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Isolation of Oleanolic Acid and Saponin from Trefoil (Lotus corniculatus, var. viking)
Notes Isolation of Oleanolic Acid and Saponin from Trefoil (Lotus corniculatus, var. uiking) B~ E.
n. WALTER
Oleanolic acid was isolated from the alcohol extract of fresh trefoil (Lotus cornicdatus, var. Viking) and identified by comparison of its physical and chemical properties with authentic material. A small quantity of crude saponin was also isolated, which upon hydrolysis yielded soyasapogenol B.
I
AN INVESTIGATION of saponins in legume forages, the method used for the isolation of saponin from bur clover (1) and from ladino clover (2) was applied t o fresh trefoil. When ether extraction was applied to the concentrated alcohol extract only a very sniall fraction of saponin appeared at the ether-water interface, but most of the material was soluble in ether. This indicated the presence of sapogenin rather than saponin, and that under the conditions of extraction i t occurred uncombined. The melting point 304305' of the crystallized material and the infrared spectrum were identical with those of authentic oleanolic acid. This is another addition t o the long list of plants that contain oleanolic acid either free or combined as saponin, which have been reported. Filtration of the solid material that appeared a t the above mentioned water-ether interface yielded 22 Gm. of crude saponin, or about 0.06% of the dry weight of trefoil. The low yield of saponin (water insoluble) and the occurrence of free oleanolic acid instead of saponin may account for nonexistence of bloat in ruminants from feeding on trefoil if it is assumed that saponins are one cause of bloat. No water-soluble saponin was obtained. N
EXPERIMENTAL
Fresh trefoil (164 Kg.) was placed in a stainless steel tank and covered with 750 L. of 957, alcohol and allowed to stand for five days. The extract was drained and treated with charcoal (12 Gm. per L.) and filtered, with filter aid, in a filter press. The alcohol was distilled a t atmospheric pressure until the volume remaining was about 90 L. Upon cooling white, microcrystalline material appeared. This was centrifuged in a continuous centrifuge. The solids were removed from the bowl and were suspended in water and again centrifuged and washed with water several times to remove watersoluble materials. The crude product was susReceived August 5 . 1980. from Western Krgi(ma1 Research I.aboratory, Western Utilization Rewarch arid Development Division, AbTiculturnl Ilesearrh Service, V. S. Department of Agriculture, Albany, Calif. Accepted for publication August 10, 1960.
pended in water and sufficient ether was added to dissolve most of the solids when shaken gently in a separatory funnel. A small quantity of insoluble material (saponin) appeared at the interface. The ether solution was clarified with charcoal and evaporated t o near dryness. When recrystallized from methanol the product melted a t 304305'. Comparison with a known sample showed it to be oleanolic acid. Yield, 57 Gm. or about 0.17% of the dry weight of trefoil. Soyasapogenol B.-The saponin was hydrolyzed by boiling for sixty hours in normal sulfuric acid in 65% ethanol. Water was added and the precipitated sapogenin was extracted with ether. Crystallization in methanol and chloroform gave a product melting a t 260'. When mixed with soyasapogenol B the melting point was unchanged. The infrared spectrum was identical with that of known soyasapogenol B. Acetyl Oleanolic Acid.-The oleanolic acid was acetylated with acetic anhydride. When recrystallized from ethanol and chloroform and dried a t 120" in a vacuum it melted a t 268", [a]? 73.7". And-Calcd. for CazHa~Oa: C, 77.06; H, 10.01. Found: C, 76.7; H, 9.89. Methyl Acetyloleano1ate.-Thc acetyl oleanolic acid (1Gm.) was dissolved in about 25 ml. of ether, and to this was added an ether solution containing an excess of diazomethane. The mixture was allowed to stand for twenty-four hours. Upon evaporation of the ether the product crystallized. Recrystallization in methanol and chloroform gave a product melting a t 217-219', [ a ] y = +70.5' in chloroform. Frazier and Noller (3) reported the melting point 219-220" and [a]? = f70.4" for this compound. And-Calcd. for C33H&: C, 77.29; H, 10.22. Found: C, 77.2; H, 10.0.
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REFERENCES (11 Walter, E. D . , THISJOURNAL,^^, 466(1987). (2) Walter E. D. Bickoff E. M., Thompson C. R . Robinson, C.' H., and Djeras& C., J . A m . Chem. 'Soc., 77: 4936(1955) (3) Frazter, D . , and Noller, C. R., ibid.,66, 1267(1944).
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n. WALTER
Oleanolic acid was isolated from the alcohol extract of fresh trefoil (Lotus cornicdatus, var. Viking) and identified by comparison of its physical and chemical properties with authentic material. A small quantity of crude saponin was also isolated, which upon hydrolysis yielded soyasapogenol B.
I
AN INVESTIGATION of saponins in legume forages, the method used for the isolation of saponin from bur clover (1) and from ladino clover (2) was applied t o fresh trefoil. When ether extraction was applied to the concentrated alcohol extract only a very sniall fraction of saponin appeared at the ether-water interface, but most of the material was soluble in ether. This indicated the presence of sapogenin rather than saponin, and that under the conditions of extraction i t occurred uncombined. The melting point 304305' of the crystallized material and the infrared spectrum were identical with those of authentic oleanolic acid. This is another addition t o the long list of plants that contain oleanolic acid either free or combined as saponin, which have been reported. Filtration of the solid material that appeared a t the above mentioned water-ether interface yielded 22 Gm. of crude saponin, or about 0.06% of the dry weight of trefoil. The low yield of saponin (water insoluble) and the occurrence of free oleanolic acid instead of saponin may account for nonexistence of bloat in ruminants from feeding on trefoil if it is assumed that saponins are one cause of bloat. No water-soluble saponin was obtained. N
EXPERIMENTAL
Fresh trefoil (164 Kg.) was placed in a stainless steel tank and covered with 750 L. of 957, alcohol and allowed to stand for five days. The extract was drained and treated with charcoal (12 Gm. per L.) and filtered, with filter aid, in a filter press. The alcohol was distilled a t atmospheric pressure until the volume remaining was about 90 L. Upon cooling white, microcrystalline material appeared. This was centrifuged in a continuous centrifuge. The solids were removed from the bowl and were suspended in water and again centrifuged and washed with water several times to remove watersoluble materials. The crude product was susReceived August 5 . 1980. from Western Krgi(ma1 Research I.aboratory, Western Utilization Rewarch arid Development Division, AbTiculturnl Ilesearrh Service, V. S. Department of Agriculture, Albany, Calif. Accepted for publication August 10, 1960.
pended in water and sufficient ether was added to dissolve most of the solids when shaken gently in a separatory funnel. A small quantity of insoluble material (saponin) appeared at the interface. The ether solution was clarified with charcoal and evaporated t o near dryness. When recrystallized from methanol the product melted a t 304305'. Comparison with a known sample showed it to be oleanolic acid. Yield, 57 Gm. or about 0.17% of the dry weight of trefoil. Soyasapogenol B.-The saponin was hydrolyzed by boiling for sixty hours in normal sulfuric acid in 65% ethanol. Water was added and the precipitated sapogenin was extracted with ether. Crystallization in methanol and chloroform gave a product melting a t 260'. When mixed with soyasapogenol B the melting point was unchanged. The infrared spectrum was identical with that of known soyasapogenol B. Acetyl Oleanolic Acid.-The oleanolic acid was acetylated with acetic anhydride. When recrystallized from ethanol and chloroform and dried a t 120" in a vacuum it melted a t 268", [a]? 73.7". And-Calcd. for CazHa~Oa: C, 77.06; H, 10.01. Found: C, 76.7; H, 9.89. Methyl Acetyloleano1ate.-Thc acetyl oleanolic acid (1Gm.) was dissolved in about 25 ml. of ether, and to this was added an ether solution containing an excess of diazomethane. The mixture was allowed to stand for twenty-four hours. Upon evaporation of the ether the product crystallized. Recrystallization in methanol and chloroform gave a product melting a t 217-219', [ a ] y = +70.5' in chloroform. Frazier and Noller (3) reported the melting point 219-220" and [a]? = f70.4" for this compound. And-Calcd. for C33H&: C, 77.29; H, 10.22. Found: C, 77.2; H, 10.0.
=+
REFERENCES (11 Walter, E. D . , THISJOURNAL,^^, 466(1987). (2) Walter E. D. Bickoff E. M., Thompson C. R . Robinson, C.' H., and Djeras& C., J . A m . Chem. 'Soc., 77: 4936(1955) (3) Frazter, D . , and Noller, C. R., ibid.,66, 1267(1944).
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