Chemical analysis of the chamomile mucilage

The extraction of chamomile flowers with 96% ethanol precipitates large amounts of (natural) mucilage. The mucilage has to be demineralized in order to determine its viscosity and identify individual polysaccharide components. This can be done using amberlite IR-120, an acidic cation exchanger, and amberlite IR-45, a basic anion exchanger. Comparative hydrolytic tests [120] showed that hydrolysis with trifluoroacetic (TFE) acid (1 ml of 1% solution of chamomile mucilage + 1 ml 4N TFE, boiled for 30 min) is the most suitable method. Table 10.7 lists several solvent systems for the TLC separation of monosaccharides and urone acids.

Good separations of the monosaccharides and urone acids are shown in Figures 4.28 and 4.29 in Reference 121, page 92.

A selection of spray reagents is summarized in Table 10.8. On heating the plates, color reactions occur. However, for densitometric quantification, only immersed plates yield reproducible results.

Franz et al. [25, 26] extracted the polysaccharides with cold water for 7 hours, using chamomile flowers that were pre-extracted with petrol ether and methanol, followed by precipitation with ethanol (final ethanol concentration 80% G/G). For the fractionation of the polysaccharides, both ion exchange and gel permeation chromatography (GPC) are recommended. Ion exchange chromatography is performed on DEAE-Sephacel columns in phosphated form by successive elution with water/phosphate buffer (0.25/0.5/1.0 M) and water/NaOH (0.2 M). The polysaccharide fractions are detected via an anthrone test, dialyzed (MWCO 3500 D), and lyophilized. The latter is performed by medium

Hplc Chamomile

FIGURE 10.8 Reversed phase HPLC and UV/Vis spectra of an aqueous extract of fresh Matricaria chamo-milla. A = flowers, B = leaves (eluent: diluted phosphoric acid, pH = 2,8/-acetonitrile; detection: 337 nm). 1 = chlorogenic acid, 2 = caffeic acid, 3 = umbelliferone, 4 = hydroxy-cinnamic acid derivate, 5 = luteolin-7-glucoside, 6 = apigenin-7-glucoside, 7 = herniarin, 8 = apigeninglucoside (not specified), 9 = apigenin-7-(6"-0-acetyl)-glucoside, 10 = apigenin, 11 = cis En-In-Ether, 12 = trans En-In-Ether.

FIGURE 10.8 Reversed phase HPLC and UV/Vis spectra of an aqueous extract of fresh Matricaria chamo-milla. A = flowers, B = leaves (eluent: diluted phosphoric acid, pH = 2,8/-acetonitrile; detection: 337 nm). 1 = chlorogenic acid, 2 = caffeic acid, 3 = umbelliferone, 4 = hydroxy-cinnamic acid derivate, 5 = luteolin-7-glucoside, 6 = apigenin-7-glucoside, 7 = herniarin, 8 = apigeninglucoside (not specified), 9 = apigenin-7-(6"-0-acetyl)-glucoside, 10 = apigenin, 11 = cis En-In-Ether, 12 = trans En-In-Ether.

pressure-GPC on HiLOAD 16/50 Superdex 75 or 200 columns. The fractions of polysaccharides were detected with an RI detector (e.g., ERC 7512 Benthron Scientific), dialyzed, and lyophilized. The determination of molecular weights was performed in the same GPC system [25].

10.5.1 Quantitative Spectral Densitometric Determination of the Monosaccharides and Urone Acids

Spots of samples (concentration approx. 5 |g) and the sugar test solutions were applied on the TLC plate (silica gel 60 plates Merck) by microcaps. The test substances were dissolved in 10% of isopropanol and diluted to the final concentrations of 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 |g/|l. Galacturonic and glucuronic acid were used in concentrations ranging from 0.5 to 1.25 |g/|l [121].

The TLC plates were developed twice over a maximum length of 10 cm each. The solvent system was ethyl acetate-isopropanol-glacial acetic acid-water (60 + 30 + 5 + 5). After thorough drying of the TLC plates, the spots were detected by immersion in Scheffer-Kickuth reagent for 5 sec. The plates were dried and heated for 8 min at a temperature of 120°C, immediately followed by in situ measurement. Instrument parameters were as follows:

Apparatus: Zeiss chromatogram spectral photometer KM3

Wavelength: 385 nm

Gap width: 0.5

Gap measuring head plate: 2.5

Table speed: 200 mm/min.

Recorder 120 mm/min.

Evaluation: F = h x bh/2 (h = peak height, bh/2 = peak width at half level) or by evaluation of the peak height

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