A ll r o o t t is s u e s were e x tr a c te d in w ater by th e same method, ir r e s p e c t iv e o f w hether tak en from e x p e r im e n ta lly flo o d e d p la n ts , d ir e c t ly from n a tu r a l s i t e s , or from g lu c o se in c u b a tio n f la s k s . A fte r v/ashing in c h ille d d i s t i l l e d w a ter, th e r o o ts (5 -1 0 g fr e s h w eigh t ex cep t in th e in c u b a tio n experim ents where q u a n titie s were
25
.
a id m aceration , and e x tr a c te d w ith 100-150 ml c o ld d i s t i l l e d w ater in a Waring B len d er. The e x tr a c t was p a ssed through m u slin th en c e n tr ifu g e d a t 9 ,0 0 0 r .p .m . fo r 20 m inutes a t 2°C. The sup ernatan t tak en a f t e r c e n tr ifu g a tio n was th e aqueous ro o t e x tr a c t used fo r organ ic a c id a ssa y a f t e r e it h e r p e r c h lo r ic a c id d e p r o te in is a tio n or io n exchange p u r if ic a t io n .
A check was made th a t t h is co ld w ater method was e x tr a c tin g a l l th e o rg a n ic a c id s from th e ro o t t i s s u e s . Some n o n -flo o d ed S en ecio v is c o s u s ro o t m a te r ia l was d iv id e d in to two p a r ts , one p a rt fo r e x tr a c tio n in c h ille d w ater as above, th e o th er p art fo r e x tr a c tio n in h o t w a ter. As Table 4 shows, th e sm all d i f f eren ces betw een th e amounts o f m a lic, l a c t i c and o x a lo a c e tic a c id s determ ined in th e two e x tr a c ts in d ic a te th a t th e c o ld w ater method was th e b e t te r o f th e tw o.
Table 4* O rganic a c id s o f n o n -flo o d ed S en ecio v is c o s u s r o o ts , e x p ressed in p mole / g fr e s h w eig h t,
m alate la c t a t e o x a lo a c e ta te c o ld w ater e x tr a c t 3*0 0 .8 7 0 ,5 0 h o t w ater e x tr a c t 2 ,6 0 .8 2 0 .3 8
The e f f ic ie n c y o f th e c o ld w ater e x tr a c tio n method was a ls o checked by fu r th e r e x tr a c tio n o f a re sid u e in b o ilin g 95/^ e th a n o l.
26
As Table 5 shows, th e r e was no sh ik im ic a c id l e f t in tho t is s u e a f t e r c o ld w ater e x tr a c tio n , th a t cou ld be fu r th e r e x tr a c te d by b o ilin g e th a n o l.
Table 5* S h ik im ic a c id c o n te n t o f Nuphar lu t e a rhizom e, ex p ressed in jug / g fr e s h w eig h t.
sh ik im ate co ld w ater e x tr a c t 3 1 .4 95% eth a n o l e x tr a c t o f r e sid u e NIL