3.2.1. Separation of Flavonoids by Paper Chromatography (34-38)
1. Dissolve about 1 g of the dried residue in 80% aqueous methanol (1 mL) and spot it on the lower right-hand corner of a sheet of Whatman 3 MM chromatographic paper (semipreparative). Use a flow of warm air for solvent evaporation between repeated applications of the solutions to the paper. The final spot, which appears deep purple when viewed under a 366-nm UV lamp, is about 2 cm in diameter and 8 cm from each edge of the paper.
2. Develop the chromatogram containing flavonoids in a descendent mode in the longest dimension into a chromatocab using TBA as the first solvent (see Note 4).
3. Remove the chromatogram from the cabinet, dry it, trim off the folded portion, and then refold it for descending chromatography in the second dimension with AcOH 15% solvent (see Note 5).
4. The dry two-dimensional developed chromatogram should be revealed under UV light alone and in the presence of ammonia fumes. It can be also be revealed with NA reagent in order to detect the ortho dihydroxylated system in the B-ring (see Note 6).
220.127.116.11. Fractionation of Total Extract in Different Polarity Solvents (37)
1. Partition the total flavonoid extract obtained (50-100 mL) between methanol/water (8:2) and chloroform (50-100 mL) in a separation funnel. Separate the chloroform phase (lower phase) and repeat the extraction three times with 25 and 12.5 mL, respectively. Concentrate the chloroform phases and store for antimicrobial assays on the rotary evaporator. This fraction contains chlorophyll, lipids, terpenoids, and less polar aglycones.
2. Add to the rest of the methanolic extract an equal volume of ethyl acetate in a separation funnel and proceed as in step 1. In this case the upper phase should be separated. Glycosides in this fraction, are mono- and diglycosylated in the B-ring.
3. The residue extract exhausted with solvents represents the aqueous fraction, which contains diglycosides of flavonoids, tryglycosides, sulphated flavonoids, alkaloids, amino acids, phenolic acids, and cumarines.
18.104.22.168. Preparative 2D Paper Chromatography Isolation, and Purification of Flavonoids
Cut out the area containing the flavonoid alone on a two-dimensional chromato-gram, about 20-50/sheet, to obtain sufficient material for identification of the flavonoid (see Note 7).
22.214.171.124. Partial Purification of Flavonoid: 1D Paper Chromatography
1. After a two-dimensional run (TBA and AcOH 15 or 30%), make a selection of the most effective solvent system to separate the mixture. The selected one will be the solvent to be used in one-dimensional paper chromatographic purification of the flavonoid.
2. Separate the flavonoid mixture from the two-dimensional run (about 20 sheets) by cutting out the related area, dissolve it in MeOH 80%, and apply it as a band (see Note 8).
3. A descending run in the selected solvent gives UV-detectable bands. Cut out the major one and elute it with spectroscopic methanol for a few minutes for spectral analysis according to the method developed by Mabry et al. (34).
3.2.2. Purification of Flavonoids by Column Chromatography (CC)
The classical chemical procedure for obtaining organic constituents from dried plant tissue, when working on the preparative scale, involves continuously extracted powdered material in a Soxhlet apparatus with a range of solvents, starting with n-hexane and chloroform and then using methanol, ethanol or ethyl acetate. The extracts obtained are concentrated in a vacuum; they may deposit crystals. Mixtures of substances present in those crystals must be redissolved in a suitable solvent and separated from the constituents by chromatography (37). We now describe a protocol for purification by CC.
Sephadex LH-20 is used for the final clean-up of flavonoid aglycones and glycosides previously separated by paper, cellulose, silica, or polyamide (see Note 9) (36). MeOH is generally an adequate solvent, although some water may be needed initially to dissolve the flavonoid. Presoaking of the gel in MeOH is necessary (37). To avoid a long and tedious run, an automated fraction collector could be used
Concentrate each cut-out spot from the paper on a rotary evaporator, dissolve the residue in a minimum of MeOH, vortex the mixture and purify the individual components as follows:
1. Apply 1 mL of the mixture to a 50 x 1-cm LH20 column equilibrated with MeOH and elute with decreased quantities of MeOH in water (9:1, 8:2, and 7:3) flowing down three column volumes for each system.
2. Elute the flavonoids from the column slowly (0.1 mL/min) to allow good resolution. If the band is not visible or detectable under UV light (366 nm), then analyze each fraction by TLC (silica G60 or cellulose) to determine which fractions should be combined. Run an analytical TLC sheet (4 x 10 cm) in a glass tank with 50 mL of suitable solvent (described in Subheading 2.3., items 3 and 7), previously equilibrated for 20 min. Remove the plates from the glass tank, mark the solvent front with a pencil, and allow them to dry in the fume hood for approx 15 min. Spray the plates with NA reagent or visualize the spots under UV light (366 nm) alone and under ammonia vapors.
3. Concentrate each eluate and test it for antimicrobial activity.
4. Each active fraction must be rerun in an LH20 column to isolate the active compound. Repeat steps 2 and 3.
When separation by the methods described above is not convenient for the type of flavonoid, then HPLC separation should be used. The technique described here is based on a previous procedure described by Corzier et al. (39) and the chromatographic conditions given in ref. 40 with modifications (31). Classical HPLC procedures for flavonoids are carried out after glycoside hydrolysis (39,40). However, the following methodology is for mono- and diglycosylated flavonol without previous hydrolysis.
1. After LH-20 column separation, concentrate the eluate in a rotary evaporator, resuspend it in the initial phase (15% B/85% A; 0.1 mg/L) and filter through a Millipore membrane before injection.
2. Inject 100 ^L of solution into the HPLC apparatus (Gilson) and run in a linear gradient 025 min of 15-35% B. The column is a C18 (Phenomenex). The solvents used are given in Subheading 2.3., items 11 and 12.
3. Monitor the flavonoids at 365 nm under a UV detector.
4. Concentrate the fractions containing pure flavonoids and prepare them for antimicrobial assays.
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