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S t u d i e s o n u r c u m i n a n d u r c u m i n o i d sV I . K i n e t ic s o f C u r c u m i n D e g r a d a t i o n i n A q u e o u s S o l u t i o nH a n n e H j o r t h T o n n e s e n a n d J a n K a r ls e nDepartmen t of Galenical Pharmacy, Institute of Pharmacy, Univers ity of Oslo, P.O. Box 1068 Blindern, 0316 Oslo 3, Norway

St udi en f i ber C ureumi n und C urcumi no i deV I . K i n e t ik d e s C u r e u m i n A b b a u sin w~iBrigen LiJsungenZ u s a m m e n f a s s u n g . E s wu rd e d i e K i n e t i k d e s p H-a b -h / i n g i g e n C u rc u m i n -Ab b a u s u n t e r s u c h t . E i n e Da r -s t e ll u n g d e r Ge s c h w i n d i g k e i t s k o n s ta n t e g e g e n d ie p H-W e r t e l i e fe r t d i e p Ka -W e r t e d e s s a u re n P ro t o n e n . D i e -se Kurve ze ig t aber g le ichze i t ig d ie Komplex i t f i t desC u r c u m i n - A b b a u s a n .S u m m a r y . T h e k i n et i cs o f th e p H-d e p e n d a n t d e g ra d a -t i o n o f c u rc u m i n h a s b e e n i n v e s t ig a t e d . A p l o t o f t h era t e c o n s t a n t a g a i n s t p H i n d i c a t e s t h e p Ka v a l u e s o ft h e a c i d p ro t o n s . T h e g ra p h a l s o i n d i c a t e s t h e c o m -p l e x it y o f t h e c u rc u m i n d e g ra d a t i o n .

Int roduct i onM a n y c o u n t r i e s h a v e i s s u ed a b a n o n t h e u s e o f s y n -the t ic dyes in food and d rugs , and the in te res t in na t -u ra l co lour ing mat te r i s increas ing . The rh izomes o fCurcuma longa L. Zingiberaceae) con ta in th ree ye l -l o w c o m p o u n d s . T h e rh i z o m e s a n d e x t r a c t s o f t h erh i z o m e s a r e c o m m e rc i a l p ro d u c t s u s e d a s c o l o u r i n gm a t t e r i n fo o d p ro c e s s i n g a ro u n d t h e wo r l d [1 ,2 ]. T h ey e l lo w c o m p o u n d s o f C ure uma l onga b e l o n g t o t h eg ro u p o f d i a ry l h e p t a n o id s . T h e m a i n c o l o u re d c o m -po und i s iden t i f i ed as cu rcumin [1 ,7 -b i s 4 -hydroxy-3 -methoxypheny l ) - l ,6 -hep tad iene-3 ,5 -d ione] [3 , 4 ] .L i t tl e is k n o wn a b o u t t h e s t a b il i ty o f c u rc u m i n . A s an a t u ra l c o l o u r i n g a g e n t i t i s k n o w n t o b e u n s t a b l e a n dh a s b e e n r e p l a c e d b y s t a b l e s y n t h e t i c d y e s wh e n e v e rposs ib le . I t i s apparen t tha t cu rcumin i s no t su i tab lefo r u s e i n m a n y p ro d u c t s b e c a u s e i t l o s e s i t s c o l o u ru p o n s t o r a g e u n d e r a l k a l i n e c o n d i t i o n s . M a n y fo o dp ro d u c t s a r e a v a i l a b l e a s d ry m i x e s t h a t r e q u i r e t h eOffprint req uests to J. KarlsenZ LebensmUnters Forsch (1985) 180:402404 Springer-Verlag1985

u s e o f a l k a li n e c o m p o n e n t s fo r p ro p e r p r e p a ra -tion [5].S u c h p ro d u c t s d e s t ab i l iz e c u rc u m i n , m a k i n g i t l o s ei t s des i red ye l low hue . I t i s therefo re o f in te res t to lo okc l o s e r a t t h e s t a b i l it y o f c u rc u m i n u n d e r v a r i o u s p Hc o n d i t i o n s . T h e p re s e n t wo rk wa s u n d e r t a k e n t o o b -t a i n k n o w l e d g e o f t h e k i ne t ic s o f t h e h y d ro l y t i c d e -g ra d a t i v e r e a c t i o n s o f c u rc u m i n . W i t h s u c h i n fo rm a -t i o n fo rm u l a t i n g c o n d i t i o n s l e a d i n g t o o p t i m u m s t a -b i l it y fo r t h e d y e i n a q u e o u s m e d i a m a y b e p re d i c t e d .

T h e s t a b i l it y o f c u rc u m i n i n t h e p H r a n g e f ro m 1to 11 was s tud ied . I t i s shown tha t the degrada t ionp ro d u c t s o f c u rc u m i n i n a l k a l i m i g h t b e a s o u rc e o f e r -ro r i n t h e q u a n t i t a t i v e a n a l y s e s o f c u rc u m i n b y t h es t a n d a rd a n a l y t ic a l m e t h o d s [6 ] . F o r t h a t r e a s o n w eu s e d a n H P L C - m e t h o d f o r th e d e t e r m i n a ti o n o f c u r -cum in in the so lu t ions [71 .T h e c o n c e n t r a t i o n o f c u rc u m i n p re s e n t i n a s ol u -t i o n a t a g i v e n p H a s a fu n c t i o n o f t im e wa s d e t e r -m i n e d a n d t h e o rd e r o f t h e o v e r -a l l d e g ra d a t i o n r e a c -t ion , the va lue o f k ra te cons tan ts ) , the ha l f - l ifes o fc u rc u m i n a n d t h e p Ka v a l u e s fo r t h e d i s s o c i a t i o n o ft h e e n o l a n d t h e p h e n o l s w e re d e t e rm i n e d .

E x p e r i m e n t a lSample PreparationSolutions 1 ml of curcumin in methanol (2.10-~ g/ml and 4.10 - 5 g/ml) were diluted with 9ml of buffers and incubated at31.5 C _ 0.5 C protec ted from light. At specified time intervals, thedegradation reactions were stopped by the addition of 1 n-HC1 topH 2. The samples were immediately extracted with ethyl acetate(5 ml) for 5 min. The ethyl acetate layer was diluted to 5.0 ml (20of the ethyl acetate remains in the buf fer layer) and the samples wereanalysed for the curcumin co ntent. To follow the decrease in curcu-min conten t, the chro matograph ic method already described [7] wasapplied. Three to 5 parallell portions of each stock solution weremeasured at each time interval. The curcumin content at 7-10 timeintervals were measured at each pH.BuffersThe buf fer systems used were:pH 1-3 KC1/HC1. - pH 6-9 KH2PO4/NaOH. - pH 9-10 NaHCO3/NaOH. - The ionic strength were 0.10-0.15 M.

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ReagentsA l l t h e c h e m i c a l s u s e d w e r e o f a n a l y t i c a l g ra d e . C u r c u m i n w a s s y n -t h e s iz e d fo l l o w in g t h e m e t h o d o f P a b o n a t r o o m t e m p e r a t u r e [ 8] . N op u r e c u r c u m i n c o u l d b e o b t a i n e d f r o m c o m m e r c i a l s o u rc e s .

hromatographic Instrumentation and onditionsA S p e c t r a - P h y s i c s m o d e l S P 8 7 0 0 h i g h - p e r f o r m a n c e l i q u i d c h r o m a -t o g r a p h w a s u s e d . A r h e o d y n e i n j e c t o r w i t h a 2 0 g l l o o p w a s u s e d .T h e d e t e c t o r w a s a S c h o e f f e l L . C . F l u o r i m e t e r F S 9 70 . D e t e c t i o nc o n d i t i o n s : E x c i t a t i o n w a v e l e n g t h 4 2 0 n m , e m i s s i o n w a v e l e n g t h4 7 0 n m . T h e s t a t i o n a r y p h a s e w a s N u c l e o s i l N H 2 C h r o m p a c k ) , p a r -t i c le s iz e 5 g i n , p r e - p a c k e d i n a 2 5 0 m m x 4 . 6 m m I . D . c o l u m n . T h em o b i l e p h a s e w a s e t h a n o l p . a . T h e a n a l y s e s w e r e c a r r i e d o u t a t a m -b i e n t t e m p e r a t u r e .

R e s u l t s a n d i s c u s s i o nTh e r e p ro d u c i b i l i t y o f t h e m e t h o d i s g o o d . Th e e x t r a c -t i o n p ro c e d u re g i ve s a c u rc u m i n r e c o v e ry o f > 9 0 .Th i s i s a c c e p t a b l e b e c a u s e o f t h e s h o r t e x t r a c t i o n t im e .B y re p e a t e d e x t r a c t i on s , c u r c u m i n w o u l d b e e x p o s e dto l igh t , ac id and e thy l ace ta te fo r a longer per iod .Th i s s h o u l d b e a v o i d e d s i n c e c u rc u m i n m a y p o l y m e r -ize on the su r face be tween the e thy l ace ta te and thebuffer l ayers . Ethy l ace ta te i s s ti ll the bes t cho ice as ex -t rac t ion so lven t because the resu l t ing sample can bei n j e c t e d d i r e c t l y o n t o t h e c o l u m n a t t h e c h ro m a t o -g ra p h i c c o n d i t i o n s u s e d , a n d e v a p o ra t i o n o f t h esample i s avo ided . A samp le o f cu rcu min in e thy l ace-ta te i s s t ab le fo r a t l eas t 10 hours (p ro tec ted f roml igh t ) a f t e r separa t ion f rom the bu ffer l ayer .

F l u o re s c e n c e d e t e c t i o n w a s u s e d b e c a u s e o f t h elow de tec t ion l imi t fo r cu rcumin in th i s sys tem [7 ] .Th i s m a k e s i t p o s s i b l e t o fo l l o w t h e d e g ra d a t i o n fo r ac o n v e n i e n t ti m e a t t h e p H w h e re t h e d e g ra d a t i o n p ro -cess i s very fas t . Poss ib le degrada t ion p roduct s gaven o i n t e r f e r en c e i n t h e c h ro m a t o g ra p h i c s y s t e m u s e d .

I t w a s d i f f ic u l t t o f i n d a c o m p o u n d s u i t a b le a s a ni n t e rn a l s t a n d a rd i n t h es e e x p e r im e n t s . H o w e v e r , s o -l u t io n s o f c u rc u m i n i n m e t h a n o l s t o r e d a t - 7 0 C fo ro n e y e a r a r e a p p a re n t l y u n c h a n g e d w i t h r e g a rd t o t h ec u rc u m i n c o n t e n t . To c o n t ro l t h e r e p ro d u c e a b i l i t y o ft h e m e t h o d , 1 m l o f a s t o c k s o l u t io n o f c u rc u m i n i nm e t h a n o l k e p t a t - 7 0 C w a s m ix e d w i t h 9 m l o fw a t e r a n d e x t r a c t e d w i t h 5 m l o f e th y l a c e ta t e , a n d t h el a y e r w a s s e p a ra t e d , d i l u t e d t o 5 . 0 m l a n d a n a l y s e dfo r t h e c u rc u m i n c o n t e n t . Th i s p ro c e d u re w a s r e -p e a t e d a t r e g u l a r i n t e rv a l s t h ro u g h o u t t h e e x p e r i -men t .

Tw o s t o c k s o l u t io n s o f c u rc u m i n i n m e t h a n o l(2 .1 0 -4 g / m l a n d 4 . 1 0 -5 g / m l ) w e re u s e d t o o b s e rv ew h e t h e r t h e c o n c e n t r a t i o n o f c u rc u m i n i s o f i m p o r -tance fo r the degrada t ion ra te and o rder . No s ign i f i -c a n t d i f f e r e n c e w a s d e t e c t e d i n r a t e o r o rd e r f o r t h et w o c o n c e n t r a t i o n s u s e d a t p H > 7 . A t p H < 7 , t he d e g -rad at io n rea ct io n is ,-~ 100 t imes fa s ter in the di lu te d

T a b l e 1 . T h e r a t e c o n s t a n t s a n d t h e h a l f - l iv e s f o r t h e o v e r a l ld e g r a d a t i o n o f c u r c u m i n a c c o r d i n g t o s e c o n d o r d e r k i n e t i cs a tv a r i o u s p H v a l u e spH k M th-~) q/2 h) pH k M-~ h - t ) q/2 h)1.23 2.8 6.6 103 8.08 8.6 104 2.1 10-13.21 5.2 3.5 103 8.20 6.3 104 2. 9. 10 15.97 4.4 4.2 103 8.45 6.0.1 0'* 3.1 10 16.94 1.2.10 3 1.5 l0 8.55 9.8 104 1.9. 10 17.00 1.2 103 1.5.101 8.60 1.2.105 1.5 .10 -17.30 4.0 103 4.6 8.75 1.6- 105 1.2- 10 17.75 2.2,104 8.4.1 0 1 9.01 3.9,105 4.8.1 0 a7.81 3.2-105 5.8- 10 -z 9.15 6.2 ,10 s 3.0- 10 27.84 2.2- 105 8.4 .10 -2 9.44 8 5 10 ~ 2.2- 10 -27.98 5.2 105 3.5 10 2 10.84 7.4 , 105 2. 5. 10 2

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F i g . 1 . P l o t o f t h e p H - p r o f i l e f o r t h e d e g r a d a t i o n o f c u r c u m i n i na q u e o u s s o l u t i o n a t 3 1 .5 C p H > 7 ). O , @ , a n d @ i n d i c a t e t h e p K ,v a l u e s o f t h e d i s s o c i a t e d p r o t o n s

s o l u t io n . T h i s i s p ro b a b l y d u e t o t h e l o w s o l u b i li t y o fc u rc u m i n i n th i s p H r a n g e .The c ho ice o f bu ffe r sys tems is c r i ti ca l . Cu rcum ins e e m s to fo rm c o m p l e x e s w i t h m a n y s a l ts u s e d i n c o m -m o n b u f f e r s y s t e m s (b o ra t e , c i t r a te , p h t h a l a te ) . C u r -c u m i n i s a p p a re n t l y i n e r t t o w a rd s K C 1 , K H 2 P O 4 , a n dN a H C O 3 . A t c o n s t a n t p H a n d i o n i c s t r e n g t h , t h e r ew a s n o s i g ni f ic a n t d i f f er e n c e in t h e d e g ra d a t i o n r a t e o fc u rc u m i n i n p h o s p h a t e a n d c a rb o n a t e b u f f e r s a tp H 9 . V a r i a t i o n i n b u f f e r c o n c e n t r a t i o n a t p H 7 .0a n d 7 .8 (p h o s p h a t e ) f r o m 0 .2 M t o 0 .05 M s h o w e d n os i g ni f ic a n t c h a n g e i n r e a c t i o n r a t e o f th e d e g ra d a t i o np ro c e s s a s a f u n c t i o n o f t h e b u f f e r c o n c e n t r a t i o n .Th e d e g ra d a t i o n o f c u rc u m i n w a s s t u d i e d o v e r t h ep H r a n g e 1 -1 1 . A t f i x e d v a l u e s o f p H , t e m p e ra t u r eand ion ic s t reng th the ov er-a l l lo s s o f cu rcuminshow ed second o rde r k ine ti cs . A co rre la t ion coeff ic ien tb e t t e r t h a n 0 .9 3 w a s a c c e p t e d fo r t h e l i n e a r g r a p h o f1 /c aga ins t t ime a t var ious pH-values (c i s t thec o n c e n t r a t i o n o f c u rc u m i n ) . Th e o b s e rv e d p s e u d o -seco nd-o r der ra te cons ta n t s (kous ) fo r the over-a l l deg -

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r a d a t i o n o f c u rc u m i n w e re c a l c u la t e d f ro m t h e s l o p e so f the s t ra igh t l ines ob ta in ed by p lo t t ing 1 /c vs t wh erec i s res idua l cu rcum in in mo lar c once n t ra t ion and t i st ime. Values o f /Cob a t severa l pH va lues a re l i s t ed inTa b l e 1. A p l o t o f t h e p H p ro f i le f o r t h e d e g ra d a t i o no f c u rc u m i n i n a q u e o u s s o l u t i o n a t 3 1.5 C i s s h o w nin Fig . 1 .

Th e s h a p e o f t h e p H p ro f i le c u rv e fo r th e h y d ro -l y t ic d e g ra d a t i o n o f c u rc u m i n i n d ic a t e s t h a t s e v e ra lac id -ba se equ i l ib r ia mu s t be invo lved F ig .2 ) [9 ].W h e n t h e p H i s < 1 , c u r c u m i n h a s a r e d c o lo u r , w h i c hi n d ic a t e s th e p ro t o n a t e d fo rm H 4 A + ) p o s t u l a t e d i nFig . 2 [9 ]. Curc um in in so lu t ion a t pH 1 -7 has a ye l lowc o l o u r . I n t h i s p H r a n g e , t h e m a j o r i t y o f t h e c u rc u m i nm o l e c u l e s a r e i n t h e n e u t r a l f o rm H 3 A ) . A t t h e g iv e nc o n c e n t r a t i o n s , t h e w a t e r s o l u b il i ty o f c u rc u m i n i s l o wi n th i s p H a r e a . C u rc u m i n i s p r e c i p i t a te d a f t e r a b o u t3 d a y s s to r a g e a n d t h e c u rc u m i n d e g ra d a t i o n i s s lo w .Th e h a l f- l iv e s o f c u rc u m i n i n t h o s e s o l u t i o n s a r e o b -t a i n e d b y a s s u m i n g c o n s t a n t r e a c t i o n o rd e r a n d e x t r a -po la t ing the l inear g raph to the t ime wh ere c = Co 2C o = in i ti a l c o n c e n t r a t i o n o f c u rc u m i n i n m o l a r ). A tp H > 7 . 5 t h e c o l o u r c h a n g e t o o r a n g e r e d . T h e p K a

value fo r the d i s soc ia t ion o f the eno l , wh ich i s ca lcu -l a t e d f ro m t h e f i r st in f l e c ti o n p o i n t o n t h e p H p ro f i lec u rv e p H = p K a i n t h e i n fl e c t io n p o i n t w h e n k i s p lo t -t ed aga ins t p H [10]) , i s fou nd to be 7 .75 -7 .80 , w h ichi s i n g o o d a g re e m e n t w i t h t h e v i s u a l ly o b s e rv e d t r a n -s i t ion range 7 .75 -7 .90 ) . The ra te cons ta n t fo r the deg-r a d a t i o n r e a c t i o n o f c u rc u m i n i n t h is p H - ra n g e i sh igh , and g ives a ha l f - l ife o f cu rcu min accord ing tosecond o rde r k ine t ics ) o f l ess than 10 min Tab le 1 ).

T h e p H p r o fi le c u r v e sh o w s a m i n i m u m i n th e p Ha re a 8 .2 0 -8 .5 0 . C u rc u m i n i s h e r e p o s t u l a t e d t o b e i ne q u il i br i u m b e t w e e n th r e e f or m s ; H 3 A , H z A - , a n dH A 2 - F ig . 2 ) . A se cond in f lec t ion po in t o f the cu rvei s observed a t p H = 8 .55 _+ 0 .05 and a th i rd i s observe da t pH 9 .05_+0 .05 . These ind ica te the pK a va lues fo rt h e d i s s o c i a t io n o f t h e p h e n o l s i n t h e c u rc u m i n m o l e -cule Fig . 2).

T h e p r i m a r y d e g r a d a t io n p r o d u c t s o f c u rc u m i n a tp H 7 t o p H 1 0 a r e s h o w n t o b e f e ru li c a c i d a n dfe ru l o y l m e t h a n e [7] . Th e m i n i m u m o f t h e p H c u rv e isl ike ly to be due to a se l f -stab i li z ing e f fec t o f the cu rcu -m i n m o l e c u le in t he H z A - f o r m r a t h e r t h a n t o a n -o t h e r r e a c t i o n m e c h a n i s m .onclusion

C u rc u m i n i n a q u e o u s s o l u t i o n s i s e x p o s e d t o h y d ro -l y ti c d e g ra d a t i v e r e a c t i o n s. T o o b t a i n o p t i m u m s t a bi l -i t y o f t h e p r e p a ra t i o n s , t h e p H s h o u l d b e m a i n t a i n e db e l o w 7 . H o w e v e r , i n t hi s p H r e g i o n t h e s o l u b i li t y o fc u rc u m i n i n a q u e o u s m e d i a i s l o w . A t p H > 7 , th e c u r -c u m i n m o l e c u l e s a r e e x t re m e l y u n s ta b l e . Th e p H p ro -f il e c u rv e s h o w s a m i n i m u m i n th e a r e a p H 8 .2 0 -8.5 0 ,b u t e v e n i n t h i s r e g io n t h e d e g ra d a t i o n o f c u rc u m i n i sfas t . In i t s na t ive fo rm, cu rcumin i s no t su i t ab le as ac o l o u r i n g a g e n t i n a q u e o u s s o l u t i o n s o f p H > 7 u n l e s sa su i t ab le s tab i l i z ing agen t i s p resen t .References

1. Shankaracharaja NB (1974) Indian Spices 10:72 . T ur mer i c , Cur cu min ( 1975) W HO Fo od A dd i t i ves Seri es No . 63 . Gov indar a j an VS ( 1980) CR C Cr Re v Food Sc i Nu t r 12 :1994 . T onnesen HH, Kar l sen J , Mostad A ( 1982) Ac ta Chem ScandB36:4755. Leshik RR (1981) Eur Pat Appl EP 37:204 (C1.A. 23L1/275)6 . T onnesen HH, Kar l sen J ( 1985) Z L ebensm Unte r s Fo r sch ( i npress)7 . T onnesen H H, Kar l sen J ( 1983) J Chr om atogr 259 :3678. P abo n H JJ (1964) Rec uei l 83:3799 . D y r s s e n D W , N o v i k o v Y P , U p p s t r 6 m L R ( 19 7 2) A n a l C la imActa 60:13910 . Conn or s KA , Amido n GL , K enno n L ( 1979) I n : Chemica l s t a -b i l i ty o f phar maceu t i ca l s . John Wi ley Sons, New Yo r k

Rece ived Decembe r 4 , 1984

404