Betalains isolation

Until 2004, no betalain standards were commercially available and current products lack the required purity. Preparative isolation from plant material is laborious and costly and the resulting standard substances vary in relative humidity, crystal water, and salts, resulting in over- or under-estimation of pigment contents... 

The special pigments of Centrospermae have attracted considerable interest during the past 40 years the pigment composition of red beet has especially been the topic of many investigations. Initially the betalains of red beet were separated and isolated using paper electrophoresis (244) and column chromatography (216) their chemical structure was elucidated by means of chemical tests and spectroscopic methods (214). The traditional methods for the quantitative determination of betacyanins and betaxanthins in beet root were spectrophotometry, mainly the Nilsson method (216). More recently, HPLC has become the method of choice for the separation and quantification of beet pigments (247). 

Progress in the chemistry of betacyanins depended on the development of efficient methods for their isolation and separation. Betalains often occur as complex mixtures and are easily decomposed during the purification steps, which render the isolation of larger amounts of material for structural studies difficult. It is therefore understandable that some of the compounds described during the 1960s need reinvestigation by modem techniques. 

The first separations of individual betacyanins were carried out by paper chromatography (55). The observation that betacyanins migrate on paper electrophoresis at pH 2 to 7 toward the anode was used for analyses of the betalain pigments from red beet (36,37) and other plants (38). This method was subsequently applied by two groups (3,4) for the isolation of crystalline betanin from a crude pigment preparation from red beet (39). 

Reaction of indoline with 66 yields a more stable condensation product (76), which was isolated as its crystalline perchlorate salt (97) (Scheme 5). Saponification of 76 with concentrated HCl afforded the dicarboxylic acid (77), which was cleaved with aqueous ammonia to ( )-betalamic acid (3) and then characterized as its dimethyl ester (58). Interestingly, it was not possible to cleave ester 76 directly because of smooth oxidative aromatization to the neo derivative 78. This proves that the catechol unit is of no importance for the formation of neo compounds from betalains. 

Betalains.—The dihydropyridine moiety of the betalains, e.g. betanin (177), has been shown to be generated from dopa, modification of which entails extra-diol cleavage of its aromatic ring. The general assumption that betalamic acid (176) is an intermediate in the biosynthesis of the dihydopyridine fragment of the betalains is supported by its isolation from betalain-synthesizing plants. ... 

The concentrated extract is applied as a streak (S cm) on a cellulose plate (0.1 mm, Merck) and developed twice (2 x IS cm) with 2-propanol-ethanol-water-acetic acid (6 7 6 1) as the mobile phase. The plate is dried in a nitrogen stream and five sharply yellow bands (Rf- 0.66,0.S6,0.48, 0.43, and 0.36) are observed. The bands with values at 0.48 and 0.43 are tentatively identiHed as vulgaxanthin I and vulgaxanthin n, respectively. The violet betanin band appears at Rj= 0.21 and some trailing may be observed. For semipreparative isolation of betalains in beetroot, homemade cellulose plates (0.3 nun, Macherey Nagel 300) give similar results. 

Approximately 1500 colored compounds, also known as natural food pigments, have been isolated from foodstuffs. On the basis of their chemical structure, these food pigments can be grouped in the following six classes heme pigments, chlorophylls, carotenoids, flavonoids, betalains, and miscellaneous pigments. 

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