O c t . 2 3 , 1962 H . L .

CRAIG ETAL 3 , 060, 165

PREPARATIO N O F TO XIC RICIN
Filed July 3 , 1952
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S a lly h . D ie/ r e
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PREPARATIO N O F TBXIC RICIN -
H a r r y L . Cr a ig , Cin c in n a t i, a n d O t t o H . Alder k s, W yo
min g , O h io , a n d Also p h H . Co r w in a n d S a lly H . D iek e,
Ba lt imo r e, a n d Ch a r lo t t e L . K a r el, S ilv er S p r in g , M d. ,
a ssig n o r s t o t h e Un it ed S t a t es o f Amer ic a a s r ep r esen t ed
by t h e S ec r et a r y o f t h e Ar my
Filed July 3 , 1952 , S er . No . 2 97 , 14 2
2 Cla ims. ( Cl. 2 60- 12 3 5)
Th is in v en t io n r ela t es t o t h e met h o d o f p r ep a r in g t o x ic
l lClIl.
Ric in is a p r o t o p la smic p o iso n p r ep a r ed f r o m c a st o r
bea n s a f t er t h e ex t r a c t io n o f c a st o r o il t h er ef r o m. It is
mo st ef f ec t iv e a s a p o iso n w h en in j ec t ed in t r a v en o usly o r
in h a led, t h e la t t er r eq uir in g ex t r eme c o mmin ut io n a n d
sma ll p a r t ic le siz e t o be ef f ec t iv e. It is believ ed t h a t t h e
t o x ic a c t io n is c a t a lyt ic r a t h er t h a n st o ic h io met r ic w h ic h
p r o ba bly a c c o un t s f o r t h e h ig h t o x ic it y o f t h e a g en t .
Bec a use o f it s r ela t iv e in st a bilit y, r ic in must be h a n dled
w it h ex t r eme c a r e. In n eut r a l a q ueo us so lut io n it is st a ble
o n ly up t o 60~7 5 C. , a n d in so lid f o r m up t o 100110
0, a lt h o ug h f o r sh o r t ex p o sur es, t emp er a t ur es up t o 13 0
ma y be t o ler a t ed. It is sen sit iv e t o a c ids, a lk a lis a n d
h a lo g en a n d ma y a lso be in a c t iv a t ed by mec h a n ic a l w o r k
in g suc h a s g r in din g o r p ulv er iz in g . Th ese f a c t o r s a r e o f
g r ea t imp o r t a n c e in dev elo p in g a sa t isf a c t o r y met h o d f o r
p r ep a r in g t h e ma t er ia l.
Alt h o ug h r ic in h a s been p r ep a r ed in c r yst a llin e c o n di
t io n in t h e la bo r a t o r y in sma ll q ua n t it ies, it bec o mes n ec es
sa r y, f o r p ur p o ses o f t o x ic o lo g ic a l w a r f a r e, t o p r ep a r e
r ela t iv ely la r g e q ua n t it ies in a h ig h st a t e o f p ur it y. Th is
n ec essit a t es t h a t a s muc h a s p o ssible o f t h e n o n - t o x ic ma
t er ia l p r esen t be r emo v ed in t h e p r o c ess.
In p r ep a r in g t h e p r o t ein ma t er ia l, t h e c a st o r bea n s a r e
? r st g r o un d a n d p r essed t o r emo v e mo st o f t h e o il. Th e
p r essed c a k e st ill r et a in s a bo ut 15% o il a n d t h is ma y be
r emo v ed by mea n s o f so lv en t s w h ic h w ill ex t r a c t a n a ddi
t io n a l 150 p o un ds o f o il p er t o n o f bea n s a n d r educ e t h e
o il r et a in ed in t h e c a k e t o a lit t le o v er 1%. In t h e ev en t
t h a t t h e ex p r essin g st ep is sup p lemen t ed by so lv en t ex t r a c
t io n , it is imp o r t a n t t o p r ev en t det o x i? c a t io n o f t h e p r o
t ein dur in g t h e so lv en t r emo v a l st ep . If r esidua l so lv en t
is r emo v ed f r o m t h e g r o un d bea n s by blo w in g w it h st ea m,
c o n sider a ble det o x i? c a t io n r esult s. Blo w in g w it h n it r o g en
e? ec t iv ely p r ev en t s det o x i? c a t io n but is ex p en siv e w h en
c a r r ied o ut o n a la r g e sc a le.
Af t er t h e o il h a s been r emo v ed, t h e p r essed c a k e o r
p o ma c e is ex t r a c t ed by a g it a t in g w it h w a t er a t a p H o f
3 . 8 101 a t 2 5 C. w h ic h r emo v es subst a n t ia lly a ll o f t h e
t o x ic p r o t ein . Th e ex t r a c t io n p r o c ess is o p er a t iv e w it h in
a p H r a n g e o f a bo ut 3 t o 4 . 5 a lt h o ug h t h e p r ef er r ed
r a n g e is a bo ut 3 . 5 t o 4 . , Th e o p t imum o p er a t in g p o in t
is a p H o f 3 . 8 - 1. 1, a s in dic a t ed a bo v e. A c a r ef ul p H
c o n t r o l is essen t ia l in o r der t h a t a s muc h n o n - t o x ic p r o t ein
a s p o ssible ma y be elimin a t ed a n d a lso t h a t t h e ? lt r a t io n
r a t e ma y be h eld a t a sa t isf a c t o r y v a lue. Eit h er H Cl o r
H 2 8 04 ma y be used t o g et t h e desir ed p H f o r t h e ex t r a c t io n
w a t er , but H 2 504 is p r ef er r ed due t o it s lo w er c o r r o sio n
r a t e a n d ea se o f h a n dlin g in c o n c en t r a t ed f o r m. Th e a c id
sh o uld be used in r ea so n a bly dilut e f o r m t o p r ev en t un due
lo c a l c o n c en t r a t io n s dur in g it s a ddit io n . A 5% c o n c en
t r a t io n is sa t isf a c t o r y.
Fo llo w in g t h e ex t r a c t io n , t h e slur r y is ? lt er ed usin g
eit h er a c o n v en t io n a l r ec essed p la t e ? lt er o r a c o n t in uo us
st r in g disc h a r g e v a c uum ? lt er . W it h t h e la t t er a bo ut 7 %
o f ? lt er a id, ba sed o n mea l w eig h t , w a s f o un d n ec essa r y
f o r sa t isf a c t o r y ? lt r a t io n .
Th e ? lt r a t e f r o m t h e w a t er ex t r a c t io n st ep , w h ic h c o n
t a in s t h e r ic in , w a s t r ea t ed w it h a 16. 7 % so lut io n o f
Na 2 S O 4 t o p r ec ip it a t e t h e p r o t ein . Th is so lut io n is c o m
t
10
15
2 0
2 5
3 0
3 5
4 5
50
55
60
65
7 0
3 , 060, 165
Pa t en t ed O c t . 2 3 , 1962
" ic e
2
p o sed o f 2 0 p o un ds o f sa lt in 100 p o un ds o f w a t er a n d t h e
a mo un t used w a s suc h t h a t t h e sa lt c o n t en t eq ua lled 2 0%
o f t h e ? lt r a t e w eig h t . Th is a mo un t a n d c o n c en t r a t io n o f
sa lt so lut io n w a s a bo ut o p t imum c o n sider in g t h e f a c t o r s
o f c o st a n d t o x in r ec o v er y. S o mew h a t h ig h er c o n c en t r a
t io n s a n d la r g er a mo un t s o f so lut io n c a n be used, h o w ev er .
Th e p r ec ip it a t io n p r o c ess is n o t limit ed t o t h e use o f
Na 2 S O 4 , sin c e a sa t ur a t ed so lut io n o f Na Cl c a n be used
suc c essf ully, but Na 2 S O 4 so lut io n g iv es bet t er n it r o g en
f r a c t io n a t io n , mo r e r a p id p r ec ip it a t io n , a n d c a n be o p er
a t ed un der w ider p H limit s. It is desir a ble t o r a ise t h e
p H t o a bo ut 7 - 8 bef o r e p r ec ip it a t io n a s t h is g iv es bet t er
ec o v er y a n d g r ea t er n o n - t o x ic n it r o g en r emo v a l. Th e p H
w a s r a ised t o t h is v a lue by usin g Na O H o r Na 2 CO 3 , t h e
la t t er bein g p r ef er r ed. Th e ba se used w a s q uit e dilut e
in o r der t o p r ev en t det o x i? c a t io n due t o h ig h lo c a l c o n
c en t r a t io n s in t h e so lut io n . A 5% so lut io n o f Na O H w a s
used, w h er ea s w it h Na 2 CO 3 a 12 % so lut io n w a s p r ef er r ed.
In g en er a l, t h is h ig h er p H dur in g p r ec ip it a t io n g a v e a
g r ea t er n o n - t o x ic n it r o g en f r a c t io n a t io n a n d a t t h e sa me
t ime ma in t a in ed t h e t o x in lo ss a t less t h a n 2 %.
Af t er p r ec ip it a t io n , t h e slur r y w a s ? lt er ed usin g f r o m
1 t o 4 % ? lt er a id, ba sed o n slur r y w eig h t , f o r sa t isf a c t o r y
? lt r a t io n ; t h e a mo un t o f ? lt er a id n eeded bein g dep en den t
o n t h e t yp e o f p r ess used. W a sh in g t h e ? lt er c a k e w it h
Na 2 S O 4 so lut io n r emo v ed a ddit io n a l n o n - t o x ic n it r o g en
w h ic h is desir a ble. In t h is w a sh in g st ep a 16. 7 % so lu
t io n o f Na 2 S O 4 w a s a g a in used. Th is w a sh in g st ep r e
mo v ed . a n a ddit io n a l 15% o f n o n - t o x ic n it r o g en f r o m t h e
c a k e.
Af t er ? lt r a t io n t h e ? lt er c a k e, w h ic h c o n t a in s t h e r ic in
in c o mbin a t io n w it h t h e Na 2 S O 4 , ma y be dr ied a n d slur r ied
w it h CCL , t o sep a r a t e t h e r ic in by ? o t a t io n . S ep a r a t io n
o f t h e r ic in a f t er a sin g le p r ec ip it a t io n a n d w a sh in g st ep
is p o ssible, but it is p r ef er r ed t o c a r r y t h e p r o c ess t h r o ug h
a n a ddit io n a l ex t r a c t io n a n d p r ec ip it a t io n st ep . Th is is
a c c o mp lish ed by slur r yin g t h e ? lt er c a k e in t h r ee t imes it s
w eig h t o f w a t er a n d t h e p H o f t h e slur r y is a g a in br o ug h t
t o 3 . 8 : . 1 by mea n s o f 5% H 2 8 04 . Th e slur r y is ? lt er ed
a n d a sec o n d p r ec ip it a t io n is br o ug h t a bo ut by a ddin g
Na 2 S O 4 so lut io n . Alt h o ug h p H c o n t r o l h er e is n o t w h o lly
essen t ia l it is a dv a n t a g eo us t o br in g t h e p H t o a p p r o x ima t e
n eut r a lit y by a ddin g 12 % Na 2 CO 3 . A p r ec ip it a t io n t ime
o f 4 5 min ut es w a s n ec essa r y t o o bt a in c o mp let e r emo v a l
o f t h e t o x in . In ? lt er in g o ut t h e p r ec ip it a t e, n o ? lt er a id
w a s used a n d t h e ? lt er c a k e w a s w a sh ed w it h Na 2 S O 4 so lu
t io n o n t h e ? lt er w h er eby a n a ddit io n a l a mo un t o f n o n
t o x ic n it r o g en w a s r emo v ed f r o m t h e c a k e. Th is w a sh in g
w a s ef f ec t iv e o n ly t h e ? r st t ime a n d r ep ea t ed w a sh in g s h a d
lit t le ef f ec t in r emo v in g f ur t h er n o n - t o x ic n it r o g en .
Th e r ic in - Na 2 S O 4 p r ec ip it a t e w a s dr ied a t a bo ut 50 t o
60 C. o n a h o t a ir t r a y dr yer . Th e dr ied p r o duc t w a s
g r o un d t o p a ss a 4 0 mesh sc r een a n d a g it a t ed w it h 5 t imes
it s w eig h t o f CCl. ; , w h ic h ser v ed t h e sep a r a t e t h e r ic in
f r o m t h e Na 2 S O 4 by ? o t a t io n . Af t er set t lin g , t h e r ic in
w a s sk immed o f f t h e t o p . Th is r educ ed t h e Na 2 S O 4 c o n
t en t o f t h e mix t ur e f r o m a p r ev io us 4 0 t o 50% do w n t o
15 t o 18 %. Abo ut 1 t o 2 % o f n it r o g en r ema in ed in t h e
Na 2 S O 4 sa lt w h ic h c o uld t h en be used f o r subseq uen t
p r ec ip it a t io n s.
Th e ? n a l p r ec ip it a t io n p r o duc ed a p a r t ic le siz e o f 1- 2
mu. O n dr yin g t h e w et c a k e, h o w ev er , t h e r ic in c emen t ed
t o g et h er f o r min g la r g er p a r t ic les. Th ese c o uld n o t be
br o k en do w n t o t h eir o r ig in a l siz e by o r din a r y g r in din g
met h o ds a n d sin c e a v er y ? n e p a r t ic le siz e w a s n ec essa r y
in o r der t h a t t h e p r o duc t mig h t be used a s a t o x ic w ea p o n ,
it w a s t h o ug h t desir a ble t o seek so me met h o d t o p r ev en t
t h e a g g lo mer a t io n o r c emen t in g p r o c ess t h a t t o o k p la c e
o n dr yin g . .
To a t t emp t t o a f f ec t t h is r esult , p h ysic a l c o n dit io n s p r e
v a ilin g un der t h e p r ec ip it a t io n p r o c ess w er e c h a n g ed.
3 , 08 6, 165
3
Th is in c luded c h a n g in g t h e t emp er a t ur e o f p r ec ip it a t io n
a n d t h e r a t e o f a g it a t io n . O t h er c h a n g es in c luded p r ec ip i
t a t io n w it h o n y p a r t ia l sa t ur a t io n o f Na 2 S O 4 a n d t h e use
o f w et t in g a n d seedin g a g en t s. No n e o f t h ese ex p edien t s
p r o duc ed a n y sig n i? c a n t imp r o v emen t in p a r t ic le siz e.
O r din a r y dr y ba ll a n d h a mmer millin g o f t h e dr ied r ic in
p r o duc ed c o n sider a ble det o x i? ia t io n p er h a p s due t o t h e
g en er a t io n o f ex c ess h ea t . Th e use o f CCl4 slur r y p lus
t h e use o f lo w t emp er a t ur e a n d lo w v mo ist ur e c o n t en t o f t h e
r ic in r educ ed det o x i? c a t io n dur in g ba ll millin g .
S p r a y dr yin g p r o v ed t o be a n ev en bet t er met h o d o f
sec ur in g a r ea so n a bly sma ll p a r t ic le siz e. Best r esult s
W er e a c h iev ed by usin g a so lut io n h a v in g a bo ut 2 0% so lids,
a n in let t emp er a t ur e o f 150 C. a n d a n a t o miz in g a ir
p r essur e o f 150 t o 18 0 p si. Th e p a r t ic le siz e sec ur ed w a s
6 t o 8 mu.
Th e best mea n s o f sec ur in g a sma ll p a r t ic le siz e w a s by
a ir g r in din g . Th is w a s c a r r ied o ut in a n a p p a r a t us h a v in g
a c h a mber w it h c o n ic a l t o p a n d bo t t o m. Th e ma t er ia l
t o be g r o un d h a s been f ed in t o t h is c h a mber a n d is w it h
dr a w n f r o m t h e bo t t o m a n d f o r c ed ba c k in t o t h e c en t er
o f t h e c h a mber t a n g en t ia lly t h r o ug h a v en t ur i. Co m
p r essed a ir o f a bo ut 100 p si. w a s f ed t o t h e v en t ur i t o
p r o v ide t h e g r in din g f o r c e. Th e ? n es a r e dr a w n o f f t h e
t o p a n d t h e la r g e p a r t ic les set t le t o t h e bo t t o m t o be r e
c ir c ula t ed a n d r eg r o un d. Th is p r o c ess p r o duc ed p a r t ic les
h a v in g a ma ss media n dia met er o f 2 . 5 t o 3 . 5 mu.
15
2 5
4
Numer o us v a r ia t io n s a r e p o ssible in t h e sev er a l st ep s o f
t h e p r o c ess c o mmen c in g w it h t h e w a t er ex t r a c t io n a n d p r e
c ip it a t io n w h ic h ma y be a sin g le o r mult ip le st ep . Al
t h o ug h a sin g le ex t r a c t io n st ep c a n be used, a s in dic a t ed
bef o r e, so me p r o c ess mo di? c a t io n s a r e n ec essa r y f o r
it s suc c essf ul o p er a t io n o n a p la n t sc a le. D o uble ex t r a c
t io n p r o v ed t o be q uit e e? ic ien - t but a ddit io n a l st ep s beyo n d
t h e sec o n d ex t r a c t io n st ep w er e n o t f o un d n ec essa r y.
Th e dr a w in g is self - desc r ip t iv e a n d sh o w s t h e v a r io us
st ep s o f t h e p r o c ess desc r ibed.
W e c la im:
1. In a met h o d o f p r ep a r in g t o x ic r ic in f r o m c a st o r
bea n s c o mp r isin g slur r yin g a n ex p r essed c a st o r bea n c a k e
w it h w a t er t o r emo v e t h e w a t er so luble r ic in a n d p r ec ip i
t a t in g t h e r ic in f r o m t h e ? lt r a t e, t h e f ur t h er st ep s w h ic h
in c lude slur r yin g t h e p r ec ip it a t e w it h CCl4 a n d sep a r a t in g
t h e r ic in by ? o t a t io n . '
2 . A p r o c ess in a c c o r da n c e w it h c la im 1 in w h ic h t h e
p r ec ip it a t e is dr ied p r io r t o slur r yin g .
Ref er en c es Cit ed in t h e f ile o f t h is p a t en t
K a ba t et a l. : J. Bio l. Ch em, v o l. 168 , 194 7 , p a g es 62 9
3 9.
K un it z et a l. : J. Gen . Ph ysio l, v o l. 3 2 ( 194 8 ) , p a g es
2 5- 3 1. .