Phenanthrene-Enriched Extract from Eulophia Macrobulbon Using Subcritical Dimethyl Ether for Phosphodiesterase-5A1 Inhibition

Abstract

Eulophia macrobulbon (E.C.Parish & Rchb.f.) Hook.f. contains a natural PDE5A1 inhibitor, the phenanthrene, 1-(4'-hydroxybenzyl)-4,8- dimethoxyphenanthrene-2,7-diol (HDP) a potential treatment for erectile dysfunction. This investigation aimed to improve extraction efficiency of HDP from E. macrobulbon by using greener extraction methodology, subcritical fluid dimethyl ether extraction (sDME) rather than classical solvent extraction (CSE) and ultrasound-assisted extraction (UAE). The efficiency and quality of obtained extracts were evaluated by: %process yield; solvent amount; extraction period; temperature; %HDP content by LC-MS assay, bioactivity as inhibition of phosphodiesterase-5A1 (PDE5A1) by radio-enzymatic assay; and chemical profiles by LC-QTOF-MS analysis. sDME yielded the highest content of HDP in the extract at 4.47%, much higher than using ethanol (0.4-0.5%), ethyl acetate (1.2-1.7%), or dichloromethane (0.7-1.4%). Process yield for sDME (1.5-2.7%) was similar or less than that observed with other solvents (0.9-17%), but providing that process yield is not prohibitively low, concentration is a more important metric for clinical application. Optimal sDME extraction conditions were: extraction period, 40 mins; 200% water as a cosolvent; sample-to-solvent ratio of 1:8; temperature, 35°C. Phenanthrene aglycone and glycoside derivatives were major constituents in sDME extracts and lesser amounts of phenolic compounds and sugars. Inhibition of PDE5A1 by sDME (IC50 0.67±0.22 µg/mL) was 10-fold more potent than the ethanolic extract and other extraction methods, suggesting a high likelihood of clinical efficacy. Thus, sDME was more efficient, faster, solvent-sparing, greener extraction methodology and more selective for phenanthrene when extracted from E. macrobulbon.