[CONTRIBUTION FROM THE BUREAUOF ENTOMOLOGY AND PLANT QUARANTINE, AGRICULTURAL RESEARCHADMINISTRATION, U. S. DEPARTMENT OF AGRICULTURE ]
AMORPHIX, A GLYCOSIDE IN AMORPHA FRUTICOSA L. FRED ACREE, JR., MARTIN JACOBSON,
AND
H. L. HALLER
Received September 9.8,194.8
In 1937 Moore (4) reported the presence of rotenone in the roots, stem bark, and seeds of the legume Amorpha fruticosa L. from Nebraska, because they gave a positive Durham test (3). This test has been accepted as a qualitative test for rotenone and some of the rotenoids (5) when applied to leguminous plants. I n 1942 Featherly (l), of the Oklahoma Agricultural and Mechanical College, suggested that, inasmuch as A .fruticosa is so abundant throughout the Mississippi River Valley, its seed might serve as a source of rotenone during the war emergency. At his request the Bureau of Entomology and Plant Quarantine examined samples of seed from widely different locations for their supposed rotenone content. Although all the samples tested gave a positive Durham reaction, no rotenone or any of the rotenoids could be isolated from any of them. However, fractionation of the chloroform extractives of the seed collected at Vermillion, S. Dak., by the Soil Conservation Service, yielded a slightly hygroscopic glycoside that melted a t 151-151.5’ and gave a positive Durham test. Analysis showed the compound, for which the name “amorphin” is suggested, to correspond to the formula C33H4001B. It gave a positive orcinol test, but it did not reduce Fehling’s solution. When warmed in concentrated hydrochloric acid, amorphin readily dissolved, and when further heated a product separated that after purification melted at 191-192’. This aglycon gave a positive Durham test, and analysis showed it to correspond to the formula CZHnO,. This compound, tentatively designated “amorphigenin,” also mas obtained from the ether extractives of the seed. The acid filtrate obtained in the hydrolysis of amorphin readily reduced Fehling’s solution. It thus appears that Amorpha fruticosa contains a glycoside which, as well as its aglycon, behaves similarly to rotenone in the Durham color test. EXPERIMENTAL
Extraction of seed. Belyaev (2) showed that the seed of A m o r p h a fruticosa contain considerable oil, and his findings were confirmed in this laboratory. Since large amounts of oil might inhibit the crystallization of any rotenone present in the extractive, the seed was first extracted for a short time with petroleum ether. The extractive contained a small amount of material positive to the Durham test. There remained in the marc a t least two compounds that reacted positively to the Durham test. One of these compounds was soluble in ether, and both were soluble in chloroform. Because i t was difficult to separate the mixture obtained by extracting only with chloroform, the following typical scheme was considered the most practical method of extraction. The coarsely ground seed (738 grams) was extracted for 3 hours with petroleum ether (b.p. 30-60”). After removal of the solvent, the marc was finely ground and exhaustively extracted, first with ether and then with acid-free chloroform. The extracts were worked up separately. Fractionation of petroleum ether extractive. During concentration the petroleum ether 572
GLYCOSIDE OF AMORPHA FRUTICOSA
573
extract partly crystallized. There separated 6.2 grams of material which gave a slightly positive Durham test. On recrystallization from methanol, 780 mg. of a colorless crystalline product was obtained, which melted a t 190-191" and was negative in the Durham test. From the methanol mother liquor the neutral fraction, which was only slightly positive in the Durhrtm test, was obtained, but nothing definite could be isolated. Complete removal of the solvent from the original petroleum ether extract yielded 76 grams of oil. This gave a positive Durham test. The active material was extracted with methanol. The concentrated methanol extract yielded 150 mg. of a colorless crystalline product, imp. 163-169", which gave a negative Durham test. The neutral fraction of the mother liquor was dissolved in ether, from which 30 mg. of yellow needles crystallized. The product melted a t 235-237", with decomposition, and did not give the Durham reaction. S o active material could be separated from the mother liquor, which still gave a slightly positive Durham test. Separarion of amorphigenin. After removal of the solvent, the ether extract yielded 11.9 grams of oil, which was dissolved in acetone and cooled. Some inactive amorphous material precipitated and was separated. The acetone was removed by evaporation, and the residual oil partly crystallized after solution in methanol. The amorphigenin thus obtained (200 mg.) was thrice recrystallized from methanol, and then melted a t 191-192'. It gave a positive Durham test but failed to reduce Fehling's solution either before or after acid hydrolysis. It gave a negative phenol test. Anal. Calc'd for CzzHzzOl: C, 66.32; H , 5.56; 2 OCHs, 15.56; mol. wt., 398. Found (avg. 2 anal.): C , 66.63; H , 5.63; OCHa, 15.55; mol. wt. (Rast), 396, 400. The mother liquor from methanol recrystallization yielded 45 mg. of colorless crystalline material, which melted a t 218" after one recrystallization. It was positive to the Durham test, but sufficient material was not available for further study. S e p a r a f i o n of amorphin. After removal of the solvent under reduced pressure, the chloroforra extractive weighed 5.7 grams. It was dissolved in a small amount of boiling acetone. Upon cooling, 950 mg. separated, which was boiled with chloroform and filtered while hot The insoluble residue, upon being recrystallized twice from water and twice froin methanol, yielded 450 mg. of pure amorphin, which melted to a glass at 151-151.5'. T h r mother liquor contained more of this compound, but was lost during further purification. Amorphin gave a positive Durham test, a positive orcinol test, and a negative phenol test. It did not reduce Fehling's solution until after acid hydrolysis. For analysis the compound was dried a t 100" (0.5 mm.) for 30 minutes. Anal. Calc'd for CssH40016: C, 57.22; H , 5.82; 2 OCH3, 8.95. Found (avg. 2 anal.): C, 57.44; H, 5.97; OC&, 8.85. Hydrolysis of amorphin. When boiled with 1 cc. of concentrated hydrochloric acid, 50 mg of amorphin readily dissolved; on further heating a produrt precipitated which was separated from the diluted and cooled reaction mixture. This aglycon x a s recrystallized from methanol and melted at 191-192". When mixed with an equal part of amorphigenin obtained as described above, the melting point showed no depression. The acid filtrate readily reduced Fehling's solution, but the sugar portion of the molecule could not be isolated. SUMMARY
The seed of Amorpha fruticosa L. gives the color reaction in the Durham test which heretofore has been considered specific for rotenone and the rotenoids, but no compounds of this class could be isolated from them. The product that is responsible for the positive reaction in the Durham test is amorphigenin, C~H2207, the aglycon of the glycoside amorphin, C33H40016. HELTSVILLE,Mn.
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REFERENCES
(1) Agriouhral Znsecticide and Fungicide Assoc. Bul. No. D-23,2 pp. Oct. 7,1942. [Processed.]
(2) BELYAEV, Masloboino-Zhirovoe Delo, 9, No. 6, 26 (1933). (3) JONES, A N D SMITH, Znd. Eng. Chem., Anal. Ed., 6, 75 (1933). (4)MOORE,Puerto Rico Agr. Erpt. Sta. Rpt., 1937, 115 pp. (5) ROARK,J. Econ. Ent., 33, 416 (1940).
