Arch. Pharm. Chem. Life Sci. 2014, 347, 1–6
1
Full Paper Synthesis and Biological Evaluation of Glucosyl Curcuminoids Adari Bhaskar Rao1, Ernala Prasad1, Seelam S. Deepthi1, and Imtiaz A. Ansari1,2 1
2
Medicinal Chemistry and Pharmacology Division, CSIR – Indian Institute of Chemical Technology, Tarnaka, Hyderabad, India Department of Chemistry, King Khalid University, Abha, Saudi Arabia
Medicinal plants proved to be a rich source in exploring a variety of lead structures in the development of new drugs. The natural curcuminoids have gained considerable attention in recent years for their multiple beneficial pharmacological and biological activities. Clinical application of these curcuminoids is often impaired due to their poor water solubility, resulting in low in vivo bioavailability of the active compound in humans. The objective of the present study is to synthesize glucosyl conjugates of curcumin 1 and tetrahydrocurcumin 4 and to evaluate their biological activities. The study highlights the synthesis of curcumin-b-di-glucoside 3 (yield 71%) and tetrahydrocurcumin-bdi-glucoside 6 (yield 64%) in good yields in a biphasic reaction medium using a phase transfer catalyst under simple and ecofriendly conditions. Both the glucosyl curcuminoids showed enhanced antioxidant, tyrosinase enzyme inhibitory, antimicrobial and potent cytotoxic activity. The improved biological activity may be due to the increased solubility of the glucosyl conjugated compounds compared to the native curcuminoids; this was further confirmed by partition coefficient studies. Thus, the synthesized glucosyl curcumin may serve as promising future therapeutic molecule in the management of cancer, whereas glucosyl tetrahydrocurcumin can be a useful ingredient in achromatic food and in cosmetic applications. Keywords: Antioxidant / Cosmetic / Hyperpigmentation / Tetrahydrocurcumin / Tyrosinase Received: May 15, 2014; Revised: July 8, 2014; Accepted: July 9, 2014 DOI 10.1002/ardp.201400195
Introduction Natural compounds derived from plants, animals, and microorganisms proved to be an excellent source for therapeutic agents. Phytochemicals once served the humankind as the source of new drug development, today these natural products or their analogs still represent as preventive and therapeutic agents against a wide range of human diseases [1, 2]. Curcuminoids are naturally occurring hydrophobic polyphenolic compounds isolated from the rhizomes of Curcuma longa Linn. (Zingiberaceae) [3]. Curcumin is a predominant compound, which has attracted a lot of attention due to its wide applications in traditional medications, food coloration, cosmetic utility, and fabric Correspondence: Dr. Adari Bhaskar Rao, Medicinal Chemistry and Pharmacology Division, CSIR – Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 007, AP, India. E-mail: [email protected] Fax: þ91 40 27193189
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
dying industry. In addition, curcuminoids possess antiinflammatory, antioxidant, antiangiogenic, anticancer, and many more therapeutic activities [4, 5]. Tetrahydrocurcumin is one of the colorless hydrogenated metabolites of the curcumin that demonstrates broad physiological and pharmacological activities similar to that of native curcumin [6]. Despite wide range of food processing and pharmacological activities, the major barrier for clinical applications of these curcuminoids is their poor solubility in water, thus limiting the bioavailability and in vivo therapeutic concentration of the compound in the humans. In addition, a relative short gastric emptying time results in an incomplete release of curcuminoids from the dosage and to the site of absorption, leading to a diminished efficacy of the compound [7, 8]. Attempts were made to enhance the bioavailability of curcuminoids through structural modifications of the molecule and/or through new formulations (nanoparticles, adjuvants, nanoparticles and liposomes) but have not gained much attention due to various limitations in their
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min) 3 and a colorless product tetrahydrocurcumin-di-bglucoside (glucosyl-THC) 6 (Scheme 1).
methodologies [9, 10]. It is well known that the glycosylation of hydrophobic phytochemicals has improved the bioavailability and biological activity of the compounds [11, 12]. In the present study, it is planned to synthesize glucosylconjugated curcuminoids and to evaluate their biological activity.
Curcumin-4,4-b-di-glucoside (3) Yellow colored solid, yield 71%. 1H NMR (DMSO-d6): 3.1–3.4 (m, 8H), 3.6 (m, 3H), 3.77 (s, 6H, OCH3), 4.31 (br, s, 1H), 4.5 (t, 2H, J ¼ 5.5Hz), 4.93 (d, 2H, J ¼ 7 Hz), 4.98 (d, 2H, J ¼ 5 Hz), 5.06 (br, s, 2H), 5.25 (d, 2H, J ¼ 4 Hz), 6.04 (s, 1H), 6.81(d, 2H, J ¼ 16 Hz), 7.05 (d, 2H, J ¼ 8.5 Hz), 7.18 (d, 2H, J ¼ 8 Hz), 7.32 (s, 2H), 7.52 (d, 2H, J ¼ 16 Hz); ESI-MS (m/z): 715 (MþNa)þ.
Results and discussion Chemistry Synthesis of glucosyl curcuminoids
Tetrahydrocurcumin-4,4-b-di-glucoside (6)
Synthesis of curcumin-4,4-b-di-D-glucoside(glucosyl-curcumin) 3 and tetrahydrocurcumin-4,4-b-di-glucoside (glucosyl-THC) 6 was achieved in higher yields, in a simple economical process [13]. The glucosyl conjugation reaction was facilitated by a phase transfer catalyst (tetrabutylammonium bromide), that promotes better mass transfer of reactants between two immiscible liquid phases, resulting in high product formation with selectivity. The progress of the reaction was monitored through HPLC and the single product peak was obtained at the end of the optimum reaction period. The structures of the glucosyl-conjugated curcuminoids were confirmed by spectral analysis. From 1H NMR studies, it was confirmed that the configurations of the anomeric carbons were defined as b for all the glucose molecules therefore the conjugated glucosylations of the two curcuminoids show bconfiguration. The SN2 nucleophilic substitution at the anomeric C-1 position of the activated sugar moiety resulted in the formation of curcumin-di-b-glucoside (glucosyl-curcu-
AcO O
O
MeO HO
AcO OMe
Br OAc
KOH TBAB
OAc
45°C/2 h
OH
1
O
Half white colored solid, yield: 64%; m.p. ¼ 184°C, TLC rf ¼ 0.27 (DCM/MeOH, 9.5:0.5), UV (EtOH) lmax 258, 220 nm. IR(KBr): nmax 3453, 3017, 2927, 2854, 1753, 1605, 1514, 1370, 1229, 1037, 757 cm1; 1H NMR (DMSO-d6 and D2O): 6.86–6.59 (m, 6H, –CH2); 5.64–5.50 (m, 2H), 5.16 (t, J ¼ 9.8 Hz, 2H), 4.37– 4.26 (m, 6H), 4.17–4.10 (m, 4H), 3.87 (s, 6H), 2.86–2.70 (m, 8H); 13 C NMR (DMSO): 193.1, 146.3, 143.9, 132.5, 120.7, 114.3, 110.9, 99.7, 79.1, 76.8, 76.1, 72.6, 70.3, 61.9, 55.8, 40.3, 31.2. LC MS (m/z): 719.13 (MþNa)þ. Natural curcuminoids are yellow colored compounds resulting in staining of the skin and dress materials during topical application. Tetrahydrocurcumin is a colorless metabolite of curcumin that exhibits pharmacological and biological activities similar to that other curcuminoids. It is planned to synthesize the conjugated glucosyl-curcumin and glucosyltetrahydrocurcumin compounds to increase hydrophilicity of these molecules and improve their biological activity.
O
2
CH 3 ONa
OMe
O
O OH
O
O
O
MeO HO
AcO OMe
4
O
OH
OH
O
OH OH
AcO
O
MeO
3
OH
OH OH
OH
Br OAc
OAc
45°C/2 h
KOH TBAB
O
5
CH3 ONa
O
MeO
OMe
O
O OH
O
OH OH
OH
OH
O
6
OH OH
OH
Scheme 1. Synthesis of curcumin-4,4-b-di-glucoside 3 and tetrahydrocurcumin-4,4-b-di-glucoside 6.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–6
Figure 1. Partition coefficient (log P o/w) of potent compounds.
Partition coefficient From the results (Fig. 1), it was observed the glucosyl conjugated curcuminoids 3 and 6 show much lower lipophilicity compared to compounds 1 and 4, and thus these glucosyl-curcuminoids were more water soluble and show better bioavailability and pharmacological activities.
Biological results The radical scavenging and tyrosinase inhibition activities The test compounds curcumin 1, glucosyl-curcumin 3, THC 4, and glucosyl-THC 6 show potent antioxidant activity by inhibiting oxidation of the added 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Fig. 2). The results revealed that the glucosyl conjugated curcuminoids show promising antioxidant activity when compared to non-conjugated curcuminoids studied. The IC50 value of glucosyl-curcumin was 20.25 mM and for glucosyl-THC it was 18.25 mM. The improved free radical scavenging activity is maybe due to increased water solubility of glucosylated hydrophilic curcuminoid compounds. The
Glucosyl Curcuminoids
3
free radical scavenging activity of the curcuminoids studied may be attributed mainly due to the compound’s pharmacophore, which contains phenolic hydroxyl group and b-diketone structure. The chemoprotective nature of the natural polyphenolic compound curcumin (diferuloylmethane) against oxidative stress-mediated disorders is through the anti-oxidative functioning [14]. Natural products are known to inhibit tyrosinase enzyme activity that participates in melanin formation thereby lightening the skin tone [15]. The natural curcuminoids are of particular interest both in skin medications and in cosmetics as these molecules show a potent tyrosinase inhibition activity. The glucosylated curcumin show enhanced tyrosinase inhibition activity with IC50 52.12 mM for glucosylcurcumin 3, whereas for glucosyl-THC 6 it was IC50: 48.12 mM, nearly comparable to conventional tyrosinase inhibitors.
Antimicrobial activity The curcuminoids 1, 3, 4, and 6 tested show broad antimicrobial and antifungal activity against following bacterial strains Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, and Staphylococcus aureus and against two fungal strains Aspergillus fumigatus and Candida albicans. From the results, it was inferred that both the glucosyl-conjugated curcumin 3 and glucosyl-THC 6 have shown enhanced antimicrobial activity, with lower MIC values when compared to curcumin 1 and THC 4 (Fig. 3). Curcumin has not shown antimicrobial activity against B. subtilis whereas both curcuminoids have no activity against the fungal strain A. fumigatus. The glucosyl-curcumin and glucosyl-THC exhibited potential anti-microbial activity as
Figure 2. IC50 values of curcuminoids on DPPH radical scavenging and tyrosinase inhibition activity.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 3. Antimicrobial activity of curcumin, THC, glucosyl-THC (MIC mmol/L).
confirmed by low MIC values, this may be due to the increased aqueous solubility of these hydrophilic curcuminoids.
Cytotoxicity The cytotoxicity of the four curcuminoids curcumin 1, THC 4, curcumin-b-di-glucoside 3, and THC-b-di-glucoside 6 were studied in selected human cancer cell lines (HT-29 colon cancer, A549 lung cancer, MCF-7 breast cancer) by MTT assay. From the results, it was observed that all the tested curcuminoids have shown cytotoxic activity on the four cell lines and compounds 4 and 6 exhibited marked activity on colon cancer cell line (HT-29). The IC50 value on colon cancer
cells for curcumin 1 was 66.94 mM, THC 4 it was 77.17 mM whereas for curcumin-b-di-glucoside 3 and for THC-b-diglucoside 6 IC50 values were 41.56 mM and 32.13 mM. Thus, it was confirmed from the results that on glucosidation of the curcuminoids the inhibition of colon cancer cells (HT-29) activity was increased by twofolds when compared with free curcuminoids (Fig. 4). The conjugated curcumin has also showed a noticeable inhibition activity on breast cancer cells (MCF-7), whereas no significant activity was found in liver cancer cells (A549). Curcuminoids possess a wide spectrum of antitumor properties but, due to its poor bioavailability the curcuminoids are not yet been clinically used routinely in
Figure 4. Evaluation of cytotoxicity (IC50 mM) of curcuminoids on different cancer cell lines by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay method.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–6
anticancer chemotherapy [16–18]. Glycosylation of hydrophobic curcuminoids increases solubility of the compound and thus improving the anticancer activity as confirmed by their lower IC50 values. Natural products are known to possess potent anticancer activity and are also used topically against widespread skin diseases and cosmetics. Curcuminoids attracted considerable attention in the recent years due to their broad pharmacological activities. The poor circulating bioavailability of these curcuminoids attributed due to low water solubility thus impairing the compounds biological activity. The synthesized glucosyl-conjugated compounds curcumin-b-di-glucoside 3 and tetrahydrocurcumin-b-di-glucoside 6 have shown potent antioxidant and cytotoxic activities when compared to native curcuminoids. The results obtained clearly substantiate the beneficial effects of glucosyl-curcumin as a potent anticancer agent and colorless glucosyl-THC shows superior antioxidant and inhibit tyrosinase activity thereby helping in lightening the skin tone. Therefore, the conjugated THC acts as useful ingredients in anti-ageing and other cosmetic topical formulations designed to maintain general skin health [19].
Conclusion The study confirms a novel synthesis of curcumin-b-di-glucoside 3 and tetrahydrocurcumin-b-di-glucoside 6 in good yields and selectivity (b-form) in simple and eco-friendly reaction conditions. The results confirm the increased hydrophilicity and biological activity of glucosyl conjugated curcuminoids when compared to their native un-conjugated curcuminoids; this was further confirmed by partition coefficient studies. The data also reveal the compounds glucosyl-curcumin and glucosyl-THC showing enhanced antioxidant, antimicrobial, and tyrosinase inhibition activities, when compared to nonconjugated curcuminoids studied. In summary, the glucosylcurcumin shows potent antioxidant and cytotoxic activity, and thus the molecule may be of therapeutic importance against several types of cancer diseases. Whereas, the non-staining glucosyl-conjugated THC molecule acts as a potent ingredient in achromatic food and in cosmetic applications. Thus, the study concludes both the synthesized conjugated curcuminoids, help in improving the medical value of the natural curcuminoids and hope that future clinical trials will lead to the development of potent preventive and therapeutic agent for skin photo aging and UV induced cancer diseases.
Experimental Materials and methods Chemicals and materials Curcumin and THC were gift from Ashian Herbex Ltd., Hyderabad, India. DPPH, and bovine serum albumin (BSA), ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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L-DOPA, tyrosinase T-3824, 25 KU were procured from Sigma Chemical Co. (St. Louis, MO, USA). The cancer cell lines A549 (lung cancer), MCF-7 (breast cancer) and HT-29 (colon cancer) used were obtained from National Center for Cell Science (NCCS), Pune, India. All the microbial strains and the Mueller Hinton broth, Dulbeccos modified Eagles agar medium (DMEM) and other media components used in these studies were procured from Hi Media Ltd (Mumbai, India). All the reagents and solvents (n-octanol) used in the experiments were of analytical grade and were purchased from Merck (India).
Equipment Spectrophotometric analyses were carried out using UV visible spectrophotometer (Perkin Elmer). 1H and 13C NMR spectra for the products were recorded on a 300, 500 MHz NMR spectrometer (Bruker, Avance, Germany). Mass spectral analyses of compounds were performed using MS (Waters Q-Tof Ultima, Manchester, UK) in the ESI positive mode.
HPLC The concentration of curcumin, THC, glucosyl-curcumin, and glucosyl-THC were quantified through HPLC (Gilson LC), using reverse phase analytical C18 column (150 mm 3.9 mm, 5 mm particle size). The system was eluted isocratically using mobile phase acetonitrile/water (85:15 v/v; pH adjusted to 3.0 with 0.01% acetic acid) at a flow rate of 1 mL/min and the sample detection was at 280 nm. The mobile phase was filtered through 0.5 mm nylon membrane filter and the solvent was degassed ultrasonically before use. Salbutamol (0.2 mg/mL) was added to each test sample as an internal standard. Peak area was used as a measure to calculate the respective concentrations.
Chemistry General procedure for the synthesis of glucosylcurcuminoids The general method used for synthesis of glucosyl conjugated compounds 3 and 6 are summarized in Scheme 1. A modified procedure of Koenings–Knorr was followed in synthesis of glucosyl-conjugated curcuminoids [20, 21]. The reaction was initiated by adding aqueous solution of KOH (10.8 mmol) to 20 mL of DCM solvent containing disallowed compounds 1 or 4 (2.71 mmol), followed by the addition of 2,3,4,6-tetra-O-acetyl-bD-glucopyranosyl bromide (5.41 mmol) and molecular sieves (4 A). The reaction mixture was stirred for 3 h at 45°C under the nitrogen atmosphere. On completion of the reaction, the compounds curcumin-b-di-glucoside tetraacetate 2 or tetrahydrocurcumin-b-di-glucoside tetraacetate 5 were isolated and these compounds were deacetylated by adding 2% sodium methoxide in dichloromethane. The two products formed were isolated and purified through column chromatography silica gel (100–200 mesh). The purity of the final products was determined by HPLC and the structures of the compounds obtained curcumin-b-di-glucoside 3 or tetrahydrocurcumin-b-di-glucoside 6 were confirmed by NMR (1H, 13C), mass and LC–MS studies.
Partition coefficient (log P value) The concentration of curcuminoids present in two-phase noctanol/water system was determined by HPLC [22]. The curcuminoids 50 mM (curcumin, THC, glucosyl-curcumin, and glucosyl-THC) were dissolved in known volumes of n-octanol (2, 4, www.archpharm.com
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6, 8, and 10 mL)/water (O/W ratio) in a shake-flask and incubated at 37°C for 2 h. Lipophilicity of the compound quantified by the logarithm of the 1-octanol/water partition: log P ¼ log[Co/Cw], where Co was the concentration of compound in the octanol phase and Cw its concentration in the aqueous water phase, when the system was at equilibrium. log P values presented were an average of three measurements.
New Delhi for award of fellowship; the authors also thank MD, Ashian Herbix Ltd, Hyderabad for gift sample of curcuminoids.
Biological assays Antioxidant activity by DPPH free radical-scavenging method
[1] N. Civjan, Chemical Biology: Approaches to Drug Discovery and Development to Targeting Disease, John Wiley & Sons, Hoboken 2012. [2] D. A. Dias, S. Urban, U. Roessner, Metabolites 2012, 2, 303–336. [3] L. Shen, H. F. Ji, Trends Mol. Med. 2012, 18, 138–144. [4] M. Labbozzetta, M. Notarbartolo, P. Poma, A. Maurici, L. Inguglia, P. Marchetti, M. Rizzi, R. Baruchello, D. Simoni, N. D’Alessandro, Ann. N. Y. Acad. Sci. 2009, 1155, 278–283. [5] R. L. Thangapazham, S. Sharad, R. K. Maheshwari, Biofactors 2013, 39, 141–149. [6] M. H. Pan, T. M. Huang, J. K. Lin, Drug Metab. Dispos. 1999, 27, 486–494. [7] P. Asawanonda, S. O. Klahan, Photomed. Laser Surg. 2010, 28, 679–684. [8] G. C. Jagetia, G. K. Rajanikant, Int. Wound J. 2012, 9, 76–92. [9] H. Kharkwal, K. Bala, D. D. Joshi, D. P. Katare, Der Pharm. Lett. 2012, 4, 1698–1711. [10] J. R. Manjunatha, B. K. Bettadaiah, P. S. Negi, P. Srinivas, Food Chem. 2013, 139, 332–338. [11] S. H. Thilakarathna, H. P. V. Rupasinghe, Nutrients 2013, 5, 3367–3387. [12] C. Manach, et al., Am. J. Clin. Nutr. 2004, 79, 727–747. [13] A. Bhaskar Rao, E. Prasad, S. S. Deepthi, S. Ramakrishna, Y. S. Venkat Rao, Indian Patent No. C0123NF201, 2013. [14] D. Huang, B. Ou, R. L. Prior, J. Agric. Food Chem. 2005, 53, 1841. [15] Z. M. Hu, Q. Zhou, T. C. Lei, S. F. Ding, S. Z. Xu, J. Dermatol. Sci. 2009, 55, 179–184. [16] R. A. Sharma, et al., Cancer Res. 2001, 7, 1894–1900. [17] H. Chen, Z. S. Zhang, Y. L. Zhang, D. Y. Zhou, Anticancer Res. 1999, 19, 3675–3680. [18] M. Lopez-Lazro, Mol. Nutr. Food Res. 2008, 52, S103–S127. [19] M. Majeed, V. Badmaev, U. Shivakumar, R. Rajendran, Curcuminoids: Antioxidant Phytonutrients, Nutriscience Publishers, New Jersey 1995. [20] Z. Wimmer, L. Pechova, D. Saman, Molecules 2004, 9, 902–912. [21] K. S. Parvathy, P. S. Negi, P. Srinivas, Food Chem. 2009, 115, 265–271. [22] S. K. Poole, C. F. Poole, J. Chromatogr. B 2003, 797, 3–19. [23] P. Ronald,L X. Wu, K. Scaich, J. Agric. Food Chem. 2005, 53, 4290–4302. [24] M. Li, Y. Ma, M. O. Ngadi, Food Chem. 2013, 141, 1504–1511. [25] E. W. C. Chan, et al., Food Chem. 2008, 109, 477–483. [26] Y. Jiang, et al., Molecules 2013, 18, 3948–3961. [27] P. Magiatis, A. L. Skaltsonmis, I. Chinou, S. A. Haroutounian, Z. Naturforsch. 2002, 57, 287–290. [28] M. Ferrari, M. C. Fornasiero, A. M. Isetta, J. Immunol. Methods 1990, 131, 165–172.
The hydrogen donating or free radical-scavenging activity of the curcumin 1, glucosyl-curcumin 3, THC 4, and glucosyl-THC 6 was evaluated by the reduction of DPPH as described in the literature [23]. Trolox (0.1 mg/mL) was used as a standard antioxidant agent [24]. The experiments were performed in triplicates for each concentration and IC50 values (scavenge 50% of the DPPH radicals) were determined by the interpolation of the dose.
Tyrosinase enzyme inhibition assay The tyrosinase enzyme inhibition assay was performed using LDOPA as the substrate as reported, with slight modifications [25, 26]. Kojic acid was used as the standard/reference inhibitor compound to study tyrosinase enzyme activity. All the analyses were performed in triplicates for each concentration and the IC50 values of tyrosinase inhibition activity were determined.
Antimicrobial assay The antimicrobial activities of the curcumin 1, glucosyl-curcumin 3, THC 4, and glucosyl-THC 6 were carried out using the dilution technique and minimum inhibitory concentrations (MIC) of the compounds were determined. The following microbial strains S. aureus (ATCC 29737), B. cereus ATCC 14603, E. coli (ATCC 10536), K. pneumoniae (ATCC 13883), and P. aeruginosa (ATCC 25619), and the fungi C. albicans (ATCC 53324) and A. fumigatus (ATCC 204305) were evaluated for antimicrobial activity. Standard antibiotics gentamycin sulfate and fluconazole were used in the study for comparison of antimicrobial activity of glucosyl-conjugated curcuminoids [27].
In vitro cytotoxicity study The cytotoxicity activity for the test compounds curcumin 1, glucosyl-curcumin 3, THC 4, and glucosyl-tetrahydrocurcumin 6, against human cancer cells were determined by MTT (3-(4,5dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay. The cell lines used in the study were MCF-7 breast cancer cells, HT-29 colon cancer cells, and A549 lung cancer cells. The study was initiated by plating 104 cells per well in 96-well plates, and addition of DNR solutions with or without P85 (0.01%) to the cells (100 mL/well). Cells were incubated with test compounds dissolved in DMSO, for 4 days at 37°C and 5% carbon dioxide. After the incubation period, the compound’s cytotoxic activity was evaluated using the MTT assay at 570 nm [28]. All experimental points were carried out in triplicate and IC50 values of the test compounds were determined.
The authors express sincere thanks to DST, New Delhi, India for financial support for this project SR/SO/BB-54/2008; E.P. thanks DST, ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The authors have declared no conflict of interest.
References
www.archpharm.com
1
Full Paper Synthesis and Biological Evaluation of Glucosyl Curcuminoids Adari Bhaskar Rao1, Ernala Prasad1, Seelam S. Deepthi1, and Imtiaz A. Ansari1,2 1
2
Medicinal Chemistry and Pharmacology Division, CSIR – Indian Institute of Chemical Technology, Tarnaka, Hyderabad, India Department of Chemistry, King Khalid University, Abha, Saudi Arabia
Medicinal plants proved to be a rich source in exploring a variety of lead structures in the development of new drugs. The natural curcuminoids have gained considerable attention in recent years for their multiple beneficial pharmacological and biological activities. Clinical application of these curcuminoids is often impaired due to their poor water solubility, resulting in low in vivo bioavailability of the active compound in humans. The objective of the present study is to synthesize glucosyl conjugates of curcumin 1 and tetrahydrocurcumin 4 and to evaluate their biological activities. The study highlights the synthesis of curcumin-b-di-glucoside 3 (yield 71%) and tetrahydrocurcumin-bdi-glucoside 6 (yield 64%) in good yields in a biphasic reaction medium using a phase transfer catalyst under simple and ecofriendly conditions. Both the glucosyl curcuminoids showed enhanced antioxidant, tyrosinase enzyme inhibitory, antimicrobial and potent cytotoxic activity. The improved biological activity may be due to the increased solubility of the glucosyl conjugated compounds compared to the native curcuminoids; this was further confirmed by partition coefficient studies. Thus, the synthesized glucosyl curcumin may serve as promising future therapeutic molecule in the management of cancer, whereas glucosyl tetrahydrocurcumin can be a useful ingredient in achromatic food and in cosmetic applications. Keywords: Antioxidant / Cosmetic / Hyperpigmentation / Tetrahydrocurcumin / Tyrosinase Received: May 15, 2014; Revised: July 8, 2014; Accepted: July 9, 2014 DOI 10.1002/ardp.201400195
Introduction Natural compounds derived from plants, animals, and microorganisms proved to be an excellent source for therapeutic agents. Phytochemicals once served the humankind as the source of new drug development, today these natural products or their analogs still represent as preventive and therapeutic agents against a wide range of human diseases [1, 2]. Curcuminoids are naturally occurring hydrophobic polyphenolic compounds isolated from the rhizomes of Curcuma longa Linn. (Zingiberaceae) [3]. Curcumin is a predominant compound, which has attracted a lot of attention due to its wide applications in traditional medications, food coloration, cosmetic utility, and fabric Correspondence: Dr. Adari Bhaskar Rao, Medicinal Chemistry and Pharmacology Division, CSIR – Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 007, AP, India. E-mail: [email protected] Fax: þ91 40 27193189
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
dying industry. In addition, curcuminoids possess antiinflammatory, antioxidant, antiangiogenic, anticancer, and many more therapeutic activities [4, 5]. Tetrahydrocurcumin is one of the colorless hydrogenated metabolites of the curcumin that demonstrates broad physiological and pharmacological activities similar to that of native curcumin [6]. Despite wide range of food processing and pharmacological activities, the major barrier for clinical applications of these curcuminoids is their poor solubility in water, thus limiting the bioavailability and in vivo therapeutic concentration of the compound in the humans. In addition, a relative short gastric emptying time results in an incomplete release of curcuminoids from the dosage and to the site of absorption, leading to a diminished efficacy of the compound [7, 8]. Attempts were made to enhance the bioavailability of curcuminoids through structural modifications of the molecule and/or through new formulations (nanoparticles, adjuvants, nanoparticles and liposomes) but have not gained much attention due to various limitations in their
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min) 3 and a colorless product tetrahydrocurcumin-di-bglucoside (glucosyl-THC) 6 (Scheme 1).
methodologies [9, 10]. It is well known that the glycosylation of hydrophobic phytochemicals has improved the bioavailability and biological activity of the compounds [11, 12]. In the present study, it is planned to synthesize glucosylconjugated curcuminoids and to evaluate their biological activity.
Curcumin-4,4-b-di-glucoside (3) Yellow colored solid, yield 71%. 1H NMR (DMSO-d6): 3.1–3.4 (m, 8H), 3.6 (m, 3H), 3.77 (s, 6H, OCH3), 4.31 (br, s, 1H), 4.5 (t, 2H, J ¼ 5.5Hz), 4.93 (d, 2H, J ¼ 7 Hz), 4.98 (d, 2H, J ¼ 5 Hz), 5.06 (br, s, 2H), 5.25 (d, 2H, J ¼ 4 Hz), 6.04 (s, 1H), 6.81(d, 2H, J ¼ 16 Hz), 7.05 (d, 2H, J ¼ 8.5 Hz), 7.18 (d, 2H, J ¼ 8 Hz), 7.32 (s, 2H), 7.52 (d, 2H, J ¼ 16 Hz); ESI-MS (m/z): 715 (MþNa)þ.
Results and discussion Chemistry Synthesis of glucosyl curcuminoids
Tetrahydrocurcumin-4,4-b-di-glucoside (6)
Synthesis of curcumin-4,4-b-di-D-glucoside(glucosyl-curcumin) 3 and tetrahydrocurcumin-4,4-b-di-glucoside (glucosyl-THC) 6 was achieved in higher yields, in a simple economical process [13]. The glucosyl conjugation reaction was facilitated by a phase transfer catalyst (tetrabutylammonium bromide), that promotes better mass transfer of reactants between two immiscible liquid phases, resulting in high product formation with selectivity. The progress of the reaction was monitored through HPLC and the single product peak was obtained at the end of the optimum reaction period. The structures of the glucosyl-conjugated curcuminoids were confirmed by spectral analysis. From 1H NMR studies, it was confirmed that the configurations of the anomeric carbons were defined as b for all the glucose molecules therefore the conjugated glucosylations of the two curcuminoids show bconfiguration. The SN2 nucleophilic substitution at the anomeric C-1 position of the activated sugar moiety resulted in the formation of curcumin-di-b-glucoside (glucosyl-curcu-
AcO O
O
MeO HO
AcO OMe
Br OAc
KOH TBAB
OAc
45°C/2 h
OH
1
O
Half white colored solid, yield: 64%; m.p. ¼ 184°C, TLC rf ¼ 0.27 (DCM/MeOH, 9.5:0.5), UV (EtOH) lmax 258, 220 nm. IR(KBr): nmax 3453, 3017, 2927, 2854, 1753, 1605, 1514, 1370, 1229, 1037, 757 cm1; 1H NMR (DMSO-d6 and D2O): 6.86–6.59 (m, 6H, –CH2); 5.64–5.50 (m, 2H), 5.16 (t, J ¼ 9.8 Hz, 2H), 4.37– 4.26 (m, 6H), 4.17–4.10 (m, 4H), 3.87 (s, 6H), 2.86–2.70 (m, 8H); 13 C NMR (DMSO): 193.1, 146.3, 143.9, 132.5, 120.7, 114.3, 110.9, 99.7, 79.1, 76.8, 76.1, 72.6, 70.3, 61.9, 55.8, 40.3, 31.2. LC MS (m/z): 719.13 (MþNa)þ. Natural curcuminoids are yellow colored compounds resulting in staining of the skin and dress materials during topical application. Tetrahydrocurcumin is a colorless metabolite of curcumin that exhibits pharmacological and biological activities similar to that other curcuminoids. It is planned to synthesize the conjugated glucosyl-curcumin and glucosyltetrahydrocurcumin compounds to increase hydrophilicity of these molecules and improve their biological activity.
O
2
CH 3 ONa
OMe
O
O OH
O
O
O
MeO HO
AcO OMe
4
O
OH
OH
O
OH OH
AcO
O
MeO
3
OH
OH OH
OH
Br OAc
OAc
45°C/2 h
KOH TBAB
O
5
CH3 ONa
O
MeO
OMe
O
O OH
O
OH OH
OH
OH
O
6
OH OH
OH
Scheme 1. Synthesis of curcumin-4,4-b-di-glucoside 3 and tetrahydrocurcumin-4,4-b-di-glucoside 6.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–6
Figure 1. Partition coefficient (log P o/w) of potent compounds.
Partition coefficient From the results (Fig. 1), it was observed the glucosyl conjugated curcuminoids 3 and 6 show much lower lipophilicity compared to compounds 1 and 4, and thus these glucosyl-curcuminoids were more water soluble and show better bioavailability and pharmacological activities.
Biological results The radical scavenging and tyrosinase inhibition activities The test compounds curcumin 1, glucosyl-curcumin 3, THC 4, and glucosyl-THC 6 show potent antioxidant activity by inhibiting oxidation of the added 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Fig. 2). The results revealed that the glucosyl conjugated curcuminoids show promising antioxidant activity when compared to non-conjugated curcuminoids studied. The IC50 value of glucosyl-curcumin was 20.25 mM and for glucosyl-THC it was 18.25 mM. The improved free radical scavenging activity is maybe due to increased water solubility of glucosylated hydrophilic curcuminoid compounds. The
Glucosyl Curcuminoids
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free radical scavenging activity of the curcuminoids studied may be attributed mainly due to the compound’s pharmacophore, which contains phenolic hydroxyl group and b-diketone structure. The chemoprotective nature of the natural polyphenolic compound curcumin (diferuloylmethane) against oxidative stress-mediated disorders is through the anti-oxidative functioning [14]. Natural products are known to inhibit tyrosinase enzyme activity that participates in melanin formation thereby lightening the skin tone [15]. The natural curcuminoids are of particular interest both in skin medications and in cosmetics as these molecules show a potent tyrosinase inhibition activity. The glucosylated curcumin show enhanced tyrosinase inhibition activity with IC50 52.12 mM for glucosylcurcumin 3, whereas for glucosyl-THC 6 it was IC50: 48.12 mM, nearly comparable to conventional tyrosinase inhibitors.
Antimicrobial activity The curcuminoids 1, 3, 4, and 6 tested show broad antimicrobial and antifungal activity against following bacterial strains Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, and Staphylococcus aureus and against two fungal strains Aspergillus fumigatus and Candida albicans. From the results, it was inferred that both the glucosyl-conjugated curcumin 3 and glucosyl-THC 6 have shown enhanced antimicrobial activity, with lower MIC values when compared to curcumin 1 and THC 4 (Fig. 3). Curcumin has not shown antimicrobial activity against B. subtilis whereas both curcuminoids have no activity against the fungal strain A. fumigatus. The glucosyl-curcumin and glucosyl-THC exhibited potential anti-microbial activity as
Figure 2. IC50 values of curcuminoids on DPPH radical scavenging and tyrosinase inhibition activity.
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Figure 3. Antimicrobial activity of curcumin, THC, glucosyl-THC (MIC mmol/L).
confirmed by low MIC values, this may be due to the increased aqueous solubility of these hydrophilic curcuminoids.
Cytotoxicity The cytotoxicity of the four curcuminoids curcumin 1, THC 4, curcumin-b-di-glucoside 3, and THC-b-di-glucoside 6 were studied in selected human cancer cell lines (HT-29 colon cancer, A549 lung cancer, MCF-7 breast cancer) by MTT assay. From the results, it was observed that all the tested curcuminoids have shown cytotoxic activity on the four cell lines and compounds 4 and 6 exhibited marked activity on colon cancer cell line (HT-29). The IC50 value on colon cancer
cells for curcumin 1 was 66.94 mM, THC 4 it was 77.17 mM whereas for curcumin-b-di-glucoside 3 and for THC-b-diglucoside 6 IC50 values were 41.56 mM and 32.13 mM. Thus, it was confirmed from the results that on glucosidation of the curcuminoids the inhibition of colon cancer cells (HT-29) activity was increased by twofolds when compared with free curcuminoids (Fig. 4). The conjugated curcumin has also showed a noticeable inhibition activity on breast cancer cells (MCF-7), whereas no significant activity was found in liver cancer cells (A549). Curcuminoids possess a wide spectrum of antitumor properties but, due to its poor bioavailability the curcuminoids are not yet been clinically used routinely in
Figure 4. Evaluation of cytotoxicity (IC50 mM) of curcuminoids on different cancer cell lines by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay method.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–6
anticancer chemotherapy [16–18]. Glycosylation of hydrophobic curcuminoids increases solubility of the compound and thus improving the anticancer activity as confirmed by their lower IC50 values. Natural products are known to possess potent anticancer activity and are also used topically against widespread skin diseases and cosmetics. Curcuminoids attracted considerable attention in the recent years due to their broad pharmacological activities. The poor circulating bioavailability of these curcuminoids attributed due to low water solubility thus impairing the compounds biological activity. The synthesized glucosyl-conjugated compounds curcumin-b-di-glucoside 3 and tetrahydrocurcumin-b-di-glucoside 6 have shown potent antioxidant and cytotoxic activities when compared to native curcuminoids. The results obtained clearly substantiate the beneficial effects of glucosyl-curcumin as a potent anticancer agent and colorless glucosyl-THC shows superior antioxidant and inhibit tyrosinase activity thereby helping in lightening the skin tone. Therefore, the conjugated THC acts as useful ingredients in anti-ageing and other cosmetic topical formulations designed to maintain general skin health [19].
Conclusion The study confirms a novel synthesis of curcumin-b-di-glucoside 3 and tetrahydrocurcumin-b-di-glucoside 6 in good yields and selectivity (b-form) in simple and eco-friendly reaction conditions. The results confirm the increased hydrophilicity and biological activity of glucosyl conjugated curcuminoids when compared to their native un-conjugated curcuminoids; this was further confirmed by partition coefficient studies. The data also reveal the compounds glucosyl-curcumin and glucosyl-THC showing enhanced antioxidant, antimicrobial, and tyrosinase inhibition activities, when compared to nonconjugated curcuminoids studied. In summary, the glucosylcurcumin shows potent antioxidant and cytotoxic activity, and thus the molecule may be of therapeutic importance against several types of cancer diseases. Whereas, the non-staining glucosyl-conjugated THC molecule acts as a potent ingredient in achromatic food and in cosmetic applications. Thus, the study concludes both the synthesized conjugated curcuminoids, help in improving the medical value of the natural curcuminoids and hope that future clinical trials will lead to the development of potent preventive and therapeutic agent for skin photo aging and UV induced cancer diseases.
Experimental Materials and methods Chemicals and materials Curcumin and THC were gift from Ashian Herbex Ltd., Hyderabad, India. DPPH, and bovine serum albumin (BSA), ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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L-DOPA, tyrosinase T-3824, 25 KU were procured from Sigma Chemical Co. (St. Louis, MO, USA). The cancer cell lines A549 (lung cancer), MCF-7 (breast cancer) and HT-29 (colon cancer) used were obtained from National Center for Cell Science (NCCS), Pune, India. All the microbial strains and the Mueller Hinton broth, Dulbeccos modified Eagles agar medium (DMEM) and other media components used in these studies were procured from Hi Media Ltd (Mumbai, India). All the reagents and solvents (n-octanol) used in the experiments were of analytical grade and were purchased from Merck (India).
Equipment Spectrophotometric analyses were carried out using UV visible spectrophotometer (Perkin Elmer). 1H and 13C NMR spectra for the products were recorded on a 300, 500 MHz NMR spectrometer (Bruker, Avance, Germany). Mass spectral analyses of compounds were performed using MS (Waters Q-Tof Ultima, Manchester, UK) in the ESI positive mode.
HPLC The concentration of curcumin, THC, glucosyl-curcumin, and glucosyl-THC were quantified through HPLC (Gilson LC), using reverse phase analytical C18 column (150 mm 3.9 mm, 5 mm particle size). The system was eluted isocratically using mobile phase acetonitrile/water (85:15 v/v; pH adjusted to 3.0 with 0.01% acetic acid) at a flow rate of 1 mL/min and the sample detection was at 280 nm. The mobile phase was filtered through 0.5 mm nylon membrane filter and the solvent was degassed ultrasonically before use. Salbutamol (0.2 mg/mL) was added to each test sample as an internal standard. Peak area was used as a measure to calculate the respective concentrations.
Chemistry General procedure for the synthesis of glucosylcurcuminoids The general method used for synthesis of glucosyl conjugated compounds 3 and 6 are summarized in Scheme 1. A modified procedure of Koenings–Knorr was followed in synthesis of glucosyl-conjugated curcuminoids [20, 21]. The reaction was initiated by adding aqueous solution of KOH (10.8 mmol) to 20 mL of DCM solvent containing disallowed compounds 1 or 4 (2.71 mmol), followed by the addition of 2,3,4,6-tetra-O-acetyl-bD-glucopyranosyl bromide (5.41 mmol) and molecular sieves (4 A). The reaction mixture was stirred for 3 h at 45°C under the nitrogen atmosphere. On completion of the reaction, the compounds curcumin-b-di-glucoside tetraacetate 2 or tetrahydrocurcumin-b-di-glucoside tetraacetate 5 were isolated and these compounds were deacetylated by adding 2% sodium methoxide in dichloromethane. The two products formed were isolated and purified through column chromatography silica gel (100–200 mesh). The purity of the final products was determined by HPLC and the structures of the compounds obtained curcumin-b-di-glucoside 3 or tetrahydrocurcumin-b-di-glucoside 6 were confirmed by NMR (1H, 13C), mass and LC–MS studies.
Partition coefficient (log P value) The concentration of curcuminoids present in two-phase noctanol/water system was determined by HPLC [22]. The curcuminoids 50 mM (curcumin, THC, glucosyl-curcumin, and glucosyl-THC) were dissolved in known volumes of n-octanol (2, 4, www.archpharm.com
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6, 8, and 10 mL)/water (O/W ratio) in a shake-flask and incubated at 37°C for 2 h. Lipophilicity of the compound quantified by the logarithm of the 1-octanol/water partition: log P ¼ log[Co/Cw], where Co was the concentration of compound in the octanol phase and Cw its concentration in the aqueous water phase, when the system was at equilibrium. log P values presented were an average of three measurements.
New Delhi for award of fellowship; the authors also thank MD, Ashian Herbix Ltd, Hyderabad for gift sample of curcuminoids.
Biological assays Antioxidant activity by DPPH free radical-scavenging method
[1] N. Civjan, Chemical Biology: Approaches to Drug Discovery and Development to Targeting Disease, John Wiley & Sons, Hoboken 2012. [2] D. A. Dias, S. Urban, U. Roessner, Metabolites 2012, 2, 303–336. [3] L. Shen, H. F. Ji, Trends Mol. Med. 2012, 18, 138–144. [4] M. Labbozzetta, M. Notarbartolo, P. Poma, A. Maurici, L. Inguglia, P. Marchetti, M. Rizzi, R. Baruchello, D. Simoni, N. D’Alessandro, Ann. N. Y. Acad. Sci. 2009, 1155, 278–283. [5] R. L. Thangapazham, S. Sharad, R. K. Maheshwari, Biofactors 2013, 39, 141–149. [6] M. H. Pan, T. M. Huang, J. K. Lin, Drug Metab. Dispos. 1999, 27, 486–494. [7] P. Asawanonda, S. O. Klahan, Photomed. Laser Surg. 2010, 28, 679–684. [8] G. C. Jagetia, G. K. Rajanikant, Int. Wound J. 2012, 9, 76–92. [9] H. Kharkwal, K. Bala, D. D. Joshi, D. P. Katare, Der Pharm. Lett. 2012, 4, 1698–1711. [10] J. R. Manjunatha, B. K. Bettadaiah, P. S. Negi, P. Srinivas, Food Chem. 2013, 139, 332–338. [11] S. H. Thilakarathna, H. P. V. Rupasinghe, Nutrients 2013, 5, 3367–3387. [12] C. Manach, et al., Am. J. Clin. Nutr. 2004, 79, 727–747. [13] A. Bhaskar Rao, E. Prasad, S. S. Deepthi, S. Ramakrishna, Y. S. Venkat Rao, Indian Patent No. C0123NF201, 2013. [14] D. Huang, B. Ou, R. L. Prior, J. Agric. Food Chem. 2005, 53, 1841. [15] Z. M. Hu, Q. Zhou, T. C. Lei, S. F. Ding, S. Z. Xu, J. Dermatol. Sci. 2009, 55, 179–184. [16] R. A. Sharma, et al., Cancer Res. 2001, 7, 1894–1900. [17] H. Chen, Z. S. Zhang, Y. L. Zhang, D. Y. Zhou, Anticancer Res. 1999, 19, 3675–3680. [18] M. Lopez-Lazro, Mol. Nutr. Food Res. 2008, 52, S103–S127. [19] M. Majeed, V. Badmaev, U. Shivakumar, R. Rajendran, Curcuminoids: Antioxidant Phytonutrients, Nutriscience Publishers, New Jersey 1995. [20] Z. Wimmer, L. Pechova, D. Saman, Molecules 2004, 9, 902–912. [21] K. S. Parvathy, P. S. Negi, P. Srinivas, Food Chem. 2009, 115, 265–271. [22] S. K. Poole, C. F. Poole, J. Chromatogr. B 2003, 797, 3–19. [23] P. Ronald,L X. Wu, K. Scaich, J. Agric. Food Chem. 2005, 53, 4290–4302. [24] M. Li, Y. Ma, M. O. Ngadi, Food Chem. 2013, 141, 1504–1511. [25] E. W. C. Chan, et al., Food Chem. 2008, 109, 477–483. [26] Y. Jiang, et al., Molecules 2013, 18, 3948–3961. [27] P. Magiatis, A. L. Skaltsonmis, I. Chinou, S. A. Haroutounian, Z. Naturforsch. 2002, 57, 287–290. [28] M. Ferrari, M. C. Fornasiero, A. M. Isetta, J. Immunol. Methods 1990, 131, 165–172.
The hydrogen donating or free radical-scavenging activity of the curcumin 1, glucosyl-curcumin 3, THC 4, and glucosyl-THC 6 was evaluated by the reduction of DPPH as described in the literature [23]. Trolox (0.1 mg/mL) was used as a standard antioxidant agent [24]. The experiments were performed in triplicates for each concentration and IC50 values (scavenge 50% of the DPPH radicals) were determined by the interpolation of the dose.
Tyrosinase enzyme inhibition assay The tyrosinase enzyme inhibition assay was performed using LDOPA as the substrate as reported, with slight modifications [25, 26]. Kojic acid was used as the standard/reference inhibitor compound to study tyrosinase enzyme activity. All the analyses were performed in triplicates for each concentration and the IC50 values of tyrosinase inhibition activity were determined.
Antimicrobial assay The antimicrobial activities of the curcumin 1, glucosyl-curcumin 3, THC 4, and glucosyl-THC 6 were carried out using the dilution technique and minimum inhibitory concentrations (MIC) of the compounds were determined. The following microbial strains S. aureus (ATCC 29737), B. cereus ATCC 14603, E. coli (ATCC 10536), K. pneumoniae (ATCC 13883), and P. aeruginosa (ATCC 25619), and the fungi C. albicans (ATCC 53324) and A. fumigatus (ATCC 204305) were evaluated for antimicrobial activity. Standard antibiotics gentamycin sulfate and fluconazole were used in the study for comparison of antimicrobial activity of glucosyl-conjugated curcuminoids [27].
In vitro cytotoxicity study The cytotoxicity activity for the test compounds curcumin 1, glucosyl-curcumin 3, THC 4, and glucosyl-tetrahydrocurcumin 6, against human cancer cells were determined by MTT (3-(4,5dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay. The cell lines used in the study were MCF-7 breast cancer cells, HT-29 colon cancer cells, and A549 lung cancer cells. The study was initiated by plating 104 cells per well in 96-well plates, and addition of DNR solutions with or without P85 (0.01%) to the cells (100 mL/well). Cells were incubated with test compounds dissolved in DMSO, for 4 days at 37°C and 5% carbon dioxide. After the incubation period, the compound’s cytotoxic activity was evaluated using the MTT assay at 570 nm [28]. All experimental points were carried out in triplicate and IC50 values of the test compounds were determined.
The authors express sincere thanks to DST, New Delhi, India for financial support for this project SR/SO/BB-54/2008; E.P. thanks DST, ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The authors have declared no conflict of interest.
References
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