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Curcumin antifungal and antioxidant activities are increased in the presence of ascorbic acid
Food Chemistry 133 (2012) 1001–1005
Contents lists available at SciVerse ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Curcumin antifungal and antioxidant activities are increased in the presence of ascorbic acid Omar Arafat Kdudsi Khalil a,b, Olga Maria Mascarenhas de Faria Oliveira b, José Carlos Rebuglio Vellosa c, Andreza Urba de Quadros d, Loriangela Marceli Dalposso d, Thaysa Ksiaskiewcz Karam d, Rubiana Mara Mainardes d, Najeh Maissar Khalil d,⇑ a
Instituto Federal de Goiás – IFG, Formosa, Goiás, Brazil Departamento de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista ‘‘Julio de Mesquita Filho’’, Araraquara, São Paulo, Brazil c Departamento de Análises Clínicas, Universidade Estadual de Ponta Grossa, Ponta Grossa, Paraná, Brazil d Departamento de Farmácia, Universidade Estadual do Centro-Oeste, Guarapuava, Paraná, Brazil b
a r t i c l e
i n f o
Article history: Received 24 January 2011 Received in revised form 5 December 2011 Accepted 2 February 2012 Available online 11 February 2012 Keywords: Curcumin Ascorbic acid Antifungal Antioxidant
a b s t r a c t The objective of this study was to evaluate the antifungal and antioxidant activities of curcumin, ascorbic acid and the mixture of these two compounds. For the antifungal assay, the minimum inhibitory concentrations (MIC) were determined using Candida strains (ATCC and clinical isolates). Curcumin alone inhibited growth of Candida albicans yeast cells, whereas ascorbic acid did not present effects. However, when the mixture of ascorbic acid and curcumin was assayed to determine the association of the two compounds, the curcumin MIC decreased 5- to 10-fold. In the antioxidant assays, the sum of the alone activities of curcumin and ascorbic acid were lower than the activity of the two-compound mixture. This study highlights the importance of the association between two common antioxidants in foods, to improve the antifungal and antioxidant activities of curcumin (in vitro), and can be applied to Candida spp. infection and diseases associated with oxidative stress. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione; commonly known as diferuloylmethane] is the major component of turmeric and is obtained from the rhizome of Curcuma longa Linn. Curcumin is mainly used in the food industry as a spice and a food-colouring agent. Curcumin has been shown to possess several biological activities such as the suppression of carcinogenesis, by suppressing the proliferation of a wide variety of tumour cells from the skin, lung, colon, stomach and breast (Agarwal, Kumar, & Bharti, 2003; Sharma, Gescher, & Steward, 2005), as well as anti-inflammatory (Zhang, Altorki, Mestre, Subbaramaiah, & Dannenberg, 1999) and antioxidant activities (Ak & Gulcin, 2008; Mohammadi et al., 2005). Beyond the cited activities, curcumin possesses activity on different microorganisms. The antifungal activities of curcumin against Paracoccidioides brasiliensis are more potent than those of fluconazole, and curcumin inhibits the adhesion of Candida species isolated from AIDS patients to human buccal epithelial cells ⇑ Corresponding author. Address: Departamento de Farmácia, Universidade Estadual do Centro-Oeste, Rua Simeão Camargo Varelá de Sá 03, Guarapuava, Paraná 85040-080, Brazil. Tel.: +55 4236298160; fax: +55 4236298102. E-mail address: [email protected] (N.M. Khalil). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.02.009
(Martins et al., 2009). Currently, the mechanism of the antimicrobial activity has not been entirely elucidated; the most accepted mechanism suggests that the methoxyl and hydroxyl groups are the ones that are mainly responsible for this activity (Gotoh, Saitoh, & Miyake, 1998; Han & Choi, 2002; Liu, Koya, Furuta, & Matsuzaki, 1996). In alkaline or neutral solution, curcumin is quickly degraded into ferulic acid and vanillin (Tonnesen & Karlsen, 1985; Oetari, Sudibyo, Commandeur, Samhoedi, & Vermeukn, 1995); this condition can decrease or increase its diverse biological activities. The degradation of curcumin is prevented by the addition of reducing agents such as glutathione, N-acetyl-L-cysteine and ascorbic acid (Oetari et al., 1996). Some works have described the benefits of the association between curcumin or ascorbic acid with other compounds. Ascorbic acid displays a synergic effect with 1,4-naphthoquinone to cause cell death in the McCoy cell line, most likely due to the generation of hydrogen peroxide by ascorbate (Kitagawa et al., 2008). The association of ascorbic acid and oxovanadium(II) complexes is highly active against fungal species (Moamen, 2010). Curcumin, at a non-antifungal concentration, interacts synergistically with azoles and polyenes, and this synergy is related to the generation of reactive oxygen species and apoptosis (Sharma, Manoharlal, Negi, & Prasad, 2010). The objective of the present study was to evaluate the antifungal and antioxidant activities of the interaction
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O.A.K. Khalil et al. / Food Chemistry 133 (2012) 1001–1005
of curcumin and ascorbic acid, comparing these activities with the studied compounds alone. 2. Materials and methods 2.1. Reagents and chemicals Curcumin, ascorbic acid, Liquid RPMI 1640 (RPMI) medium with and without sodium bicarbonate, amphotericin B and 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) were purchased from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade and of the highest commercially available purity. L-glutamine
2.2. Antioxidant assay 2.2.1. General The compounds alone or their combinations were tested in 50 mM potassium phosphate buffer (pH 7.4) at 37 °C, on light protection and by the determined times. Some aliquots of the solutions were used for the activity assays. Concentrations of curcumin used do not interfere in the spectrophotometric analysis of these assays. 2.2.2. 2,20 -Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical cation decolourisation assay Briefly, a mixture of ABTS (7 mM) and potassium persulphate (2.45 mM) were prepared and allowed to stand at room temperature for 12 h in the dark (Re et al., 1999). The ABTS+ solution was diluted to an absorbance of 0.70 at 734 nm in 50 mM phosphate-buffered saline, pH 7.4. Different concentrations of curcumin and ascorbic acid (alone or in combination) were incubated for 0, 1 and 3 h before the assay. The ABTS+ was incubated with these solutions for 30 min at room temperature before measuring the absorbance at 734 nm. The control used in this assay was the ethanol, solvent used for stock solution of curcumin. 2.2.3. Hypochlorous acid (HOCl) scavenging activity 2.2.3.1. Synthesis of HOCl. For the assay, 50 lM HOCl was prepared immediately before use by adjusting a solution of NaOCl to pH 12.0 with diluted NaOH. The concentration of HOCl was further determined spectrophotometrically at 292 nm using the molar absorption coefficient of 350 M1 cm1. 2.2.3.2. HOCl scavenging assay. This assay was based on the reaction of KI with HOCl to produce the oxidation product I3 (Shacter, Lopez, & Pati, 1991). Different concentrations of curcumin and ascorbic acid (alone or in addition) were incubated for 0, 0.5 and 2 h before the assay. The HOCl (50 lM) was incubated with these solutions for 30 min at room temperature before the addition of KI (1.0 M) and the measurement of the absorbance at 350 nm to evaluate the amount of remaining HOCl. The assay was carried out in 50 mM potassium phosphate buffer (pH 7.4) at 37 °C, protected from light. In both the assays, the scavenging activities were calculated as the percentage of inhibition by:
% inhibition ¼
Absorbance of control Absorbance of test Absorbance of control 100
The absorbance of the control corresponds to the test with the same volume of ethanol or buffer used in the experiments with curcumin or ascorbic acid, respectively. The absorbance of curcumin at the concentrations used did not interfere in the analysis.
2.3. Antifungal activity 2.3.1. Strains Candida albicans (ATCC 14053 and 6458) and Candida krusei (ATCC 4258) obtained from the American Type Culture Collection (ATCC) were used as a reference fungal strain and two different clinical isolates: C. albicans (IC) and Candida tropicalis also were evaluated. This study was approved by the Institutional Ethical Committee of the University. The antifungal activities were determined by broth microdilution using 2-fold serial dilutions in RPMI 1640 medium, as described by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS), method M27-A (2002). 2.3.2. Medium Liquid RPMI 1640 medium with L-glutamine and without sodium bicarbonate (R-6504; Sigma Chemical Co.), buffered with 0.165 M MOPS (morpholinepropanesulphonic acid; 34.54 g/l), was used for in vitro anticandidal analyses. The medium was adjusted to pH 7.0 with 1.0 M NaOH and was filter sterilised. A stock solution of curcumin was prepared in 99.5% ethanol, and a stock solution of ascorbic acid was prepared in NaCl (0.85%). The concentration of ethanol used did not significantly affect the growth of the strains. The medium with the fungal strain, without any sample, was used as a growth control, and the blank control was composed of only the medium. Amphotericin B (12.5 lg/ml) was used as the standard drug control. Sterile 96-well microtitration plates were prepared with serial dilutions of the compounds. Cell suspensions were prepared in RPMI medium and adjusted to give final inoculum concentrations of 0.5 103–2.5 103 colony-forming units (CFU)/ml. The cells were incubated with different concentrations of curcumin (range 5.0–0.3125 lg/ml) and/or ascorbic acid (250.0–25 lg/ml) at 37 °C for 24 h. The MIC was defined as the lowest concentration that does not result in any visible growth of the yeast compared with the growth in the control well. In this assay also evaluated the possible synergistic effect of ascorbic acid in combination with vanillin and ferulic acid; degradation products of curcumin (Wang et al., 1997). For C. albicans (ATCC 14053) and C. tropicalis portion of 10 ll of cell suspensions (previously diluted 20-fold) was withdrawn at predetermined time point (24 h after incubation), and spread on a Sabouraud dextrose agar plate. Colony counts were determined after incubation at 37 °C for 24 h. The experiment was performed in triplicate. 2.4. Statistical analysis Data are presented as the mean ± SD. The differences between the groups (association curcumin-ascorbic acid and the sum of curcumin and ascorbic acid isolated) were examined using one-way ANOVA test. A p-value of less than 0.05 was considered statistically significant. 3. Results and discussion 3.1. Antioxidant activity As shown in Fig. 1, ascorbic acid and curcumin possesses scavenger activity on ABTS+ at the concentrations used. Despite presenting a good inhibitory potential, when the isolates were incubated at 0, 1 or 3 h before the assay, curcumin presented a decreased inhibitory action from 48% at time zero to 16% in 3 h (decrease of 66%). Ascorbic acid was able to inhibit the system by 37% (time zero) and 25% (3 h) at a 2.5 lg/ml dose, and 6% (time zero) and 0% (3 h) at a 1.0 lg/ml dose. When ascorbic acid was added
O.A.K. Khalil et al. / Food Chemistry 133 (2012) 1001–1005
ABTS radical inhibition (%)
100
curcumin 10 µg/mL
90
ascorbic acid 2.5 µg/mL
80
ascorbic acid 1.0 µg/mL
70
curcumin 10 µg/mL + ascorbic acid 2.5 µg/mL curcumin 10 µg/mL + ascorbic acid 1.0 µg/mL
60 50 40 30 20 10 0 60
0
120
180
time (min) Fig. 1. Inhibitory effect of curcumin, ascorbic acid and curcumin plus ascorbic acid on ABTS+. Compounds were incubated at different times in 50 mM potassium phosphate buffer (pH 7.4), at 37 °C, protected from light.
to the solution of curcumin, there was a better preservation in the curcumin inhibitory activity over the ABTS+ radical, presenting an inhibitory effect that decreased from 86% (zero time) to 59% (3 h; decrease of 31%), with 2.5 lg/ml of acid ascorbic and from 62% (zero time) to 18% (3 h; decrease of 71%) with 1.0 lg/ml ascorbic acid. These results indicate that the mixture of ascorbic acid and curcumin is more active than the sum of their individual effects in the ABTS+ inhibition, when ascorbic acid was used at a concentration of 2.5 lg/ml (p > 0.05). Curcumin and ascorbic acid presented activities on HOCl (Fig. 2). The dosage of curcumin tested was higher than the dosages of ascorbic acid and showed a higher inhibition percentage. For all dosages of the two studied samples, it was observed that when the HOCl assay was incubated for variable times (30 min to 2 h), its inhibitory activity decreases compared to the initial time. The association of different curcumin and ascorbic acid concentrations has also been assayed. When incubated with ascorbic acid, curcumin had an inhibitory action that was better preserved than in the absence of ascorbic acid. At the initial time, curcumin promoted inhibition by approximately 70%, either alone or in the presence of ascorbic acid. When incubated for 2 h, the efficiency of curcumin was reduced to 7%; however, in the presence of 2.5 or 0.5 lg/ml
curcumin 12.5 µg/mL ascorbic acid 2.5 µg/mL
100
ascorbic acid 0.5 µg/mL
HOCl inhibition (%)
90
curcumin 12.5 µg/mL + ascorbic acid 2.5 µg/mL
80
curcumin 12.5 µg/mL + ascorbic acid 0.5 µg/mL
70 60 50 40 30 20 10 0 0
20
40
60
80
100
120
time (min) Fig. 2. Inhibitory effect of curcumin, ascorbic acid and curcumin plus ascorbic acid on HOCl. Assay was carried through in 50 mM potassium phosphate buffer (pH 7.4), at 37 °C, protected from light. Remainder HOCl was revaluated by KI.
1003
ascorbic acid, the efficiency of curcumin was reduced from 70% to 22% and 32%, respectively. This demonstrates the protective effect of ascorbic acid for the HOCl scavenging activity of curcumin. It is important to note that ascorbic acid at a concentration of 2.5 lg/ml inhibited the HOCl system from 43% (zero time) to 9% (2 h), whereas 0.5 lg/ml ascorbic acid had no effect. These results indicate that the mixture of ascorbic acid and curcumin has a higher activity in HOCl inhibition than the sum of their individual effects when the concentration of ascorbic acid used is 12.5 lg/ml (p > 0.05). Oxidative stress is a pathophysiological process that can be defined as a change between oxidants and antioxidants and may lead to cancer, diabetes and cardiovascular diseases (Milaeva, 2011; Park & Oh, 2011; Styskal, Van Remmen, Richardson, & Salmon, 2011). According to the results, curcumin has increased antioxidant properties in the presence of ascorbic acid and can expand its diverse biological effects, which are dependent on these properties. HOCl is a reactive oxygen specie produced naturally in the human body and carries the role of the body’s vital defense. In addition, HOCl has high potential for tissue damage as a result of its biological oxidation activity. The association between curcumin and ascorbic acid is more effective than the sum of these two compounds in suppressing HOCl, leading to an increase in biological activities.
3.2. Antifungal activity Due to limitations of the current antifungal drugs, it has been of increasing interest to develop new and more effective antifungal agents, especially of natural product origin. With this in mind, the potential effects of the association of curcumin and ascorbic acid on fungal strains of clinical interest were analysed. In vitro antifungal activities of curcumin and ascorbic acid compared with the association of these two compounds were investigated against C. albicans (ATCC 14053 and 6458) and C. krusei (ATCC 4258) (Table 1). As shown, ascorbic acid does not present effects on the studied microorganisms in the tested concentrations. In C. albicans ATCC 14053 and 6458, the curcumin MIC was 5.0 and 0.625 lg/ml, respectively; however, when used in combination with ascorbic acid, these values were reduced to 0.625 and 0.3125 lg/ml, respectively. In relation to C. krusei, the curcumin MIC was 5.0, and in combination with ascorbic acid, this concentration was reduced to 1.25. In all experiments where the MIC values of curcumin were analysed in the presence of different concentrations of ascorbic acid, there was no change in values for all dosages of ascorbic acid assayed (250, 50 and 25 lg/ml). For the clinical isolates, also ascorbic acid does not present effects on the studied microorganisms in the tested concentrations. In C. albicans (IC), the curcumin MIC was 25.0 lg/ml, when used in combination with ascorbic acid, these values were reduced to 1.0 (ascorbic acid 250 lg/ml) and 10.0 lg/ml (ascorbic acid 50 lg/ml), respectively. In relation to C. tropicalis, the curcumin MIC was 5.0 lg/ml, and in combination with ascorbic acid, this concentration was reduced to 1.25 lg/ml (ascorbic acid 250 and 50 lg/ml) and 2.5 lg/ml (ascorbic acid 250 lg/ml). Evaluating the effectiveness of the combination of ascorbic acid plus vanillin and ferulic acid (the same concentrations of the test curcumin) we observed no effect of the compounds alone or in combination on the microorganisms tested (data not shown). Representative plots CFU for curcumin, ascorbic acid and combination are presented in Fig. 3. It was observed that ascorbic acid was unable to kill C. albicans and C. tropicalis cells. Curcumin showed effect on the CFU count, and this effect was amplified in the presence of ascorbic acid.
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Table 1 Antifungal activity of curcumin, ascorbic acid and curcumin plus ascorbic acid against Candida spp. MIC (lg/mL)
Compound
Curcumin (CUR) Ascorbic acid (AA) CUR + AA (250.0) CUR + AA (50.0) CUR + AA (25.0) a
C. albicans ATCC 14053
C. albicans ATCC 6458
C. kruzei ATCC 4258
C. tropicalisa
C. albicansa
5.0 >250.0 0.625 0.625 0.625
0.625 >250.0 0.3125 0.3125 0.3125
>5.0 >250.0 1.25 1.25 1.25
5.0 >250.0 1.25 1.25 2.5
25.0 >250.0 1.0 10.0 25.0
Clinical isolates.
1600
A
1400 1200
CFU
1000 800 600 400
*
200 0 Control 1400
ascorbic acid (250 µg/mL)
B
curcumin curcumin + ascorbic acid (3 µg/mL)
4. Conclusion
1200 1000
CFU
presence of ascorbic acid. Oetari and co-workers (1996) demonstrated spectrophotometrically that curcumin is degraded rapidly at pH 7.4, and this effect was prevented in the presence of ascorbic acid. The degradation products of curcumin in buffer can be related to the possible mechanism related to decreased antioxidant activity of curcumin. As described (Wang et al., 1997), the main products formed are: trans-6-(4-hydroxy-3-methoxyphenyl)-2,4dioxo-5-hexenal was predicted as major degradation product and vanillin, ferulic acid and feruloylmethane. Comparing the structure of the compounds (Feng & Liu, 2009), it is observed that curcumin has more enolic and phenolic hydroxyl groups and a conjugative system, which enhance the antioxidant activity. Both degradation products of curcumin in buffer pH 7.4 (vanillin and ferulic acid) were found to have no activity against strains of Candida, even in combination with ascorbic acid. The instability of curcumin at pH 7.4 represents a problem for its clinical use, because of its short half-life. Alternatives that can improve its stability can result in an increase in its biological effects.
800 600
*
400 200 0 Control
ascorbic acid (250 µg/mL)
curcumin curcumin + ascorbic acid (3 µg/mL)
Fig. 3. Effect of curcumin, ascorbic acid or association on mean growth (CFU) of C. tropicalis (A) and C. albicans (B) in sabouraud dextrose agar plate. Colony counts were determined after incubation at 37 °C for 24 h, of triplicate cultures ± standard deviation. ⁄Combination of curcumin + ascorbic acid was significantly different from all other groups (p < 0.05).
The MIC concentration of curcumin was shown to be reduced by the combination of curcumin and ascorbic acid against the Candida strains under consideration. Amphotericin showed an inhibitory effect on all microorganisms tested. The study of the biological effects of a mixture of two or more compounds is an area of growing interest. Isolated compounds that do not present a determined effect when associated with another compound can increase the efficacy of the latter compound. This is the case of the association described in this work in regard to the antifungal activity. It is also possible that when two isolated compounds display the same effect, the mixture of the two compounds would present a greater effect than the sum of the effects of each individual compound. This is the case of the association described in this work in regard to the antioxidant activity. The possible mechanism of the effects described above can be related to the increase of the stability of curcumin in pH 7.4 buffer in the
It was shown that the combination of ascorbic acid with curcumin reduces the MIC against Candida strains and also increases the antioxidant activity of curcumin. Curcumin is a potent antioxidant mechanism of action that is not yet well understood. Unfortunately, the antioxidant activity of curcumin rapidly decrease with pH 7.4 buffer; however, this decreasing is minimised in presence of ascorbic acid. Thus, it is suggested that the association between curcumin and ascorbic acid amplifies the previously reported antifungal and antioxidant activities of curcumin. The association represents a promissory way to use the curcumin therapeutically with application on candidal infections and diseases associated with oxidative stress, but in vivo tests are necessary to confirm the potential of this association. Acknowledgements A part of this work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Processo 577183/2008-7) and Fundação Araucária (Chamada de Projetos 08/2009). References Agarwal, B. B., Kumar, A., & Bharti, A. C. (2003). Anticancer potential of curcumin: Preclinical and clinical studies. Anticancer Research, 23, 363–398. Ak, T., & Gulcin, I. (2008). Antioxidant and radical scavenging properties of curcumin. Chemical and Biological Interactions, 174, 27–37. Feng, J. Y., & Liu, Z. Q. (2009). Phenolic and enolic hydroxyl groups in curcumin: Which plays the major role in scavenging radicals? Journal of Agricultural and Food Chemistry, 57, 11041–11046. Gotoh, Y., Saitoh, H., & Miyake, T. (1998). Chemical transformation of tylosin 0 derivatives into macrolides having a 3 -methoxyl group. Carbohydrates Research, 309, 45–55. Han, S. Y., & Choi, S. C. (2002). Antibacterial activity and identification of the active compound from turmeric extract. Journal of the Korean Society of Dyers and Finishers, 14, 11–17.
O.A.K. Khalil et al. / Food Chemistry 133 (2012) 1001–1005 Kitagawa, R. R., da Fonseca, L. M., Ximenes, V. F., Khalil, N. M., Vilegas, W., & Raddi, M. S. G. (2008). Ascorbic acid potentiates the cytotoxicity of the naphthoquinone 5-methoxy-3,4-dehydroxanthomegnin. Phytochemistry, 69, 2205–2208. Liu, M., Koya, S., Furuta, H., & Matsuzaki, S. (1996). Growth-inhibiting activity of anthraquinones and benzoanthraquinones against methicillin-resistant Staphylococcus aureus (MRSA). Dokkyo Journal of Medical Science, 23, 85–93. Martins, C. V. B., da Silva, D. L., Neres, A. T., Magalhães, T. F., Watanabe, G. A., Modolo, L. V., et al. (2009). Curcumin as a promising antifungal of clinical interest. Journal of Antimicrobial Chemotherapy, 63, 337–339. Milaeva, E. R. (2011). Metal-based antioxidants – potential therapeutic candidates for prevention the oxidative stress – related carcinogenesis: Mini-review. Current Topics in Medicinal Chemistry, 11, 2703–2713. Moamen, S. R. (2010). Synthesis, characterization, thermal and antimicrobial studies of diabetic drug models: Complexes of vanadyl(II) sulfate with ascorbic acid (ascorbic acid), riboflavin (vitamin B2) and nicotinamide (vitamin B3). Journal of Molecular Structure, 969, 163–171. Mohammadi, K., Thompson, K. H., Patrick, B. O., Storr, T., Martins, C., Polishchuk, E., et al. (2005). Synthesis and characterization of dual function vanadyl, gallium and indium curcumin complexes for medicinal applications. Journal of Inorganic Biochemistry, 99, 2217–2225. National Committee for Clinical Laboratory Standards. (2002). Reference method for broth dilution antifungal susceptibility testing of yeast (2nd ed.), Approved Standard M27-A2, Wayne, PA, USA. Oetari, S., Sudibyo, M., Commandeur, J. N. M., Samhoedi, R., & Vermeukn, N. P. E. (1995). Effects of curcumin on cytochrome P450 and glutathione S-transferase activities in rat liver. Biochemical Pharmacology, 51, 39–45.
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Park, J. G., & Oh, G. T. (2011). The role of peroxidases in the pathogenesis of atherosclerosis. Biochemistry and Molecular Biology Reports, 44, 497–505. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolourization assay. Free Radical Biological and Medicine, 26, 1231–1237. Shacter, E., Lopez, R. L., & Pati, S. (1991). Inhibition of the myeloperoxidase–H2O2– Cl system of neutrophils by indomethacin and other non-steroidal antiinflammatory drugs. Biochemical Pharmacology, 41, 975–984. Sharma, M., Manoharlal, R., Negi, A. S., & Prasad, R. (2010). Synergistic anticandidal activity of pure polyphenol curcumin I in combination with azoles and polyenes generates reactive oxygen species leading to apoptosis. FEMS Yeast Research, 10, 570–578. Sharma, R. A., Gescher, A. J., & Steward, W. P. (2005). Curcumin: The story so far. European Journal of Cancer, 41, 1955–1968. Styskal, J., Van Remmen, H., Richardson, A., & Salmon, A. B. (2011). Oxidative stress and diabetes: What can we learn about insulin resistance from antioxidant mutant mouse models? Free Radical Biology and Medicine. doi:10.1016/ j.freeradbiomed.2011.10.441. Tonnesen, H. H., & Karlsen, J. (1985). Studies of curcumin and curcuminoids. V. Alkaline degradation of curcumin. Zeitschrift für Lebensmitteluntersuchung und – Forschung A, 180, 132–134. Wanga, I. J., Pana, M. H., Chengb, A. L., Lina, L. I., Hoa, Y. S., Hsiehb, C. Y., et al. (1997). Stability of curcumin in buffer solutions and characterization of its degradation products. Journal of Pharmaceutical and Biomedical Analysis, 15, 1867–1876. Zhang, F., Altorki, N. K., Mestre, J. R., Subbaramaiah, K., & Dannenberg, A. J. (1999). Curcumin inhibits cyclooxygenase-2 transcription in bile acid and phorbol ester-treated human gastrointestinal cells. Carcinogenesis, 20, 445–451.
Contents lists available at SciVerse ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Curcumin antifungal and antioxidant activities are increased in the presence of ascorbic acid Omar Arafat Kdudsi Khalil a,b, Olga Maria Mascarenhas de Faria Oliveira b, José Carlos Rebuglio Vellosa c, Andreza Urba de Quadros d, Loriangela Marceli Dalposso d, Thaysa Ksiaskiewcz Karam d, Rubiana Mara Mainardes d, Najeh Maissar Khalil d,⇑ a
Instituto Federal de Goiás – IFG, Formosa, Goiás, Brazil Departamento de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista ‘‘Julio de Mesquita Filho’’, Araraquara, São Paulo, Brazil c Departamento de Análises Clínicas, Universidade Estadual de Ponta Grossa, Ponta Grossa, Paraná, Brazil d Departamento de Farmácia, Universidade Estadual do Centro-Oeste, Guarapuava, Paraná, Brazil b
a r t i c l e
i n f o
Article history: Received 24 January 2011 Received in revised form 5 December 2011 Accepted 2 February 2012 Available online 11 February 2012 Keywords: Curcumin Ascorbic acid Antifungal Antioxidant
a b s t r a c t The objective of this study was to evaluate the antifungal and antioxidant activities of curcumin, ascorbic acid and the mixture of these two compounds. For the antifungal assay, the minimum inhibitory concentrations (MIC) were determined using Candida strains (ATCC and clinical isolates). Curcumin alone inhibited growth of Candida albicans yeast cells, whereas ascorbic acid did not present effects. However, when the mixture of ascorbic acid and curcumin was assayed to determine the association of the two compounds, the curcumin MIC decreased 5- to 10-fold. In the antioxidant assays, the sum of the alone activities of curcumin and ascorbic acid were lower than the activity of the two-compound mixture. This study highlights the importance of the association between two common antioxidants in foods, to improve the antifungal and antioxidant activities of curcumin (in vitro), and can be applied to Candida spp. infection and diseases associated with oxidative stress. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione; commonly known as diferuloylmethane] is the major component of turmeric and is obtained from the rhizome of Curcuma longa Linn. Curcumin is mainly used in the food industry as a spice and a food-colouring agent. Curcumin has been shown to possess several biological activities such as the suppression of carcinogenesis, by suppressing the proliferation of a wide variety of tumour cells from the skin, lung, colon, stomach and breast (Agarwal, Kumar, & Bharti, 2003; Sharma, Gescher, & Steward, 2005), as well as anti-inflammatory (Zhang, Altorki, Mestre, Subbaramaiah, & Dannenberg, 1999) and antioxidant activities (Ak & Gulcin, 2008; Mohammadi et al., 2005). Beyond the cited activities, curcumin possesses activity on different microorganisms. The antifungal activities of curcumin against Paracoccidioides brasiliensis are more potent than those of fluconazole, and curcumin inhibits the adhesion of Candida species isolated from AIDS patients to human buccal epithelial cells ⇑ Corresponding author. Address: Departamento de Farmácia, Universidade Estadual do Centro-Oeste, Rua Simeão Camargo Varelá de Sá 03, Guarapuava, Paraná 85040-080, Brazil. Tel.: +55 4236298160; fax: +55 4236298102. E-mail address: [email protected] (N.M. Khalil). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.02.009
(Martins et al., 2009). Currently, the mechanism of the antimicrobial activity has not been entirely elucidated; the most accepted mechanism suggests that the methoxyl and hydroxyl groups are the ones that are mainly responsible for this activity (Gotoh, Saitoh, & Miyake, 1998; Han & Choi, 2002; Liu, Koya, Furuta, & Matsuzaki, 1996). In alkaline or neutral solution, curcumin is quickly degraded into ferulic acid and vanillin (Tonnesen & Karlsen, 1985; Oetari, Sudibyo, Commandeur, Samhoedi, & Vermeukn, 1995); this condition can decrease or increase its diverse biological activities. The degradation of curcumin is prevented by the addition of reducing agents such as glutathione, N-acetyl-L-cysteine and ascorbic acid (Oetari et al., 1996). Some works have described the benefits of the association between curcumin or ascorbic acid with other compounds. Ascorbic acid displays a synergic effect with 1,4-naphthoquinone to cause cell death in the McCoy cell line, most likely due to the generation of hydrogen peroxide by ascorbate (Kitagawa et al., 2008). The association of ascorbic acid and oxovanadium(II) complexes is highly active against fungal species (Moamen, 2010). Curcumin, at a non-antifungal concentration, interacts synergistically with azoles and polyenes, and this synergy is related to the generation of reactive oxygen species and apoptosis (Sharma, Manoharlal, Negi, & Prasad, 2010). The objective of the present study was to evaluate the antifungal and antioxidant activities of the interaction
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of curcumin and ascorbic acid, comparing these activities with the studied compounds alone. 2. Materials and methods 2.1. Reagents and chemicals Curcumin, ascorbic acid, Liquid RPMI 1640 (RPMI) medium with and without sodium bicarbonate, amphotericin B and 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) were purchased from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade and of the highest commercially available purity. L-glutamine
2.2. Antioxidant assay 2.2.1. General The compounds alone or their combinations were tested in 50 mM potassium phosphate buffer (pH 7.4) at 37 °C, on light protection and by the determined times. Some aliquots of the solutions were used for the activity assays. Concentrations of curcumin used do not interfere in the spectrophotometric analysis of these assays. 2.2.2. 2,20 -Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical cation decolourisation assay Briefly, a mixture of ABTS (7 mM) and potassium persulphate (2.45 mM) were prepared and allowed to stand at room temperature for 12 h in the dark (Re et al., 1999). The ABTS+ solution was diluted to an absorbance of 0.70 at 734 nm in 50 mM phosphate-buffered saline, pH 7.4. Different concentrations of curcumin and ascorbic acid (alone or in combination) were incubated for 0, 1 and 3 h before the assay. The ABTS+ was incubated with these solutions for 30 min at room temperature before measuring the absorbance at 734 nm. The control used in this assay was the ethanol, solvent used for stock solution of curcumin. 2.2.3. Hypochlorous acid (HOCl) scavenging activity 2.2.3.1. Synthesis of HOCl. For the assay, 50 lM HOCl was prepared immediately before use by adjusting a solution of NaOCl to pH 12.0 with diluted NaOH. The concentration of HOCl was further determined spectrophotometrically at 292 nm using the molar absorption coefficient of 350 M1 cm1. 2.2.3.2. HOCl scavenging assay. This assay was based on the reaction of KI with HOCl to produce the oxidation product I3 (Shacter, Lopez, & Pati, 1991). Different concentrations of curcumin and ascorbic acid (alone or in addition) were incubated for 0, 0.5 and 2 h before the assay. The HOCl (50 lM) was incubated with these solutions for 30 min at room temperature before the addition of KI (1.0 M) and the measurement of the absorbance at 350 nm to evaluate the amount of remaining HOCl. The assay was carried out in 50 mM potassium phosphate buffer (pH 7.4) at 37 °C, protected from light. In both the assays, the scavenging activities were calculated as the percentage of inhibition by:
% inhibition ¼
Absorbance of control Absorbance of test Absorbance of control 100
The absorbance of the control corresponds to the test with the same volume of ethanol or buffer used in the experiments with curcumin or ascorbic acid, respectively. The absorbance of curcumin at the concentrations used did not interfere in the analysis.
2.3. Antifungal activity 2.3.1. Strains Candida albicans (ATCC 14053 and 6458) and Candida krusei (ATCC 4258) obtained from the American Type Culture Collection (ATCC) were used as a reference fungal strain and two different clinical isolates: C. albicans (IC) and Candida tropicalis also were evaluated. This study was approved by the Institutional Ethical Committee of the University. The antifungal activities were determined by broth microdilution using 2-fold serial dilutions in RPMI 1640 medium, as described by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS), method M27-A (2002). 2.3.2. Medium Liquid RPMI 1640 medium with L-glutamine and without sodium bicarbonate (R-6504; Sigma Chemical Co.), buffered with 0.165 M MOPS (morpholinepropanesulphonic acid; 34.54 g/l), was used for in vitro anticandidal analyses. The medium was adjusted to pH 7.0 with 1.0 M NaOH and was filter sterilised. A stock solution of curcumin was prepared in 99.5% ethanol, and a stock solution of ascorbic acid was prepared in NaCl (0.85%). The concentration of ethanol used did not significantly affect the growth of the strains. The medium with the fungal strain, without any sample, was used as a growth control, and the blank control was composed of only the medium. Amphotericin B (12.5 lg/ml) was used as the standard drug control. Sterile 96-well microtitration plates were prepared with serial dilutions of the compounds. Cell suspensions were prepared in RPMI medium and adjusted to give final inoculum concentrations of 0.5 103–2.5 103 colony-forming units (CFU)/ml. The cells were incubated with different concentrations of curcumin (range 5.0–0.3125 lg/ml) and/or ascorbic acid (250.0–25 lg/ml) at 37 °C for 24 h. The MIC was defined as the lowest concentration that does not result in any visible growth of the yeast compared with the growth in the control well. In this assay also evaluated the possible synergistic effect of ascorbic acid in combination with vanillin and ferulic acid; degradation products of curcumin (Wang et al., 1997). For C. albicans (ATCC 14053) and C. tropicalis portion of 10 ll of cell suspensions (previously diluted 20-fold) was withdrawn at predetermined time point (24 h after incubation), and spread on a Sabouraud dextrose agar plate. Colony counts were determined after incubation at 37 °C for 24 h. The experiment was performed in triplicate. 2.4. Statistical analysis Data are presented as the mean ± SD. The differences between the groups (association curcumin-ascorbic acid and the sum of curcumin and ascorbic acid isolated) were examined using one-way ANOVA test. A p-value of less than 0.05 was considered statistically significant. 3. Results and discussion 3.1. Antioxidant activity As shown in Fig. 1, ascorbic acid and curcumin possesses scavenger activity on ABTS+ at the concentrations used. Despite presenting a good inhibitory potential, when the isolates were incubated at 0, 1 or 3 h before the assay, curcumin presented a decreased inhibitory action from 48% at time zero to 16% in 3 h (decrease of 66%). Ascorbic acid was able to inhibit the system by 37% (time zero) and 25% (3 h) at a 2.5 lg/ml dose, and 6% (time zero) and 0% (3 h) at a 1.0 lg/ml dose. When ascorbic acid was added
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ABTS radical inhibition (%)
100
curcumin 10 µg/mL
90
ascorbic acid 2.5 µg/mL
80
ascorbic acid 1.0 µg/mL
70
curcumin 10 µg/mL + ascorbic acid 2.5 µg/mL curcumin 10 µg/mL + ascorbic acid 1.0 µg/mL
60 50 40 30 20 10 0 60
0
120
180
time (min) Fig. 1. Inhibitory effect of curcumin, ascorbic acid and curcumin plus ascorbic acid on ABTS+. Compounds were incubated at different times in 50 mM potassium phosphate buffer (pH 7.4), at 37 °C, protected from light.
to the solution of curcumin, there was a better preservation in the curcumin inhibitory activity over the ABTS+ radical, presenting an inhibitory effect that decreased from 86% (zero time) to 59% (3 h; decrease of 31%), with 2.5 lg/ml of acid ascorbic and from 62% (zero time) to 18% (3 h; decrease of 71%) with 1.0 lg/ml ascorbic acid. These results indicate that the mixture of ascorbic acid and curcumin is more active than the sum of their individual effects in the ABTS+ inhibition, when ascorbic acid was used at a concentration of 2.5 lg/ml (p > 0.05). Curcumin and ascorbic acid presented activities on HOCl (Fig. 2). The dosage of curcumin tested was higher than the dosages of ascorbic acid and showed a higher inhibition percentage. For all dosages of the two studied samples, it was observed that when the HOCl assay was incubated for variable times (30 min to 2 h), its inhibitory activity decreases compared to the initial time. The association of different curcumin and ascorbic acid concentrations has also been assayed. When incubated with ascorbic acid, curcumin had an inhibitory action that was better preserved than in the absence of ascorbic acid. At the initial time, curcumin promoted inhibition by approximately 70%, either alone or in the presence of ascorbic acid. When incubated for 2 h, the efficiency of curcumin was reduced to 7%; however, in the presence of 2.5 or 0.5 lg/ml
curcumin 12.5 µg/mL ascorbic acid 2.5 µg/mL
100
ascorbic acid 0.5 µg/mL
HOCl inhibition (%)
90
curcumin 12.5 µg/mL + ascorbic acid 2.5 µg/mL
80
curcumin 12.5 µg/mL + ascorbic acid 0.5 µg/mL
70 60 50 40 30 20 10 0 0
20
40
60
80
100
120
time (min) Fig. 2. Inhibitory effect of curcumin, ascorbic acid and curcumin plus ascorbic acid on HOCl. Assay was carried through in 50 mM potassium phosphate buffer (pH 7.4), at 37 °C, protected from light. Remainder HOCl was revaluated by KI.
1003
ascorbic acid, the efficiency of curcumin was reduced from 70% to 22% and 32%, respectively. This demonstrates the protective effect of ascorbic acid for the HOCl scavenging activity of curcumin. It is important to note that ascorbic acid at a concentration of 2.5 lg/ml inhibited the HOCl system from 43% (zero time) to 9% (2 h), whereas 0.5 lg/ml ascorbic acid had no effect. These results indicate that the mixture of ascorbic acid and curcumin has a higher activity in HOCl inhibition than the sum of their individual effects when the concentration of ascorbic acid used is 12.5 lg/ml (p > 0.05). Oxidative stress is a pathophysiological process that can be defined as a change between oxidants and antioxidants and may lead to cancer, diabetes and cardiovascular diseases (Milaeva, 2011; Park & Oh, 2011; Styskal, Van Remmen, Richardson, & Salmon, 2011). According to the results, curcumin has increased antioxidant properties in the presence of ascorbic acid and can expand its diverse biological effects, which are dependent on these properties. HOCl is a reactive oxygen specie produced naturally in the human body and carries the role of the body’s vital defense. In addition, HOCl has high potential for tissue damage as a result of its biological oxidation activity. The association between curcumin and ascorbic acid is more effective than the sum of these two compounds in suppressing HOCl, leading to an increase in biological activities.
3.2. Antifungal activity Due to limitations of the current antifungal drugs, it has been of increasing interest to develop new and more effective antifungal agents, especially of natural product origin. With this in mind, the potential effects of the association of curcumin and ascorbic acid on fungal strains of clinical interest were analysed. In vitro antifungal activities of curcumin and ascorbic acid compared with the association of these two compounds were investigated against C. albicans (ATCC 14053 and 6458) and C. krusei (ATCC 4258) (Table 1). As shown, ascorbic acid does not present effects on the studied microorganisms in the tested concentrations. In C. albicans ATCC 14053 and 6458, the curcumin MIC was 5.0 and 0.625 lg/ml, respectively; however, when used in combination with ascorbic acid, these values were reduced to 0.625 and 0.3125 lg/ml, respectively. In relation to C. krusei, the curcumin MIC was 5.0, and in combination with ascorbic acid, this concentration was reduced to 1.25. In all experiments where the MIC values of curcumin were analysed in the presence of different concentrations of ascorbic acid, there was no change in values for all dosages of ascorbic acid assayed (250, 50 and 25 lg/ml). For the clinical isolates, also ascorbic acid does not present effects on the studied microorganisms in the tested concentrations. In C. albicans (IC), the curcumin MIC was 25.0 lg/ml, when used in combination with ascorbic acid, these values were reduced to 1.0 (ascorbic acid 250 lg/ml) and 10.0 lg/ml (ascorbic acid 50 lg/ml), respectively. In relation to C. tropicalis, the curcumin MIC was 5.0 lg/ml, and in combination with ascorbic acid, this concentration was reduced to 1.25 lg/ml (ascorbic acid 250 and 50 lg/ml) and 2.5 lg/ml (ascorbic acid 250 lg/ml). Evaluating the effectiveness of the combination of ascorbic acid plus vanillin and ferulic acid (the same concentrations of the test curcumin) we observed no effect of the compounds alone or in combination on the microorganisms tested (data not shown). Representative plots CFU for curcumin, ascorbic acid and combination are presented in Fig. 3. It was observed that ascorbic acid was unable to kill C. albicans and C. tropicalis cells. Curcumin showed effect on the CFU count, and this effect was amplified in the presence of ascorbic acid.
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Table 1 Antifungal activity of curcumin, ascorbic acid and curcumin plus ascorbic acid against Candida spp. MIC (lg/mL)
Compound
Curcumin (CUR) Ascorbic acid (AA) CUR + AA (250.0) CUR + AA (50.0) CUR + AA (25.0) a
C. albicans ATCC 14053
C. albicans ATCC 6458
C. kruzei ATCC 4258
C. tropicalisa
C. albicansa
5.0 >250.0 0.625 0.625 0.625
0.625 >250.0 0.3125 0.3125 0.3125
>5.0 >250.0 1.25 1.25 1.25
5.0 >250.0 1.25 1.25 2.5
25.0 >250.0 1.0 10.0 25.0
Clinical isolates.
1600
A
1400 1200
CFU
1000 800 600 400
*
200 0 Control 1400
ascorbic acid (250 µg/mL)
B
curcumin curcumin + ascorbic acid (3 µg/mL)
4. Conclusion
1200 1000
CFU
presence of ascorbic acid. Oetari and co-workers (1996) demonstrated spectrophotometrically that curcumin is degraded rapidly at pH 7.4, and this effect was prevented in the presence of ascorbic acid. The degradation products of curcumin in buffer can be related to the possible mechanism related to decreased antioxidant activity of curcumin. As described (Wang et al., 1997), the main products formed are: trans-6-(4-hydroxy-3-methoxyphenyl)-2,4dioxo-5-hexenal was predicted as major degradation product and vanillin, ferulic acid and feruloylmethane. Comparing the structure of the compounds (Feng & Liu, 2009), it is observed that curcumin has more enolic and phenolic hydroxyl groups and a conjugative system, which enhance the antioxidant activity. Both degradation products of curcumin in buffer pH 7.4 (vanillin and ferulic acid) were found to have no activity against strains of Candida, even in combination with ascorbic acid. The instability of curcumin at pH 7.4 represents a problem for its clinical use, because of its short half-life. Alternatives that can improve its stability can result in an increase in its biological effects.
800 600
*
400 200 0 Control
ascorbic acid (250 µg/mL)
curcumin curcumin + ascorbic acid (3 µg/mL)
Fig. 3. Effect of curcumin, ascorbic acid or association on mean growth (CFU) of C. tropicalis (A) and C. albicans (B) in sabouraud dextrose agar plate. Colony counts were determined after incubation at 37 °C for 24 h, of triplicate cultures ± standard deviation. ⁄Combination of curcumin + ascorbic acid was significantly different from all other groups (p < 0.05).
The MIC concentration of curcumin was shown to be reduced by the combination of curcumin and ascorbic acid against the Candida strains under consideration. Amphotericin showed an inhibitory effect on all microorganisms tested. The study of the biological effects of a mixture of two or more compounds is an area of growing interest. Isolated compounds that do not present a determined effect when associated with another compound can increase the efficacy of the latter compound. This is the case of the association described in this work in regard to the antifungal activity. It is also possible that when two isolated compounds display the same effect, the mixture of the two compounds would present a greater effect than the sum of the effects of each individual compound. This is the case of the association described in this work in regard to the antioxidant activity. The possible mechanism of the effects described above can be related to the increase of the stability of curcumin in pH 7.4 buffer in the
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