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 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. , 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 -3,3,4,5,7,8-hexahydroxyflavone means of extracting the flavonoids. In either -3,4,5,7-tetrahydroxyflavone case, high temperature extraction, usually at the -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 -3,3,4,5,6,7-hexahydroxyflavone removed from the extraction liquor by evapora -3,3,4,5,7-pentahydroxyflavone tion (under vacuum, if necessary, to prevent, de -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 quent ion-exchange step. Gossypetin-8- 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 -3-rhamnoside of quercetin solvent to approximately 1 kilogram of ground -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 -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 -5,7-dihydroxy-4'-methoxyflavone comminuted imaterial is generally preferred. -4,5,7-trihydroxyflavone 30 Obviously, in using a ground naterial Such Aulanetin-a pentamethoxyflavone as mashed grapes, the already reiatively large -5,7-dihydroxyflavone liquid volume contribution by the water in the -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 -5,7,8-trihydroxyflavone ployed. -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 -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 fiannel, cheese cloth, muslin and nylon are pre -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 - 7 - rhamnoglucoside of 4,5,7 - tri ents are available for use With my invention. hydroxyflavanone 50 However, particularly advantageous results may -Arhamnosidoglucoside of hesper be obtained by utilizing a weak acid cation-ex itin change resin in which the activity is principally due to carboxylic groups (e.g. commercially avail able in such forms as Amberlite IRC-50 manu " methyl '--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, of course, dependent phenyl) - 8,8 - dimethyl - 6 - (3 - methyl - 2 - upon the Concentration of adsorbable materials butenyl) - 4,8 - benzol (12 - b; 3,4 - b') di - 65 in the fiavonoid-containing extraction liquor, it pyran-4-One is merely necessary that care be taken to supply Such ilavonoid compounds may be present in enough resin capacity to prevent the break portions of certain plants, for example, locoweed through of the fiavonoid compound from the leaves, grapes, crude potato extract and various resin before the adsorption step is completed. plant rootS and Woods. To facilitate extraction It is generally sufficient, for each kilogram of of the fiavonoid compounds, the flavonoid-con plant Solid initially present, to use from about taining portion of the plant is usually ground, i.5 liters to about 7.5 liters of comminuted resin by conventional means, into a finely divided (on a wet-settled basis) while an average pre State. The extracting liquid may initially com ferred quantity is about 3 liters. prise either an Organic solvent or water. How 75 As is generally customary in ion-exchange 2,681,907 5 6 resin processes, it is preferred to utilize the resin my resin adsorption-elution procedure, the metal in comminuted form (about 40-100 mesh and in cation may be retained on the resin and further a bed-like, columnar arrangement. Although not purified flavonoids eluted With an Organic Sol critical, I find it suitable to employ a column di Went. ameter of approximately 4 centimeters to ap The following Specific examples illustrate my proximately 8 centimeters with a diameter of ap invention in greater detail. proximately 5 centimeters being preferred. The EXAMPLE I number of columns employed is usually merely a A Pyrex tube, 5 centimeters x 120 centimeters, matter of convenience, the combined length and fitted with No. 12 rubber stoppers was packed resulting capacity constituting the important Con O With a 5 centimeter layer of glass Wool, followed sideration. by 5 centimeters of purified Sea, Sand, and then Adsorption of the flavonoid values from the filled to a height of 92 centimeters with a slurry aqueous extract; nay, therefore, be achieved by of a cation-exchange resin (Amberlite RC-50 simply contacting a Sufficient quantity of com manufactured by Rohm and Haas Co., Phila minuted resin. With the extract. Suitable flow 5 delphia, Pa.). For preparation of the hydrogen rates thru a bed of the resin, in the preferred form of the resin, 3 to 4 liters of 2%, hydrochloric columnar arrangement, for effecting adsorption, acid was passed through the column. The col are from about 10 liters/hour/square decimeter umn was then washed with distilled water until of Column cross Section (i. e. resin bed area) to the liquid came through neutral. about 30 liters/hour/square decimeter of column 20 Two grams of rutin Were dissolved in 6 liters of cross section While a rate of approximately 20 Water COntaining 1 gram Of alurhinum chloride liters/hours/square decimeter of column cross hexahydrate, resulting in the formation of a Section is preferred. bright, yellow aluminum-flavonoid compound in Numerous Organic Solvent eluting agentS are Solution. This Solution was thein paSSed through Suitable for renoving flavonoid compounds 25 the column. The aluminuin-fiavonoid compound which are adsorbed on resins as herein described. was retained on the column in the form of a In generai, any relatively low molecular weight, light yellow band, approximately 25 centimeters aliphatic, organic Solvent. Such as, for example, in width at the top of the resin bed. The column acetone, ethanol, ethyl acetate, butanol or iso Was Washed. With 2 liters of distied water. The propyl alcohol may be employed. However, in 30 irutin was then eiuted by Washing the column view of its SoiréWhat in Ore efficient and more uni versal eluting action and relative cheapness, With ethanol. The aluminum reinlained on the ethanol is preferred. Column. When utilizing the preferred condition set EXAMPLE II forth above, it is generally Satisfactory to use ap The ion-exchange system described in Ex proximately 4 of a liter of eluting Solvent/kilo ample I was employed. gram of starting material to approximately 6 One gram. Of rutin WaS Suspended in 2 liters liters of eluting Solvent/kilogram of Starting ma of distilled Water and 1 grani of potassium ace terial, the preferred volume depending upon the tate was added, with stirring, until the potassiuin. amount of resin erployed and the quantity of 40 acetate dissolved. A few drops of annonii flavonoids adsorbed thereon. An eluting Solvent hydroxide were added, drop by drop, with con flow rate of approximately 10 liters/hour/square tinued Stirring until the rutin Was completely decimeter of column CrOSS Section to approxi dissolved as the bright yellow potassium-rutin mately 30 liters/hour/square decimeter of col Compound. The resulting Solution was then umn cross section is usually suitable, While a flow paSSed through a Column of Amberlite IRC-5) rate of approximately 20 liters/hour/square 45 (H) at a flow rate of approximately 4 liters per decimeter of column croSS section is preferred. hour. The potassium-rutin was adsorbed in a In a preferred embodiment of my invention, 1 tight band (5 centimeters to 8 centimeters wide) kilogram of a Substantially Solid flavonoid-con at the top of the resin bed. The column was taining portion of a plant is finely ground in a thoroughly washed with distilled water-the ball mill. The resulting comminuted material rutin. Still remaining on the column. is then extracted four times With 30 lite portions Two methods were used in the elution of rutin of boiling water. The resulting combined flavo from the column: noid-containing Supernatant extract is then Sep (1) The rutin was eluted directly with a 1 arated from the remaining Solid residues by fil 5 5 liter Wash of ethanol, with the potassium remain tration thru a fiannel bag. The filtered extract ing on the column, or is then percolated thru a 5 centimeter diameter (2) The potassium was removed by a liter column containing approximately 3 liters of wet wash with 0.1 N hydrochloric acid solution, foll settled, comminuted cation-exchange resin (ap lowed by 1 liter of distilled water. Finally, to proximately 60-100 mesh), characterized by a 60 remove the rutin, 1 liter of ethanol was used. plurality of Carboxylic groups, at a rate of ap The second method is generally preferred. in proximately 4 liters/hour. The adsorbed flavo Spection of the Column after direct elution with noid compound inay then be eluted from the ethanol revealed a Sinall residual zone of rutin resin by percolating 4 liters of ethanol thru the at the top of the coluinn; but by filst disrupting multiple columns in approximately One hour. 65 the potassium-rutin, as in the second method, the Occasionally, it is desired to further purify the Subsequent alcoholic elution was complete. flavonoids eluted from the resin. The method of my invention is particularly useful for such a EXAMPLE III purpose. In rary CaSeS, the Organic eluting The ion-exchange System described in Example Solvent may be evaporated, the resulting flavo 70 I was employed. noid-containing residue taken up in hot water, The lead salt of morin Was prepared by adding the metal salt of the flavonoid precipitated by a Solution of basic lead acetate to an acetone so addition of the appropriate metal cation, the lution of 5 grams of impure morin. The result resulting precipitate separated and an aqueous ing precipitate was filtered, using Suction, and solution formed therefrom. Then, by repeating Washed on the filter with distilled water. 2,681,907 7 8 This iead salt precipitate was dissolved in 250 terface. This material precipitated on leaving milliliters glacial acetic acid and diluted to 1 the column. It was discarded since it gave nega liter With distilled Water. The resulting Solution tive tests for flavonoid material. The alcoholic was passed slowly through the column. A yellow filtrates from the four columns were combined band was formed, covering the upper one-third 5. and concentrated to about 275 milliliterS. Con of the resin. A brown solution came through into siderable yellow-brown solid material precipitated the filtrate. The column was washed with about during the concentration. An equal volume of 2 liters of distilled water, until the Washings water was added to the concentrate and the SO changed from brown to colorless. The Inorin Was lution set in the refrigerator overnight to allow then eluted from the column in 500 milliliters of li further precipitation to take place. The yield WaS denatured ethanol. 26.4 grams or 2.64% of crude morin. The used column was regenerated, with 5% The crude morin was recrystallized from 900 nitric acid, rather than with the hydrochloric niliiliters of 60% acetic acid solution. The yield acid, to prevent formation of lead chloride. was 12 giams or 1.2%. The dried morin Was dis EXAMPLE IW solved in a minimu.in quantity of 95% ethyl alco The ion-exchange system described in Example hol and 15 grams of solid potassium acetate I was employed. added. A bright, yellow precipitate of the potaS sium salt of morin Separated at once. The pota S One kilogram of powdered Galanga root (the sium salt was suspended in approximately 20 rhizome of Alpinia officinarium) was boiled for 20 two hours with 30 liters of distilled water. The liters of distilled Water and a few drops of pota S solution was filtered twice through a flannel bag, sium hydroxide solution were added to complete then 2 liters of the extract was passed through a solution. The pHi of the resulting solution was resin column at a rate of 4 liters per hour. The approximately neutral. original aqueous extract had given a positive re 5 The solution of the potassium Salt Was then duction test. The solution that passed through passed through two fresh columns in order to de the column had about the same OutWard appear compose the complex. The potassium Was ex ance as on entry, but it now gave a negative re changed for hydrogen ion and the adsorbed norin duction test. Subsequently eluted with ethyl alcohol. Concen The extract was followed on the column. With a ; tration of the aicoholic solution at reduced pres wash of about 4 liters of distilled Water. This re Sure and Subsequent addition of Water yielded moved some color, but no detectable pale yellow noirin. The yield Was 9.5 grams or flavonoid. The filtrate gave a negative reduction 0.95%. test. The column was then washed with 1 liter Faper partition chronatography of an alcoholic of deratured ethanol. The first alcohol to come solution of the finai product revealed no contam through contained some red impurity which gave ination by other flavonoid or other visible or a negative reduction test. The flavonoid coin fluorescent pigments. Mixed chromatograms of pounds were eluted in the next 600 milliliters of this riorin With an authentic specimen of morin alcohol, containing some Water. produced only one pigment Zone. The alcohol-water solution was concentrated to 40 EXAMPLE WI 200 milliliters by distillation. Some finely di Four columns of the ion-exchange system de vided solid precipitated on cooling. Water (300 Scribed in Example I were employed. Five hun milliliters) was added, and the flask placed in the dred grains of powdered yerba Santa, leaves was refrigerator for four days to allow complete pre SucceSSively extracted with three 38 liter portions cipitation to occur. After filtration, this light tan of boiling distilled water in two hour periods. The colored precipitate was re-dissolved in 1 liter of last extract gave only a very weak test; for favo boiling water and again filtered. A small amount noid materials. The extracts were filtered of blown impurity was removed on the filter through a flannel bag and 21 liters of the extract paper. On cooling the filtrate, a relatively pure Was paSSed through each column at a rate of 7.5 flavonoid fraction ( and ) liters/hour. precipitated. The columns were washed with distilled water aS long as any color was removed (about 4 EXAMPILE W liters/column). The flavonoid material Was then Four columns of the ion-exchange system de eluted by Washing each column with approxi scribed in Example I were employed. 5 5 mately i/2 liters of ethyl alcohol. The alcohol Cone kilogram of ground heartwood of the Solutions were coinbined, concentrated to 3/2 Moris tinctoria, tree was extracted four times liters in a fiash evaporator, diluted with 2% liters With 45 liter portions of distilled Water. Each ex of water, and set in the refrigerator. A brown tract was boiled for two hours and then filtered ish-colored tally naSS of crude homoeriodictyol through flannel bags. 60 (5, 7,4'-trihydroxy-3-methoxyflavanone) sepa Approximately 45 liters of the cooled, red rated from the solution on standing. brown extract was passed through each Column. The solution was filtered, and the tarry residue The filtrate from the columns was only slightly extracted With 1.2 litel's of ethyl ethel. The ether less colored. The resin bed gradually assumed a solution was decanted from a snail amount of un yellow-brown tint as the solution passed through. dissolved black Soig and shaken. With four 50 The columns were next washed with distilled milliliter portions of 10% ammonium carbonate Water until the filtrate was clear (3 liters). The Solution. This treatment removed a considerable adsorbed material, including morin, was then amount of acidic tarry inaterials. The homoerio eluted with 95% ethyl alcohol. Each couinn re dictyol was then precipitated as the in Soluble So quired about one liter of alcohol. The alcohol dium salt by extracting the ether solution with wash was followed by distilled water in order to five 90-milliliter portions of 10% sodium carbo flush the last of the alcoholic solution through nate solution. The sodium homoeriodictyate was the resin bed. collected by filtration of the sodium carbonate so Some yellow-brown material moved down the lution and washed with a small quantity of dis column just in advance of the alcohol-water in tilled Water. 2,681,907 10 The sodium salt was dissolved in 3 liters of dis bed of a comminuted cation-exchange resin char'- tilled water and passed through a fresh column. acterized by a plurality of carboxylic groups, A bright-yellow band of homoeriodictyol formed eluting the resulting adsorbed flavonoid values at the top of the resin bed as the complex was de with an organic solvent, adding a metal cation composed by hydrogen-sodium exchange. As ad 5 to the resulting flavonoid-containing Solvent, ditional solution was passed through the column, separating the resulting metal-flavonoid precipi the band broadened in width. The column Was tate from the resulting supernatant solvent, dis washed with 3 liters of distilled water, by which solving said separated precipitate in Water, pass time the filtrate was clear and colorless. The ing the resulting solution through a fresh bed of homoeriodictyol was then eluted from the column O said comminuted resin and eluting the resulting with ethyl alcohol. The alcohol solution was con adsorbed flavonoids with an organic Solvent. centrated to approximately 600 milliliters and 5. The method of claim 1 where the resin is a then diluted with an equal volume of distilled weak acid cation-exchange resin characterized water. A pale-yellow precipitate of homoeriodic by a plurality of active carboxylic groupS. tyol separated on standing in the refrigerator. 6. The method of claim 1 wherein the Organic The yield was 14.0 grams to 14.5 grams or 2.8% solvent is ethanol. to 2.9%. 7. The method of claim 3 wherein the metal Paper partition chromatography of the final is selected from the group consisting of alumi product revealed no evidence of contamination num, potassium, sodium and lead. by or other flavonoids. Mixed 20 8. The method of claim 1 wherein the fla chromatograms and mixed melting point deter vonoids are selected from the group consisting minations with authentic homoeriodictyol. Sub of rutin, morin, homoeriodictyol, galangin and stantiated the purity and identity of the product. kaempferide. In general, it may be said that the above ex 9. An improved method of Separating fia" amples are merely illustrative and should not be vonoids from an aqueous Solution containing construed as limiting the scope of my invention said flavonoids and extraneous impurities which which should be understood to be limited only as comprises passing said aqueous Solution through indicated by the appended claims. a bed of approximately 1.5 liters to approxi I claim: mately 7.5 liters of a wet-settled comminuted i. An improved method of separating fla 30 cation-exchange resin characterized by a plu vonoids from an aqueous solution containing rality of active carboxylic groups for about each said flavonoids and extraneous impurities which 5 grams to about 25 grams of flavonoids initially comprises contacting said aqueous Solution. With in said extract, at a rate of approximately 10 a comminuted cation-exchange resin, Separat liters/hour/square decimeter of bed area, to about ing the resulting flavonoid-depleted Solution from 35 30 liters/hour/square decimeter of bed area, the resulting fiavonoid-retaining resin and elut thereafter passing approximately 42 liter to ap ing the flavonoids from said resin. With an Organic proximately 6 liters of ethanol for about each 5 solvent. grams to about 25 grams of adsorbed flavonoids 2. An improved method of obtaining flavonoid through the resulting bed of flavonoid re values from flavonoid-containing vegetation 40 taining resin at a rate of approximately 10 Which comprises comminuting said vegetation, liters/hour/square decimeter of bed area to 30 deriving a flavonoid-containing aqueous extract liters/hour/square decimeter of bed area, and from the resulting comminuted vegetation, paSS collecting the resulting ethanol elutriant cOn ing said extract through a comminuted, cation taining Said flavonoids. exchange resin bed until the flavonoid values in 10. An improved method of Separating fia said extract are Substantially completely ad vonoids from an aqueous solution containing sorbed on said resin and eluting the resulting ad said flavonoids and extraneous impurities which sorbed flavonoid values with an organic Solvent. comprises passing said aqueous Solution though 3. An improved method of separating a fla a bed of approximately 3 liters of a Wet-settled vonoid compound from an aqueous solution conminuted cation-exchange resin character containing a metal compound of Same and eX ized by a plurality of active carboxylic groups traneous impurities which comprises contacting for about each 10 grains of favonoids initially said aqueous solution with a comminuted cation in said extract, at a rate of approximately 20 exchange resin, separating the resulting metal liters/hour/square decimeter of bed area, there flavonoid-depleted solution from the resulting 5 after passing approximately 4 liters of ethanol metal flavonoid-retaining resin, contacting the for about each 10 grams of adsorbed flavonoids resulting separated resin with an organic solvent through the resulting bed of flavonoid-re and Separating the resulting flavonoid-contain taining resin at a rate of approximately 10 ing organic solvent from the resulting metal-re liters/hour/square decimeter and collecting the taining resin. 6. resulting ethanol elutriant containing said 4. An improved method of obtaining flavonoid flavonoids. Values from a flavonoid-containing portion of a References Cited in the file of this patent plant which comprises comminuting said plant portion, deriving an aqueous extract from the Nachod: “Ion Exchange' (1949), pp. 277 and resulting comminuted portion, passing said 65 299. flavonoid-containing aqueous extract through a