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Oleanolic Acid from Cranberries*
Oleanolic Acid from Cranberries* By BETTY Y. T. WUt and LLOYD M. PARKS A modified procedure, involvin decolorization of the ammonium salt, is feescribed for the purification of crude ursolic acid obtained from cranberries. Oleanolic acid was isolated from the mother liquors remaining from the final recrystallization of the purified product. N THEIR REPORT of
the isolation of ursolic acid from cranberry pomace, Vaccinium mucrocarpurn Ait., Markley and Sando (1) noted the presence of another unidentified resin acid in the ether-soluble fraction. In the course of purification of crude ursolic acid,' obtained by extraction of cranberries, we have isolated and identified oleanolic acid, whose presence in t h a t plant has not heretofore been reported. Kremers, et al. (2), using a procedure based on the fact that sodium ursolate is only slightly soluble i n aqueous alkali while most of the accompanying impurities are soluble i n this solvent, reported better success in the purification of crude ursolic acid than by use of the methods of Dodge (3) and Sando (4). Considerable difficulty was encountered by us with the Kremers procedure in decolorizing the deeply colored sodium ursolate and a yield of only 15 per cent of pure ursolic acid (based o n weight of the crude) was obtained. I n a modified procedure ammonium ursolate, prepared by saturation of a methanolic solution of crude ursolic acid, was decolorized with Norit and the free ursolic acid obtained by acidification. In this way yields of 22 t o 30 per cent of pure ursolic acid were obtained with much less manipulation. Oleanolic acid was isolated from the mother liquors remaining from crystallization of the purified ursolic acid. EXPERIMENTAL
Purification of Ursolic Acid by the Modified Procedure.--Three hundred grams of crude ursolic acid was suspended in 10 L. of methatiol ;tnd a stream of ammonia gas was passed into it. At the end of one hour most of the solid had dissolved and filtration of the warm mixture through a cloth left only 20 Gm. of insolubles, which consisted chiefly of fibrous plant material and dirt. The dark brown filtrate was boiled for one and one-half hours with 80 Gm. of Norit with vigorous
*
Received March 16, 1953. from the School of Pharmacy, lIniversity of Wisconsin, Madison Supported in part by a research grant froni the National Cranberry Association. Hansun, Massachusetts. t National Cranberry Association Fellow in Pharmaceutical Chemistry, 1951-52. 1 Kindly supplied by the National Cranberry Association.
stirring. After allowing t o stand overnight the mixture was heated t o boiling and filtered while hot through a 2 cm. layer of filter cel. Treatment of the filtrate twice more with Norit yielded a pale yellow colored solution. This solution was concentrated t o about 3 L. to the appearance of a white precipitate, acidified with dilute hydrochloric acid and diluted with water to 6 L. The free ursolic acid which was precipitated was filtered by suction and washed well with water. The semidried ursolic acid was dispersed in 4 L. of water, to the suspension was added a solution of 20 Gm. of sodium hydroxide in water, and the mixture was heated with stirring a t its boiling point for one hour. The white insoluble sodium ursolate was then separated by centrifuging, washed with hot water, dissolved jn 1 1 / 2 L. of methanol and the solution acidified with dilute hydrochloric acid. The precipitated ursolic acid was filtered by suction, washed with water until free from chloride, and dried for twenty-four hours at 85'; it weighed 148 Gm., m. p. 270-276".* This semipure product was suspended in 2 L. of ethanol-acetone (3: 1 ) and the mixture heated a t its boiling point, with most of the ursolic acid remaining insoluble and settling t o the bottom of the container. The hot saturated supernatant liquid was separated by decantation and immediately upon cooling small white needle crystals of ursolic acid formed. The crystalline product was filtered by suction, the mother liquor from this crystal crop was diluted with 200 ml. of fresh solvent and used to treat the insoluble residue of ursolic acid in the same manner as before. By repetition of this treatment several fractions of crystalline product were obtained as follows: Weight, Fraction
Gm.
z.C.P.,
1
26.40 17.35 21.30 23.20 27.10 25.00
281-282 280-281.5 ~ _ . 280-282 279-282 250-260 249-255
2 3 4 5 6
Fractions 1 and 2 were combined and after two recrystallizations from acetone-alcohol yielded 40.4 Gin. of white needles, in. p. 283-283.5'. Fractions 3 and 4 after four recrystallizations yielded 32.3 Gin. of the same product, ni. p. 282.5-283.5". No attempt was made a t further purification of Fractions 5 and 6. Additional purification runs on the original crude ursolic acid by the procedure described gave the pure crystalline acid in yields of 22, 22, and 30%, respectively. Isolation of Oleanolic Acid.-Concentration of the mother liquors from the final recrystallizations of Fractions 3 and 4, above, t o a volume of 30 ml. resulted in the separation of 11 Gin. of yellow crystal2 All melting points were obtained with Anschutz total immersion thermometers.
602
October, 1953
603
SCIENTIFIC EDITION
line powder. This was acetylated in the usual manner with acetic anhydride and anhydrous sodium acetate, the solid reaction product dissolved by refluxing with 300 ml. of 7070 alcohol, and the resulting solution diluted with 500 ml. of water. The acetate separated as a white precipitate and was crystallized from absolute methanol to yield 4.25 Gm. of long white needles, m. p. 266-268", which showed no depression (267-269O) when mixed with an authentic sample of acetyl oleanolic acid ( 5 ) ; = +75.4" (chloroform, C = 1.366). One gram of the acetate was hydrolyzed by refluxing with alcoholic potassium hydroxide, the hydrolysis mixture acidified with diluted hydrochloric acid, diluted with water, and the white
precipitate shaken out with several portions of ether. Evaporation of the ether solution left a white residue which was crystallized from absolute methanol to yield 0.65 Gm. of white needles of oleanolic acid, m. p. 312314". which showed no depression (311-313') when mixed with an authentic sample (5). [a]';' = +80.2" (chloroform, C = 0.988). REFERENCES (1) Markley. K. S., and Sando, C. E., J . B i d . Chem.. 105 643(1934). ($) Kremers, R. E., Sell, H. M., and Stookey. A. D., unpubfished data. (3) Dodge F. D. J . A m Chcm. SOC. 40 1932(1918). (4) Sando'C. E. ' J . Biol.'Chcm.. 90. k77j1931). (5) Rowe,'B. J., kt al., THISJOURNAL, 38, 122(1949).
Chemical Studies on Ursolic Acid* By BETTY Y. T. WUt and LLOYD M. PARKS
CaH46J1gg$&228), b
From ursolic there was prepared acetyl ursolyl amide and acetyl ursolyl nitrile, 0th of which were reduced by lithium aluminum hydride to an ursolyl amine (C-28), C&46 {-OH2NHp. -cH By a similar reduction of ursonic
acid oxime there was obtained an isomeric ursolyl amine (C-2),
C d h
ursonate9 C 2 9 H 4 s ( ~ ~ O O C H was 3 , reduced, by the Huang-Minlon modification of the Wolff-Kishner method, to ursanic acid, which was reduced by lithium aluminum hydride to the primary alcohol, ursanol,
an isomer of the naturally occurring secondary alcohol, a-amyrin,
HE COMBINATION of certain amines, such as "procaine, with penicillin to form salts of low water solubility to prolong the action of penicillin in the body is well known. Since ursolic acid itself is of extremely low solubility it appeared possible that an amine obtained from it might be used in the Same way. Each of the two reactive groups in ursolic acid, namely the carboxyl at C-28 and the secondary alcohol at C-2, offers points for introduction of the amino group; one of the objects of this study was to prepare these two indicated amino derivatives. Among the known triterpene compounds in nature there has been shown to exist relationships which are of biogenetic significance. Thus, in addition to the hydroxy acid, ursolic acid, there
*
Received March 16 1953 from the School of Pharmacy, University of Wiseonsih, Mahison. Supported in part by a research grant from the National Cranberry Association Hanson Massachusetts. t NOtiqnal C-b& Assmihion Fellow in Pharmaceutical Chermstrg, 1951-52.
is also found the corresponding diol, uvaol, and the secondary alcohol, a -amyrin. There has not yet been found (nor may it necessarily exist in nature) the corresponding isomeric primary alcohol; another object of this study was to prepare the indicated primary alcohol, which we have designated as ursanol. Acetyl ursolyl amide was obtained in consistently good yields by the action of ammonia gas on a dioxane solution of acetyl ursolyl chloride and the presence of the amide group was confirmed by its infrared spectrum (Fig. 1). By the action of acetic anhydride acetyl ursolyl amide was converted to acetyl wsolyl nitrile, presence of the nitrile group being confirmed by its infrared spectrum (Fig. 2). Both acetyl ursolyl amide and acetyl ursolyl nitrile were reduced in good yields to an ursolyl amine (C-28) by lithium aluminum hydride. An isomeric ursolyl amine (C-2) was prepared by reduction of ursonic acid oxime, and its methyl
the isolation of ursolic acid from cranberry pomace, Vaccinium mucrocarpurn Ait., Markley and Sando (1) noted the presence of another unidentified resin acid in the ether-soluble fraction. In the course of purification of crude ursolic acid,' obtained by extraction of cranberries, we have isolated and identified oleanolic acid, whose presence in t h a t plant has not heretofore been reported. Kremers, et al. (2), using a procedure based on the fact that sodium ursolate is only slightly soluble i n aqueous alkali while most of the accompanying impurities are soluble i n this solvent, reported better success in the purification of crude ursolic acid than by use of the methods of Dodge (3) and Sando (4). Considerable difficulty was encountered by us with the Kremers procedure in decolorizing the deeply colored sodium ursolate and a yield of only 15 per cent of pure ursolic acid (based o n weight of the crude) was obtained. I n a modified procedure ammonium ursolate, prepared by saturation of a methanolic solution of crude ursolic acid, was decolorized with Norit and the free ursolic acid obtained by acidification. In this way yields of 22 t o 30 per cent of pure ursolic acid were obtained with much less manipulation. Oleanolic acid was isolated from the mother liquors remaining from crystallization of the purified ursolic acid. EXPERIMENTAL
Purification of Ursolic Acid by the Modified Procedure.--Three hundred grams of crude ursolic acid was suspended in 10 L. of methatiol ;tnd a stream of ammonia gas was passed into it. At the end of one hour most of the solid had dissolved and filtration of the warm mixture through a cloth left only 20 Gm. of insolubles, which consisted chiefly of fibrous plant material and dirt. The dark brown filtrate was boiled for one and one-half hours with 80 Gm. of Norit with vigorous
*
Received March 16, 1953. from the School of Pharmacy, lIniversity of Wisconsin, Madison Supported in part by a research grant froni the National Cranberry Association. Hansun, Massachusetts. t National Cranberry Association Fellow in Pharmaceutical Chemistry, 1951-52. 1 Kindly supplied by the National Cranberry Association.
stirring. After allowing t o stand overnight the mixture was heated t o boiling and filtered while hot through a 2 cm. layer of filter cel. Treatment of the filtrate twice more with Norit yielded a pale yellow colored solution. This solution was concentrated t o about 3 L. to the appearance of a white precipitate, acidified with dilute hydrochloric acid and diluted with water to 6 L. The free ursolic acid which was precipitated was filtered by suction and washed well with water. The semidried ursolic acid was dispersed in 4 L. of water, to the suspension was added a solution of 20 Gm. of sodium hydroxide in water, and the mixture was heated with stirring a t its boiling point for one hour. The white insoluble sodium ursolate was then separated by centrifuging, washed with hot water, dissolved jn 1 1 / 2 L. of methanol and the solution acidified with dilute hydrochloric acid. The precipitated ursolic acid was filtered by suction, washed with water until free from chloride, and dried for twenty-four hours at 85'; it weighed 148 Gm., m. p. 270-276".* This semipure product was suspended in 2 L. of ethanol-acetone (3: 1 ) and the mixture heated a t its boiling point, with most of the ursolic acid remaining insoluble and settling t o the bottom of the container. The hot saturated supernatant liquid was separated by decantation and immediately upon cooling small white needle crystals of ursolic acid formed. The crystalline product was filtered by suction, the mother liquor from this crystal crop was diluted with 200 ml. of fresh solvent and used to treat the insoluble residue of ursolic acid in the same manner as before. By repetition of this treatment several fractions of crystalline product were obtained as follows: Weight, Fraction
Gm.
z.C.P.,
1
26.40 17.35 21.30 23.20 27.10 25.00
281-282 280-281.5 ~ _ . 280-282 279-282 250-260 249-255
2 3 4 5 6
Fractions 1 and 2 were combined and after two recrystallizations from acetone-alcohol yielded 40.4 Gin. of white needles, in. p. 283-283.5'. Fractions 3 and 4 after four recrystallizations yielded 32.3 Gin. of the same product, ni. p. 282.5-283.5". No attempt was made a t further purification of Fractions 5 and 6. Additional purification runs on the original crude ursolic acid by the procedure described gave the pure crystalline acid in yields of 22, 22, and 30%, respectively. Isolation of Oleanolic Acid.-Concentration of the mother liquors from the final recrystallizations of Fractions 3 and 4, above, t o a volume of 30 ml. resulted in the separation of 11 Gin. of yellow crystal2 All melting points were obtained with Anschutz total immersion thermometers.
602
October, 1953
603
SCIENTIFIC EDITION
line powder. This was acetylated in the usual manner with acetic anhydride and anhydrous sodium acetate, the solid reaction product dissolved by refluxing with 300 ml. of 7070 alcohol, and the resulting solution diluted with 500 ml. of water. The acetate separated as a white precipitate and was crystallized from absolute methanol to yield 4.25 Gm. of long white needles, m. p. 266-268", which showed no depression (267-269O) when mixed with an authentic sample of acetyl oleanolic acid ( 5 ) ; = +75.4" (chloroform, C = 1.366). One gram of the acetate was hydrolyzed by refluxing with alcoholic potassium hydroxide, the hydrolysis mixture acidified with diluted hydrochloric acid, diluted with water, and the white
precipitate shaken out with several portions of ether. Evaporation of the ether solution left a white residue which was crystallized from absolute methanol to yield 0.65 Gm. of white needles of oleanolic acid, m. p. 312314". which showed no depression (311-313') when mixed with an authentic sample (5). [a]';' = +80.2" (chloroform, C = 0.988). REFERENCES (1) Markley. K. S., and Sando, C. E., J . B i d . Chem.. 105 643(1934). ($) Kremers, R. E., Sell, H. M., and Stookey. A. D., unpubfished data. (3) Dodge F. D. J . A m Chcm. SOC. 40 1932(1918). (4) Sando'C. E. ' J . Biol.'Chcm.. 90. k77j1931). (5) Rowe,'B. J., kt al., THISJOURNAL, 38, 122(1949).
Chemical Studies on Ursolic Acid* By BETTY Y. T. WUt and LLOYD M. PARKS
CaH46J1gg$&228), b
From ursolic there was prepared acetyl ursolyl amide and acetyl ursolyl nitrile, 0th of which were reduced by lithium aluminum hydride to an ursolyl amine (C-28), C&46 {-OH2NHp. -cH By a similar reduction of ursonic
acid oxime there was obtained an isomeric ursolyl amine (C-2),
C d h
ursonate9 C 2 9 H 4 s ( ~ ~ O O C H was 3 , reduced, by the Huang-Minlon modification of the Wolff-Kishner method, to ursanic acid, which was reduced by lithium aluminum hydride to the primary alcohol, ursanol,
an isomer of the naturally occurring secondary alcohol, a-amyrin,
HE COMBINATION of certain amines, such as "procaine, with penicillin to form salts of low water solubility to prolong the action of penicillin in the body is well known. Since ursolic acid itself is of extremely low solubility it appeared possible that an amine obtained from it might be used in the Same way. Each of the two reactive groups in ursolic acid, namely the carboxyl at C-28 and the secondary alcohol at C-2, offers points for introduction of the amino group; one of the objects of this study was to prepare these two indicated amino derivatives. Among the known triterpene compounds in nature there has been shown to exist relationships which are of biogenetic significance. Thus, in addition to the hydroxy acid, ursolic acid, there
*
Received March 16 1953 from the School of Pharmacy, University of Wiseonsih, Mahison. Supported in part by a research grant from the National Cranberry Association Hanson Massachusetts. t NOtiqnal C-b& Assmihion Fellow in Pharmaceutical Chermstrg, 1951-52.
is also found the corresponding diol, uvaol, and the secondary alcohol, a -amyrin. There has not yet been found (nor may it necessarily exist in nature) the corresponding isomeric primary alcohol; another object of this study was to prepare the indicated primary alcohol, which we have designated as ursanol. Acetyl ursolyl amide was obtained in consistently good yields by the action of ammonia gas on a dioxane solution of acetyl ursolyl chloride and the presence of the amide group was confirmed by its infrared spectrum (Fig. 1). By the action of acetic anhydride acetyl ursolyl amide was converted to acetyl wsolyl nitrile, presence of the nitrile group being confirmed by its infrared spectrum (Fig. 2). Both acetyl ursolyl amide and acetyl ursolyl nitrile were reduced in good yields to an ursolyl amine (C-28) by lithium aluminum hydride. An isomeric ursolyl amine (C-2) was prepared by reduction of ursonic acid oxime, and its methyl