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, phenanthrene, 1-(4'-hydroxybenzyl)-4,8- dimethoxyphenanthrene-2,7-diol (HDP), a potential agent for the treatment of erectile dysfunction. The aim of this study was to improve the extraction efficiency of HDP from E. macrobulbon by using a more environmentally friendly extraction method, subcritical liquid dimethyl ether extraction (sDME), instead of classical solvent extraction (CSE) and ultrasound-assisted extraction (UAE). The efficiency and quality of the extracts obtained were evaluated using the following criteria: %process yield; solvent amount; extraction time; temperature; %HDP content by LC-MS, bioactivity as inhibition of phosphodiesterase-5A1 (PDE5A1) by radio-enzymatic assay; and chemical profiles by LC-QTOF-MS. sDME provided the highest content of HDP in the extract at 4.47%, much higher than the use of ethanol (0.4-0.5%), ethyl acetate (1.2-1.7%), or dichloromethane (0.7-1.4%). The process yield for sDME (1.5-2.7%) was similar to or lower than the other solvents (0.9-17%), but as long as the process yield is not prohibitively low, the concentration is a more important measure for clinical use. The optimal conditions for sDME extraction were: Extraction time, 40 min; 200% water as co-solvent; sample-to-solvent ratio of 1:8; temperature, 35 °C. Phenanthrene aglycone and glycoside derivatives were the major constituents of the sDME extracts and lesser amounts of phenolic compounds and sugars. The inhibition of PDE5A1 by sDME (IC₅₀ 0.67 ± 0.22 µg/ml) was tenfold more potent than ethanolic extract and other extraction methods, suggesting a high probability of clinical efficacy. Thus, sDME was a more efficient, faster, solvent-saving and environmentally friendly extraction method and more selective for phenanthrene when extracted from E. macrobulbon.

Figure 1

Parameters influencing dimethyl ether (sDME) extraction of E. Macrobulbon root powder as total yield (black bars) and its content of the bioactive ingredient, HDP measured LC/MS (open bars). Values deemed optimal for each parameter were used for the next parameter measures (b–e) which were (a) sDME volume (1:8), (b) (40 min extraction period), and (c) (35 °C). Each bar is a single determinations.

Figure 2

Total ion count LC–MS chromatograms (TIC) from of sample extracts of E. macrobulbon with 50 µg/ml. All chromatograms have the same y-scales but only A and D scales shown. The numbered peaks correspond with compounds identified in Table 2. Extraction protocols were: (A) 10 g water added 5 g powdered E. Macrobulbon root and extracted with 40 g DME (method of sample no. 31, Table 1); (B) cDCM, method of sample no. 3 (Table 1); (C) cEtOAc, method of sample no. 12 (Table 1), (D) cEtOH, method of sample no. 12 (Table 1).

Figure 3

Identifiable compounds of aglycone phrenanthrene structure from E. Macrobulbon root extract using LC-QTOF-MS.