Studies on Curcumin and Curcuminoids

VI. Kinetics of Curcumin Degradation in Aqueous Solution Hanne Hjorth Tonnesen and Jan Karlsen
Department of Galenical Pharmacy, Institute of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, 0316 Oslo 3, Norway

Studien fiber Cureumin und Curcuminoide VI. Kinetik des Cureumin-Abbaus in w~iBrigen LiJsungen Zusammenfassung. Es wurde die Kinetik des pH-abh/ingigen Curcumin-Abbaus untersucht. Eine Darstellung der Geschwindigkeitskonstante gegen die pHWerte liefert die pKa-Werte des sauren Protonen. Diese Kurve zeigt aber gleichzeitig die Komplexitfit des Curcumin-Abbaus an. Summary. The kinetics of the pH-dependant degradation of curcumin has been investigated. A plot of the rate constant against pH indicates the pKa values of the acid protons. The graph also indicates the complexity of the curcumin degradation.

Introduction Many countries have issued a ban on the use of synthetic dyes in food and drugs, and the interest in natural colouring matter is increasing. The rhizomes of Curcuma longa L. (Zingiberaceae) contain three yellow compounds. The rhizomes and extracts of the rhizomes are commercial products used as colouring matter in food processing around the world [1,2]. The yellow compounds of Cureuma longa belong to the group of diarylheptanoids. The main coloured compound is identified as curcumin [1,7-bis(4-hydroxy3-methoxyphenyl)-l,6-heptadiene-3,5-dione] [3, 4]. Little is known about the stability of curcumin. As a natural colouring agent it is known to be unstable and has been replaced by stable synthetic dyes whenever possible. It is apparent that curcumin is not suitable for use in many products because it loses its colour upon storage under alkaline conditions. Many food products are available as dry mixes that require the
Offprint requests to: J. Karlsen
Z LebensmUnters Forsch (1985) 180:402404 Springer-Verlag1985

use of alkaline components for proper preparation [5]. Such products destabilize curcumin, making it lose its desired yellow hue. It is therefore of interest to look closer at the stability of curcumin under various pH conditions. The present work was undertaken to obtain knowledge of the kinetics of the hydrolytic degradative reactions of curcumin. With such information formulating conditions leading to optimum stability for the dye in aqueous media may be predicted. The stability of curcumin in the pH range from 1 to 11 was studied. It is shown that the degradation products of curcumin in alkali might be a source of error in the quantitative analyses of curcumin by the standard analytical methods [6]. For that reason we used an HPLC-method for the determination of curcumin in the solutions [71. The concentration of curcumin present in a solution at a given pH as a function of time was determined and the order of the over-all degradation reaction, the value of k (rate constants), the half-lifes of curcumin and the pKa values for the dissociation of the enol and the phenols were determined.

Experimental
Sample Preparation
Solutions 1 ml of curcumin in methanol (2.10-~ g/ml and 4.10- 5 g/ ml) were diluted with 9ml of buffers and incubated at 31.5 C _ 0.5 C protected from light. At specified time intervals, the degradation reactions were stopped by the addition of 1 n-HC1 to pH 2. The samples were immediately extracted with ethyl acetate (5 ml) for 5 min. The ethyl acetate layer was diluted to 5.0 ml (20% of the ethyl acetate remains in the buffer layer) and the samples were analysed for the curcumin content. To follow the decrease in curcumin content, the chromatographic method already described [7] was applied. Three to 5 parallell portions of each stock solution were measured at each time interval. The curcumin content at 7-10 time intervals were measured at each pH.

Buffers
The buffer 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.

Reagents
All the chemicals used were of analytical grade. Curcumin was synthesized following the method of Pabon at room temperature [8]. No pure curcumin could be obtained from commercial sources.

Table 1. The rate constants and the half-lives for the overall degradation of curcumin according to second order kinetics at various pH values pH
1.23 3.21 5.97 6.94 7.00 7.30 7.75 7.81 7.84 7.98

k(M th-~)
2.8 5.2 4.4 1.2.103 1.2" 103 4.0" 103 2.2,104 3.2-105 2.2- 105 5.2" 105

q/2(h)
6.6" 103 3.5" 103 4.2" 103 1.5" l0 t 1.5.101 4.6 8.4.10 1 5.8- 10 - z 8.4.10 -2 3.5" 10 2

pH
8.08 8.20 8.45 8.55 8.60 8.75 9.01 9.15 9.44 10.84

k(M-~h -t)
8.6" 104 6.3" 104 6.0.10'* 9.8" 104 1.2.105 1.6- 105 3.9,105 6.2,10 s 8,5,10 ~ 7.4, 105

q/2(h)
2.1 10-1 2.9.10 1 3.1 10 1 1.9.10 1 1.5.10-1 1.2- 10 1 4.8.10 a 3.0-10 2 2.2- 10 -2 2.5.10 2

Chromatographic Instrumentation and Conditions

A Spectra-Physics model SP 8700 high-performance liquid chromatograph was used. A rheodyne injector with a 20 gl loop was used. The detector was a Schoeffel L.C. Fluorimeter FS 970. Detection conditions: Excitation wavelength 420 nm, emission wavelength 470 nm. The stationary phase was Nucleosil N H 2 (Chrompack), particle size 5 gin, pre-packed in a 250 m m x 4.6 mm I.D. column. The mobile phase was ethanol p.a. The analyses were carried out at ambient temperature.

Results and Discussion

106

k (M -1 h -1 )

The reproducibility of the method is good. The extraction procedure gives a curcumin recovery of > 90%. This is acceptable because of the short extraction time. By repeated extractions, curcumin would be exposed to light, acid and ethyl acetate for a longer period. This should be avoided since curcumin may polymerize on the surface between the ethyl acetate and the buffer layers. Ethyl acetate is still the best choice as extraction solvent because the resulting sample can be injected directly onto the column at the chromatographic conditions used, and evaporation of the sample is avoided. A sample of curcumin in ethyl acetate is stable for at least 10 hours (protected from light) after separation from the buffer layer. Fluorescence detection was used because of the low detection limit for curcumin in this system [7]. This makes it possible to follow the degradation for a convenient time at the pH where the degradation process is very fast. Possible degradation products gave no interference in the chromatographic system used. It was difficult to find a compound suitable as an internal standard in these experiments. However, solutions of curcumin in methanol stored at - 70 C for one year are apparently unchanged with regard to the curcumin content. To control the reproduceability of the method, 1 ml of a stock solution of curcumin in methanol kept at - 7 0 C was mixed with 9 ml of water and extracted with 5 ml of ethyl acetate, and the layer was separated, diluted to 5.0ml and analysed for the curcumin content. This procedure was repeated at regular intervals throughout the experiment. Two stock solutions of curcumin in methanol (2.10 -4 g/ml and 4.10-5 g/ml) were used to observe whether the concentration of curcumin is of importance for the degradation rate and order. No significant difference was detected in rate or order for the two concentrations used at pH > 7. At pH < 7, the degradation reaction is ,-~ 100 times faster in the diluted

10 5

lO 4

/
,4 /
7

10 3

9 pH

10

11

Fig. 1. Plot o f the pH-profile for the degradation of curcumin in aqueous solution at 31.5 C (pH > 7). O , @, and @ indicate the p K , values of the dissociated protons

solution. This is probably due to the low solubility of curcumin in this pH range. The choice of buffer systems is critical. Curcumin seems to form complexes with many salts used in common buffer systems (borate, citrate, phthalate). Curcumin is apparently inert towards KC1, KH2PO4, and NaHCO3. At constant pH and ionic strength, there was no significant difference in the degradation rate of curcumin in phosphate and carbonate buffers at pH 9. Variation in buffer concentration at pH 7.0 and 7.8 (phosphate) from 0.2 M to 0.05 M showed no significant change in reaction rate of the degradation process as a function of the buffer concentration. The degradation of curcumin was studied over the pH range 1-11. At fixed values of pH, temperature and ionic strength the over-all loss of curcumin showed second order kinetics. A correlation coefficient better than 0.93 was accepted for the linear graph of 1/c against time at various pH-values (c ist the concentration of curcumin). The observed pseudosecond-order rate constants (kous) for the over-all deg403

@r~Nina|

Pa#ers

HO"~'~i

r~
O ~ O C H

OH+
3

H4A+

HO OH

H3A

HO"~,.~ A ~,. A ~ A . , ~
HaCO. ~ v

""

"IT T v - . . - o c . 3 O OH

HO~r~

~T/OH O O_

H2A-

H 3 C O ~ o C H

HA2-

.o.f.
H 3 C O ~ O C H O O_ 3

value for the dissociation of the enol, which is calculated from the first inflection point on the pH profile curve (pH = pK a in the inflection point when k is plotted against pH [10]), is found to be 7.75-7.80, which is in good agreement with the visually observed transition range (7.75-7.90). The rate constant for the degradation reaction of curcumin in this pH-range is high, and gives a half-life of curcumin (according to second order kinetics) of less than 10 min (Table 1). The pH profile curve shows a minimum in the pH area 8.20-8.50. Curcumin is here postulated to be in equilibrium between three forms; H3A, H z A - , and HA 2 - (Fig. 2). A second inflection point of the curve is observed at pH = 8.55 _+0.05 and a third is observed at pH 9.05_+0.05. These indicate the pKa values for the dissociation of the phenols in the curcumin molecule (Fig. 2). The primary degradation products of curcumin at pH 7 to pH 10 are shown to be ferulic acid and feruloylmethane [7]. The minimum of the pH curve is likely to be due to a self-stabilizing effect of the curcumin molecule in the H z A - form rather than to another reaction mechanism.

Conclusion
O O-

Fig. 2. Proposed dissociation of curcumin in aqueous solution

radation of curcumin were calculated from the slopes of the straight lines obtained by plotting 1/c vs t where c is residual curcumin in molar concentration and t is time. Values of/Cobs at several pH values are listed in Table 1. A plot of the pH profile for the degradation of curcumin in aqueous solution at 31.5 C is shown in Fig. 1. The shape of the pH profile curve for the hydrolytic degradation of curcumin indicates that several acid-base equilibria must be involved (Fig.2) [9]. When the pH is < 1, curcumin has a red colour, which indicates the protonated form (H4A +) postulated in Fig. 2 [9]. Curcumin in solution at pH 1-7 has a yellow colour. In this pH range, the majority of the curcumin molecules are in the neutral form (H3A). At the given concentrations, the water solubility of curcumin is low in this pH area. Curcumin is precipitated after about 3 days storage and the curcumin degradation is slow. The half-lives of curcumin in those solutions are obtained by assuming constant reaction order and extrapolating the linear graph to the time where c = Co/2 (Co = initial concentration of curcumin in molar). At p H > 7 . 5 the colour change to orange red. The pKa

Curcumin in aqueous solutions is exposed to hydrolytic degradative reactions. To obtain optimum stability of the preparations, the pH should be maintained below 7. However, in this pH region the solubility of curcumin in aqueous media is low. At pH > 7, the curcumin molecules are extremely unstable. The pH profile curve shows a minimum in the area pH 8.20-8.50, but even in this region the degradation of curcumin is fast. In its native form, curcumin is not suitable as a colouring agent in aqueous solutions of pH > 7 unless a suitable stabilizing agent is present.

References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Shankaracharaja NB (1974) Indian Spices 10:7 Turmeric, Curcumin (1975) WHO Food Additives Series No. 6 Govindarajan VS (1980) CRC Cr Rev Food Sci Nutr 12:199 Tonnesen HH, Karlsen J, Mostad A (1982) Acta Chem Scand B36:475 Leshik RR (1981) Eur Pat Appl EP 37:204 (C1.A. 23L1/275) Tonnesen HH, Karlsen J (1985) Z Lebensm Unters Forsch (in press) Tonnesen HH, Karlsen J (1983) J Chromatogr 259:367 Pabon HJJ (1964) Recueil 83:379 Dyrssen DW, Novikov YP, Uppstr6m LR (1972) Anal Claim Acta 60:139 Connors KA, Amidon GL, Kennon L (1979) In: Chemical stability of pharmaceuticals. John Wiley & Sons, New York

Received December 4, 1984

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