- 2,738,346 United States Patent Office Patented Mar. 13, 1956 a 2 Using our invention, a mixture of flavonoid compounds 2,738,346 may be rapidly separated in good yields into individual, relatively pure flavonoids. Our method does not require METHOD FOR THE SEPARATION OF FLAVONOD careful control or readjustment of conditions or the COMPOUNDS selection of different solvents and elutriants for each Simon H. Wender, Norman, Okla., and Clark H. Ice, specific separation. Aiken, S. C., assignors to the United States of America Table I, following, is illustrative, but by no means as represented by the United States Atomic Energy exclusive, of the flavonoid mixtures which may be sepa Commission rated into their individual components utilizing the method No Drawing. Application December 1, 1952, O herein described. Serial No. 323,505 . . . . . TABLE I Mixture 1: 8 Claims. (CI. 260-210) a. Quercitrin (3-rhamnoside of quercetin) Our invention relates to an improved method for the b. Rutin (3-rutinoside of quercetin) separation of flavonoid compounds and more particularly c. Quercetin (3, 3, 4, 5, 7-pentahydroxyflavone) to the separation of closely related flavonoid compounds. Mixture 2: The flavonoid compounds comprise a very important class of plant pigments which are widely distributed in a. Xanthorhamnin (3-rhamninoside of rhamnetin) the vegetable kingdom. Interest is shown in a number 20 b. Quercitrin (3-rhamnoside of quercetin) of these compounds due to their vitamin-like action in c. Quercetin (3, 3, 4, 5, 7-pentahydroxyflavone) increasing the resistance of blood capillaries to rupture. Mixture 3: The term vitamin “P” is sometimes applied to flavonoids having this property. Rutin, a member of this class a, Xanthorhamnin (3-rhamninoside of rhamnetin) of pigments, enjoys widespread use as a drug for blood 25 b. Rutin (3-rutinoside of quercetin). vessel treatment. In addition, it is anticipated that flavo c. Quercetin (3, 3, 4, 5, 7-pentahydroxyflavone) noids will be of use in the control of radiation injury, Mixture 4: and considerable experimental effort is being expended a. Naringin (7-rhamnoglucoside of 4, 5, 7- tri in this direction. hydroxyflavone) There is, therefore, considerable demand for such types 30 of compounds for both practical and experimental pur b. Hesperidin (7-rhamnoglucoside of hesperitin) poses. Co-pending patent application S. N. 283,749, Mixture 5: filed April 22, 1952, in the name of Simon H. Wender, a. Morin (2, 3, 4, 5, 7-pentahydroxyflavone) which matured into Patent No. 2,681,907 on June 22, b. Quercetin (3, 3, 4, 5, 7-pentahydroxyflavone) 1954, discloses a method for separating substantially pure 35 flavonoid compounds in relatively concentrated form Numerous organic solvents are suitable for dissolving from extraneous organic and inorganic impurities. Co the flavonoid mixture which is to be separated into its pending patent application S. N. 285,046, filed May 12, individual components by the method described herein. 1952, in the names of Simon H. Wender, Thomas B. In general, any anhydrous, relatively low molecular Gage, Clark H. Ice, and Quentin L. Morris discloses 40 weight, aliphatic organic solvent, such as, for example, methods of separating general groups of flavonoid com ethanol, ethyl acetate, butanol, isopropyl alcohol, amyl pounds from one another or individual compounds of alcohol, ethyl methyl ketone and acetone may be em different groups from one another. For example, flavo ployed. In general, however, we prefer to employ noid aglycones may be separated from flavonoid glyco acetone. sides. However, the method is not sufficiently precise 45 Many inorganic adsorbents are not suitable for use to permit separation of closely related compounds within with our invention. However, we find that hydrous metal the same family group. silicate adsorbents are unexpectedly satisfactory, and Previous methods for the separation of flavonoid com that hydrous magnesium silicate adsorbents are particu pounds within a single group include fractional crystal larly satisfactory. One such hydrous magnesium silicate lization and solven extraction techniques. The employ 50 adsorbent is commercially available as "Magnesol,” which ment of these methods usually results in low yields, is is sold by the Food Machinery and Chemical Corpora cumbersome and tedious, and in many cases does not tion, Westvaco Chemical Division, New York. effect good separation of individual flavonoids. The quantity of adsorbent required for the optimum An object of our invention, therefore, is to provide separation of the flavonoid mixture into individual com an improved method for the separation of flavonoid 55 ponents is, of course, dependent upon the amount and compounds...... concentration of the feed solution. it is merely neces Another object of our invention is to provide a method sary that care be taken to supply enough adsorbent ca for the separation of closely related flavonoid compounds. pacity to prevent break-through before the adsorption Still another object of our invention is to provide a step is completed. method for the separation of individual flavonoid com 60 As is generally customary in adsorption chromatography pounds in high yields. separations, it is preferred to utilize the adsorbent in a comminuted form (about 40-100 mesh) and in a bed Other objects and advantages of our invention will be like, columnar arrangement. Although not critical, we apparent from the following description. find it satisfactory to employ columns whose lengths are In accordance with our invention, a mixture of flavo approximately four times to approximately six times noid compounds may be resolved into its individual com 65 their diameters. pounds by passing an organic solution of said mixture The flavonoid mixture dissolved in the organic solvent through a bed of a comminuted hydrous metal silicate may be adsorbed onto the column by simply passing the adsorbent, chromatographically eluting the resulting ad solvent solution through the column. The particular sorbed compounds from said bed with an aqueous-or flow-rate selected is not critical, provided it is slow, ganic solvent and separately collecting the resulting in 70 enough to prevent flavonoid break-through. dividual flavonoid compounds. We find, in general, that at least partially water-miscible 2,788,846 3 4 solvents selected from the herein above-described organic quercetrin and quercetin from lemon flavin; and to re solvents, when saturated with water, or when mixed solve a flavonoid mixture separated from the leaves of with water in the proper proportions, are suitable for Vaccinium myrtillus into its individual components. chromatographically eluting the adsorbed flavonoid com Therefore, the scope of our invention should be under pounds from the hydrous metal silicate column, while 5 stood to be limited only as indicated by the appended the ethyl acetate-water system is greatly preferred. Since claims. water-saturated ethyl acetate is greatly preferred, our What we claim is: specification will be further illustrated specifically with 1. An improved method for the separation of flavonoid respect to this elutriant. compounds, which comprises passing a solution of said As used herein and in the appended claims, the term 0 compounds in a relatively low molecular weight, an "aqueous-organic solvent,” as applied to the elutriant, hydrous aliphatic organic solvent through a bed of a is intended to designate an at least partially water-mis comminuted hydrous magnesium silicate adsorbent, cible organic solvent containing readily measurable quan chromatographically eluting the resulting adsorbed com tities of water. The optimum amount of water in each pounds from said bed with an aqueous-relatively low aqueous-organic solvent system may be easily deter 5 molecular weight aliphatic organic solvent, and separately mined by routine testing. However, when employing the collecting the individual flavonoid compounds. ethyl acetate-water system, it is preferred to saturate the 2. The method of claim 1 wherein the anhydrous rela ethyl acetate with respect to water. tively low molecular weight, aliphatic organic solvent is In a preferred embodiment of our invention, an an acetone. hydrous acetone solution containing about 1.0 gram of 3. The method of claim 1 wherein the aqueous-rela a flavonoid mixture is passed through a Pyrex column 18 tively low molecular weight aliphatic organic solvent is millimeters in diameter and 80 millimeters in length which an aqueous Solution of ethyl acetate. contains a wet-settled, comminuted hydrous magnesium 4. The method of claim 3, wherein the aqueous solu silicate. The adsorbed flavonoids are then chroma tion of ethyl acetate is saturated with respect to water. tographically eluted with an ethyl acetate solution satu 5. The method of claim 4 wherein the aqueous solu rated with Water, and containing traces of acetic acid tion of ethyl acetate saturated with respect to water con (approximately 0.1% to approximately 3% acetic acid, tains a small amount of acetic acid. by volume, is satisfactory for this purpose), and the in 6. The method of claim 5 wherein the aqueous solu dividual flavonoid compounds are separately collected. tion of ethyl acetate Saturated with respect to water con The following specific example illustrates our inven 30 tains approximately 0.1% to approximately 3% acetic tion in greater detail. acid, by volume. Example I 7. A method for the separation of flavonoid aglycones which comprises passing a solution of said aglycones in Six hundred milliliters of anhydrous acetone was added a relatively low molecular weight, anhydrous aliphatic to 150 grams of "Magnesol' and the mixture stirred to 35 organic solvent through a bed of comminuted hydrous give a thin slurry. The slurry was added at once to magnesium silicate, chromatographically eluting the re a Pyrex column, 60 millimeters in diameter and 160 sulting adsorbed flavonoid aglycones from said bed with millimeters in length, and the sides of the column were an aqueous-relatively low molecular weight aliphatic or then rinsed down with 200 milliliters of acetone. An ganic solvent, and separately collecting the individual anhydrous acetone solution containing about 0.5 gram 40 flavonoid aglycones. of a mixture of morin and quercetin (both flavonol 8. A method for the separation of flavonoid glycosides aglycones) was passed through the column. After the which comprises passing a solution of said glycosides in solution had passed through the column, a filter paper a relatively low molecular weight, anhydrous aliphatic circle was placed on top of the column, and an ethyl organic solvent through a bed of comminuted hydrous acetate solution Saturated with water and containing magnesium silicate, chromatographically eluting the re traces of acetic acid was passed through the column until Sulting adsorbed flavonoid glycosides from said bed with two individual bands, which were visible under ultra an aqueous-relatively low mclecular weight aliphatic or violet light, passed down the column and were separately ganic solvent, and separately collecting the individual collected. Paper chromatography showed that the first flavonoid glycosides. fraction was pure quercetin while the second was pure morin. References Cited in the file of this patent In general, it may be said that the above example is merely illustrative and should not be construed as limit UNITED STATES PATENTS ing the scope of our invention. Thus, since our process 2,500,930 Couch et al. ------Mar. 21, 1950 may be used to separate closely related flavonoid com 5 5 2,520,127 Couch et al. ------Aug. 29, 1950 pounds, it has wide applicability to the separation of less 2,524,414 Wolfrom et al. ------Oct. 3, 1950 closely related flavonoids. In this regard, we have found OTHER REFERENCES that our process is significantly superior to those pre viously employed for the separation of flavonoid com Gage et al.: Science, 113, 522. pounds in different general groups. For example, our 60 McNeely et al.: J. Am. Chem. Soc., 67, 527-9 (1945). process may be used to substantially completely remove. Wender et al.: "Science," Vol. 109 (1949), pages 287 quercetin, a flavonol aglycone, from commercial rutin, 289. a fiavonol glycoside; to isolate pure isoquercetrin from C. A. (1953), 5212 c. grapes and from black currants; to separately recover