Preparative Separation of Chlorogenic Acid by Centrifugal Partition Chromatography from Highbush Blueberry Leaves (Vaccinium Corymbosum L.)

Introduction: Blueberries (genus Vaccinium) have gained worldwide focus because of the high anthocyanin content of their fruits. In contrast, the leaves of blueberry have not attracted any attention, even though they contain large quantities of chlorogenic acid, a strong antioxidant compound.

Objective: The aim of this investigation was the quantification and preparative isolation of chlorogenic acid (5-caffeoylquinic acid, 5-CQA) from blueberry leaves using a new separation scheme, centrifugal partition chromatography (CPC).

Methodology: A water fraction containing a high concentration of 5-CQA (14.5% of dry weight extract) was obtained by defatting a crude methanol extract from blueberry leaves. CPC was applied to isolate 5-CQA from this water fraction using a two-phase solvent system of ethyl acetate-ethanol-water at a volume ratio 4:1:5 (v/v/v). The flow-rate of mobile phase was 2 mL/min with the ascending mode while rotating at 1200 rpm. The eluate was monitored at 330 nm. The structure of chlorogenic acid in the CPC fraction was confirmed with HPLC, UV, ESI/MS and NMR spectra.

Results: The HPLC chromatogram showed that the fractions collected by CPC contained chlorogenic acid with 96% purity based on peak area percentage. The total amount of chlorogenic acid isolated from 0.5 g of a water fraction was 52.9 mg, corresponding to 10.6% of the water fraction. The isolated compound was identified successively as 5-CQA with MS (parent ion at m/z 355.1 [M + H](+)) and (1)H NMR spectra [caffeoyl moiety in the down field (δ 6.0-8.0 ppm) and quinic acid moiety in the up field (δ 2.0-5.5 ppm)].

Conclusion: 5-CQA was successfully isolated from blueberry leaves by the CPC method in a one-step procedure, indicating a further potential use for blueberry leaves.