12.1
Lepii ni iriosue Alkaloids. !I. Alkaloids of LirpinrLs westiunrrs Small'
Juiiuury 1967
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of average control group activity to the average activity of each drugged group was found t o be 0.69, 0.40, and 0.41. These results showed the varied effects characteristic of minor depressants which produce ataxia. I n the hexobarbital sleeping time test, the alkaloid mixture was administered (intraperitoneal) in doses of 5 mg/kg to groups of 10 mice. One hour later, hexobarbital (100 mg/kg) was administered, arid the resulting sleeping time was measured arid compared to a saline control group. The results showed a poteiitiatiori of hexobarbital sleeping time in that the average sleeping time of the drugged mice was fourid to be 50 miri while that of the control group was 13 min. Both of these preliminary tests gave, therefore, indication of C S S activity and may be interpreted to mean that the mixture of alkaloids obtained from L. ~ r e s t i a m scoritailis orie or more depressant coiistituents of a weak nature. 111addition, pure, crystalline lupinine was evaluated for it5 action on isolated uterine strips. The results of this assay showed lupinine to possess oxytocic activity only to a minor degree. A pure sample of (-)li-oxosparteine was also tested for its oxytocic activity arid found to exhibit only about 207, of the oxytocic activity of sparteine sulfate. Experimental Section6 Alkaloids of Lupinus westianus Nutt.-The whole plants (including roots) which were collected while in full bloom during April 1962, near Panama City, Fla., were air-dried for 2 weeks and then milled to about 80 mesh. This material was used for all of our extraction work. I t was determined to possess an average moisture content of iC;. Three kilograms was moistened with 6 SH&H solution and loosely packed into a sack made of bleached muslin. T h e sack and contents were immersed in chloroform, and the extract>ioti was allowed to proceed during 24 hr with occasional stirring of the CHCla infusion. I n this manner three 1-day batch extractions were carried out. ilfter the three extracts were combined and condensed to approximately 4 l., the dark green residiie was successively washed with 6 HCl. T h e combiiied acidic washes were made strongly basic with 6 S KaOH aiid theii extracted (CHCl,). T h e chloroform extracts were combiiied, dried fiYa2S0.f),filtered, and evaporated in e'actto i o provide t h e mixtrire of crude alkaloids as a red-brown colored. viscous oil. 4 total of 80 g of criide alkaloids was acciimulated i l l this way by the batchwise extraction of 9.T kg of I,. westianus. Paper chromatographic examination of the crude alkaloid mixtiire gave three spots: Rf 0.53, 0.42, and 0.22, shown later to be sparteine, lripinine, and mriltiflorine, respectively. Fractional distillation of the mixt,ure of crude alkaloids provided sparteine and lupinine. IIultiflorine was obtained by coliimii chromatography of the distillation residue on IT-oelm. noiialkaline, alLimina. T h e lower boiling fraction [bp 94" (0.9 mm)] exhibited an infrared spectrum (neat) which was found to he siiperimposable ripon t h a t obtaiiied from authentic sparteine.* Both hubstances also displayed identical chromatographic behavior, and the specific rotatioil ( [ c Y ] ~ ~ -16.8 D f 0.2" (c 6.03, ethaiiol)) determined from the plant alkaloid closely matched (6) Temperature readings are uncorrected. Melting points were determined with samples in evacuated sealed capillary tubes. Combustion analyses a r e by t h e Schwarzkopf Xlicroanalytical Laboratory, 11-oodside, N . Y. W h a t m a n No. 1 paper with a solvent system of 1-butanol-acetic acid (10:1, I./\.) t h a t had been saturated ivith water was used for t h e descending paper chromatoeraphy reported herein. T h e papergrams x e r e allowed t o develop until t h e solvent front had pone approximately 24 cm from t h e starting line. Spots were risiialised b y application (spraying) of Draeendnrff reagent.' ( 7 ) "The l I e r c k Index," j t l i ed, XIerck a n d Co., Inc., Ralin-ay, S . J., 1940, p 696. (8) Prepared from cornmercially ai-ailable (Inland .ilkaloid Co.) sparteine zulfate.
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that ( [ C Y ] D - 1 T . O O (ethanol)) previo~islyreported9 for sparteine I n addition, a monoperchlorate salt imp 169.0-169.5') and a monohydriodide salt (mp 229-231') were prepared from the plant alkaloid: mixture melting point with the corresponding aalts prepared from authentic sparteine,* showed no depression. T h e higher boiling fraction [bp 113' (0.8 mm)] obt,ained from distillation of the mixture of crude plant alkaloids slowly cryatallized after several hours. This material was recrystallized from hexane several timeb to provide a constaiit melting product. T h e product was shown to be lupinine by means of elemental analysis and comparison of its properties (mp 68.0-68.5", [ C Y ] 231) -23.2 i 0.3' (c 3.T7, ethanol), hydrochloride m p 213.5-214.0') with those reportedg for lupinine ( m p iO-Tl", [ C Y ] D -21.3" (ethanol), hydrochloride m p 20i-209'). In addition, an infrared spectrum (CC1,) determined from the plant alkaloid was foiind to be siiperimposable iipoii that obtained from a sample of aiithentic lupiiIine.'0 dnal. Calcd for CloHi,SO: C, 70.96; H, 11.31: X, 8.28. Fo1ind: C, T0.62; H, 11.67: S , 8.5i. T h e individual base (Rf 0.22)obtained from t h e ethyl acetate eluates of aliiminia chromatography of the distillation rejidue was shown t o be miiltiflorine. T h e material, initially obtained as a viscous, slightly yellow oil, was moleciilarlp distilled [ 160196" (air bath, 0.06 m m ) ] to yield a colorless oil which was induced to crystallize from hexane. T h e prodlict %vasrecrystallized until constant m p 107-108". This melting point, along with its specific rotation ( [CY!% -310 =t 1.0" (c 5.03, methanol)) and spectral properties [infrared, YZ,"~ 1640 (conjugated amide carbonyl)" and 1590 em-' fconjrigated olefin);" ultraviolet, x:A:n" 248 and 325 mp ( e 7800 and 12,400):nmr, B C D C 1 3 6.89 (1H doriblet, J = 13 cps) and 4.94 (1 H doiiblet, J = 1 3 cps)] were strongly reminibcent of properties displayed by miiltifloriiie, an alkaloid recently foiind in this laboratory' to be among those elaborated by L . cliffusus. T h e identity with multiflorine was readily confirmed by means of mixtiire melting point and superimposable infrared spectra. T h e weight percentage of each alkaloid was determined by rarefiil extraction of 30.0 g of dry, milled whole plant. T h e criide mixture of alkaloids obtained amoiinted to 0.316 g, or 1.14c< of the weight of the moisture-free plant. A sample (0.26Tg ) of this material was pawed throiigh a column of \Toelm, nonalkaline aliimina (Brockmann grade I ) in ethyl acetate to remove the highly polar colored impurities. T h e column was washed with et,hyl acetate until no more basic material was obtained in the eluent. Evaporation of the ethyl acetate left 0.180 g of the slightly yellow alkaloid mixture. Based upon this determination, the alkaloid content of L . westianiis was calculated as 0.7';; of the weight of the whole, moisture-free plant. T h e relative amounts of each alkaloid were then determined by measurement of the peak areas obtained from gas-liquid partition chromatographic analysis1* of the alkaloid mixtiire. I t was previously established, using a kiiowii mixtnre of the three alkaloids, that the ratio of integrated peak areas acciirately reflected the weight ratio of the alkaloids. I n this manner it was determined t h a t sparteine, lupinine, and miiltiflorine occurred in L. zcestianzis to the extent, of 0.16,0.09, and 0.52C;;, respectively, of the weight of the moisture-free whole plant.
Acknowledgment.-This work was supported in part by a Kational Institute of Mental Health grant (MH07314) for which the authors express siricere thanks. We are also pleased t'o acknowledge our thanks to Professor William J. Iiirinard, Jr., University of Pit tsburgh School of Pharmacy, through whose court,esy the physicological test results relmted herein were obtained. ( 9 ) ?i. .J. Leonard, M k a l u i d s , 3, 126 (1953). (10) TT-e are indebted t o Dr. Brj-ce Douglas, Bmitli Kline and Frencli Laboratories, Philadelphia, P a . , for this sample. (11) L.J. Eellamy, " T h e Infrared Spectra of Complex llolecules," 2nd ed, John \Vile? a n d Sons, Inc.. N e n . Tork, 3.. I-.,1958; Ii. Nakanishi, "Infrared Absorption Rpectroscop)-," Holden-Day, Inr., San Francisco, Calif., 1962. (12) Carried out on a I: & 11 Scientific Corp., A I ~ d e l500, gas cliromatograph with an 8 f t X 0 . 2 5 in. cooper column packed with l)iatoport-\V containing 5 % (n. w) General Electric, SE-30 silicone rubber. Helium, 3.5 kgIcm2. v a s used as carrier gas, and the column \vas temperature programmed bet\\ een 150 and 200' during the analyses.
