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Tox i eoa , 1966 , Vo l . 4 . pp . 890 . Pe rQamon Prcaa L td . , Pr in ted in Grca t B r i t a i n

THE ISOLAT ION AND IDENT I F ICAT ION OF THREEALKALO I DS FROM ACAC I A BERLAND IER I*

HERMAN R . ADAMS t and BENN IE I . CAMPDepa r tmen t o f B iochem i s t r y and Nu t r i t ion , Te xas A and M Un i ve r s i t y ,

Co l l ege S t a t i on , Texas , U .S . A .(Accep t ed jo r pu b l i ca t i on 5 May 1966)

Aba f rac t -A me thod i s desc r ibed for the i so l a t i on and i de n t i f i ca t i on o f three ~pheny l e thy l -am ine a l ka lo ids f rom Acac i a be r t a r rd i e r i (gua j i l l o ) . The i so l a t ed am i ne s we r e i de n t i f i ed bymeans o f pape r , t h i n - l aye r , and gas~h roma tog r ap hy as t y ram ine , N -me thy l t y tam i ne andho rden i ne .

I NTRODUCT I ONAcac i a be r l and i e r i (gu a j i l l o ) i s a membe r o f the f am i l y Legum i no sae . I t i s a de c i d i oussh rub 3 . to 14 f t in he igh t w i th p i nna t e l eaves and f ew o r no pr i ck l es . Th i s p l an t growsin i ts grea tes t dens i t y i n the Edwa rds P l a t eau o f Te xas and No r t he rn Mex i co .

Dur ing pe r i ods o f ex t ended d rough t , sheep and goa t s may inges t the p l an t exc lus i ve l yfo r pe r iods o f 6 to 12 mon ths be fo re deve l op i ng a l ocomo to r a t ax i a o f the h i ndquar t e r s .The cond i t ion i s co l loqu i a l l y ca l l ed "gua j i l lo wobb l es " o r " l i mbe r l eg " .

CAMP and LYMAN [1 ] i so l a ted the sympa t hom i me t i c am i ne , N -me thy l - -pheny l e thy l -am i ne , f rom gu a j i l l o . Fu r the r inves t iga t ion revea l ed the p resence o f a l ka lo ids o the r thanN -me thy l - -pheny l e thy l am i ne . The presen t s tudy was i n i t i a t ed to e luc i da te the chem i ca lna tu r e o f these un iden t i f i ed a l ka lo ids .

MATER I ALS AND METHODSEx t rac t i on p rocedu r eTh i r t y - f i ve pounds o f g round l eaves f rom gu a j i l l o were ex t rac ted w i th 70% e thano l

ad j us t ed to a pH o f 2 w i th concen t ra t ed HC I . The ex t rac t ion p rocedu re was repea t ed3 t imes , and a f i na l ex t rac t ion was made us ing 33% e thano l .

The comb ined l iqu id ex t rac t s we r e concen t ra t ed unde r r educed p ressu re to a vo lumeo f 5 L , then d i lu ted to 10 I . w i th wa t e r . A p l an t p i gmen t prec ip i ta ted upon the add i t i ono f wa t e r and was removed by f i l t ra t i on . The f i l t r a t e was concen t ra t ed unde r reducedpressu re to 4 I . A sa tu ra t ed so lu t i on o f sod ium hyd rox i de was added to the f i l t r a t e and aye l l ow prec ip i ta te formed wh i ch was removed by cen t r i fuga t ion .

The bas i c supe rna t an t so lu t ion was fur the r c l a r i f i ed by the add i t ion o f l ead ace ta te andl ead subace t a t e [2 ] , and the heavy prec ip i t a t e was removed by cen t r i fuga t ion . Concen-t ra ted HC l was added to the so l u t ion to prec ip i ta te the excess l ead as l ead ch lor ide , and"Suppo r t ed i n pa r t by a g ran t f rom the He rman F rosch Founda t i on , New Yo r k , N.Y . Con t r i bu t i on o f theTe xas Ag r iw l t tus l Expe r i men t S ta t ion .tBased i n pa r t upon a the s i s p resen t ed to the G r adua t e Schoo l o f Te xasAand M Un i ver s i t y by He rman R .Adams i n pa r t i a l f u l f i l lmen t o f the requ i remen t s f o r the deg ree o f Mas t e r o f Sc i ence .

85

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86 HERMAN R . ADAMS and BENN I E J CAMPthe p r ec i p i t a t e was r emoved by f i l t ra t i on . The ac i d i c f i l t ra te was ad jus ted t o pH 8 w i thsod i um ca rbona te , and the pheno l s we re ex t r ac t ed i n t o ch l o ro fo rm . The ch l o ro fo rmex t r ac t was ex t r ac t ed repea ted l y w i th an aqueous sod i um hydrox i de so l u t i on to sepa r a t ethe pheno l i c am i nes f rom the non -pheno l i c am i nes .The a l ka l i ne aqueous phase was made ac i d i c w i t h concen t ra ted HC l and then a l ka l i neonce aga in w i th sod i um ca rbona t e . Then the pheno l i c am i nes we re ex t r ac t ed i n to ch l o ro -fo rm o r e t he r .

Ch roma tog r aph i c techn iquesPape r ch roma tog r aphy . A samp l e o f the c rude p l an t ex t r ac t was ch roma tog raphedtwo-d imens iona l l y on Wha tman No . 1 pape r us ing the fo l low ing so l ven t syst ems : (1 )n -bu tano l sa t u r a t ed w i th wa t e r : ace t i c ac i d (10 :1) ; (2 ) n -bu t ano l : e thano l : ammon i umhydrox ide (90 : 10 :1) . Ascend i ng ch roma tog raphy was used to deve l op the pape r s t r ipssuspended by g l ass rods in sea l ed ch roma tog r aphy chambe r s .The hydroch lor ide sa l t s o f the i so l a t ed am ines and r e f e r ence am i nes we re d i sso l vedi n ch l o ro fo rm or me thano l and spo t ted on Wha tman No . 1 pape r . The pape r s t r ips we redeve l oped ascend ing l y i n t he so l ven t syst ems : (1 ) sec . -bu t ano l : wa t e r : form i c ac i dace t i c ac i d (30 :4 :3 :1) ; (2 ) i so-propano l : ace t i c ac i d : wa t e r (15 :3 :2 ) .Th i n l aye r ch roma tog raphy . S i l i ca Ge l G (E . Me rck ) p l a t es o f 025 mm t h i ckness we reused to ch roma tog r aph t he r e f e r ence compounds and the p l an t am ines . The p l a t es we reac t i va ted and s t o r ed a t 100 i n an e l ec t r i c oven . Top and s i de bounda r i es we re cu ti n the p l a t e ma te r i a l w i th a penc i l t o f ac i l i t a t e smoo the r so l ven t f ron t s and to s t op theso l ven t f ron t s a t a des i r ed he igh t .The p l an t am ines and r e f e r ence am i nes we re app l i ed as the hydroch lo r ide sa l t s on t h i nl aye r p l a t es . The so l ven t systems t ha t ga ve sa t i s f ac t o r y r esu l t s we re : (1 ) sec . -bu t ano l :

wa t e r : g l ac i a l ace t i c ac i d (15 : 3 : 2 ) , (2 ) n -bu t ano l : g l ac i a l ace t i c ac i d : wa t e r ( 12 : 3 : 5 ) , (3 )i so-propano l : g l ac i a l ace t i c ac i d : wa t e r (15 :3 : 2 ) , and (4 ) n-bu tano l sa t u r a t ed w i th wa t e r .Cas - l iqu id ch roma tog r aphy . A Wa rne r -Ch i l co t t Company Mode l 604 gas ch roma to -graph , equ i pped w i th a f l ame i on i z a t i on de t ec t o r , and a 10 /1 ou tpu t sp l i t t e r for prepara t i ve

wo rk , was used to ch roma tog r aph the p l an t am ines and r e f e r ence samp l es . The co l umnwas p repa red accord ing to the me t hod o f BROCHMANN e t a / . [3 ] . The co l umn cons i s t edo f ch romoso rb W (Johns-Manv i l l e ) 60 -80 mesh , wh i ch was deac t i va ted w i th hexame thy l - i s i l i z an e [4 ] and coa ted w i th 30 pe r cen t SE -30 [5] . The co l umn ma t e r i a l was packed i ns t a i n l ess s tee l tub ing (3 / 8 i n . x 6 f t ) . The co l umn was ope ra ted i so t he rma l l y a t 202 andthe ou tpu t sp l i t t e r t empe ra tu re was ma in t a ined a t 268 . The ca r r i e r gas (n i t rogen ) f lowr a t e was 296 m l pe r m in . A l l o f the samp l es we re ch roma tog raphed as the f ree base i nme thano l . Co l l ec t ions we re made i n U shaped g l ass tubes a t room tempe ra tu re .Ch romogen i c reagen t s

A 0~1% so l u t i on o f n inhyd r i n in 95 % e thano l was u t i l i zed to t es t f o r p r ima r y andseconda ry am i nes . Eh r l i ch ' s reagen t [6 ] was used to tes t f o r t he presence o f t he indo l eg roup . Dragendo r f f ' s reagent [7 ] was used because i t was found t o be spe c i f i c for ce r t a i n o fthe a l ka l o i ds presen t . Pheno l i c compounds we re de t ec t ed w i th Pau l y ' s [8] reagen t .A spray reagen t f o r secondary a l i ph a t i c am ines [9 ] con s i s t ed o f a 1% n i t rop russ i deso l u t i on to wh i ch 10 % , by vo l ume o f ace ta ldehyde was added . The ch roma tog ram was

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I so l a t ion o f A l ka lo ids f rom Acac i a be r l and i e r i 8 7sprayed w i th th i s r eagen t , and then i t was sprayed w i t h a 2 % so l u t i on o f sod i um ca rbona teand hea ted a t l 00 for 5 m in . A b lue co l o r was pos i t i ve f o r seconda ry a l i ph a t i c am i nes .The sod i um p i c r a t e reagen t [10 ] f o r hyd rocyan i c ac i d and cyan ides was p repa red bycomb i n i ng 1 m l o f a sa t u r a t ed so l u t i on o f p i c r i c ac i d w i th 3 m l o f 10% sod i um hyd rox i de .Re f e rence samp l esTyram i ne , 3-hydroxytyram ine , and ho rden i ne we re ob t a ined comme rc i a l l y . N -me thy l - -pheny l e thy l am ine was prepa red by the me t hod O f CAMP and MooRE [11 ] .r r -me thy l t y ram ine was syn thes i zed by the procedu re o f K IRKWOOD and I V IAR I ON [12] ,s t a r t i ng w i th t he in termed i a te - (4me thoxypheny l ) -e thy l am i ne . The me thod was a l t e r edi n t ha t the x-me thy l t y ram i ne was p r ec i p i t a t ed f rom anhyd rous e t he r as the hydroch lo r idesa l t by us i ng a l coho l i c hyd rogen ch l o r i de .

RESULTS AND D I SCUSS I ONA samp l e o f the c rude ac i d i c p l an t ex t r ac t was ch roma tog raphed two -d imens iona l l yon a se r i es o f pape r ch roma tog r ams . A concen t ra ted spo t t ha t was pos i t i ve w i t h D ragen -do r f f ' s reagen t and n inhydr i n was i den t i f i ed by compa ra t i ve ch roma tog raphy as t v -me thy l - -pheny l e thy l am ine , as repo r ted by CAMP and LYMAN [1] . Two un i den t i f i ed spo t s o fl owe r concen t ra t ion t ra i l ed beh i nd the N -me thy l - -pheny l e thy l am ine and gave po s i t i ven inhyd r in and Pau l y ' s t es t s bu t nega t i ve Dragendor f f ' s , Eh r l i ch ' s and sod i um p i c r a t e tes t s .The pos i t i ve n inhydr in and Pau l y ' s t es t i nd i ca t ed t ha t the compounds we re pheno l i c am i nes .When the ch l o ro fo rm ex t r ac t con ta in ing the pheno l i c compounds was ch roma tog raphedon th i n l aye r p l a t es , f ou r pheno l i c compounds we re de t ec t ed w i th Pau l y ' s reagent . Twoo f the compounds gave a po s i t i v e n inhydr i n tes t , a t h i r d gave nega t i ve n inhydr i n t es t bu ta pos i t i ve Dragendo r f f ' s tes t . The four th pheno l was nega t i ve w i th n i nhyd r in and D ragen -

do r f f ' s t es t s .The a l ka l o i ds we re ex t r ac t ed f rom the p l an t ma t e r i a l i n m inu t e quan t i t i es , and compa ra -t i ve ch roma tog r aphy was emp l oyed i n an a t t emp t to i de n t i f y some o r a l l o f the compounds .The r e f e r ence compounds we re se l ec t ed f rom the -pheny l e thy l am ine se r i es o f a l ka l o i ds [13] ,because N -me thy l - -pheny l e thy l am ine had prev ious l y been i so l a t ed f rom gua j i l l o [1] . Thepheno l i c compounds i n the -pheny l e thy l am ine se r i es t ha t cou l d have been i so l a t ed by theex t r ac t i on procedu res and a l so g i ven pos i t i ve Pau l y ' s tes t s we re tyram ine , x -me thy l -t y ram i ne and ho rden i ne . 3 -Hyd roxy t y ram i ne gave a nega t i ve Pau l y ' s tes t and was e l i m i -na ted as one o f the pheno l i c am i nes .

Re f e rence samp l es o f t y ram ine and N -me thy l t y ram i ne we re ch roma tog raphed on pape ra l ong w i th t he ch l o ro form ex t r ac t con ta in ing the pheno l i c a l ka l o i ds o f gua j i l l o . Whensprayed w i th Pau l y ' s r eagen t , two o f the p l an t am ines had R ! va l ues co r respond i ng t ot y ram ine and N -me thy l t y ram i ne . Ty ram i ne gave a cha r ac t e r i s t i c purp l e co l o r and N -me thy l -t y ram ine gave a grey co lor when t es t ed w i th n inhydr i n . The p l an t am i nes o f compa rab l e R fva l ues gave i de n t i ca l co l o r s . One o f t he pheno l i c compounds co r respond i ng to N -me thy l -t y ram ine gave a pos i t i ve t es t f o r a seconda ry am i ne when t r ea t ed w i th the n i t ropruss idereagent (Tab l e 1 ) .

Gas - l i qu id ch roma tog raphy was emp l oyed bo th as an an a l y t i ca l t oo l and as a prepa ra -t i ve me thod o f pu r i f i ca t i on . A compa r i son o f r e t en t i on va lues be tween s tandards ( F i g . 1)and a ch lo ro form ex t r ac t con ta in ing bo th pheno l i c and non -pheno l i c am ines (F ig . 2 )demons t ra t ed the p l an t am i nes to be tv-me thy l --pheny l e thy l am ine , t y ram ine and N -me thy l -t y ram ine . I t was imposs ib l e t o de t ec t ho rden i ne i n the presence o f N -me thy l t y ram ine on

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88 HERMAN R . ADAMS and BENN I E J . CAMPTABLE 1 . PAPER CHR OMATOGRAPHY OF AM INE4 ISOLATED FROM OUAJ ILLO

l 1CnW

W

OUW

z

Oa I

"Samp l e was spo t ted as hyd roch lor ide sa l t .tSo l ven t syst ems : (1 ) sec :BuOH : H , O : HCOOH : HOAc (75 :10 :7 " 5 :2 "

(2 ) i so -P rOH : HOAc : H , O (75 :15 :10 ) .

N -METHYLTYRAM I NE

TYRAM I NE

HOROENNE

N -METHYL -B -PHENYLETHYLAMWE

4 8 12 16 20 24T IME I N M INUTES

F IG . 1 . GAS -L IQU ID CHR OMATOG RAPHY OF RBFERENCE SAMPLES ON A CHROMOSORB W COLUMNCOATED w ITH 30~ sE-30 .

Vapo r i ze r t empe ra tu re was 268 and co l umn t empe ra ture was 202 ' .

Samp l e " So l ven t syst em t Sp ray reagen t

( I ) (2 ) Pau l y ' sN i t rop russ ide

andac e t a ldehyde N i nhyd r i n

R fTy ram i ne 056 073 Red Nega t i ve Purp l eN -me thy l -

t y ram i ne 064 0 " 76 Red B l ue G r eyP l an t am i ne s 0 " 58 0 " 70 Red Nega t i ve Purp l e

0 " 64 0 " 76 Red B l ue G r ey

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WUZOaW

W

OUW

I so l a t ion o f A l ka l o i ds f rom Acac i a be r l and i e r i 8 9zW

A

T IME IN M INUTESF10 . 2 . GAS -L IQU ID CHROMATOGRAPHY OF PLANT AM INF3 ON A CHROMOSORB W COLUMN

COAT W I TH 3O~ 3&-3O.Vapo r i ze r t empe ra tu re was 268 and co l umn tempe ra t lnro was 202 . The ma j o r peaks a re

(A) N -me thy l - -pheny l e thy l am i ne , (B) t y ram i ne , (O N -me thy l t y ram i ne and ho rden i ne .the an a l y t i ca l co l umn because o f the m i nu t e quant i t y o f ho rden i ne presen t i n the p l an tex t r ac t .A ch l o ro form ex t r ac t con ta in ing the pheno l i c a l ka l o i ds o f gu a j i l l o was sub j ec ted t oprepa ra t i ve gas - l iqu id ch roma tog raphy and the peaks co r respond i ng to tyram ine , N -me thy l -t y ram i ne and ho rden i ne we re co l l ec t ed . The pheno l i c p l an t am ines co l l ec t ed f rom gas-l i qu i d ch roma tog r aphy we re spo t ted w i th s tandards on th i n l aye r p l a t es . The ch roma to -g r ams we re deve l oped i n 4 so l ven t syst ems and t he pheno l i c compounds we re de t ec t ed

w i th Pau l y ' s reagen t . In each so l ven t system , the R~ va l ues f o r the t h r ee pheno l i c am i nesi so l a t ed f rom gua j i l l o we re compa rab l e to the R f va lues ob ta ined w i th r e f e r ence samp l eso f t y ram ine , N -me thy l t y ram ine and ho rden ine (Tab l e 2 ) .The ex t r ac t i on p rocedu re and ch romogen i c reagents c l as s i f i ed the i so l a t ed compoundsas pheno l i c am i nes . The r e t en t i on va l ues o f gas - l iqu id ch roma tog raphy f o r r e f e r encesamp l es and the i so l a t ed p l an t am i nes we re compa r ab l e . F rom these f i nd i ng s i t wasconc l uded t ha t the i so l a t ed p l an t a l ka l o i ds we re tyram ine , x -me thy l t y ram ine and ho rden i ne .

SUMMARY(1 ) A procedu re i s descr ibed for the i so l a t i on o f t h r ee pheno l i c am i nes f rom Acac i abe r l and i e r i by means o f so l ven t ex t r ac t i on and gas ch roma tog raphy .(2 ) The ch romogen i c reagent s and ch roma tog raphy t echn iques used t o i de n t i f y andi so l a t e the am ines a re presen ted .(3 ) The p l an t am ines we re found to be i n the -pheny l e thy l am ine se r i es o f a l ka l o i ds .

Compa r a t i ve paper , t h i n l aye r , and gas - l i qu i d ch roma tog raphy i de n t i f i ed the i so l a t edpheno l i c am i nes as t yram ine , x -me thy l t y ram i ne , and ho rden i ne .

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90 HERMAN R . ADAMS and BENN i E J . CAMPTAHLE 2 . Tmmv LAYER C f IROMATOORAPHY OF GLC PUR I F I ED PLANT AM INES*" 0 "2S mm S i l i ca Ge l G P l a t es .tSamp l es we re spo t t ed as hyd roc h l o r i de sa l t .$So l ven t sys t ems :

(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 t u ra t ed w i th H , O .

RE FERENCES[1] CAMP , B . J . and LYMAN , C . M . , The i so l a t i on o f N -me t hy l - - pheny l e t hy l am i ne f rom Aca c i a be r / an d i e r i .J. Am . pha rm. Ass . , Sc i . Ed . , 25 , 719 , 1956 .[2]

W I I r rE , E . P . , A l ka l o i ds o f the l egum i nosae . N. Z . J . Sc i . TechnoJ . (B ) , 25 , 137 , 1944 .[3] BROCHMANN , E . , HANSSEN , A . and $VENDSEN , B . , Sep a r a t i on and i den t i f i ca t i on o f sympa thom ime t i c

am ines by ga s - l i qu i d ch roma tog raphy . J . pha rm. Sc i . , 51 , 938 , 1962.[4 ]

Bo I IE t in : r t , J . , LANGER , S . H . , PERRUT , R . H . and PURNELL , J . J . , A s t udy o f the adso rp t i ve p rop e r t i es o ff i reb r i ck i n re l a t i on to i t s p rope r t i es as a so l id suppo r t i n gas - l i qu i d ch roma tog raphy . J . cheer .

Soc . , 2440 , 1960 .[5] HORN ING , E . C . , M08CATELLY , E . A . and $WEELY , C . C . , Po l yes t e r l i qu i d ph ases i n ga s - l i qu i d ch roma -tography . Chem . Ind . , 751 , 1959 .[6] BLOCK , R . J . , D I rnRUM , E . L . and GUNTER , Z . , A Manua l o j Pap e r Ch rou r tog raphy and Pap e r E l ec t ro -pho res i s . 2nd ed . , Academ i c Press , New York , p . 137 , 1958 .[7] BLOCx , R . J . , DURRUM , E . L . and GUNTER , Z . , A Manua l o j Pap e r Ch ro ra tog raphy and Pap e r E l e c t r o -pho res i s . 2nd ed . , Academ i c Press , New York , p . 361 , 1958 .[8] BLOCx , R . J . , DUARUM , E . L . and GUNTER , Z . , A Manua l o f Pap e r Ch ro ra tog raphy and Pap e r E l ec t ro -pho res i s . 2nd ed . , Academ i c Press , New York , p . 132 , 1958 .[9 ]

FE I OL , F . , Spo t Te s t s , Vo l . I I , E l sev i e r , New York , p . 189 , 1954 .[10] Snun~oxs , J . S . and GENTZxow , C . J . , Med i ca l and Pub l i c Hea l th Labora to ry Me t ho ds .

Lea andFe b i ge r , Ph i l ad e l ph i a , p . 532 , 1955.[ l l ]

CAMP , B . J . and MOORS , J . M . , A quan t i t a t i ve me thod fo r the a l ka l o i d o f Aca c i a be r l and i e r i . J . Am .pha rm. Ass . , Sc i . Ed . , 49 , 158 , 1960 .[12 ] I {nexwooD , S . and MAR ION , L . , The b i ogenes i s o f a l ka l o i ds , L -The i so l a t i on o f N -me t hy l t y r am i nef rom ba r l ey . J. Am . them . Soc . , 72 , 2522 , 1950 .[13 ]

MANSKE , R . H . F . and HOLMES , H . L . , 71 re A l k a l o i ds , Vo l . I I I , Academ i c Press , New York , p . 313 ,1953 .

Samp l e t (1 )So l ven t

(2 )syst ems$

(3) (4 )Spray r eagen t

Pau l y ' sR I

GLC pu r i f i edp l an t am ines 0 " 10 0 "42 0 " 14 0 " 06 Red0" 39 0 " 56 0 "51 0 "27 Red0"28 0 "51 0 " 31 0 " 17 RedHo rden ine 0 " 10 0 " 42 0 " 14 0 -06 RedTyram i ne 0 " 39 0 "56 0 " 51 027 RcdN-me thy l t y ram ine 0 " 28 0 " 51 0 "31 0 " 17 Red