THE ISOLATION AND IDENTI F ICATION OF THREE ALKALOIDS FROM ACACIA BERLANDIERI*

Transcription

1 Toxieoa, 1966, Vol. 4. pp.890. PerQamon Prcaa Ltd., Pr inted in Grcat Br i tain THE ISOLATION AND IDENTI F ICATION OF THREE ALKALOIDS FROM ACACIA BERLANDIERI* HERMAN R. ADAMSt and BENNIE I. CAMP Depar tment of Bi ochemi st ry and Nut r i t i on, Texas A and M Un i vers i ty, Co l l ege Stat i on, Texas, U.S.A. (Accepted j or pub l i ca t i on 5 May 1966) Aba f rac t -A me thod i s descr i bed for the i so l a t i on and i dent i f i ca t i on of three ~pheny l e thy l - ami ne a l ka l o i ds f rom Acac i a ber tar rd i er i (gua j i l l o). The i so l a ted ami nes were i dent i f i ed by means of paper, thin- l ayer, and gas~hroma tography as tyrami ne, N-methy l tytami ne and horden i ne. INTRODUCTION Acac i a ber l and i er i (gua j i l l o) i s a member of the fami l y Legumi nosae. I t i s a dec i d i ous shrub 3. to 14 f t i n he i ght w i th p i nna te l eaves and f ew or no pr i ck l es. Th i s p l ant grows i n i ts grea test dens i ty i n the Edwards Pl a teau of Texas and Nor thern Mex i co. Dur i ng per i ods of extended drought, sheep and goa ts may i ngest the p l ant exc l us i ve l y for per i ods of 6 to 12 months be fore deve l op i ng a l ocomotor a tax i a of the h i ndquar ters. The cond i t i on i s co l l oqu i a l l y ca l l ed "gua j i l l o wobb l es" or " l i mber l eg". CAMP and LYMAN [1] i so l a ted the sympa thomi me t i c ami ne, N-me thy l -ß-pheny l e thy l - ami ne, f rom gua j i l l o. Fur ther i nvest i ga t i on revea l ed the presence of a l ka l o i ds other than N-me thy l -ß-pheny l e thy l ami ne. The present study was i n i t i a ted to e l uc i da te the chemi ca l na ture of these un i dent i f i ed a l ka l o i ds. MATERIALS AND METHODS Ext rac t i on procedure Th i r ty- f i ve pounds of ground l eaves f rom gua j i l l o were ext rac ted w i th 70% e thano l ad j usted to a ph of 2 w i th concent ra ted HCI. The ext rac t i on procedure was repea ted 3 t i mes, and a f i na l ext rac t i on was made us i ng 33% e thano l. The comb i ned l i qu i d ext rac ts were concent ra ted under reduced pressure to a vo l ume of 5 L, then d i l uted to 10 I. w i th wa ter. A p l ant p i gment prec i p i tated upon the add i t i on of wa t er and was removed by f i l t rat ion. The f i l t rate was concent ra t ed under reduced pressure to 4 I. A sa tura ted so l ut i on of sod i um hydrox i de was added to the f i l t ra te and a ye l l ow prec i p i tate formed wh i ch was removed by cent r i fuga t i on. The bas i c superna tant so l ut i on was fur ther c l ar i f i ed by the add i t i on of l ead ace tate and l ead subace tate [2], and the heavy prec i p i tate was removed by cent r i fuga t i on. Concent ra ted HCl was added to the so l ut i on to prec i p i tate the excess l ead as l ead ch l or i de, and "Suppor ted i n par t by a grant f rom the Herman Frosch Founda t i on, New York, N.Y. Cont r i but i on of the Texas Agr i w l t tus l Exper i ment Stat i on. tbased i n par t upon a thes i s presented to the Gradua te Schoo l of TexasA and M Un i vers i ty by Herman R. Adams i n par t i a l fu l f i l l ment of the requ i rements for the degree of Master of Sc i ence. 85

2 86 HERMAN R. ADAMS and BENNIE J. CAMP the prec i p i tate was removed by f i l t ra t i on. The ac i d i c f i l t ra te was ad j usted to ph 8 w i th sod i um carbona te, and the pheno l s were ext rac ted i nto ch l oroform. The ch l oroform ext rac t was ext rac ted repea ted l y w i th an aqueous sod i um hydrox i de so l ut i on to separa te the pheno l i c ami nes f rom the non-pheno l i c ami nes. The a l ka l i ne aqueous phase was made ac i d i c w i th concent ra ted HCl and then a l ka l i ne once aga i n w i th sod i um carbona te. Then the pheno l i c ami nes were ext rac ted i nto ch l oroform or e ther. Chroma tograph i c techn i ques Paper chroma tography. A samp l e of the crude p l ant ext ract was chroma tographed two-d i mens i ona l l y on ha tman No. 1 paper us i ng the fo l l ow i ng so l vent systems : (1) n-butano l sa tura ted w i th wa ter : ace t i c ac i d (10 :1) ; (2) n-butano l : e thano l : ammon i um hydrox i de (90 : 10 :1). Ascend i ng chroma tography was used to deve l op the paper st r ips suspended by gl ass rods in sea l ed chroma tography chambers. The hydroch l or i de sa l ts of the i so l a ted ami nes and re ference ami nes were d i sso l ved i n ch l oroform or me thano l and spot ted on ha tman No. 1 paper. The paper st r i ps were deve l oped ascend i ng l y i n the so l vent systems : (1) sec. -butano l : wa ter : formi c ac i d ace t i c ac i d (30 :4 :3 :1) ; (2) i so-propano l : ace t i c ac i d : wa ter (15 :3 :2). Th i n l ayer chroma tography. S i l i ca Ge l G (E. Merck) p l a tes of 025 mm th i ckness were used to chroma tograph the re ference compounds and the p l ant am i nes. The pl ates were act i vated and stored at 100 in an e l ect r i c oven. Top and s ide boundar i es were cut i n the p l a te ma ter i a l w i th a penc i l to fac i l i tate smoother so l vent f ronts and to stop the so l vent f ronts a t a des i red he i ght. The p l ant ami nes and re ference ami nes were app l i ed as the hydroch l or i de sa l ts on th i n l ayer p l ates. The so l vent systems that gave sat i sfactory resul ts were : (1) sec. -butano l : wa ter : g l ac i a l ace t i c ac i d (15 :3 :2), (2) n-butano l : g l ac i a l ace t i c ac i d : wa ter (12 :3 :5), (3) i so-propano l : g l ac i a l ace t i c ac i d : wa ter (15 :3 :2), and (4) n-butano l sa tura ted w i th wa ter. Cas- l i qu i d chroma tography. A arner -Ch i l cot t Company Mode l 604 gas chroma tograph, equ i pped w i th a f l ame i on i za t i on de tec tor, and a 10/1 output sp l i t ter for prepara t i ve work, was used to chroma tograph the p l ant ami nes and re ference samp l es. The co l umn was prepared accord i ng to the me thod of BROCHMANN e t a /. [3]. The co l umn cons i sted of chromosorb (Johns-Manv i l l e) mesh, wh i ch was deac t i va ted w i th hexame thy l - ä i s i l i zane [4] and coa ted w i th 30 per cent SE-30 [5]. The co l umn ma ter i a l was packed i n sta i n l ess stee l tub i ng (3/8 i n. x 6 f t ). The co l umn was opera ted i sotherma l l y a t 202 and the output sp l i t ter tempera ture was ma i nta i ned a t 268. The car r i er gas (n i t rogen) f l ow ra te was 296 ml per mi n. Al l of the samp l es were chroma tographed as the f ree base i n me thano l. Co l l ec t i ons were made i n U shaped g l ass tubes a t room tempera ture. Chromogen i c reagents A 0~1% so l ut i on of n i nhydr i n i n 95 % e thano l was ut i l i zed to test for pr i mary and secondary am i nes. Ehr l i ch ' s reagent [6] was used to test for the presence of the i ndo l e group. Dragendor f f ' s reagent [7] was used because i t was found to be spec i f i c for cer ta i n of the a l ka l o i ds present. Pheno l i c compounds were de tec ted w i th Pau l y ' s [8] reagent. A spray reagent for secondary a l i pha t i c ami nes [9] cons i sted of a 1% n i t ropruss i de so l ut i on to wh i ch 10%, by vo l ume of ace ta l dehyde was added. The chroma togram was

3 Iso l a t i on of Al ka l o i ds f rom Acac i a ber l and i er i 8 7 sprayed w i th th i s reagent, and then i t was sprayed w i th a 2 % so l ut i on of sod i um carbona te and hea ted a t l 00 for 5 mi n. A b l ue co l or was pos i t i ve for secondary a l i pha t i c ami nes. The sod i um p i cra te reagent [10] for hydrocyan i c ac i d and cyan i des was prepared by comb i n i ng 1 ml of a sa tura ted so l ut i on of p i cr i c ac i d w i th 3 ml of 10% sod i um hydrox i de. Re ference samp l es Tyrami ne, 3-hydroxytyrami ne, and horden i ne were obta i ned commerc i a l l y. N-me thy l - ß-pheny l e thy l ami ne was prepared by the me thod Of CAMP and MooRE [11]. r r -me thy l tyrami ne was synthes i zed by the procedure of KIRKOOD and IVIARION [12], star t i ng w i th the i ntermed i a te ß- (4me thoxypheny l ) -e thy l ami ne. The me thod was a l tered i n tha t the x-me thy l tyrami ne was prec i p i tated f rom anhydrous e ther as the hydroch l or i de sa l t by us i ng a l coho l i c hydrogen ch l or i de. RESULTS AND DISCUSSION A samp l e of the crude ac i d i c p l ant ext rac t was chroma tographed two-d i mens i ona l l y on a ser i es of paper chroma tograms. A concent ra ted spot tha t was pos i t i ve w i th Dragendor f f ' s reagent and n i nhydr i n was i dent i f i ed by compara t i ve chroma tography as tv-me thy l - ß-pheny l e thy l ami ne, as repor ted by CAMP and LYMAN [1]. Two un i dent i f i ed spots of l ower concent ra t i on t ra i l ed beh i nd the N-me thy l -ß-pheny l e thy l ami ne and gave pos i t i ve n i nhydr i n and Pau l y ' s tests but nega t i ve Dragendor f f ' s, Ehr l i ch ' s and sod i um p i cra te tests. The pos i t i ve n i nhydr i n and Pau l y ' s test i nd i ca ted tha t the compounds were pheno l i c ami nes. hen the ch l oroform ext rac t conta i n i ng the pheno l i c compounds was chroma tographed on th i n l ayer p l a tes, four pheno l i c compounds were de tec ted w i th Pau l y ' s reagent. Two of the compounds gave a pos i t i ve n i nhydr i n test, a th i rd gave nega t i ve n i nhydr i n test but a pos i t i ve Dragendor f f ' s test. The four th pheno l was nega t i ve w i th n i nhydr i n and Dragendor f f ' s tests. The a l ka l o i ds were ext rac ted f rom the p l ant ma ter i a l i n mi nute quant i t i es, and comparat i ve chroma tography was emp l oyed i n an a t tempt to i dent i fy some or a l l of the compounds. The re ference compounds were se l ec ted f rom the ß-pheny l e thy l ami ne ser i es of a l ka l o i ds [13], because N-me thy l -ß-pheny l e thy l ami ne had prev i ous l y been i so l a ted f rom gua j i l l o [1]. The pheno l i c compounds i n the ß-pheny l e thy l ami ne ser i es tha t cou l d have been i so l a ted by the ext rac t i on procedures and a l so g i ven pos i t i ve Pau l y ' s tests were tyrami ne, x-me thy l - tyrami ne and horden i ne. 3-Hydroxytyrami ne gave a nega t i ve Pau l y ' s test and was e l i mi - na ted as one of the pheno l i c ami nes. Re ference samp l es of tyrami ne and N-me thy l tyrami ne were chroma tographed on paper a l ong w i th the ch l oroform ext rac t conta i n i ng the pheno l i c a l ka l o i ds of gua j i l l o. hen sprayed w i th Pau l y ' s reagent, two of the p l ant ami nes had R! va l ues cor respond i ng to tyrami ne and N-me thy l tyrami ne. Tyrami ne gave a charac ter i st i c purp l e co l or and N-me thy l - tyrami ne gave a grey co l or when tested w i th n i nhydr i n. The p l ant ami nes of comparab l e R f va l ues gave i dent i ca l co l ors. One of the pheno l i c compounds cor respond i ng to N-me thy l - tyrami ne gave a pos i t i ve test for a secondary ami ne when t rea ted w i th the n i t ropruss i de reagent (Tab l e 1). Gas- l i qu i d chroma tography was emp l oyed both as an ana l yt i ca l too l and as a preparat i ve me thod of pur i f i ca t i on. A compar i son of re tent i on va l ues be tween standards (F i g. 1) and a ch l oroform ext rac t conta i n i ng both pheno l i c and non-pheno l i c ami nes (F i g. 2) demonst ra ted the p l ant ami nes to be tv-me thy l -ß-pheny l e thy l ami ne, tyrami ne and N-me thy l - tyrami ne. I t was i mposs i b l e to de tec t horden i ne i n the presence of N-me thy l tyrami ne on

4 88 HERMAN R. ADAMS and BENNIE J. CAMP TABLE 1. PAPER CHROMATOGRAPHY OF AMINE4 ISOLATED FROM OUAJILLO Samp l e " So l vent systemt ( I ) (2) Pau l y ' s Spray reagent N i t ropruss i de and ace ta l dehyde N i nhydr i n Rf Tyram i ne Red Negat i ve Purp l e N-me thy l - tyrami ne " 76 Red Bl ue Grey P l ant ami nes 0 " 58 0 " 70 Red Nega t i ve Purp l e 0" 64 0" 76 Red Bl ue Grey " Samp l e was spot ted as hydroch l or i de sa l t. tso l vent systems : (1) sec:buoh : H,O : HCOOH : HOAc (75 :10 :7 " 5 :2" (2) i so-proh : HOAc : H,O (75 :15 :10). N-METHYLTYRAMINE TYRAMINE l 1 Cn HOROENNE O U z O a I N-METHYL-B-PHENYLETHYLAME TIME IN MINUTES F IG. 1. GAS-LIQUID CHROMATOGRAPHY OF RBFERENCE SAMPLES ON A CHROMOSORB COLUMN COATED with 30 ~ se-30. Vapor i zer tempera ture was 268 and co l umn tempera ture was 202'.

5 Iso l a t i on of Al ka l o i ds f rom Acac i a ber l and i er i 8 9 A U Z O a z O U TIME IN MINUTES F GAS-LIQUID CHROMATOGRAPHY OF PLANT AMINF3 ON A CHROMOSORB COLUMN COAT ITH 3O~ 3&-3O. Vapor i zer tempera ture was 268 and co l umn tempera t l nro was 202. The ma j or peaks are (A) N-me thy l -ß-pheny l e thy l ami ne, (B) tyrami ne, (O N-me thy l tyrami ne and horden i ne. the ana l yt i ca l co l umn because of the mi nute quant i ty of horden i ne present i n the p l ant ext rac t. A ch l oroform ext rac t conta i n i ng the pheno l i c a l ka l o i ds of gua j i l l o was sub j ec ted to prepara t i ve gas- l i qu i d chroma tography and the peaks cor respond i ng to tyrami ne, N-me thy l - tyrami ne and horden i ne were co l l ec ted. The pheno l i c p l ant ami nes co l l ec ted f rom gasl i qu i d chroma tography were spot ted w i th standards on th i n l ayer p l a tes. The chroma tograms were deve l oped i n 4 so l vent systems and the pheno l i c compounds were de tec ted w i th Pau l y ' s reagent. In each so l vent system, the R~ va l ues for the three pheno l i c ami nes i so l a ted f rom gua j i l l o were comparab l e to the Rf va l ues obta i ned w i th re ference samp l es of tyrami ne, N-me thy l tyrami ne and horden i ne (Tab l e 2). The ext rac t i on procedure and chromogen i c reagents c l ass i f i ed the i so l a ted compounds as pheno l i c ami nes. The re tent i on va l ues of gas- l i qu i d chroma tography for re ference samp l es and the i so l a ted p l ant ami nes were comparab l e. From these f i nd i ngs i t was conc l uded tha t the i so l a ted p l ant a l ka l o i ds were tyrami ne, x-me thy l tyrami ne and horden i ne. SUMMARY (1) A procedure i s descr i bed for the i so l a t i on of three pheno l i c ami nes f rom Acac i a ber l and i er i by means of so l vent ext rac t i on and gas chroma tography. (2) The chromogen i c reagents and chroma tography techn i ques used to ident i fy and i so l a te the ami nes are presented. (3) The p l ant ami nes were found to be i n the ß-pheny l e thy l ami ne ser i es of a l ka l o i ds. Compara t i ve paper, th i n l ayer, and gas- l i qu i d chroma tography i dent i f i ed the i so l a ted pheno l i c ami nes as tyrami ne, x-me thy l tyrami ne, and horden i ne.

6 90 HERMAN R. ADAMS and BENN i E J. CAMP TAHLE 2. Tmmv LAYER Cf IROMATOORAPHY OF GLC PURIFIED PLANT AMINES* So l vent systems$ Spray reagent Samp l e t (1) " 0"2S mm S i l i ca Ge l G P l a tes. tsamp l es were spot ted as hydroch l or i de sa l t. $So l vent systems : (1) sec. -BuOH : HOAc : H,O (75 :15 :10). (2) n-buoh : HOAc : H,O (60 :15 :25) (3) i so-proh : HOAc : H,O (75 :15 :10) (4) n-buoh sa tura ted w i th H,O. (2) (3) (4) Pau l y ' s GLC pur i f i ed p l ant ami nes 0 " 10 0 "42 0" 14 0" 06 Red 0" 39 0" 56 0"51 0 "27 Red 0"28 0"51 0 "31 0" 17 Red Horden i ne 0" 10 0"42 0 " Red Tyram i ne 0 " 39 0"56 0 " Rcd N-me thy l tyrami ne 0 " 28 0 " 51 0"31 0 " 17 Red RI REFERENCES [1] CAMP, B. J. and LYMAN, C. M., The i so l a t i on of N-me thy l -ß-pheny l e thy l ami ne f rom Acac i a ber / and i er i. J. Am. pharm. Ass., Sc i. Ed., 25, 719, [2] I I r re, E. P., Al ka l o i ds of the l egumi nosae. N.Z. J. Sc i. TechnoJ. (B), 25, 137, [3] BROCHMANN, E., HANSSEN, A. and $VENDSEN, B., Separa t i on and i dent i f i ca t i on of sympa thomi me t i c ami nes by gas- l iquid chroma tography. J. pharm. Sc i., 51, 938, [4] BoI IEt i n : r t, J., LANGER, S. H., PERRUT, R. H. and PURNELL, J. J., A study of the adsorpt i ve proper t i es of f i rebr i ck i n re l a t i on to i ts proper t i es as a so l i d suppor t i n gas- l i qu i d chroma tography. J. cheer. Soc., 2440, [5] HORNING, E. C., M08CATELLY, E. A. and $EELY, C. C., Po l yester l i qu i d phases i n gas- l i qu i d chromatography. Chem. Ind., 751, [6] BLOCK, R. J., DI rnrum, E. L. and GUNTER, Z., A Manua l o j Paper Chro»ur tography and Paper E l ec t rophores i s. 2nd ed., Academi c Press, New York, p. 137, [7] BLOCx, R. J., DURRUM, E. L. and GUNTER, Z., A Manua l o j Paper Chro»ra tography and Paper E l ec t rophores i s. 2nd ed., Academi c Press, New York, p. 361, [8] BLOCx, R. J., DUARUM, E. L. and GUNTER, Z., A Manua l of Paper Chro»ra tography and Paper E l ec t rophores i s. 2nd ed., Academi c Press, New York, p. 132, [9] FEIOL, F., Spot Tests, Vo l. I I, E l sev i er, New York, p. 189, [10] Snun~oxs, J. S. and GENTZxow, C. J., Med i ca l and Publ i c Hea l th Laboratory Me thods. Lea and [ l l ] Feb i ger, Ph i l ade l ph i a, p. 532, CAMP, B. J. and MOORS, J. M., A quant i tat i ve me thod for the a l ka l o i d of Acac i a ber l and i er i. pharm. Ass., Sc i. Ed., 49, 158, J. Am. [12] I {nexwood, S. and MARION, L., The b i ogenes i s of a l ka l o i ds, L-The i so l a t i on of N-me thy l tyrami ne f rom bar l ey. J. Am. them. Soc., 72, 2522, [13] MANSKE, R. H. F. and HOLMES, H. L., 71re Al ka l o i ds, Vo l. I I I, Academi c Press, New York, p. 313, 1953.