Journal List > Nat Prod Sci > v.24(4) > 1120282

Rho and Yoon: Efficient Isolation of Dihydrophaseic acid 3′-O-β-D-Glucopyranoside from Nelumbo nucifera Seeds Using High-performance Countercurrent Chromatography and Reverse-phased High-performance Liquid Chromatography

Abstract

High-performance countercurrent chromatography (HPCCC) coupled with reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to isolate dihydrophaseic acid 3′-O-β-D-glucopyranoside (DHPAG) from the extract of Nelumbo nucifera seeds. Enriched DHPAG sample (2.3 g) was separated by HPCCC using ethyl acetate/n-butanol/water system (6:4:10, v/v/v, normal-phase mode, flow rate: 4.0 mL/min) to give 23.1 mg of DHPAG with purity of 88.7%. Further preparative RP-HPLC experiment gave pure DHPAG (16.3 mg, purity > 98%). The current study demonstrates that utilization of CCC method maximizes the isolation efficiency compared with that of solid-based conventional column chromatography.

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Fig. 1.
Chemical structure of dihydrophaseic acid 3′-O-β-D-glucopyranoside (DHPAG).
nps-24-288f1.tif
Fig. 2.
HPLC analyses of NNE samples. (A) 25% ethanol extract, (B) ethyl acetate soluble extract, (C) n-butanol soluble extract and (D) enriched DHPAG extract.
nps-24-288f2.tif
Fig. 3.
HPCCC experiment on enriched DHPAG extract (A), HPLC analyses of target HPCCC fraction (B) and purified DHPAG by preparative RP-HPLC (C). HPCCC parameters are described in Experimental section.
nps-24-288f3.tif
Table 1.
Content of dihydrophaseic acid 3′-O-β-D-glucopyranoside (DHPAG) in each NNE sample.
Sample Content Sample mass Total amount of DHPAG in sample extract Recovery (%) of DHPAG
NNE-B 4.25 mg/g 8.0 g 34.0 mga -
En-DHPAG 9.89 mg/g 2.3 g 22.8 mgb 67.1%d
CCC fraction of DHPAG 0.887 mg/mg 23.1 mg 20.4 mgc 89.5%e

a, b, c content × ∼sample mass

d b ÷ a × 100

e e(c ÷ b) × 100

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