Natural Product Research Center, KIST Gangneung Institute, Techno Valley, Gangneung, Gangwon 210-340, Korea.
Phytochem Anal. 2010 Sep-Oct;21(5):457-62. doi: 10.1002/pca.1218.
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.
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).
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.
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)].
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.
蓝莓（Vaccinium 属）因其果实中含有高浓度的花青素而受到全球关注。相比之下，蓝莓的叶子却没有引起任何关注，尽管它们含有大量的绿原酸，这是一种很强的抗氧化化合物。
本研究旨在采用一种新的分离方案——离心分配色谱（CPC），从蓝莓叶中定量和制备分离绿原酸（5-咖啡酰奎宁酸，5-CQA）。
通过对蓝莓叶粗甲醇提取物进行脱脂，得到一种含有高浓度 5-CQA（占干重提取物的 14.5%）的水相。采用乙酸乙酯-乙醇-水（体积比 4:1:5，v/v/v）两相溶剂体系，通过 CPC 从该水相分离 5-CQA。流动相的流速为 2 mL/min，以 1200 rpm 的转速进行上行模式。洗脱液在 330nm 处监测。用 HPLC、UV、ESI/MS 和 NMR 谱图确认 CPC 馏分中绿原酸的结构。
HPLC 色谱图表明，通过 CPC 收集的馏分中含有绿原酸，其纯度基于峰面积百分比为 96%。从 0.5g 水相分离得到的绿原酸总量为 52.9mg，相当于水相的 10.6%。分离得到的化合物通过 MS（母离子在 m/z 355.1 M + H）和（1）H NMR 谱[咖啡酰部分在低场（δ 6.0-8.0ppm）和奎尼酸部分在高场（δ 2.0-5.5ppm）]依次鉴定为 5-CQA。
通过 CPC 方法一步法成功地从蓝莓叶中分离出 5-CQA，表明蓝莓叶有进一步的潜在用途。
