Betacyanins are plant-based dyes with potential as histological stains

Interest is increasing in certain parts of the world in replacing synthetic dyes with dyes from natural sources, particularly from plants. Although textile dyers have used various groups of natural dyes, microscopists generally have restricted their use to anthocyanins. Recently, however, another class of plant-based dyes has found some favor, the betacyanins. Betacyanins are a group of red and violet betalain dyes found only in certain plants of the order Caryophyalles and in Basidiomycetes mushrooms. Although the chemical structures of betacyanins are known, little use has been made of that information to understand or predict their behavior with biomedical specimens. We investigated two common, widely distributed betacyanin-containing plants, edible beets (Beta vulgaris) and wild pokeweed (Phytolacca americana). Aqueous alcoholic extracts were made from beet root and pokeweed berries, adjusted to pH 4.1 or 5.3 and used together with Harris' hematoxylin to stain histological sections. We used a methanolic extract of pokeweed berries, pH 3.0, to stain cultured mycological specimens. Both extracts produced satisfactory staining that was equivalent to that of eosin Y, although the colors were more muted with the beet root extract. Epithelial cytoplasm, muscle, collagen and erythrocytes were well demonstrated. Betanin is the predominant component of beet root extract; it possesses one delocalized positive charge and three carboxylic acid substituents. The dyes are weak acids and the carboxylate anions are more diffuse than for eosin Y; this produces weaker bonding to tissue cations. The principal colored component of pokeweed berries, prebetanin, possesses a sulfonic acid group as well as carboxylic acids, which favors acid dyeing and more intense coloration. Both dyes show potential for hydrogen bonding and to a much lesser extent for some types of van der Waals forces. Complex formation with metals such as aluminum to create a nuclear stain is not likely with beet root dyes nor is it possible with pokeweed dyes. Betacyanins are suitable for staining microscopy preparations in place of other red acid dyes such as eosin. Of the two dyes tested here, prebetanin from pokeweed berries was superior to betanin from red beet roots. These berries are widely distributed and readily collected; the extraction procedure is simple and does not require expensive solvents.