Patented June 22, 1954 2,681,907 UNITED STATES PATENT OFFICE 2,681,907 ISOATION OF FILAWONOD COMPOUNDS Simon H. Wender, Norman, Okla., assignor to the United States of America as represented by the United States Atomic Energy Commission No Drawing. Application April 22, 1952, Serial No. 283,749 10 Claims. (C. 260-210) 1. 2 My invention relates to a method of purifying vide an improved method for isolating flavonoid flavonoids and more particularly to the recovery compounds. of quantities of substantially pure flavonoids Another object is to provide a method for iso from their naturally occurring source materials. lating relatively large quantities of a flavonoid The flavonoid compounds comprise a very in compound in Substantially pure form. portant class of plant pigments which are widely Still another object is to provide an improved distributed in the vegetable kingdom. Interest proceSS for Separating relatively pure flavonoids is shown in a number of these compounds due to in concentrated form from the original solid their vitamin-like action in increasing the resist source materials. ance of blood capillaries to rupture. The term O Further objects and advantages of my inven “vitamin P' is sometimes applied to flavonoids tion will be apparent from the following descrip having this property. Rutin, a member of this tion. class of plant pigments enjoys widespread use as In accordance with my invention, substantially a drug for blood vessel treatment. In addition, pure flavonoids may be separated in relatively it is anticipated that flavonoids will be of use in concentrated form from extraneous organic and the control of radiation injury, and considerable inoi'ganic impurities by preparing a water ex experimental effort is being expended in this tract of same, contacting said Water extract with direction. a cation-exchange resin, separating the resulting There is, therefore, considerable demand for flavonoid-depleted water extract from the result such types of compounds, for both practical and 20 ing flavonoid-retaining resin and eluting the experimental purposes. Since they usually occur favonoids retained on Said resin with an organic in trace quantities, the isolation of flavonoids in Solvent. substantially-pure form and in relatively large Using my invention, flavonoids in a highly quantity has been exceedingly difficult to achieve. dilute, voluminous Water extract may readily be Alcohol extracts of naturally occurring Source 25 concentrated, via the ion-exchange resin, into a materials have often been used in conjunction much smaller volume, in addition to being highly with subsequent crystallization procedures. How purified, to an unexpected degree, of sugars and ever, alcohol also extracts many resinous-like Scores of other impurities co-extracted by the niaterials which interfere with crystallization. Water from the Original Source material. No Water extracts have sometimes been employed to 30 evaporation, Crystallization or involved addi avoid the latter disadvantage, but, because of the tional steps are required. Furthermore, with the usual lesser Solubilities of flavonoids in Water, methods devised in this invention, the metallic unusually large volumes of water are necessary, salts of the flavonoids, such as the lead Salts, can with Subsequently costly evaporations. In addi be decomposed and the metals removed by use of tion, interfering inorganic ions and Sonne Sugars 3 5 a cation-exchange resin, rather than by the prior are often coextracted. Nevertheless, regardless art hydrogen Sulfide step. of the solvent used, the steps necessary to obtain Miy invention may be Satisfactorily utilized a relatively pure concentrate are usually compli with substantially any impure a queous Solution of cated, and considerable flavonoid losses occur. flavonoid compounds, including those resulting In addition, a purification step often used in the 40 as intermediate steps in other flavonoid purifica isolation of flavonoids from natural products in tion processes. Thus, for example, my method voives the preparation of the lead Salt of the may be used either directly with aqueous extracts flavonoid. This precipitate is Separated, then of portions of plants containing flavonoid type suspended in a solvent such as alcohol, and de compounds or with Solutions containing netal composed with hydrogen Sulfide. The resulting 45 compounds of the flavonoids. In either case, the lead sulfide precipitate is then separated from concentration of impurities in these Solutions is the remaining Supernatant flavonoid Solution. usually considerably higher than the concen However, not only is the use of hydrogen sulfide tration of the flavonoids. disagreeable, but flavonoid losses are significant. Table I, following, is illustrative of the fla An object of my invention, therefore, is to pro 50 vonoid compounds which may be separated from 2,681,907 3 4 extraneous impurities utilizing the methods here ever, since an aqueous solution is preferred for in described. contacting the resin with the extracted material, TABLE I Water is preferred for the extraction step. OC casionally, however, an organic Solvent Such as Flavonol aglycones acetone or ethanol is required as a more drastic Gossypetin-3,3,4,5,7,8-hexahydroxyflavone means of extracting the flavonoids. In either Kaempferol-3,4,5,7-tetrahydroxyflavone case, high temperature extraction, usually at the Morin-2',3,4,5,7-pentahydroxyflavone boiling point of the extracting liquid, is preferred. Nortangeretin-3,4,5,6,7-pentahydroxyflavone When an organic Solvent is employed, at a rela Fatuletin-3,3,4,5,7-pentahydroxy - 6 - methoxy tively high temperature, to extract flavonoids fiaVOne from plaint material, the organic Solvent may be Quercetagetin-3,3,4,5,6,7-hexahydroxyflavone removed from the extraction liquor by evapora Quercetin-3,3,4,5,7-pentahydroxyflavone tion (under vacuum, if necessary, to prevent, de Rhamnetin-3,3,4,5 pentahydroxy-7-methoxy composition of the flavonoids) and the resulting fia,VOne 5 flavonoid-containing residue taken up With a rel Robinetin-3,3,4,5,7 pentahydroxyflavone atively small volume of hot water, thus produc ing the desired aqueous solution for the Subse Flavonol glycosides quent ion-exchange step. Gossypetin-8-glucoside of goSSypetin The latio of extracting Solvent to ground ima Gossypitrin-7-glucoside of goSSypetin 20 terial is not critical, although an excess of Sol Isoquercitrin-3-glucoside of quercetin vent is generally preferred. Thus, Suitable ratioS Quercemeritrin-7-glucoside of quercetin are from about 25 liters to about 250 liters of Quercitrin-3-rhamnoside of quercetin solvent to approximately 1 kilogram of ground Robinin-3-robinoside of kaempferol material (representing about 5 grams to about Rutin-3-rutinoside of quercetin 25 grams of flavonoid values and an average of Xanthorhamnin-3-trirhamnoside of rhamnetin about 10 grams). However, Where the extra C tion is one of normal difficulty, a ratio of about Flavone diglycones 125 liters of solvent to about each kilogram of Acacetin-5,7-dihydroxy-4'-methoxyflavone comminuted imaterial is generally preferred. Apigenin-4,5,7-trihydroxyflavone 30 Obviously, in using a ground naterial Such Aulanetin-a pentamethoxyflavone as mashed grapes, the already reiatively large Chrysin-5,7-dihydroxyflavone liquid volume contribution by the water in the Genkwanin-4,5-dihydroxy-7-methoxyflavone grapes must be considered when selecting the isowogonin-5,8-dihydroxy-7-methoxyflavone volume of additional extracting Solvent to be emi NorwOgonin-5,7,8-trihydroxyflavone ployed. Oroxylin A-5,7-dihydroxy-6-methoxyflavone Any Suitable filtration method may be utilized Wogonii-5,7-dihydroxy-8-methoxyflavone to Separate Supernatant fiavonoid-containing ex Flaianone aglycones traction liquors from remaining Solid residues. Sometimes filter paper filtration may be difficult Butin-3',4',7-trihydroxyflavanone 40 due to clogging of the filter paper pores by the Hesperitin - 3',5,7 trihydroxy - 4 - methoxy - extract and, therefore, filter materials such as flavanone fiannel, cheese cloth, muslin and nylon are pre Homoeriodictyol-4,5,7 - trihydroxy-3-meth ferred. Occasionally, more than One Such na oxyflavanone terial may be used consecutively, starting with Tiquiritiginin-4,7-dihydroxyflavanone 5 the One containing the larger pore size and ter Fladnone glycosides rhinating With the One containing the Smaller pore size. deSperidin-7-rhamnoSide of hesperitin Numerous Suitable cation-exchange adsorb Naringin - 7 - rhamnoglucoside of 4,5,7 - tri ents are available for use With my invention. hydroxyflavanone 50 However, particularly advantageous results may Neohesperidin-Arhamnosidoglucoside of hesper be obtained by utilizing a weak acid cation-ex itin change resin in which the activity is principally Chalcones due to carboxylic groups (e.g. commercially avail able in such forms as Amberlite IRC-50 manu "Hesperidin methyl chalcone'--a product ob factured by Roihin and Haas Co., Philadelphia, tained from the methylation of hesperidin chal Pa. and Duolite Cation Selector CS-100 manul COe factured by chemical Process Co., San Francisco, Phloretin - 3 - (p - hydroxyphenyl) phloropro California). Satisfactory results may be achieved piophenone with the sodium, potassium, or other forms of Related compounds 60 the resins. However, the hydrogen form is pre ferred. Although the quantity of resin required ESCulletin-6,7-dihydroxycoumarin for optimum recovery of a flavonoid compound Poniferin - 5 - hydroxy - 3 - (3,4, - dihydroxy - from extracting liquor is,