Flavonoid acetates

Flavonoid acetates and methyl ethers have been separated by WEmoES and Toribio [236] using solvent II (Table 166). Catechin-resorcinol condensation products in particular can be resolved in this way [236]. Catechin acetate diastereoisomers and the acetates of oUgomeric flavonoid tannins such as the acetates of dicatechin and anhydro-catechin can be thus detected with certainty [56]. The acetates of the more highly condensed compounds remain at the start point. Plant extracts containing catechins and flavonoid tannins are best acetylated initially by 15 h treatment with acetic anhydride/absolute pyridine at room temperature. Solvent II or chloroform-ethyl acetate (90 + 10) can be used with silica gel columns for preparative separation of such mixtures. 

To detect glycosides heat the chromatograms to 130—150°C for 15 min. Blue-grey zones are produced (detection limit prunasin 0.3—0.5 pg [18]). Flavonoids are better detected with a modified reagent of the following composition phosphoric acid (85%) — acetic acid — aniline — diphenylamine (20 ml - -100 ml + 5 ml + 5 g). 

Lead(II) acetate yields colored lead salts with flavonoids and thiourea derivatives. 

Males et al. [103] used aqueous mobile phase with formic acid for the separation of flavonoids and phenolic acids in the extract of Sambuci flos. In a cited paper, authors listed ten mobile phases with addition of acids used by other investigators for chromatography of polyphenolic material. For micropreparative separation and isolation of antraquinone derivatives (aloine and aloeemodine) from the hardened sap of aloe (Liliaceae family), Wawrzynowicz et al. used 0.5-mm silica precoated plates and isopropanol-methanol-acetic acid as the mobile phase [104]. The addition of small amounts of acid to the mobile phase suppressed the dissociation of acidic groups (phenolic, carboxylic) and thus prevented band diffusions. 

The sugars attached to the anthocyanin molecule are in order of relative abundance glucose, rhamnose, galactose, xylose, arabinose, and glucuronic acid. The molecule may also contain one or more of the acyl acids p-coumaric, caffeic, and ferulic or the aliphatic acids malonic and acetic esterified to the sugar molecules. Extracts of anthocyanins invariably contain flavonoids, phenolic acids, catechins and polyphenols. The net result is that it is impossible to express the chemical composition accurately. Specifications usually present tinctorial power, acidity, per cent solids, per cent ash and other physical properties. 

Normal-phase TLC using a silica stationary phase was employed for the optimization of the separation of flavonoid content of Matricariae flos (Chamomilla recutita L. Rauschert). Air-dried and powdered plant material was extracted by refluxing for 10 min with methanol. The suspension was filtered, evaporated and the residue was redissolved in methanol. The mobile phases included in the experiments were 1 = ethyl acetate-methylethylketone-formic acid-water (50 30 10 10, v/v) 2 = ethyl acetate-methanol-water (75 15 10 v/v) 3 = ethyl acetate-formic acid-water (80 10 10, v/v) 4 = ethyl acetate-formic acid-water (100 20 30, v/v) 5 = ethyl acetate-formic acid-acetic acid-water (100 11 11 27, v/v) 6 = n-butanol-acetic acid-water (66 17 17, v/v) 7 = ethyl acetate-methanol-formic acid-water (75 10 5 10, v/v) 8 = ethyl acetate-acetic acid-water (80 10 10, v/v). Development was carried out in the linear ascending mode at... 

A new HP-TLC method has been applied for the quantitative analysis of flavonoids in Passiflora coerulea L. The objective of the experiments was the separation and identification of the compound(s) responsible for the anxiolytic effect of the plant. Samples were extracted with 60 per cent ethanol or refluxed three times with aqueous methanol, and the supernatants were employed for HPTLC analysis. Separation was performed on a silica layer prewashed with methanol and pretreated with 0.1 M K2HP04, the optimal mobile phase composition being ethyl acetate-formic acid-water (9 1 l,v/v). It was established that the best extraction efficacy can be achieved with 60 - 80 per cent aqueous methanol. The HPTLC technique separates 10 different flavonoids, which can be used for the authenticity test of this medicinal plant [121],... 

A simple TLC method has been developed for the separation and identification of flavons and flavon glycosides in the extract of Phillyrea latifolia L. The leaves (100 g) were defatted in 11 of chloroform for 24 h and then extracted with 2 X 11 of ethanol-water (80 20, v/v). The collected extracts were concentrated and extracted again with n-hexane to remove chlorophylls and other apolar constituents. Analytes were extracted with ethyl acetate. Both normal phase and RP-TLC have been used for the separation of flavonoids. The results are compiled in Table 2.36. It was concluded from the data that TLC can be successfully applied for the quality control of plant extracts containing various flavone derivatives [124],... 

TLC Si 60 Ethylacetate-formic acid-glacial acetic acid 100+11 + 11+26 Flavonoids Ca, Ra... 

The retention behaviour of flavonoids has also been extensively studied on silica stationary phases using heptane, benzene or dichloromethane as weaker components of the binary mobile phase and ethyl acetate and methyl ethyl ketone as modifier. Flavones (3-hydroxy, 5-hydroxy and 7-hydroxyflavone, tectochrysin, chrysin, apigenin, genkwanin, baicalein), flavonols (galangin, pilloin, kaempferol, rhamnetin, quercetin, robinetin,... 

Using numerical taxonomy it was found that the best separations were obtained by chloroform-methanol-(98-100 per cent) formic acid (44.1 3 2.35) and n-hexane-ethyl acetate-glacial acetic acid (31 14 5, v/v) as mobile phases. As the flavonoid profile of the propolis samples showed considerable differences, the method has been proposed for the authenticity test and traceability of various propolis products [141]. 

RP-HPLC has been employed for the determination of flavonoids and other phenolic compounds in cranberry juice. The neutral and acidic analytes were preconcentrated octadecyl silica SPE cartridges conditioned with distilled water (neutral analytes) or with 0.01 M HC1 (acidic compounds). Hydrolysis of samples was carried out in aqueous methanol solution acidified with 6 M HC1 at 35°C for 16h. Chromatographic separation was performed in an ODS column (150 X 4.6mm i.d. particle size 5/.an). Solvents A and B were water-acetic acid (97 3, v/v) and methanol, respectively. The gradient started with 0 per cent B (flow rate, 0.9 ml/min), reached 10 per cent B in lQmin (flowrate, 1.0 ml/min) and increased to 70 per cent B in 40min (flowrate, 1.0 ml/min). Analytes were detected at 280 and 360 nm. Some typical chromatograms are presented in Fig. 2.71. The concentrations of flavonoids and phenolic acids are compiled in Table 2.69. It was stated that the SPE-HPLC procedure makes possible the simultaneous determination of phenolic compounds and flavonoids, therefore, it can be employed for the measurement of these classes of analytes in other fruit juices [188],... 

As the separation characteristics of liquid chromatographic and electrophoretic techniques markedly differ from each other, combined methods using the advantages of both procedures have been successfully used for the analysis of flavonoids. Thus, the use of CZE-UV, HPTLC-UV and GC-MS for the measurement of flavonoids in seeds and root exudates of Lotus pedunculatus has been reported. The rooting solution and seed exudate were passed through cellulose acetate filters to bind the flavonoids. After extraction,... 

In the meantime, all of the higher intermediates shown in Fig. 2 have been tested and numerous comprehensive surveys of this work have appeared (24, 81—83, 91, 93, 112, 113, 132). Some of these simultaneously describe the formation of secondary aromatic substances in wood, i.e. lignans, tannins, flavonoids, etc., which arise by essentially similar routes coupled with acetate metabolism. A few outstanding recent developments may bear repetition here. 

The protective method has also been employed with 3-ketoesters. In this case, the goal is to avoid keto-enol photoisomerization that is an efficient energy-wasting channel. Scheme 74 shows that direct photorearrangement of aryl benzoyl acetates (298) to the ort/jo-hydroxydibenzoylmethanes (299) is poor, whereas irradiation of the related acetal derivatives gives higher yields [208]. The resulting ort/ o-hydroxydibenzoylmethanes are precursors for the synthesis of flavones. Related flavonoids can be obtained in similar yields by PFR of aryl dihydrocinna-mates [209]. 

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