Flavonoids PDF

## II. Flavonoide ### II.1. Noțiuni teoretice The general name of flavonoids is attributed to all natural pigments in plants present in leaves, flowers and fruits that derive from benzo-y-pirone (chromone). The basic structure of these natural pigments is flavone, hence the name flavonoids. Antoc...

## II. Flavonoide ### II.1. Noțiuni teoretice The general name of flavonoids is attributed to all natural pigments in plants present in leaves, flowers and fruits that derive from benzo-y-pirone (chromone). The basic structure of these natural pigments is flavone, hence the name flavonoids. Antocyanins (from the Greek: anthos = flower; kyanus = dark blue) are ionic flavonoids that are part of the glycoside class; their aglycones (the part that does not contain hydrates of carbon) are called anthocyanidins and represent the pigments proper. Antocyanins are plant pigments, soluble in water that are found in the hydrophilic part of cells and are responsible for the different colors of flowers and fruits: red, violet, blue and all combinations of these. The various intermediate shades are obtained by combining the fundamental colors of a relatively small number of compounds from the class of anthocyanins. These compounds have practically the same basic structure, with a skeleton of C15 atoms (the flavilium cation), the various shades being determined on the one hand by the nature and position of the substituents, and on the other hand by the type and number of glycoside units linked to the hydroxyl groups. Antocyanin pigments are amphoteric, changing their color with the variation in pH: the acidic form is usually red, while the basic form is blue, while the neutral form (amphoteric) is violet. The color of the flowers, however, also depends on the presence of copigments (flavones, isoflavones, flavonols), as well as on the possibility of complexation by chelation with trivalent metal ions (Fe³⁺ and Al³⁺). Antocyanidins (aglycones of anthocyanins) contain as a basic nucleus the benzopyrilium cation, actually being derivatives of the flavilium cation (2-phenylbenzopyrilium) which presents extended conjugation: <start_of_image> Schematic of benzopyrilium cation and flavilium cation. The majority of anthocyanidins contain the cation of 3,5,7-trihydroxyflavilium: Schematic of 3,5,7-trihydroxyflavilium cation. The hydrates of carbon contained in anthocyanins in the form of glycosides (to the hydroxyl groups in the 3,5,7-trihydroxyflavilium cation) are: glucose, galactose, rhamnose, xylose or arabinose and di- or tri-saccharides. In most cases anthocyanins are glucosides or diglucosides. Glycoside units usually bind to the hydroxyl groups in positions 3 and/or 5. Some anthocyanin pigments may also have phenolic OH groups acylated with substituted p-hydroxycinnamic acids to the aromatic nucleus. General formula of anthocyanins: - R = H, p-coumaric acid - R = OH, caffeic acid - R = OCH3, ferulic acid **Flavones** (from the Latin: flavus = yellow) are natural pigments from plants, generally yellow in color, which can be found in petals, pollen and also in plant leaves. They are solids, yellow, soluble in water, alcohol and diluted solutions of acids or alkalis. Like anthocyanidins, they appear as glycosides, but in a much smaller proportion. The basic skeleton of flavonoids is flavone. The most known flavones are luteolin (chamomile, carrot leaves) and apigenin (parsley seeds). Flavones in acid medium form oxonium salts that, due to extended conjugation, are more intensely colored than free bases. In nature, flavones only appear in a non-protonated form, as free bases. Schematic of oxonium cation and free base flavone. **Flavonols** are natural pigments yellow like flavones and are widely spread in plants either as copigments alongside anthocyanins in petals, or in the leaves of higher plants. The basic compound of this class of natural dyes is a hydroxyl derivative of flavone: flavonol or 3-hydroxyflavone. Flavonols are found in plants as glycosides with a role of yellow colorants. In the UV-VIZ spectrum, they show two main absorption bands: band I between 350 and 390 nm and band II between 250 and 270 nm, both being used for their identification. Although over a hundred aglycones of flavonols are known, three of them are the most widespread: kaempferol, quercetin and myricetin. Among quercetin glycosides, the most known is quercetin-3-rutinoside, known under the pharmaceutical name of rutin and used in the treatment of capillary fragility in humans. Rutin is extracted for commercial use from the buckwheat plant, but it can also be found in magnolia flowers, tobacco leaves, tea leaves, rhubarb. **Flavanones** are not yet discovered in nature in a free state. By contrast, hydroxylated flavanones are found in nature both in a free state and as glycosides. In plants they coexist frequently with their corresponding flavones: hesperidin with diosmin in the bark of Zanthoxylum avicenneae (Sichuan pepper or Chinese pepper) and naringin with roifolin in the peel of Citrus aurantium (bitter orange). **Isoflavones** are hydroxylated derivatives of isoflavone (3-phenyl-4-chromone). They are colorants but less widespread than flavones, found either as free compounds or as glycosides. Some examples are daidzein and genistein. While anthocyanins are the pigments from Angiospermae and Gymnospermae, **betacyanins** are the pigments from Centrospermae. Betacyanins, unlike anthocyanins, are much less stable under acidic hydrolysis. The most known betacyanin is betanin, which is the major pigment from beetroots (Beta vulgaris). The aglycone of betanin is betanidin that is linked by a glycoside bond to glucose. Betacyanins also differ from anthocyanins because they are not completely metabolized during digestion by approximately 14% of the human population, leading to their removal in urine. The excreted urine is therefore colored, a phenomenon known as "beeturia". Schematic of betanidina and betanina. ### II.2. Aplicații experimentale #### II.2.1. Lucrarea 1. Extracția, separarea şi identificarea antocianidinelor Place fresh petals in a 50-100mL flask equipped with reflux condenser, add 10-15mL of 2M HCL and heat for 40 minutes at 100 ° C (reflux). The colored extract is cooled and then separated from the petals by decanting and, if necessary, the decanted solution is filtered using a glass filter, with a cotton wool cap. The cooled extract is placed into a separating funnel twice with 4mL of ethyl acetate for the removal of flavones. The layers of ethyl acetate are removed, and the remaining aqueous layer (the bottom layer) is heated in a crucible on a water bath at 80°C for about 3 minutes (until the ethyl acetate odor is no longer felt) to remove ethyl acetate traces. The colored pigment obtained is then extracted with 3mL of amyl alcohol (1-pentanol) in a separating funnel. The amyl alcohol layer (the top layer) is then concentrated in a small crucible on a water bath at boiling point (attention: prolonged heating destroys the pigment). The residue of anthocyanidins obtained in the crucible, once cooled, is dissolved in 5-7 drops of 1% solution of HCL in methanol, then it is covered with a watch glass and then separated by thin layer chromatography on cellulose or chromatographic paper (CH). For development, the Forestal mixture is used: concentrated HCL-AcOH-H2O 3:30:10. The standards of anthocyanidins, if not commercially available, can be easily obtained from the following sources: pelargonidin from geranium flowers or red radish skin, cyanidin from red beech leaves or blackberries, peonidin from peonies, delphinidin from blue cornflowers or eggplant skin, petunidin from black grapes or red wine, malvidin from hollyhock flowers. Antocyanidins can be chromatographically separated on silica gel using as eluent the mixture ethyl acetate:formic acid:2M HCL (85:6:9), but the color of the spots fades rapidly after separation. It is important to use standard silica gel, as traces of metals help to separate peonidin and malvidin from cyanidin and delphinidin. Two-dimensional thin layer chromatography can also be performed on microcrystalline cellulose, using as eluents the mixtures formic acid-concentrated HCL-water 10:1:3 and amyl alcohol-acetic acid-water 2:1:1. - Pigments and color, Rf(x100), Forestal, color in VIS, Amax[nm], MeOH-HCl | Pigment | Rf(x100), Forestal | Color in VIS | Amax[nm], MeOH-HCl | |-----------------|--------------------:|-----------------------|-----------------------| | Pelargonidin | 68 | Red-orange | 520 | | Cyanidin | 49 | Red-dark | 535 | | Peonidin | 63 | Magenta (red fuchsia) | 532 | | Delphinidin | 32 | Red-violet | 546 | | Petunidin | 46 | Red-violet | 543 | | Malvidin | 60 | Red-violet | 542 |