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High-throughput Bioassay and HPLC-UV Methods for Determination of the Bioactivity of Atractylodes lancea and Its Bioactive Constituent Atracylodin in Biological Samples: Application for Pharmacokinetic Study in Patients with Advanced-stage Cholangiocarcinoma

Anurak Cheoymang, Nadda Muhamad, Inthuon Kulama, Kesara Na-Bangchang*

The study aimed to establish a high-throughput bioassay method for the determination of total bioactivity of Atractylodes lancea (AL) in human serum samples. In addition, a simple HPLC-UV method for the determination of plasma concentrations of Atractylodin (ATD), the main bioactive component of AL, was also developed. For the bioassay method, Staphylococcus aureus (S. aureus) ATCC 25923 strain was used as a test organism. Inhibition of bacterial growth was assessed using MTT assay. The calibration curve was prepared from the concentration response curve in serum (0, 0.39, 0.78, 1.56, 3.13, 2.56, and 50 ng/ µl), which was linear with correlation coefficients better than 0.990. The Limit of Quantification (LOQ) was 1.66 µg/ml using 20 µl serum samples. The HPLC-UV assay procedure was developed based on reversed-phase chromatography using Hypersil Gold C18 column and elution solvent consisting of acetonitrile and water at the ratio of 70:30 (v:v). The UV detection was set at the wavelength of 340 nm. The calibration curve was prepared from concentration-response curve in serum (0, 0.39, 0.78, 1.56, 3.13, 2.56, and 50 ng/µl), which was linear with correlation coefficients (r) better than 0.990. The LOQ was 2.5 ng/ml using 1 ml plasma sample. Both assay methods were specific, sensitive, accurate and reproducible quantitative analyses of serum bioactivity of AL and plasma concentrations of ATD. The methods were successfully applied for the pharmacokinetic study of total bioactivity (anticholangiocarcinoma activity) of AL extract in five patients with advanced-stage cholangiocarcinoma.