Multiple in vitro systems for assessment of metabolic profile and pharmacokinetics of the mainbioactive constituent of Eulophia macrobulbon

Abstract

Eulophia macrobulbon belongs to Orchidaceae family, commonly known as Orchids. It has been used in Thai traditional medicine, especially as an aphrodisiac. The main bioactive compound of E. macrobulbon is EM2. There are reports on its chemical composition and pharmaceutical activity but reports about its cytotoxicity and pharmacokinetic parameters are rare. The objectives of this study are to gain new knowledge in the process of structural changes and pharmacokinetic values of EM2 by in vitro methods to predict the pharmacokinetics of EM2 for further safety data for humans. Also, to screen for cytotoxicity of EM2 using Hep G2 cells and Caco-2 cells. The cytotoxicity of EM2 in Hep G2 cells and Caco-2 cells were evaluated by MTT assay. The results showed that EM2 was toxic to 50% of Hep G2 cells at a concentration (IC50) of 30.08 ± 0.62 micromolar compared with the control (the concentration of dimethyl sulfoxide (DMSO) was 0.01%). For the cytotoxicity of EM2 in Caco-2 cells, the result of IC50 was 41.64 ± 1.60 micromolar compared with the control. This, the concentrations of EM2 at 1 and 10 µM were chosen and use for further experiment to determine its effectives. Caco-2 monolayers in 12 trans-well insert plate was collected to use in permeability testing of EM2 compound. The TEER values were checked to confirm monolayers formation between apical chamber (AP) and basolateral chamber (BL). The Papp values shows the time dependence of sample EM2 at initial concentration 1 µM and 10 µM in absorptive and secretory directions across Caco-2 monolayers. The Papp values of EM2 results have shown that absorptive transport (AP-BL) from 30 to 120 minutes has decreased which means that sample EM2 at start concentration 1 µM and 10 µM has the high permeability through the Caco-2 monolayers compared to caffeine and rhodamine123. The absorptive permeability or Papp, (AP-BL) values of EM2 at initial concentrations of 1 and 10 µM were ranged 3.48~0.90 and 1.20~0.46 ×10-4 cm/s, respectively (R123 was ranged 1.01~0.50 ×10-4 cm/s and caffeine was ranged 2.25~1.38 ×10-4 cm/s), the secretory permeability or Papp, (BL-AP) values were 1.17~0.30 and 0.26~0.14 ×10-4 cm/s, respectively (R123 was ranged 0.90~6.20 ×10-4 cm/s and caffeine was ranged 2.31~0.57 ×10-4 cm/s). EM2 was used to determines the concentration of the drug in dialysis buffer compared to plasma to provides an indication of drug binding protein. The results of EM2 at initial concentration of 1 and 10 µM were obtained %bound with protein in plasma close to 100%. Metabolic study in time dependence of EM2 at initial concentration 10 µM in pooled human liver microsomes, human hepatocytes and Hep G2 cells were half-life (t1/2) at 2.71, 15.23 and 67.41 hours, respectively. The conventional clearance (CL) obtained from the comparison of hepatocytes applied to the total number of human hepatocytes were 7.56, 7.12 and 1.75 liters per hour, respectively. However, EM2 also be selected for investigation CYP-phenotyping. The results from exhibited that there were at least 4 metabolites showing in LC-MS chromatogram.

-