,„„ FLA VONOIDS AND RELATED COMPOUNDS 



lyb 



It seems to be derived from a condensation of two flavonoids but is peculiar in having no 

 hydroxylation on either B ring or C3 chain and in having an added methyl group on one of 

 the A rings. Several other bis-flavonols have been isolated from leaves of Ginkgo biloba 

 (40). Other types of bisflavonoids are discussed by Kawano (41). 



ISOLATION 



Many compounds of this group are water-soluble, especially in the glycoside forms, 

 and they are therefore present in aqueous plant extracts. Even those which are only 

 slightly soluble in water are sufficiently polar to be well extracted by methanol, ethanol 

 or acetone; and these are the solvents most frequently used for extraction of the flavonoids. 

 Re-extraction of an aqueous solution with an immiscible but rather polar organic solvent 

 is frequently of value in separating this group from more polar compounds such as carbo- 

 hydrates. Ethyl acetate is a useful solvent for dealing with catechins and leucoanthocy- 

 anidins in this way. Benzene can be used for benzophenones and stilbenes. Amyl alcohol 

 has been extensively used for the anthocyanins. Secondary butyl alcohol is the most polar 

 alcohol to be incompletely miscible with water; and if the aqueous extract is saturated with 

 sodium chloride or magnesium sulfate, it is very successfulfor removing compounds of 

 this group. Polyphenolic substances such as these are quite sensitive to air oxidation in 

 neutral and basic solution so that it is a good practice to prepare extracts in the presence 

 of a dilute acid (e.g. 0. 1 N HCl). However, hot acid or long-standing with acid in the cold 

 may cause hydrolysis of glycosides. 



Classically, various precipitating reagents have been used for these compounds. 

 Neutral or basic lead acetate has been particularly recommended. Flavonoids can be 

 freed from the lead precipitate by adding dilute sulfuric acid or hydrogen sulfide leaving 

 the lead as insoluble lead sulfate or sulfide. Other precipitating agents have been picric 

 acid, potassium acetate, barium hydroxide, pyridine, etc. These methods have been de- 

 scribed by Geissman (10), Freudenberg (42) and Schmidt (43). 



More recently column chromatography has been used for separation of these com- 

 pounds although no completely satisfactory system for all of them has been developed. 

 Magnesol and silicic acid partition columns have been used with water -saturated ethyl 

 acetate or ether as developing solvents (44, 45, 46). Karrer and Strong (47) used adsorp- 

 tion chromatography on aluminum oxide plus calcium carbonate, with water as a solvent 

 to purify anthocyanins, although this adsorbent may cause changes in more sensitive com- 

 pounds. Forsyth (48) has used a partition column of cellulose powder pulp with amyl al- 

 cohol-acetic acid-water as the mobile phase to separate polyphenols of cacao. Garber 

 et al. (49) have used a similar method for anthocyanins. Ion exchange columns have been 

 used to separate polyphenols from plant materials by Williams and Wender (50, 51) and 

 Levin and Harris (52). Chandler and Swain (53) and Neu (54) recommend the use of poly- 

 amide (Nylon) columns for purification of flavonoids. 



CHARACTERIZATION 



Classically, many different color reactions and solubility properties were used to 

 characterize the different classes of flavonoid pigments. These are weH summarized m 

 the review by Geissman (10). A few will be mentioned here. 



If interfering pigments are not present, plant tissues (e.g. white flower petals) can 

 be tested for the presence of flavones and flavonols by exposing to ammonia vapor. A 

 yellow coloration indicate the presence of these compounds. Chalcones and aurones turn 

 from yellow to red in this test. If an aqueous pigment extract is made alkaline, various 

 color changes may be observed although the changes in one pigment may mask changes 

 in another: 



