High-performance liquid chromatographic determination of oligomeric procyanidins from dimers up to the hexamer in hawthorn
Introduction
Hawthorn (Crataegus sp.) extracts have beneficial effects on the heart and blood circulation including cardiovascular protective and hypotensive effects [1], [2], [3]. Oligomeric procyanidins are considered to be the main active constituents, in addition to flavone- and flavonol-type flavonoids [4]. Procyanidins or condensed tannins including flavan-3-ol units, epicatechins and/or catechins (Fig. 1), can be categorized as oligomeric procyanidins (OPs) consisting of 2–6 flavanol units, and polymeric procyanidins (PPs) consisting of more than six flavanol units [5]. The presence of procyanidins up to the hexameric level has been reported in hawthorn extracts [6].
Procyanidins are widespread in nature as secondary metabolites [5], and they have also been found in commonly consumed foods [7]. Polyphenolic procyanidins have received attention owing to their antioxidant and radical scavenging activities [5], while polyphenolics with a low degree of polymerization increase coronary flow and cardiac contractility, and decrease blood pressure [2]. Many of the properties of procyanidins are based on their ability to form complexes with proteins and polysaccharides. They can undergo both intra- and intermolecular hydrogen bondings [5].
The standardization of hawthorn plant material and preparations is performed by determining the concentrations of either the flavonoids or oligomeric procyanidins [4]. Procyanidins are determined by colorimetric methods as cyanidinchloride [8] or as phenols [9], but these methods do not reveal the concentrations of individual oligomers but total concentrations. Rohr et al. [10] developed a high-performance liquid chromatographic (HPLC) method for the quantitative analysis of procyanidin dimers (B-2, B-4 and B-5) and procyanidin trimer C-1 in hawthorn leaves and flowers. However, larger procyanidins have not been quantified. In contrast to the case for flavonoids [11], the procyanidin content in Crataegus sp. is mainly unknown.
There is considerable interest in the procyanidin contents of plants and foods, but the analyses are difficult to perform due to the structural diversity of OPs and the lack of procyanidin standard substances commercially available. Determinations of individual OPs, including dimers and trimers, have mainly been performed by HPLC under reversed-phase conditions [10], [12], [13], [14]. Procyanidins have also been separated according to their degree of polymerization, without actually separating individual compounds, by normal-phase HPLC–mass spectrometry (MS) [15], [16]. However, HPLC–MS has not proved suitable for the quantitative determination of OPs in hawthorn, because ionization of the OPs under the same experimental conditions is different. Therefore, an unresolved question is, which ion should be selected for the quantitative analysis [6]. Quality control and standardization of hawthorn preparations and plant material have been found to be inadequate because the structures and concentrations of OPs in hawthorn are largely not known. Physiological activities of individual compounds in relation to their polymerization degrees are also unknown. Therefore, suitable methods for determining individual procyanidins are needed. The main objective of this study was to develop an HPLC method using UV diode array detection for determining OPs in hawthorn plant material and raw extracts in order to obtain detailed information on the oligomeric procyanidin profiles and the concentrations of oligomers up to the hexamer present in hawthorn.
Section snippets
Materials
The dried flowers, leaves and fruits of hawthorn (Crataegus laevigata) were kindly donated by Flachsmann, Germany. The oligomeric procyanidins used in this work were isolated from the leaves and flowers of hawthorn, and the structures of the isolated procyanidins were determined by means of spectroscopic and chemical hydrolysis methods [17]. The isolated compounds included procyanidin dimers epicatechin-(4β→8)-epicatechin (B-2), catechin-(4α→8)-epicatechin (B-4), epicatechin-(4β→6)-epicatechin (
Preparation of the sample solution
The hawthorn extracts contain, in addition to OPs, various phenolic compounds such as phenolic carboxylic acids, flavonoid glycosides and polymeric procyanidins. Because it was not possible to analyse all the compounds in one HPLC run, the phenolic compounds were fractionated. Fraction P3 contained oligomeric procyanidins from dimers up to hexamer. Part of the dimeric procyanidins were eluted together with the flavonoid glycosides and phenolic acids in fraction P2, and the dimers were then
Conclusions
The present HPLC method can be used for the qualitative and quantitative analysis of a complex series of oligomeric procyanidins of hawthorn up to the hexameric level. The compounds included procyanidin dimers epicatechin-(4β→8)-epicatechin (B-2), catechin-(4α→8)-epicatechin (B-4), epicatechin-(4β→6)-epicatechin (B-5), procyanidin trimers epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin (C-1), epicatechin-(4β→8)-epicatechin-(4β→6)-epicatechin (trimer II),
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