. The isolation and characterization of 3 aristolactams and 4,5-dioxoaporphinein Houttuynia cordata Thunb. (yuxingcao魚腥草) was reported initially in the year of 1992. Aristolactams, with a phenanthrene chromophore, are mainly found in the species of aristolochia together with4,5-dioxoaporphinesand the aristolochic acids.

Objective: Due to toxicity of Aristolochic Acids and Aristolactams, some Chinese herbs received increasingly attention. We aim to determine whether aristolochic acids exist in Houttuynia cordata Thunb. (yuxingcao魚腥草). If they do exist, we will look further how to quantify and how they react with proteins in vivo. But they did not exist, we could still use this method to quantify aristolochic acid Ⅰin another herb materials in the further study. Method: Aristolochic acid Ⅰ was extracted by Soxhlet extraction for 2hours. An attempt at the quantification of aristolochic acid Ⅰfound in Houttunya cordata was determined by High Performance Liquid Chromatography (HPLC) using an ultraviolet(UV) detector. HPLC was carried out on a Agilent ZORBAX Eclipse PlusC18column (4.6×100mm, 3.5 μm ),gradient eluting with mobile phase consisting of 0.1% methanoic acid –acetonitrile and at a flow rate of 0.5mL/min. The UV detector was set at λ=254 nm.25μL was injected each run. Total runtime was 40min. The pure compound of AAⅠcame into use for the establishment and validation of methodology. Five batches of Houttuynia cordata Thunb. (yuxingcao魚腥草) were analyzed to determinate presence of aristolochic acidⅠ. Results: The concentration of pure aristolochic acid Ⅰ demonstrated good linearity with the absorbance in the range of 0.313~19.8μg/mL, r2=0.9998. Thus the method precision was satisfactory, and in addition was measured by within-run precision and between-run precision, Within-run precision was lower than 3%, 2.8%, 2.4%, and 0.7% respectively. Between-run precision was 4.2%.Andthe average recovery rate was 96.2%(n=9). The repeatability among the same solution was good, RSD=0.49%. the corresponding peak for aristolochic acid in HPLC-UV spectrogram of 5 batches of Houttuynia cordata Thunb. (yuxingcao) was not found. Conclusions: The Soxhlet extraction can be used in extraction of AAⅠ from traditional Chinese herbs. The HPLC method can be applied to determine the concentration of aristolochic acid Ⅰ. This experiment determined the absence of aristolochic acid Ⅰ in Houttuynia cordata Thunb. (yuxingcao) over the detection limit of this method.