FLAVONOIDS - Chemistry, Biochemistry and Applications

Introduction

Extraction

However, the composition of the extract varies with the solvent - be it water, methanol, ethanol, acetone or ethyl acetate. Extraction is typically performed with magnetic stirring or shaking, but other methods have recently been introduced to increase the efficiency and speed of the extraction procedure. There is improved diffusivity of the solvent and at the same time there is the possibility to work under an inert atmosphere and with protection from light.

The lower viscosities and higher diffusion rates of supercritical fluids compared to liquids are ideal for the extraction of diffusion-controlled matrices such as plant tissues. The advantages of the method are lower solvent consumption, controlled selectivity, and less thermal or chemical degradation than methods such as Soxhlet extraction. Many applications have been reported in the extraction of natural products, with supercritical carbon dioxide being the most widely used extraction solvent.12,13 However, the extraction of polar compounds such as flavonoids requires the use of polar solvents (such as methanol). added as modifiers.

Ultrasound-assisted extraction is a rapid technique that can also be used with immiscible solvent mixtures: for example, hexane with methanol–water (9:1), is a system used for the Brazilian plant Lychnophora ericoides (Asteraceae). Microwave-assisted extraction (MAE) has been described for the extraction of various compounds from various matrices.16 It is a simple technique that can be performed in minutes.

Preparative Separation

Preliminary Purification

The use of PLE gave better results than maceration – in addition, shorter extraction times and smaller amounts of solvent were required.10 PLE of grape skins and skins from winery waste proved to be an efficient process for obtaining catechin and epicatechin with little degradation, provided the temperature is maintained under 1308C.11. As the name suggests, supercritical fluid extraction (SFE) is based on the dissolution properties of supercritical fluids. The hexane phase concentrated the less polar sesquiterpene lactones and hydrocarbons, while the aqueous alcohol phase concentrated the flavonoids and more polar sesquiterpene lactones.15.

Preparative Methods

High-Performance Liquid Chromatography
Medium-Pressure Liquid Chromatography
Centrifugal Partition Chromatography

The purpose of this chapter is not to describe the technique and instrumentation in detail, but to demonstrate the use of HPLC in the preparative separation of flavonoids. MPLC columns have a high loading capacity — up to a 1:25 ratio of sample to packing material32 — and are ideal for the separation of flavonoids. Various counter-current chromatographic techniques have been successfully used to separate flavonoids.7 Counter-current chromatography is a separation technique based on partitioning a sample between two immiscible solvents, with the relative proportion of solute passing into each of the two phases being determined by distribution coefficients of the solute components.

Countercurrent distribution, droplet countercurrent chromatography and rotational locular countercurrent chromatography are now rarely used, but CPC, also known as centrifugal countercurrent chromatography, finds extensive application for the preparative separation of flavonoids. The leaves of the African plant Tephrosiavogelii (Leguminosae) were first extracted with dichloromethane and then with methanol. Methanol extract (500 mg) was injected into a mixture of the two phases of the solvent system and elution of the three major glycosides was achieved within 3 hours.58.

Final purification of the constituent flavonol glycosides, stilbenes and catechins was done by gel filtration and semi-preparative HPLC.20. Four pure isoflavones were obtained from a crude soybean extract after CPC with the solvent system CHCl3–MeOH–H2O (4:3:2) (Figure 1.2).

FIGURE 1.1 Separation of flavonol glycosides from Tephrosia vogelii (Leguminosae) with a Quattro CPC instrument

Analytical Methods

Sample Preparation

Remove possible interferents (for either the separation or detection stage) from the sample, thereby increasing the selectivity of the analytical method. The goal of sample preparation is for the components of interest to be extracted from complex matrices with the least time and energy consumption, but with the highest efficiency and reproducibility. Conditions should be mild enough to avoid oxidation, thermal degradation and other chemical and biochemical changes.

In addition to typical sample preparation methods, such as filtration and liquid-liquid extraction,61 newer developments are now widely used. Interfering sugars can be eluted with aqueous methanol on reverse phase columns prior to elution of flavonoids with methanol. Sep-Pak C18 cartridges have been used for the fractionation of flavonol glycosides and phenolic compounds from cranberry juice into neutral and acidic fractions before HPLC analysis.63 Antimutagenic flavonoids were identified in aqueous extracts of dry spinach after removal of lipophilic compounds by SPE.64.

Few applications have been reported for polyphenols, but simpler phenols have been extracted with this method, albeit with addition of methanol to the supercritical fluid.13 Some potential can be found for online SFE, as very clean extracts (but at low extraction efficiency for phenolic compounds) can be obtained.67.

Thin-Layer Chromatography

Widely distributed flavonoid aglycones, such as apigenin, luteolin and quercetin, can be separated in chloroform-. A system widely used for flavonoid glycosides is ethyl acetate-formic acid-glacial acetic acid-water. By adding ethyl methyl ketone (ethyl acetate-ethyl methyl ketone-formic acid-.glacial vinegar-water), rutin and vitexin-2''-O-rhamnoside can be separated.71 Careful choice of solvent system also allows separation of flavonoid glucosides from their galactoside analogues.72 This is particularly important for the distinction between C-glucosides and C-galactosides.

As an illustration, 8-C-glucosylapigenin (vitexin) can be separated from 8-C-galactosylapigenin with a solvent of ethyl acetate-formic acid-water. In terms of detection, a short exposure of the TLC plate to iodine vapor produces yellow-brown spots on a white background. One of the most important of these is a "natural product reagent" that produces intense fluorescence under 365 nm UV light after spraying with a 1% solution of diphenylboric acid-b-ethylamino ester (diphenylboryloxyethylamine) in methanol.

Further details on the use of the "natural products reagent" can be found in an article by Brasseur and Angenot.73. This can be used, for example, in the case of hawthorn extracts (Crataegus monogyna and C. laevigata, Rosaceae), which contain flavone O-glycosides and flavone C-glycosides or passion flower extracts (Passiflora incarnata, Passifloraceae), which contain only flavone C-glycosides.

High-Performance Liquid Chromatography

Isoflavones, found mainly in Leguminosae (such as soy, Medicago sativa and red clover, Trifolium pratense) in the plant kingdom, are also successfully analyzed by HPLC on C18 columns.76. Flavylium cations are colored and can be selectively detected in the visible range at approx. 520 nm, thereby avoiding interference from other phenols and flavonoids that may be present in the same extracts. For aglycones and glycosides of isoflavones, some reported separations of soybean products (eg, the work of Barnes et al.82) have used C8 packings, but these are rare.

Information can also be obtained for simple polyphenols in the presence of more complex polycyclic polyphenols. In general, enriched ginkgo extracts for the preparation of ginkgo products are standardized to contain 24% flavonoids and 6% terpenes. Quality control fingerprint analysis of Ginkgo preparations has shown that it is possible to separate six flavonoid aglycones, 22 flavonoid glycosides and five biflavonoids in one run (Figure 1.4).

This was the procedure adopted for the analysis of white onions (Allium cepa, Liliaceae) and stalks of white celery (Apium graveolens, Apiaceae). In the analysis of the non-hydrolyzed celery extract (isorhamnetin as internal standard), several peaks were observed.

FIGURE 1.3 HPLC profile for 28 different polyphenols on a C 18 column. Classes of compound are shown in the upper part of the chromatogram

High-Performance Liquid Chromatography–Ultraviolet Spectrophotometry. 16

This improves the quantification possibilities because detection can be performed at the maximum wavelength of the compound in question. LC–UV is valuable for the identification of isoflavones because their spectra differ in absorption properties from most other flavonoids. It was found that for the complete characterization of phenolic compounds it is possible to use reagents that cause a shift of the UV absorption maxima.1.

In combination with shift reagents added after the column, LC-UV made it possible to determine the hydroxylation pattern of flavonols and the position of the sugars on the aglycones. A similar procedure was applied for the identification of the other flavonol glycosides - which show the presence of three different aglycones: kaempferol, quercetin and myricetin. Thermospray LC-MS provided additional information on the molecular weight of the flavonol glycosides and their aglycones.38.

Coupled HPLC-MS is one of the most important techniques of the last decade of the 20th century. Online accurate mass measurements of all MS-MS fragments were obtained on the Q-TOF instrument, while the multistage MSn capability of the IT was used to prove fragmentation pathways.

FIGURE 1.5 Flavonoid glycosides isolated from the aerial parts of Epilobium angustifolium.

High-Performance Liquid Chromatography–Nuclear Magnetic Resonance

Thus, liquid LC-NMR procedures are mostly limited to the direct measurement of the main components of the crude extract and this is often under congested HPLC conditions. The obtained LC-NMR information is derived from the 1H NMR spectra of selected peaks in the HPLC chromatogram. From LC-MS, the A or B ring substitution can be deduced from the fragmentation pattern, but the exact location of the substituent cannot be determined.

In order to obtain further information for characterization of the polyphenols in the extract, LC–NMR was performed under the same conditions used for LC–UV–MS. Thus, LC–UV–MS and stopped-flow LC–NMR were not sufficient to fully establish the structure of 5 . In order to determine the position of C-glycosylation, an LC–MS–MS experiment was performed by selecting [MþH120]. þas parent ion.

If full metabolite profiling of a plant extract is to be performed, LC-NMR can be run in on-flow mode. Applications of LC-NMR for online identification of flavonoids are still few and far between, one reason probably being the high cost of the apparatus.

FIGURE 1.11 Online mass spectra of isoorientin (1) and orientin (2) in the TSP mode. The respective TSP MS–MS (CID) analyses of the parent [M þ H 120] þ (m/z 329) ions are also shown.

Capillary Electrophoresis

Outlook

Marchart, E., Krenn, L., and Kopp, B., Quantification of the flavonoid glycosides in Passiflora incarnata by capillary electrophoresis, Planta Med. In the aromatic region of the spectra, some 2JCH couplings may be too small to be detected as cross peaks. The recent molecular characterization of the LAR and ANR was a major advance in the understanding of PA biosynthesis.

F2H was postulated to be involved in the formation of the common flavones after its identification from G.