ISOLATION OF CAFFEIC ACID FROM EUPATORIUM ADENOPHORUM SPRENG BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY AND SYNTHESIS OF CAFFEIC ACID-INTERCALATED LAYERED DOUBLE HYDROXIDE

Abstract

Preparative high-speed countercurrent chromatography (HSCCC) was successfully used for isolation and purification of caffeic acid from Eupatorium adenophorum Spreng with a solvent system composed of ethyl acetate-methanol-water at a volume ratio of 10:1:10, v/v. Using a preparative unit of the HSCCC centrifuge, about a 938 mg amount of the crude extract was separated, yielding 63.2 mg of caffeic acid at purity of 96.0%. Then, the anti-microbial and anti-virus drug caffeic acid (C(9)H(8)O(4)) was intercalated into layered double hydroxides for the first time by anion exchange under a nitrogen atmosphere. The product caffeic acid-LDH has been characterized by powder X-ray diffraction (XRD), Fourier transform-infrared (FT-IR), Scanning electron micrographs (SEM), indicating that the drug has been successfully intercalated into LDH.

Fig.1

HPLC analyses of the crude extract of Eupatorium adenophorum Spreng. HPLC conditions: a Shimadzu ODS column (150 × 4.6 mm I.D.). Mobile phase: methanol: 0.05% phosphoric acid water (1:1, v/v), flow-rate: 0.5 mL/min, monitored at 254 nm by a DAD detector. Peak 2 = caffeic acid

Fig. 2

Preparative HSCCC separation of the crude extract of Eupatorium adenophorum Spreng with solvent system composed of ethyl acetate-methanol-water (10:1:10, v/v), stationary phase: upper organic phase of solvent system, mobile phase: lower aqueous phase of solvent system; flow-rate: 2.0 mL/min; revolution speed: 800 rpm; sample: 234.5 mg dissolved in 10 mL of lower phase of the solvent system. HPLC conditions: a Shimadzu ODS column (150 × 4.6 mm I.D.). Mobile phase: methanol: 0.05% phosphoric acid water (1:1, v/v), flow-rate: 0.5 mL/min, monitored at 254 nm by a DAD detector. Peak 2 = caffeic acid.

Fig. 3

XRD patterns of NO₃^-form of LDH (a) and synthesized caffeic acid–LDH hybrid (b).

Fig.4

Fourier transform-infrared spectra of NO₃^- form of LDH (a), caffeic acid (b) and caffeic acid–LDH (c)

Fig.5

Scanning electron micrographs of NO₃^- form of LDH (a) and caffeic acid–LDH hybrid (b).