Research Article | Open Access
Synthesis, Characterization, Mössbauer Parameters, and Antitumor Activity of Fe(III) Curcumin Complex
Curcumin-Fe(III) complex was prepared from Fe(NO3)3·9H2O precursor and curcumin by refluxing a slightly basic methanolic solution of their mixture with the objective of investigating its cytotoxicity. The enol form of curcumin ligand was established by FTIR, UV/Vis, 1H NMR, and 13C NMR spectroscopy. The as-prepared product was characterized by elemental analysis, FTIR, UV, and Mössbauer spectroscopic techniques. An octahedral high-spin Fe(III) complex was obtained, δ, 0.37 mms−1; Q.S., 0.79 mms−1; no magnetic relaxation was observed at liquid N2 temperature, neither reduction of Fe(III). The tested cytotoxicity of the as-prepared complex on four cancer cell lines indicated inhibition of the curcumin activity upon complexing with iron.
The β diketone curcumin and curcuminoids (1,7-diaryl-1,6-heptadiene-3,5-diones) which are a group of naturally occurring 1,3-diketones have received considerable attention in the last few decades. This is due to the fact that they possess antitumor [1–3] and antioxidant effects  and have a good potential for metal ions complexation.
It has been reported that metal complexation alters the various physiological properties especially the cytotoxic and antitumor activities of many naturally occurring compounds . It is demonstrated that the coordination of metal ions, for example, Cu(II), Mn(II), Au(III), and so forth, with bioactive ligands can actually improve the pharmaceutical activity of drugs [6, 7].
The Mössbauer metal isotopes, for example, Fe, Au, Ru, Ir, and so forth, form stable complexes with the curcumin ligand [8, 9]. Tonnesen and Greenhill  have reported the reduction of Fe(III) to Fe(II) in the presence of curcumin. However, there is no published Mössbauer data on such complexes that could shed light on iron moiety and the correlation between the magnetic, symmetry, and oxidation states of metal ions in such chelates and their biological activity. In this paper we report the Mössbauer data of Fe(curc)3 complex, compare it to the well-known Fe(acac)3 data, and identify and correlate oxidation state of the central iron metal ion, magnetic relaxation, and structural symmetry of the as-prepared complex to its antitumor cytotoxicity on four cancer cell lines.
2.1. Chemicals and Materials
All solvents (Sigma-Aldrich) were reagent grades and were used without further purifications. Curcumin (Sigma-Aldrich and Acros Organics) was extensively analyzed and the Sigma sample form was established. Fe(NO3)3·9H2O (BDH) laboratory reagent was used.
2.2. Analytical Instruments
FTIR spectra were recorded in the solid state (KBr pellets) in the range 250–4000 cm−1 using a Shimadzu FTIR-8400S, Prestige-21 spectrophotometer. UV/Vis spectra were recorded on a Shimadzu UV-1650 PC spectrophotometer in the range 200–800 nm. 1H and 13C NMR were recorded on a Jeol Eclipse 400 MHz instrument using TMS external standard (chemical shift δ in ppm). C, H, N, O, and iron analysis was carried at Mikroanalytisches Labor Pascher, Remagen Laboratories. The 57Fe Mössbauer absorption spectra were recorded at 295 K and 78 K using a Harwell Mössbauer spectrometer. The spectra were refined using least square method.
2.3. Cytotoxic Activity Measurements
Cytotoxic activity measurements were carried out in Cairo Cancer Research Center, Egypt. The determination and counting of viable cells were achieved by adding 50 μL of 0.05% trypan blue solution to 50 μL of the single cell suspension. The cells were examined under the inverted microscope using the hemocytometer. Nonstained (viable) cells were counted and the following equation was used to calculate the cell count/mL of cell suspension:
The cells were then diluted to give the concentration of single cell suspension required for each experiment.
The percentage of cell survival was calculated as follows:(i)survival fraction = O.D. (treated cells)/O.D. (control cells),(ii)the IC50 values are the concentrations of thymoquinone required to produce 50 inhibitions of cell growth. The experiment was repeated 3 times for each cell line.
2.4. Synthesis of Fe(III)(acac)3 and Fe(III)(curc)3 Complexes
The Fe(III)(acac)3 complex was prepared by dissolving 2.1 g of finely ground iron(III) chloride hexahydrate in 50 mL distilled water in a 250 mL beaker. 0.1 M NaOH solution was added dropwise until the brown precipitate that was formed is completely redissolved. 2.4 mL of acetylacetone in 20 mL ethanol was then added dropwise with continuous magnetic string followed by the addition of 3.3 g sodium acetate in 15 mL distilled water. The whole mixture was heated in a water bath to ≈80°C and maintained at that temperature for 1 h with rapid stirring. The solution was then cooled to room temperature and cooled further in an ice bath. The red crystals were vacuum filtered and dried in a desiccator [10, 11]. The complex was characterized by elemental analysis, (found; C%, 50.6; H%, 6.3; yield%, 72), FTIR spectroscopy (see supporting information available online at http://dx.doi.org/10.1155/2013/982423), and Mössbauer spectroscopy, Figure 1 and Table 1. Fe(III)(curc)3 complex was prepared by refluxing for 3 h in a methanolic mixture solution of 0.6 mmol curcumin and 0.2 mmol Fe(III)(NO3)3·9H2O that was made slightly basic by the addition of few drops of triethylamine. The deep red brown solid product was vacuum filtered, washed with methanol, and dried in vacuum at room temperature overnight. The complex was characterized by elemental analysis, FTIR (supporting information), and Mössbauer spectroscopy, Figure 2 and Table 1. The elemental analytical data are consistent with the formulation: Fe[C21H19O6]3·2H2O. found, (calc): C%, 63.02, (63.36); H%, 5.06, (5.10); O%, 26.20, (26.79); Fe%, 5.02, (4.68).
3. Results and Discussion
The Mössbauer effect is the most reliable technique to identify the oxidation state of iron ions in complexes. The Mössbauer absorption spectrum of the as-prepared curcumin-iron(III) complexes is of similar pattern showing a quadrupole-split doublet of equal intensities and chemical isomer shifts characteristic of octahedral high-spin iron(III) [12–15], Figures 1 and 2 and Table 1.
The absence of any magnetic components at liquid nitrogen temperature rules out the existence of magnetic relaxation. There is no Mössbauer effect evidence that Fe(III) is reduced to Fe(II) in the presence of curcumin as reported in the literature . The Fe2+ ion, if present, should show a doublet having an isomer shift of 1.2 mm/s and a quadrupole of 2.9 mm/s .
The FTIR and 1H NMR spectroscopic measurements have confirmed the enol form of curcumin (supporting information). The FTIR spectrum of Fe(III)(curc)3 complex lost the 3423 cm−1 band assigned to (O–H) of the enol form with the persistence of the phenolic (OH) group vibrational band strongly indicating that it is not coordinated to the metal ion (supporting information). The chemistry of curcumin solution system is pH dependent. Tonnesen and Karlsen  have studied the stability of curcumin in the pH range from 1 to 11. They have postulated that curcumin is in equilibrium between three forms, that is, H3Curc; H2Curc−; HCurc2−, at pH area 8.2–8.5. They reported a 50% degradation of curcumin in a 0.1% NaOH solution. Pineda et al.  measured three acidity constants for curcumin. The one corresponding to the equilibrium H3Curc ↔ H2Curc− + H+  was attributed to the acetylacetone-type group. Such a dependence of curcumin system on pH of solution means that the H+ ion is involved in reaction mechanism. However, there are some controversies on which proton(s) is involved. While Tonnesen et al.  concluded that complex formation between curcumin and iron and the reduction of Fe3+ to Fe2+ in the presence of curcumin are independent of the phenolic hydroxyl groups in the curcumin molecule, Barclay et al.  reported that synthetic nonphenolic curcumin exhibited no antioxidant activity deducing that the H atoms are donated from the phenolic groups. On the other hand, Jovanovic et al.  suggested the release of H atoms from CH2 group.
Accordingly, we envisage that the number of protons released by the curcumin enol form determines the type of product. Hence, the three curcumin ligands are coordinated to the iron(III) ion in a bidentate fashion forming the octahedral geometry indicated by the measured Mössbauer parameters, Figure 3.
The antitumor activity of such an established octahedral high-spin iron(III) complex was tested against four cancer cell lines, that is, MCT-7, HepG-2, Hela, and HCT-116. Their activities were compared to doxorubicin (DOX) and pure curcumin. The calculated IC50 values indicated that metal complexation inhibits the cytotoxicity of curcumin, Tables 2 and 3. Table 2 presents the survival fractions of the four cancer cell lines at different concentrations of inhibitors, and the calculated IC50 values are presented in Table 3.
Curcumin alone showed higher antioxidant effect towards all four tumor cell lines at IC50 values of 3.02 (MCF-7), 3.43 (HepG2), 3.43 (Hela), and 3.81 (HCT-116) μg mL−1, than both curcumin-iron(III) complexes (see supporting information). These results are in contrast with the reported concept of synergistic enhancement of ligand effects by combination with ions [5–7, 21–23]. One may relate the lower cytotoxicity of the curcumin-iron complexes compared to curcumin alone to the fact that the central carbon atom with the labile hydrogen is locked and unable to produce oxyradicals unless the curcumin ligand dissociates from the complex. It is more likely that the keto-enol function but not the phenol OH group directs curcumin cytotoxic behavior.
It is worth mentioning that metal acetates or chlorides are the reagents used by research workers for the synthesis of curcumin-metal complexes although Pineda et al.  did not specify the iron salt employed in their study.
Then, one cannot rule out at this stage the role of the oxidizing nitrate groups in inhibiting the reduction of Fe3+ ions by curcumin. It is the reaction suggested by Pineda et al.  to take place prior to complex formation.
The IC50 values of both iron complexes, being of comparable values, could be attributed to the antioxidant potential of iron(III) ion. Hence, one can conclude that the diketone system of curcumin appears to be the part of the curcumin molecule involved in the scavenging of oxygen radicals.
Conflict of Interests
The authors declare that they have no conflict of interests arising from any financial relation with the commercial identity mentioned in this paper.
This project was supported by King Saud University, Deanship of Scientific Research, College of Science, Research Center.
Table1 and 2 represent Curcumin 1H and 13C NMR data and assignments, while Figure 1(a,b,c,d) is the FTIR spectra of curcumin and different samples of the curcumin iron complex. Figure 2(a&b) present the 1H & 13C nmr epectra of curcumin. Figure 3 is a diagrametical representation of survival fractions of the different cancer cells versus concentration of inhibitors.
- R. H. Liu, “Potential synergy of phytochemicals in cancer prevention: mechanism of action,” Journal of Nutrition, vol. 134, no. 12, pp. 3479S–3485S, 2004.
- B. K. Adams, E. M. Ferstl, M. C. Davis et al., “Synthesis and biological evaluation of novel curcumin analogs as anti-cancer and anti-angiogenesis agents,” Bioorganic and Medicinal Chemistry, vol. 12, no. 14, pp. 3871–3883, 2004.
- E. R. Hahm, Y. S. Gho, S. Park, C. Park, K. W. Kim, and C. H. Yang, “Synthetic curcumin analogs inhibit activator protein-1 transcription and tumor-induced angiogenesis,” Biochemical and Biophysical Research Communications, vol. 321, no. 2, pp. 337–344, 2004.
- S. V. Jovanovic, C. W. Boone, S. Steenken, M. Trinoga, and R. B. Kaskey, “How curcumin works preferentially with water soluble antioxidants,” Journal of the American Chemical Society, vol. 123, no. 13, pp. 3064–3068, 2001.
- K. H. Thompson, K. Böhmerle, E. Polishchuk et al., “Complementary inhibition of synoviocyte, smooth muscle cell or mouse lymphoma cell proliferation by a vanadyl curcumin complex compared to curcumin alone,” Journal of Inorganic Biochemistry, vol. 98, no. 12, pp. 2063–2070, 2004.
- S. Zhang, C. Tu, and X. Wang, “Novel C ytotoxic Copper(II) complexes of 8-aminoquinoline derivatives: crystal structure and different reactivity towards Glu tathioneInorg,” European Journal of Chemistry, vol. 98, pp. 4028–4035, 2004.
- S. Dutta, A. Murugkar, N. Gandhe, and S. Padhye, “Enhanced antioxidant activities of metal conjugates of Curcumin derivatives,” Metal-Based Drugs, vol. 8, no. 4, pp. 183–188, 2001.
- H. H. Tonnesen and J. V. Greenhill, “Studies on curcumin and curcuminoids. XXII: curcumin as a reducing agent and as a radical scavenger,” International Journal of Pharmaceutics, vol. 87, no. 1–3, pp. 79–87, 1992.
- K. K. Sharma, S. Chandra, and D. K. Basu, “Synthesis and antiarthritic study of a new orally active diferuloyl methane (curcumin) gold complex,” Inorganica Chimica Acta, vol. 135, no. 1, pp. 47–48, 1987.
- A. Hantzsch and C. H. Desch, Annalen, vol. 1, American Chemical Society, Washington, DC, USA, 1902.
- J. Josepg, H. A. C. Mckay, and E. J. W. Verwey, “The infra-red spectra of metal acetylacetonates in the sodium chloride region,” Journal of Inorganic and Nuclear Chemistry, vol. 5, pp. 295–303, 1958.
- M. I. Khalil and A. I. AL-Wassil, “The Nature of iron moiety, a mossbauer spctroscopic investigation,” Journal of the Serbian Chemical Society, vol. 3, pp. 19–25, 1999.
- M. W. Hisham and F. J. Berry, “The influence of ball milling and subsequent calcination on the formation of LiFeO2,” Journal of Materials Science, vol. 37, no. 21, pp. 4621–4625, 2002.
- E. Murad and R. M. Taylor, “The Mossbauer spectra of hydroxycarbonate green rusts,” Clay Minerals, vol. 19, no. 1, pp. 77–83, 1984.
- C. W. Childs and J. H. Johnston, “Mossbauer spectra of proto-ferrihydrite at 77K and 295K,” Australian Journal of Soil Research, vol. 18, pp. 245–250, 1980.
- H. H. Tonnesen and J. Karlsen, “Studies on curcumin and curcuminoids,” Zeitschrift für Lebensmittel-Untersuchung und -Forschung, vol. 180, pp. 402–404, 1985.
- M. B. Pineda, M. T. R. Silva, M. R. Romo, E. G. Verqara, and A. R. Hennandez, “Spectroscopica,” Chemica Acta A, vol. 60, no. 5, pp. 1091–1097, 2004.
- H. H. Tonnesen, J. Karlsenand, and G. B. Henegouwer, “Studies on curcumin and curcuminoids VIII. Photochemical stability of curcumin,” Zeitschrift für Lebensmittel-Untersuchung und -Forschung, vol. 183, no. 2, pp. 116–122, 1986.
- L. R. C. Barclay, M. R. Vinqvist, K. Mukai et al., “On the antioxidant mechanism of curcumin: classical methods are needed to determine antioxidant mechanism and activity,” Organic Letters, vol. 2, no. 18, pp. 2841–2843, 2000.
- S. V. Jovanovic, S. Steenken, C. W. Boone, and M. G. Simic, “H-atom transfer is a preferred antioxidant mechanism of curcumin,” Journal of the American Chemical Society, vol. 121, no. 41, pp. 9677–9681, 1999.
- X. Sheng, X. M. Lu, Y. T. Chen et al., “Synthesis, DNA-binding, cleavage, and cytotoxic activity of new 1,7-dioxa-4,10-diazacyclododecane artificial receptors containing bisguanidinoethyl or diaminoethyl double side arms,” Chemistry, vol. 13, no. 34, pp. 9703–9712, 2007.
- A. Valentini, F. Conforti, A. Crispini et al., “Synthesis, oxidant properties, and antitumoral effects of a heteroleptic palladium(II) complex of curcumin on human prostate cancer cells,” Journal of Medicinal Chemistry, vol. 52, pp. 484–491, 2009.
- G. Federici, S. Bernadini, and D. Pucci, “Effects of a heteroleptic palladium (II) comple x of curcumin on human prostate cancer ghedini,” Journal of Solid State Chemistry, vol. 160, pp. 230–236, 2002.
Copyright © 2013 Mutasim Ibrahim Khalil et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.