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Studies on curcumin and curcuminoids. XV. Catalytic effect of demethoxy- and bisdemethoxycurcumin on the peroxidation of linoleic acid by 15-lipoxygenase
International Journal ofPharmaceutics, 51 (1989) 179-181
179
Elsevier IJP 01766
Studies on curcumin and curcuminoids. XV. Catalytic effect of demethoxy- and bisdemethoxycurcumin on the peroxidation of linoleic acid by 15-1ipoxygenase Hanne Hjorth Tonnesen Institute of Pharmacy, Universityof Oslo, Oslo (Norway) (Received 21 November 1988) (Accepted 29 November 1988)
Key words: Curcumin analog; Catalytic effect; Linoleic acid peroxidation; Drug development
Curcumin and the curcuminoids belong to the group of diarylheptanoids. The rhizomes of Curcuma longa L. (turmeric) contain 3-5% of curcumin and two related compounds; demethoxy- and bisdemethoxycurcumin. The rhizomes of Curcuma longa L. are reported to have anti-inflammatory activity, which is ascribed to the content of the curcuminoids (Mukopadhyay et al., 1982; Rao et al., 1982; Deodhar et al., 1980; Satoskar et al., 1986; Yegnanarayan et al., 1976; Srimal and Dhawan, 1973; Arora et al., 1971; Sharma and Chandra, 1987; Ghatak and Basu, 1972). Recent studies in animals indicate that the anti-inflammatory activity of pure curcumin is less than the activity observed when a mixture of the 3 curcuminoids equal to what is isolated from plant material is used (Srimal and Dhawan, personal communication). This might indicate a synergistic effect of the curcuminoids in vivo. One of the postulated action mechanisms for non-steroidal anti-inflammatory drugs (NSAID's) is the blocking of the lipoxygenase (LO) pathway
Correspondence: H.H. Tonnesen, Institute of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, 0316 Oslo 3, Norway.
or the cyclo-oxygenase pathway (Palmer and Salmon, 1985; Paulus et al., 1987). Due to the simplicity of the method, soybean lipoxygenase has been widely used for the primary screening of LO inhibitors (Alcaraz and Ferrandiz, 1987). In a previous work (Tonnesen, 1989), the possible inhibitory effect of pure, synthetic curcumin on 15-1ipoxygenase was investigated by use of soybean lipoxygenase. Linoleic acid was used as substrate. The results obtained showed that curcumin had a significant catalytic effect on the peroxidation of linoleic acid under the experimental conditions given. The catalytic effect was proportional to the curcumin concentration. Curcumin, however, is shown to have inhibitory effect on mammalian 5-1ipoxygenase and cyclo-oxygenase (Flynn et al., 1986). The results reported by Flynn et al. were obtained using commercially available curcumin. Commercially obtained curcumin is found to contain about 6% demethoxycurcumin and about 0.3% bisdemethoxycurcumin (Tonnesen, unpublished work). For further studies on the mechanism of the anti-inflammatory action of curcumin, a possible inhibitory effect of the other curcumin analogs and of different qualities of commercially ob-
180 % activation
I AA
200
6O X
O
\x 50
150
40 100
30 50
20 i
-10
4
,
-tgCcur¢.
Fig. 1. Catalytic effect on the peroxidation of linoleic acid by addition of curcuminoids to the 15-1ipoxygenase/linoleic acid system. (o e), Curcumin (synthetic); (zx zx), dernethoxycurcumin (synthetic); (o o), bisdemethoxycurcumin (synthetic); ( x x), curcumin (commercially obtained, Chr. Hansen Lab.). t a i n e d c u r c u m i n o n 15-1ipoxygenase was investigated. Pure d e m e t h o x y - a n d b i s d e m e t h o x y c u r c u m i n were synthesized after the m e t h o d of P a b o n ( P a b o n , 1964). C o m m e r c i a l l y o b t a i n e d c u r c u m i n was p r o v i d e d b y C h r i s t i a n H a n s e n ' s L a b o r a t o r i e s , D e n m a r k , a n d b y F l u k a , Switzerland. T h e c u r c u m i n o i d s were dissolved in e t h a n o l a n d m i x e d with the r e a c t i o n m e d i u m c o n t a i n i n g linoleic acid. S o y b e a n l i p o x y g e n a s e was then a d ded. T h e r e a c t i o n was followed b y m e a s u r i n g an increase in a b s o r b a n c e at 234 n m for 90 s as d e s c r i b e d p r e v i o u s l y (TBnnesen, 1989). D i f f e r e n t c o n c e n t r a t i o n s of the c u r c u m i n o i d s a n d of linoleic acid (substrate) were investigated, a n d the results are given in Figs. 1 a n d 2. D e m e t h o x y c u r c u m i n has a significant catalytic effect on the p e r o x i d a t i o n of linoleic acid in the c o n c e n t r a t i o n r a n g e 0.0133-0.0003 m g / m l . T h e c a t a l y t i c effect increases with decreasing conc e n t r a t i o n s of d e m e t h o x y c u r c u m i n .
10
l 0.5
l 1.0
i 1.5
:
20
i,
2S
,_ .~._.I07(M)
Fig. 2. Lineweaver-Burke plot of the inverse of the molar substrate concentration vs the inverse of the change in absorbance at 234 nm from t = 3 0 s to t = 9 0 s. (e O), Sample without curcumin (y = 2.49.10 -6 x +6.77, r = 0.9988); (© ©), demethoxycurcumin concentration 0.00133 mg/ml (y = 2.39- 10 -6 x + 5.68, r = 0.9965); (× ×), bisdemethoxycurcumin concentration 0.00133 mg/ml (y = 2.13.10 -6 x + 4.71, r = 0.9968). B i s d e m e t h o x y c u r c u m i n in a c o n c e n t r a t i o n of 0.0133 m g / m l inhibits the p e r o x i d a t i o n of linoleic acid. T h e e x p e r i m e n t was p e r f o r m e d with two different b a t c h e s of s y n t h e t i c b i s d e m e t h o x y curcumin. A t lower c o n c e n t r a t i o n s (0.0067-0.0003 m g / ml), b i s d e m e t h o x y c u r c u m i n has a c a t a l y t i c effect on the p e r o x i d a t i o n reaction. This effect is p r o p o r tional with the c o n c e n t r a t i o n of b i s d e m e t h o x y c u r c u m i n in the samples. T h e L i n e w e a v e r - B u r k e plots of d e m e t h o x y - a n d b i s d e m e t h o x y c u r c u m i n i n d i c a t e an u n c o m p e t i t i v e activation m e c h a n i s m (Fig. 2). This is in agreem e n t with w h a t is o b t a i n e d with p u r e c u r c u m i n ( T e n n e s e n , 1989).
181 The two qualities of commercially obtained c u r c u m i n i n c r e a s e the p e r o x i d a t i o n r a t e w i t h a f a c t o r of a b o u t 180 c o m p a r e d to a s a m p l e w i t h o u t the addition of curcuminoids. T h e results o b t a i n e d in this e x p e r i m e n t s h o w t h a t the 3 n a t u r a l l y o c c u r r i n g c u r c u m i n a n a l o g s h a v e a c a t a l y t i c e f f e c t o n t h e p e r o x i d a t i o n of linoleic acid by soybean lipoxygenase. When a m i x t u r e of t h e c u r c u m i n o i d s is u s e d ( c o m m e r c i a l l y a v a i l a b l e c u r c u m i n p r o d u c t s ) the c a t a l y t i c e f f e c t s e e m s to b e synergistic. If the a n t i - i n f l a m m a t o r y p r o p e r t i e s o f the c u r c u m i n o i d s are r e l a t e d to their c a t a l y t i c effect o n the 15-1ipoxygenase m e c h a n i s m , the s y n e r g i s t i c e f f e c t o f the c u r c u m i n o i d s c o u l d b e a p o s s i b l e e x p l a n a t i o n f o r the d i f f e r e n c e in a c t i v ity o b s e r v e d w h e n p u r e c u r c u m i n a n d c o m m e r c i a l l y o b t a i n e d c u r c u m i n are u s e d in a n i m a l s .
References Arora, R.B., Basu, N., Kapoor, V. and Jain, A.P., Anti-inflammatory studies on Cureuma Longa (Turmeric). Indian J. Med. Res., 59 (1971) 1289-1295. Alcaraz, M.J. and Ferrandiz, M.L., Modification of arachidonic metabolism by flavonoids. J. Ethnopharmacol., 21 (1987) 209-229. Deodhar, S.D., Sethi, R. and Srimal, R.C., Preliminary study on antirheumatic activity of curcumin (diferuloyl methane). Indian J. Med. Res., 71 (1980) 632-634. Flynn, D.L., Rafferty, M.F. and Boctor, A.M., Inhibition of 5-hydroxy-eicosatetraenoic acid (5-HETE) formation in intact human neutrophils by naturally occurring diarylhep-
tanoids: inhibitory activities of curcuminoids and yakuchinones. Prostaglandins Leukotriens Med., 22 (1986) 357360. Ghatak, N. and Basu, N., Sodium curcuminate as an effective anti-inflammatory agent. Indian J. Exp. Biol., 10 (1972) 235-236. Mukhopadhyay, A., Basu, N., Ghatak, N. and Gujral, P.K., Anti-inflammatory and irritant activities of curcumin analogues in rats. Agents and Actions, 12 (1982) 508-515. Pabon, H.J.J., A synthesis of curcumin and related compounds. Rec. Tray. Chim. Pays-Bas, 83 (1964) 379-386. Palmer, R.M.J. and Salmon, J.A., Inhibition of 5' lipoxygenase: relevance to inflammation. N A T O A S I Ser. A, 95 (1985) 311-330. Paulus, H.E., Furst, D.E. and Dromgoole, S.H., Drugs for Rheumatic Disease, Churchill Livingstone, London, 1987, pp. 209-211. Rao, T.S., Basu, N. and Siddiqui, H.H., Anti-inflammatory activity of curcumin analogues. Indian J. Med. Res., 75 (1982) 574-578. Satoskar, R.R., Shah, S.J. and Shenoy, S.G., Evaluation of anti-inflammatory property of curcumin (diferuloyl methane) in patients with postoperative inflammation. Int. J. Clin. PharmacoL Ther. Toxicol., 24 (1986) 651-654. Sharma, K.K. and Chandra, S., Synthesis and antiarthritic study of a new orally active diferuloyl methane (curcumin) gold complex, lnorg. Chim. Acta, 135 (1987) 47-48. Srimal, R.C. and Dhawan, B.N., Pharmacology of diferuloyl methane (curcumin), a non-steroidal anti-inflammatory agent. J. Pharm. Pharmacol., 25 (1973) 447-452. Tonnesen, H.H., Studies on curcumin and curcuminoids. XIII. Catalytic effect of curcumin on the peroxidation of linoleic acid by 15-1ipoxygenase. Int. J. Pharm., 50 (1989) 67-69. Yegnanarayan, R., Saraf, A.P. and Balwani, J.H., Comparison of anti-inflammatory activity of various extracts of Curcuma longa (L.). Indian J. Med. Res., 64 (1976) 601-608.
179
Elsevier IJP 01766
Studies on curcumin and curcuminoids. XV. Catalytic effect of demethoxy- and bisdemethoxycurcumin on the peroxidation of linoleic acid by 15-1ipoxygenase Hanne Hjorth Tonnesen Institute of Pharmacy, Universityof Oslo, Oslo (Norway) (Received 21 November 1988) (Accepted 29 November 1988)
Key words: Curcumin analog; Catalytic effect; Linoleic acid peroxidation; Drug development
Curcumin and the curcuminoids belong to the group of diarylheptanoids. The rhizomes of Curcuma longa L. (turmeric) contain 3-5% of curcumin and two related compounds; demethoxy- and bisdemethoxycurcumin. The rhizomes of Curcuma longa L. are reported to have anti-inflammatory activity, which is ascribed to the content of the curcuminoids (Mukopadhyay et al., 1982; Rao et al., 1982; Deodhar et al., 1980; Satoskar et al., 1986; Yegnanarayan et al., 1976; Srimal and Dhawan, 1973; Arora et al., 1971; Sharma and Chandra, 1987; Ghatak and Basu, 1972). Recent studies in animals indicate that the anti-inflammatory activity of pure curcumin is less than the activity observed when a mixture of the 3 curcuminoids equal to what is isolated from plant material is used (Srimal and Dhawan, personal communication). This might indicate a synergistic effect of the curcuminoids in vivo. One of the postulated action mechanisms for non-steroidal anti-inflammatory drugs (NSAID's) is the blocking of the lipoxygenase (LO) pathway
Correspondence: H.H. Tonnesen, Institute of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, 0316 Oslo 3, Norway.
or the cyclo-oxygenase pathway (Palmer and Salmon, 1985; Paulus et al., 1987). Due to the simplicity of the method, soybean lipoxygenase has been widely used for the primary screening of LO inhibitors (Alcaraz and Ferrandiz, 1987). In a previous work (Tonnesen, 1989), the possible inhibitory effect of pure, synthetic curcumin on 15-1ipoxygenase was investigated by use of soybean lipoxygenase. Linoleic acid was used as substrate. The results obtained showed that curcumin had a significant catalytic effect on the peroxidation of linoleic acid under the experimental conditions given. The catalytic effect was proportional to the curcumin concentration. Curcumin, however, is shown to have inhibitory effect on mammalian 5-1ipoxygenase and cyclo-oxygenase (Flynn et al., 1986). The results reported by Flynn et al. were obtained using commercially available curcumin. Commercially obtained curcumin is found to contain about 6% demethoxycurcumin and about 0.3% bisdemethoxycurcumin (Tonnesen, unpublished work). For further studies on the mechanism of the anti-inflammatory action of curcumin, a possible inhibitory effect of the other curcumin analogs and of different qualities of commercially ob-
180 % activation
I AA
200
6O X
O
\x 50
150
40 100
30 50
20 i
-10
4
,
-tgCcur¢.
Fig. 1. Catalytic effect on the peroxidation of linoleic acid by addition of curcuminoids to the 15-1ipoxygenase/linoleic acid system. (o e), Curcumin (synthetic); (zx zx), dernethoxycurcumin (synthetic); (o o), bisdemethoxycurcumin (synthetic); ( x x), curcumin (commercially obtained, Chr. Hansen Lab.). t a i n e d c u r c u m i n o n 15-1ipoxygenase was investigated. Pure d e m e t h o x y - a n d b i s d e m e t h o x y c u r c u m i n were synthesized after the m e t h o d of P a b o n ( P a b o n , 1964). C o m m e r c i a l l y o b t a i n e d c u r c u m i n was p r o v i d e d b y C h r i s t i a n H a n s e n ' s L a b o r a t o r i e s , D e n m a r k , a n d b y F l u k a , Switzerland. T h e c u r c u m i n o i d s were dissolved in e t h a n o l a n d m i x e d with the r e a c t i o n m e d i u m c o n t a i n i n g linoleic acid. S o y b e a n l i p o x y g e n a s e was then a d ded. T h e r e a c t i o n was followed b y m e a s u r i n g an increase in a b s o r b a n c e at 234 n m for 90 s as d e s c r i b e d p r e v i o u s l y (TBnnesen, 1989). D i f f e r e n t c o n c e n t r a t i o n s of the c u r c u m i n o i d s a n d of linoleic acid (substrate) were investigated, a n d the results are given in Figs. 1 a n d 2. D e m e t h o x y c u r c u m i n has a significant catalytic effect on the p e r o x i d a t i o n of linoleic acid in the c o n c e n t r a t i o n r a n g e 0.0133-0.0003 m g / m l . T h e c a t a l y t i c effect increases with decreasing conc e n t r a t i o n s of d e m e t h o x y c u r c u m i n .
10
l 0.5
l 1.0
i 1.5
:
20
i,
2S
,_ .~._.I07(M)
Fig. 2. Lineweaver-Burke plot of the inverse of the molar substrate concentration vs the inverse of the change in absorbance at 234 nm from t = 3 0 s to t = 9 0 s. (e O), Sample without curcumin (y = 2.49.10 -6 x +6.77, r = 0.9988); (© ©), demethoxycurcumin concentration 0.00133 mg/ml (y = 2.39- 10 -6 x + 5.68, r = 0.9965); (× ×), bisdemethoxycurcumin concentration 0.00133 mg/ml (y = 2.13.10 -6 x + 4.71, r = 0.9968). B i s d e m e t h o x y c u r c u m i n in a c o n c e n t r a t i o n of 0.0133 m g / m l inhibits the p e r o x i d a t i o n of linoleic acid. T h e e x p e r i m e n t was p e r f o r m e d with two different b a t c h e s of s y n t h e t i c b i s d e m e t h o x y curcumin. A t lower c o n c e n t r a t i o n s (0.0067-0.0003 m g / ml), b i s d e m e t h o x y c u r c u m i n has a c a t a l y t i c effect on the p e r o x i d a t i o n reaction. This effect is p r o p o r tional with the c o n c e n t r a t i o n of b i s d e m e t h o x y c u r c u m i n in the samples. T h e L i n e w e a v e r - B u r k e plots of d e m e t h o x y - a n d b i s d e m e t h o x y c u r c u m i n i n d i c a t e an u n c o m p e t i t i v e activation m e c h a n i s m (Fig. 2). This is in agreem e n t with w h a t is o b t a i n e d with p u r e c u r c u m i n ( T e n n e s e n , 1989).
181 The two qualities of commercially obtained c u r c u m i n i n c r e a s e the p e r o x i d a t i o n r a t e w i t h a f a c t o r of a b o u t 180 c o m p a r e d to a s a m p l e w i t h o u t the addition of curcuminoids. T h e results o b t a i n e d in this e x p e r i m e n t s h o w t h a t the 3 n a t u r a l l y o c c u r r i n g c u r c u m i n a n a l o g s h a v e a c a t a l y t i c e f f e c t o n t h e p e r o x i d a t i o n of linoleic acid by soybean lipoxygenase. When a m i x t u r e of t h e c u r c u m i n o i d s is u s e d ( c o m m e r c i a l l y a v a i l a b l e c u r c u m i n p r o d u c t s ) the c a t a l y t i c e f f e c t s e e m s to b e synergistic. If the a n t i - i n f l a m m a t o r y p r o p e r t i e s o f the c u r c u m i n o i d s are r e l a t e d to their c a t a l y t i c effect o n the 15-1ipoxygenase m e c h a n i s m , the s y n e r g i s t i c e f f e c t o f the c u r c u m i n o i d s c o u l d b e a p o s s i b l e e x p l a n a t i o n f o r the d i f f e r e n c e in a c t i v ity o b s e r v e d w h e n p u r e c u r c u m i n a n d c o m m e r c i a l l y o b t a i n e d c u r c u m i n are u s e d in a n i m a l s .
References Arora, R.B., Basu, N., Kapoor, V. and Jain, A.P., Anti-inflammatory studies on Cureuma Longa (Turmeric). Indian J. Med. Res., 59 (1971) 1289-1295. Alcaraz, M.J. and Ferrandiz, M.L., Modification of arachidonic metabolism by flavonoids. J. Ethnopharmacol., 21 (1987) 209-229. Deodhar, S.D., Sethi, R. and Srimal, R.C., Preliminary study on antirheumatic activity of curcumin (diferuloyl methane). Indian J. Med. Res., 71 (1980) 632-634. Flynn, D.L., Rafferty, M.F. and Boctor, A.M., Inhibition of 5-hydroxy-eicosatetraenoic acid (5-HETE) formation in intact human neutrophils by naturally occurring diarylhep-
tanoids: inhibitory activities of curcuminoids and yakuchinones. Prostaglandins Leukotriens Med., 22 (1986) 357360. Ghatak, N. and Basu, N., Sodium curcuminate as an effective anti-inflammatory agent. Indian J. Exp. Biol., 10 (1972) 235-236. Mukhopadhyay, A., Basu, N., Ghatak, N. and Gujral, P.K., Anti-inflammatory and irritant activities of curcumin analogues in rats. Agents and Actions, 12 (1982) 508-515. Pabon, H.J.J., A synthesis of curcumin and related compounds. Rec. Tray. Chim. Pays-Bas, 83 (1964) 379-386. Palmer, R.M.J. and Salmon, J.A., Inhibition of 5' lipoxygenase: relevance to inflammation. N A T O A S I Ser. A, 95 (1985) 311-330. Paulus, H.E., Furst, D.E. and Dromgoole, S.H., Drugs for Rheumatic Disease, Churchill Livingstone, London, 1987, pp. 209-211. Rao, T.S., Basu, N. and Siddiqui, H.H., Anti-inflammatory activity of curcumin analogues. Indian J. Med. Res., 75 (1982) 574-578. Satoskar, R.R., Shah, S.J. and Shenoy, S.G., Evaluation of anti-inflammatory property of curcumin (diferuloyl methane) in patients with postoperative inflammation. Int. J. Clin. PharmacoL Ther. Toxicol., 24 (1986) 651-654. Sharma, K.K. and Chandra, S., Synthesis and antiarthritic study of a new orally active diferuloyl methane (curcumin) gold complex, lnorg. Chim. Acta, 135 (1987) 47-48. Srimal, R.C. and Dhawan, B.N., Pharmacology of diferuloyl methane (curcumin), a non-steroidal anti-inflammatory agent. J. Pharm. Pharmacol., 25 (1973) 447-452. Tonnesen, H.H., Studies on curcumin and curcuminoids. XIII. Catalytic effect of curcumin on the peroxidation of linoleic acid by 15-1ipoxygenase. Int. J. Pharm., 50 (1989) 67-69. Yegnanarayan, R., Saraf, A.P. and Balwani, J.H., Comparison of anti-inflammatory activity of various extracts of Curcuma longa (L.). Indian J. Med. Res., 64 (1976) 601-608.