Curcumin : Analysis and Stability

Curcumin is a polyphenol extracted from Curcuma longa, used as a spice, in food coloring, and as a traditional herbal medicine. It has wide therapeutic platform as anti-oxidant, anticancer, anti-inflammatory and anti-infection properties. This review discusses the analytical methods used in determination of curcumin in various matrices with degradation profile, expected degradation products and stability tests.


INTRODUCTION
Curcumin, (1E,6E)-1,7-bis(4-hydroxy-3methoxyphenyl)hepta-1,6-diene-3,5-dionealso known as diferuloylmethane Figure 1, is a phenolic compound 1 present in many kinds of medicinal plants, especially in Curcuma longa (turmeric) 2 , and was first discovered and isolated in 1815 by Harvard College laboratory scientists Vogel and Pelletier 3 .The first article referring to the use of Curcumin in human diseases was published in 1937 4 .Curcumin possesses many pharmacological activities including antioxidant, anti-infection, anti-inflammation, anti-Alzheimer and anticancer [5][6][7][8][9][10][11][12][13][14][15][16][17][18] .Curcumin is a special spice which is the functional ingredient in curry powder 19 , and a potential natural food coloring 20 ; it impairs an attractive yellowish-orange color to food, authorized as a food additive by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1974, it's E number is E 100 21 .It is widely used in food applications including dairy products, fat emulsions, confectionery, soups and sauces [22][23][24][25][26][27][28][29][30][31] .In this review paper we are highlighting various analytical techniques (focusing on spectrophotometric and chromatographic techniques) used to detect curcumin in different matrices (biofluids, rhizomes, food and different pharmaceutical dosage forms) also with degradation, degradation products and stability indicating methods under different stress conditions.Moreover, this review summarizes in table forms different conditions of each analytical technique.We hope this review will be helpful to all scientists interested in curcumin therapeutic effects and /or applications on food industry.

. Infrared spectroscopy
These techniques had been widely used for determination of curcumin as they allowed rapid and sensitive, ease in sample preparation, and non destructive technique meaning that the used samples can be used for further analysis.In addition, IR spectroscopy could identify and differentiate between curcumin of different geographical origin 32,33 ,or between curcumin and other Curcuminoids in rhizomes 34,35 .Curcumin had near infrared spectroscopy at regions of 1500-2500 and 2040-2486nm for total Curcuminoids 36-42.

UV-Vis spectrophotometry
Being yellow colored; the most easy and simple method for curcumin estimation is via direct UV-Vis spectrophotometry, as the official standard AOAC method which depends on direct estimation of curcumin content in certain solvents 43 , absorption intensity at wavelength of 420 -430 nm (depending on the solvent system 44 ).The absorption band is found to have an asymmetric profile in non-polar solvents (as chloroform, acetic acid, , toluene, and carbon tetrachloride 45 ).The type and nature of the solvent affects the absorption profile of curcumin causing only a small red-shift (ca.0~20 nm) when going from nhexane to methanol 46 .The PH of solvent also affects the absorption spectrum of curcumin : λ max is 520 nm when measuring curcumin in acetone-bicarbonate buffer (pH 11) 47  giving λ max = 430 nm 49 , while curcumin metal complexes(which have better anti-oxidant therapeutic activity) show a main absorption band at 415-430 nm and compared to curcumin alone 7,50,51 .Further more curcumin may be encapsulated in nanoparticles(for better solubility) as poly(l-lactic acid) nanoparticles giving absorption band at 465 nm 52 .However, using this technique it is not possible to separate and to quantify the curcumin from raw Curcuminoids mixture 52-58 .

Chromatographic-based methods 1.2.1 Thin layer chromatography (TLC) and (HPTLC)
Developed TLC methods could be used as a technique for quality control of Curcuma rhizomes.Some of the recent published researches related to the use of TLC coupled with high performance liquid chromatography (HPTLC) for analysis of curcumin are collected in the Table 1.

High performance liquid chromatography (HPLC).
HPLC techniques are usually the methods of choice for determination of curcumin, the most common detectors used are UV or PDA (as curcumin has absorbance in the visible range).Table 2 illustrates recent reported analytical methods of curcumin either in food, pharmaceutical formulations, biological fluids (biofluids) or even alone.

Liquid chromatography coupled with mass spectrometry (LC/MS).
LC/MS can be used to detect even trace amounts of curcumin in biological fluids, food or in other complex matrices and provide fast and accurate analysis as an on-line technique.Moreover, it can be used to differentiate from other Curcuminoids.Furthermore it can be used not only to identify and quantify known Curcuminoids, but also to identify unknown Curcuminoids in extracts from turmeric or related plant material.Recent LC/MS methods and their conditions are listed in Table 3.

Curcumin Degradation 2.1. Oxidation of curcumin
The major product of the autoxidation of curcumin is a bicyclopentadione, formed by oxygenation and double cyclization of the heptadienedione chain connecting the two methoxyphenol rings of curcumin. 107,108roducts of curcumin oxidative transformation are eight compounds (Bicyclopentadione, Dihydroxy cyclopentadione, Hemiacetal cyclopentadione, Ketohydroxy cyclopentadione, Spiroepoxide cyclopentadione, Vinylether cyclopentadione, Cyclobutyl cyclopentadione and Diguaiacol). 109Six of these products had oxygen substitutions at C-1 and C-7.Two products that did not incorporate oxygen were a cyclobutyl cyclopentadione and, an obvious cleavage product.Two of the isolated products, the Spiroepoxide and the vinyl ether cyclopentadiones, were intermediates in the reaction to the Bicyclopentadione.The others were end products formed in addition to the Bicyclopentadione. 110Neither vanillin nor ferulic acid was formed in sufficient amount to be detected. 111

Degradation of curcumin in buffered solutions
Decomposition was pH dependent and occurs faster at neutral-basic conditions.The stability was proven to be more in acidic pH and decrease as the pH increases.It was more stable at pH of 1.2; less than 1% of curcumin decomposed within 6hrs of the total curcumin in the absence of light. 112,113hen curcumin was incubated in phosphate buffer, pH 7.2 at 37˚C(biological media ), it was found that 90% was degraded in 30 min.Trans-6-(4-hydroxy-3-methoxyphenyl)-2,4-dioxo-5-hexenal, vanillin, ferulic acid and feruloyl methane were identified as degradation products. 114,115urcumin exhibits a red color at pH less than 1, curcumin (due to the presence of the protonated form), and a yellow color at pH ranging from 1 to 7 (as the majority of the curcumin molecules present in the neutral form), while at pH values higher than 7.5 a distinct dark red color rapidly appeared which fade rapidly with time leaving an yellowish orange solution.Furthermore, for the buffer system being used, curcumin forms complexes with borate, citrate, and phthalate, while being inert towards KCl, KH2PO4, and NaHCO3 116 .

Photo degradation of curcumin
Exposure to visible light inflicts more degradation than UV light .The photochemical degradation of solid state curcumin exposed to sunlight for 120 h yielded vanillin (34 %), ferulic aldehyde (0.5 %), ferulic acid (0.5 %), vanillic acid (0.5 %) ,phydroxybenzaldehyde, p-hydroxybenzoic acid 117,118 .Curcumin was found to be more stable in the dried form against sunlight exposure than in solution 119 Besides the photo-sensitivity of curcumin, it is also self-degradable in the dark, this self-degradation process is enhanced in basic medium, and it was found that this process was fairly dependent on salt (NaCl) concentration. 120

Thermal degradation of curcumin
Curcumin is heat sensitive (however it is stable up to 70 °C121 ), current researches suggest that curcumin undergoes thermal degradation due to roasting (heating at 180 °C up to 70 minutes) and its degradation products are vanillin, ferulic acid, and 4vinyl guaiacol. 122If curcumin was used as food coloring agent, the processing temperature of the food should not exceed 190 °C. 123Around 27-53 % of curcumin was lost by heat processing of turmeric and major loss was observed by pressure cooking, with maximum loss in pressure cooking for 10 min. 124

Figure 1 .
Figure 1.Chemical structure of curcumin 1. Curcumin analysis 1.1 Spectroscopic techniques 1.1.1.Infrared spectroscopyThese techniques had been widely used for determination of curcumin as they allowed rapid and sensitive, ease in sample preparation, and non destructive technique meaning that the used samples can be used for further analysis.In addition, IR spectroscopy could identify and differentiate between curcumin of different geographical origin32,33 ,or