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Synthesis, characterization and antioxidant activity of chitosan-chromone derivatives
Carbohydrate Polymers 181 (2018) 812–817
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Synthesis, characterization and antioxidant activity of chitosan-chromone derivatives
T
⁎
Cahit Demetgül , Neslihan Beyazit Mustafa Kemal University, Chemistry Department, Faculty of Arts and Sciences, 31060, Hatay, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords: Chitosan derivatives Chromone Terephthalaldehyde Antioxidant activity
In this study, a new chromone-functionalized chitosan Schiff base and its cross-linked derivative were synthesized and characterized by FT-IR, UV–vis, 13C CP/MAS solid-state NMR, TGA, XRD-powder and SEM measurements and elemental analysis data. Degrees of substitution (DS) were determined from the elemental analysis by using the C/N ratios. The in vitro antioxidant activity of high molecular chitosan and its chromone derivatives was evaluated as radical scavengers against 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH%). The results showed that both of the chitosan-chromone derivatives have good antioxidant potential which might be due to the phenolic group introduced after chemical modification of chitosan with a chromone derivative. Chromone-chitosan Schiff base (CSCH) had a better ability to scavenging DPPH radical (IC50, 0.88 mg/mL) than that of its cross-linked derivative (CSCH-TP) obtained by using terephthalaldehyde (IC50, 1.32 mg/mL).
1. Introduction
fungal (Grindlay and Reynolds, 1986; Jovanovic, Steenken, Tosic, Marjanovic, & Simic, 1994), anti-cancer (Martens and Mithöfer, 2005), anti-HIV (Zhou, Shi, & Lee, 2010), anti-ulcers (Parmer, Tariq, & Ageel, 1987), antiinflammatory (Gabor, 1986), wound healing (Maho & Yoshiyuki, 2010), and anti-oxidant (Kuroda, Uchida, Watanabe, & Mimaki, 2009). It was reported that the chemical modification of chitosan molecule with a chromone derivative led to an enhancement in the biological activities such as antimicrobial (Kumar & Koh, 2012), fungicidal and insecticidal (El Badawy, 2008). But there is no any previous report on the antioxidant activity of a chitosan-chromone derivative. In this study, a new chitosan-chromone Schiff base (CSCH) and its crosslinked derivative (CSCH-TP) were synthesized by the condensation reaction of 6-formyl-7-hydroxy-5-methoxy-2-methylbenzopyran-4-one (CH) with chitosan (CS) and terephthalaldehyde (TP) was used as a crosslinking agent. Their structural characterization was carried out by means of UV–vis, FT-IR, 13C NMR spectroscopy, XRD Diffraction, TGA, SEM imaging techniques and elemental analyses. Furthermore, the antioxidant activities of the obtained materials were evaluated by using DPPH scavenging assay.
Chitosan (CS) is the second-most abundant biopolymer derived from the N-deacetylation of chitin. The design and synthesis of chitosanbased materials has been attracted by researcher groups due to their unique biological and physicochemical properties such as biocompatibility, biodegradability and film-forming ability. Various novel chitosan derivatives have been obtained by chemical modifications of reactive hydroxyl and amino/acetamido groups attached to the macromolecule backbone (Kumar, 2000; Jiao et al., 2011; Muzzarelli, 1977). For instance, Schiff bases that can be synthesized by condensation of amino groups of chitosan with aldehydes or ketones have been received extensive interests for their expanded biofunctional properties such as antibacterial, antitumor and antioxidant activities (Elshaarawy, Refaee, & El-Sawi, 2016; Tamer et al., 2017). Chitosan and especially self-assembled chitosan materials have therefore become of great interest as new functional biomaterials with significant potential in numerous fields (Guo et al., 2015; Jiao et al., 2015; Zhao et al., 2015). In numerous studies, chitosan was reported as a potential antioxidant agent (Kim & Thomas, 2007; Vinsova & Vavrikova, 2011; Kaya et al., 2014). Natural compounds isolated from plants possess biological activities can be used to improve the antioxidant effect of chitosan (Pasanphan & Chirachanchai, 2008). Chromone and its derivatives are an important class of heterocycles, mainly present in natural products and exhibit a wide range of pharmacological properties such as anti-bacterial, anti-
⁎
2. Materials and methods 2.1. Materials Chitosan with high molecular weight (> 75% deactylated) was
Corresponding author. E-mail address: [email protected] (C. Demetgül).
https://doi.org/10.1016/j.carbpol.2017.11.074 Received 2 October 2017; Received in revised form 15 November 2017; Accepted 20 November 2017 Available online 24 November 2017 0144-8617/ © 2017 Elsevier Ltd. All rights reserved.
Carbohydrate Polymers 181 (2018) 812–817
C. Demetgül, N. Beyazit
summarized in Scheme 1.
purchased from Sigma-Aldrich. Glacial acetic acid, terephthalaldehyde (TP), methanol, ethanol and the organic solvents were purchased from Merck Co. The anti-oxidant reagent 1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich. All reagents were of analytical grade and were as received without further purification. Elemental analyses (C, H and N) were performed using a Thermo Scientific Flash 2000 CHNS/O Analyzer. The FT-IR and UV–vis spectra were recorded on a Perkin Elmer Spectrum Two with U-ATR FTIR spectrometer and on a OPTIZENα UV–vis Spectrometer, respectively. 0.02 g of sample was dissolved in 25 mL of acetic acid 1%. The electronic absorbance of samples in a quartz cell scanned from 230 to 500 nm was recorded. 13C NMR spectra were obtained by a Bruker Superconducting FT/NMR Spectrometer Avance TM 300 MHz WB with CP/MAS technique (cross-polarization, magic-angle-spinning). The Xray diffraction patterns of samples were recorded at room temperature on a using a Rigaku System RadB X-Ray Diffractometer, using monochromated Cu Kα radiation in the range 2–40° (2θ), at 25 °C. The surface morphology of chitosan and derivatives were examined by using a JEOL JSM 5500 scanning electron microscope (SEM) at 10 kV. The thermogravimetry analyses (TGA) were carried out in nitrogen atmosphere using METTLER TOLEDO, heated from 30 °C to 1000 °C at a heating rate of 10 °C min−1.
2.3. Antioxidant activity 2.3.1. DPPH scavenging activity CSCH or CSCH-TP (2 mL in 1.0% (v/v) glacial acetic acid solution) was added to a methanolic solution of DPPH (2.0 × 10−4 M, 2.0 mL) and 5 mL of methanol. The mixture was shaken for 10 s and left to stand at room temperature for 30 min. The scavenging activity of CSCH or CSCH-TP on DPPH% was determined by the absorbance at 517 nm. The percentage of scavenging activity was calculated by using the following Eq. (1): % scavenging activity = (Abscontrol−Abssample)/Abscontrol × 100
(1)
where Abscontrol is the absorbance of the control (without the test sample) and Abssample is the absorbance of the sample (with the test sample). 3. Results and discussion 3.1. Elemental analysis The elemental analysis data of chitosan and its chromone derivatives (CSCH and CSCH-TP) are listed in Table 1. Carbon to nitrogen (C/ N) ratios of CS, CSCH and CSCH-TP were found to be 5.57, 8.41 and 10.62%, respectively. The increase in the C/N ratios of chitosan-chromone derivatives can be explained by the chemical modification of chitosan with a nitrogen-free chromone analog. From Table 1, the degree of substitution (DS) of the chitosan derivatives to −NH2 group on chitosan was calculated by following Eq. (2), modified with base in the model of Inukai et al. (Inukai, Chinen, Matsuda, Kaida, & Yasuda, 1998):
2.2. Preparation of chitosan-chromone derivatives, CSCH and CSCH-TP The chromone derivative (6-formyl-7-hydroxy-5-methoxy-2-methylbenzopyran-4-one, CH) was obtained by the oxidation of visnagin, according to the procedure described before by our group (Beyazit, Çatıkkaş, Bayraktar, & Demetgöl, 2016; Gönaydın & Beyazit, 2004). For the preparation of CSCH, 0.5 g of powder chitosan was dissolved in 25 mL of 1.0% (v/v) glacial acetic acid. The mixture was vigorously stirred under the reflux condition until the chitosan was completely dissolved. 0.2 g of chromone derivative in 50 mL methanol solution was added drop wise into the chitosan solution and the mixture was stirred under reflux condition for 4–5 h. The yellow color product was filtered off, washed with ethanol and dried in vacuo. For the preparation of CSCH-TP, 0.3 g of CSCH was dissolved in 40 mL of 1.0% (v/v) glacial acetic acid and 0.3 g of terephthalaldehyde (TP) in 25 mL ethanol solution were added drop wise into the CSCH solution. The mixture was stirred under reflux condition for 4–5 h. The yellow color product was filtered off, washed with ethanol and dried in vacuo. The synthesis route of chitosan-chromone derivatives are
DS =
(C / N ) m − (C / N ) o n
(2)
Where (C/N)m is the C/N of the chitosan derivative, (C/N)o is the C/N of the original chitosan, n is the number of carbon introduced after chitosan modification. The DS values obtained were about 0.33 and 0.50 for CSCH and CSCH-TP, respectively. 3.2. UV–vis spectra The electronic spectra of chitosan and its derivatives were measured
Scheme 1. The synthesis of chitosan-chromone Schiff base (CSCH) and its cross-linked derivative (CSCH-TP).
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results suggest that chitosan-chromone derivatives have less crystalline and more amorphous structure than the pure chitosan. Also the XRD spectrum of cross-linked chitosan derivative (CSHCH-TP) showed a weaker and broader crystalline peak as compare to that of CSCH. It is known that the crystalline structure of chitosan is mainly stabilized by intermolecular and intramolecular hydrogen bonding (Wang, Du, Fan, Liu, & Hu, 2005). The intramolecular hydrogen bonding of chitosan is formed between amino group at C-2 and hydroxyl group at C-3. The decreasing of crystallinity can be explained by deformation of some of the inter-molecular and intra-molecular hydrogen bonding because of the steric hindrance after Schiff base formation (Franca, Lins, Freitas, & Straatsma, 2008; Wan, Creber, Peppley, & Bui, 2004).
Table 1 Elemental analyses results of CS, CSCH and CSCH-TP. Compound
Element (%)
Chitosan (CS) CSCH CSCH-TP
C
H
N
C/N
45.34 50.66 55.22
7.03 6.10 5.68
8.23 6.01 5.20
5.57 8.41 10.62
in the range of 230–500 nm as shown in Fig. S1. It is well known that a maximum absorption band was observed at 235 nm corresponding to the n-π* transition and there is not any transition observed between 300 and 800 nm in the electronic spectrum of chitosan (Baran & Menteş, 2016; Tamer et al., 2016). On the other hand in the electronic spectra of CSCH and CSCH-TP, two new significant bands were observed at 260 and 305 nm assigned to π-π* transition of aromatic rings and n-π* transition of azomethine groups, respectively. The results revealed that a condensation reaction occurred between chitosan and chromone derivative to form an imine bond.
3.5. Thermal analysis (TGA) The thermal gravimetric analysis (TGA) of chitosan was previously reported in the literature. Chitosan exhibits two stage thermal decomposition process: the first one (T < 100 °C) is commonly associated with the loss of physisorbed water molecules, whereas the second stage occuring at ca. 300 °C corresponds to the decomposition of chitosan backbone (Corazzari et al., 2015. According to the TGA thermograms (Fig. S5 and S6), the main decomposition process of CSCH and CSCH-TP takes place at ca. 230 °C, lower than the decomposition stage of original chitosan backbone. The results suggested that chitosan derivatives are less thermally stable than the chitosan. The thermal instability of chitosan-chromone derivatives might be due to the substitution of free primary amino groups with chromone rings which is breaking the interand intra-molecular hydrogen bonding and lowering the crystallinity of chitosan (Demetgül, 2012).
3.3. FT-IR spectra FT-IR spectra of pure chitosan and its derivatives are illustrated in Fig. S2, S3 and S4. The spectrum of chitosan shows basic characteristic peaks at 3355 cm−1 (overlapped stretching vibrations between OeH and NeH groups), 2920 and 2871 cm−1 (CeH stretch), 1640 and 1589cm−1 (amide II band due to CeO and NeH stretching, respectively), 1063 and 1026 cm−1 (skeletal vibration involving CeO stretch) (Demetgül & Serin, 2008; Kumar, Koh, Kim, Gupta, & Dutta, 2012). In the FT-IR spectra of CSCH and CSCH-TP, the peak at 3355 cm−1 shifted to 3365 and 3381 cm−1, respectively and broadened due to the OeH absorption of chromone ring. The new peaks were observed in the FT-IR spectra of chitosan-chromone derivatives. The peaks observed at 3071 and 3087 cm−1 was attributed to the aromatic CeH stretch for CSCH and CSCH-TP, respectively. Furthermore the strong new peaks were also observed at 1630 and 1639 cm−1 assigned to the characteristic absorbance of azomethine (C]N) group, confirming the formation of Schiff base.
3.6.
13
C NMR analysis
It was previously reported that the typical 13C CP-MAS solid-state NMR chemical shift values of pure chitosan were observed at 23 ppm (eCH3e), 58 ppm (C2), 61 ppm (C6), 76 ppm (C5,C3), 85 ppm (C4), 106 ppm (C1), 174 ppm (C]O) [Carbohydrate Polymers Volume 173, 1 October 2017, Pages 714–720]. As compared with chitosan, new peaks were observed at 20 ppm (chromone eCH3e), 103 and 110 ppm (overlapped signals between C-1 of chitosan and chromone ring carbons), 165 ppm (overlapped signals between azomethine carbons and chromone carbons adjacent to oxygen atoms) in the spectrum of CSCH and CSCH-TP (Figs. 2 and 3). The intensity of the signal at 174 ppm was strengthened which was ascribed to carbonyl carbons of both chitosan and chromone scaffolds. When compared to CSCH, the spectrum of CSCH-TP exhibits new peaks at 129 and 137 ppm attributed to the aromatic carbons of terephthalaldehyde, confirming the formation of crosslinked Chitosan derivative.
3.4. X-Ray diffraction (XRD) study The XRD spectra of pure chitosan (CS), CSCH and CSCH-TP are given in Fig. 1. X-Ray Diffraction study of CS exhibits two sharp crystalline peaks at 2θ = 10° and 2θ = 20° in agreement with the previous reports (Baran, Açıksöz, & Menteş, 2016). In the XRD patterns of CSCH and CSCH-TP, the intensity of peaks decreased and broadening of the peaks was observed when compared with the free chitosan. These
3.7. Scanning electron microscopy (SEM) The SEM images of chitosan and chitosan-chromone derivatives (CSCH and CSCH-TP) are represented in Fig. 4. The difference of structural morphology between chitosan and its chromone derivatives was also supported by their SEM images. While the pure chitosan had a nonporous and smooth membranous phase surface, the SEM image of CSCH showed a polymorphic porous structure. On the other hand, crosslinked chitosan derivative, CSCH-TP, exhibited a more regular smooth surface compared to the surface of CSCH. 3.8. DPPH radical scavenging activity Fig. 5 shows the scavenging activity of chitosan and its chromone derivatives for the DPPH radical. The scavenging activity of chitosanchromone derivatives was enhanced compared with that of original chitosan. The IC50 value for CSCH was 0.88 mg/mL, which was slightly higher than the 1.32 mg/mL for CSCH-TP.
Fig. 1. XRD Diffractograms of chitosan and its derivatives.
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C. Demetgül, N. Beyazit Fig. 2.
13
C CP-MAS NMR Spectrum of CSCH.
from the plants (Sousa, Guebitz, & Kokol, 2009). Chromone and its derivatives are an important class of oxygencontaining natural heterocyclic compounds and they have been known as antioxidant agents (Keri, Budagumpi, Pai, & Balakrishna, 2014). In the literature, there are few studies in which chitosan was modified with a chromone derivative (Huang et al., 2013; El Badawy, 2008; Kumar and Koh, 2012). As far as we know, the antioxidant activity of a
In numerous studies, original chitosan was investigated as a potential antioxidant agent. The antioxidant activity of chitosan has been associated with its strong hydrogen-donating ability and it was reported that a low molecular weight and a higher concentration have a positive effect on the activity (Vinsova & Vavrikova, 2011). There have been many studies which intended to improve its biological activities by chemical modification of chitosan with natural compounds obtained
Fig. 3.
815
13
C CP-MAS NMR Spectrum of CSCH-TP.
Carbohydrate Polymers 181 (2018) 812–817
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Fig. 4. SEM images of CS (a), CSCH (b) and CSCH-TP (c).
enhancement in the antioxidant activity was observed after the functionalization of chitosan with a chromone which is an important scaffold in medicinal chemistry. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.2017.11.074. References Baran, T., & Menteş, A. (2016). Polymeric material prepared from Schiff base based on Ocarboxymethyl chitosan and its Cu(II) and Pd(II) complexes. Journal of Molecular Structure, 1115, 220–227. Baran, T., Açıksöz, E., & Menteş, A. (2016). Highly efficient, quick and green synthesis of biarlys with chitosan supported catalyst using microwave irradiation in the absence of solvent. Carbohydrate Polymers, 142, 189–198. Beyazit, N., Çatıkkaş, B., Bayraktar, Ş., & Demetgül, C. (2016). Synthesis, characterization and catecholase-like activity of new Schiff base metal complexes derived from visnagin: theoretical and experimental study. Journal of Molecular Structure, 1119, 124–132. Corazzari, I., Nistico, R., Turci, F., Faga, M. G., Franzoso, F., Tabasso, S., et al. (2015). Advanced physico-chemical characterization of chitosan by means of TGA coupled on-line with FTIR and GCMS: Thermal degradation and water adsorption capacity. Polymer Degradation and Stability, 112, 1–9. Demetgül, C., & Serin, S. (2008). Synthesis and characterization of a new vic-dioxime derivative of chitosan and its transition metal complexes. Carbohydrate Polymers, 72, 506–512. Demetgül, C. (2012). Synthesis of the ketimine of chitosan and 4, 6-diacetylresorcinol, and study of the catalaselike activity of its copper chelate. Carbohydrate Polymers, 89, 354–361. El Badawy, M. (2008). Chemical modification of chitosan: synthesis and biological activity of new heterocyclic chitosan derivatives. Polymer International, 57, 254–261. Elshaarawy, R. F. M., Refaee, A. A., & El-Sawi, E. A. (2016). Pharmacological performance of novel poly-(ionic liquid)-grafted chitosan-N-salicylidene Schiff bases and their complexes. Carbohydrate Polymers, 146, 376–387. Franca, E. F., Lins, R. D., Freitas, L. C. G., & Straatsma, T. P. (2008). Characterization of chitin and chitosan molecular structure in aqueous solution. Journal of Chemical Theory and Computation, 4(12), 2141–2149. Günaydın, K., & Beyazit, N. (2004). The chemical investigations on the ripe fruits of Ammi visnaga (Lam.) lamarck growing in Turkey. Natural Product Research, 18(2), 169–175. Gabor, M. (1986). Anti-inflammatory and anti-allergic properties of flavonoids. Progress in Clinical and Biological Research, 213, 471–480. Grindlay, D., & Reynolds, T. (1986). The aloe vera phenomenon: A review of the properties and modern uses of the leaf parenchyma gel. Journal of Ethnopharmacology, 16, 117–151.
Fig. 5. DPPH radical scavenging activity of chitosan and its chromone derivatives.
chitosan-chromone derivative has never been investigated before. In our study, DPPH radical scavenging activity assays showed that a considerable amount of increase in the antioxidant activity was observed for chitosan-chromone derivatives (CSCH and CSCH-TP) as compared with chitosan alone. This might be due to the phenolic group of chromone ring condensed with chitosan to afford the corresponding Schiff base. However, after the crosslinking with terephthalaldehyde, the activity was decreased slightly which might be related to the reduced active amino group content of chitosan backbone. 4. Conclusion In the present work, a new chemically modified chitosan with a chromone ring and its cross-linked derivative were synthesized and characterized by elemental analysis data and various spectroscopic methods. SEM, TGA and XRD studies demonstrated that chitosanchromone derivatives (CSCH and CSCH-TP) have polymorphic, porous surface, low thermal stability and low cristallinity as compared with original chitosan. Chitosan alone has been reported to have antioxidant activity and in order to improve its free radical scavenging activity, it has been modified in several ways. In this study, the effect of chitosan modification with chromone moiety on its antioxidant activity was evaluated by using the DPPH radical scavenging assay. As a result, an 816
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Eranthis cilicica and their antioxidant activity. Phytochemistry, 70, 288–293. Maho, S., & Yoshiyuki, K. (2010). Enhancing effects of a chromone glycoside, eucryphin: isolated from Astilbe rhizomes on burn wound repair and its mechanism. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology, 17, 820–829. Martens, S., & Mithöfer, A. (2005). Flavones and flavone synthases. Phytochemistry, 66, 2399–2407. Muzzarelli, R. A. A. (1977). Chitin. Oxford: Pergamon Press83–252. Parmer, N. S., Tariq, M., & Ageel, A. M. (1987). Effect of thromboxane A2 and leukotriene C4 inhibitors on the experimentally induced gastric lesions in the rat. Research Communications in Chemical Pathology and Pharmacology, 58, 15–25. Pasanphan, W., & Chirachanchai, S. (2008). Conjugation of gallic acid onto chitosan: An approach for green and water-based antioxidant. Carbohydrate Polymers, 72(1), 169–177. Sousa, F., Guebitz, G. M., & Kokol, V. (2009). Antimicrobial and antioxidant properties of chitosan enzymatically functionalized with flavonoids. Process Biochemistry, 44(7), 749–756. Tamer, T. M., Hassan, M. A., Omer, A. M., Baset, W. M. A., Hassan, M. E., El-Shafeey, M. E. A., et al. (2016). Synthesis, characterization and antimicrobial evaluation of two aromatic chitosan Schiff base derivatives. Process Biochemistry, 51(10), 1721–1730. Tamer, T. M., Hassan, M. A., Omer, A. M., Valachova, K., Eldin, M. S. M., Collins, M. N., et al. (2017). Antibacterial and antioxidative activity of O-amine functionalized chitosan. Carbohydrate Polymers, 169, 441–450. Vinsova, J., & Vavrikova, E. (2011). Chitosan derivatives with antimicrobial: antitumor and antioxidant activities −A review. Current Pharmaceutical Design, 17, 3596–3607. Wan, Y., Creber, K. A. M., Peppley, B., & Bui, V. T. (2004). Ionic conductivity and tensile properties of hydroxyethyl and hydroxypropyl chitosan membranes. Polymer Physics, 42(8), 1379–1397. Wang, X., Du, Y., Fan, L., Liu, H., & Hu, Y. (2005). Chitosan- metal complexes as antimicrobial agent: synthesis, characterization and Structure-activity study. Polymer Bulletin, 55, 105–113. Zhao, H., Jiao, T., Zhang, L., Zhou, X., Zhang, Q., Peng, Q., et al. (2015). Preparation and adsorption capacity evaluation of graphene oxide-chitosan composite hydrogels. Science China Materials, 58, 811–818. Zhou, T., Shi, Q., & Lee, K. H. (2010). Anti-AIDS agents 83 Efficient microwave-assistedone- pot preparation of angular 2,2-dimethyl-2H-chromone containing compounds. Tetrahedron Letters, 51, 4382–4386.
Guo, H., Jiao, T., Zhang, Q., Guo, W., Peng, Q., & Yan, X. (2015). Preparation of graphene oxide-based hydrogels as efficient dye adsorbents for wastewater treatment. Nanoscale Research Letters, 10(272), 1–10. Huang, W., Wang, Y., Zhang, S., Huang, L., Hua, D., & Zhu, X. (2013). A facile approach for controlled modification of chitosan under γ-Ray irradiation for drug delivery. Macromolecules, 46(3), 814–818. Inukai, Y., Chinen, T., Matsuda, T., Kaida, Y., & Yasuda, S. (1998). Selective separation of germanium (IV) by 2,3-dihydroxypropyl chitosan resin. Analytica Chimica Acta, 371, 187–193. Jiao, T. F., Zhou, J., Zhou, J. X., Gao, L. H., Xing, Y. Y., & Li, X. H. (2011). Synthesis and characterization of chitosan-based Schiff base compounds with aromatic substituent groups. Iranian Polymer Journal, 20(2), 123–136. Jiao, T., Zhao, H., Zhou, J., Zhang, Q., Luo, X., Hu, J., et al. (2015). Self-assembly reduced graphene oxide nanosheet hydrogel fabrication by anchorage of chitosan/silver and its potential efficient application toward dye degradation for wastewater treatments. ACS Sustainable Chemistry & Engineering, 3(12), 3130–3139. Jovanovic, S. V., Steenken, S., Tosic, M., Marjanovic, B., & Simic, M. G. (1994). Flavonoids as antioxidants. Journal of American Chemical Society, 116, 4846–4851. Kaya, M., Çakmak, Y. S., Baran, T., Asan-Özüsağlam, M., Menteş, A., & Tozak, K. O. (2014). New chitin, chitosan, and O-carboxymethyl chitosan sources from resting eggs of Daphnia longispina (Crustacea); with physicochemical characterization, and antimicrobial and antioxidant activities. Biotechnology and Bioprocess Engineering, 19(1), 58–69. Keri, R. S., Budagumpi, S., Pai, R. K., & Balakrishna, R. G. (2014). Chromones as a privileged scaffold in drug discovery: A review. European Journal of Medicinal Chemistry, 78, 340–374. Kim, K. W., & Thomas, R. L. (2007). Antioxidant activity of chitosan with varying molecular weight. Food Chemistry, 101(1), 308–313. Kumar, S., & Koh, J. (2012). Physicochemical: Optical and biological activity of chitosanchromone derivative for biomedical applications. International Journal of Molecular Sciences, 13, 6102–6116. Kumar, S., Koh, J., Kim, H., Gupta, M. K., & Dutta, P. K. (2012). A new chitosan-thymine conjugate: synthesis: Characterization and biological activity. International Journal of Biological Macromolecules, 50, 493–502. Kumar, M. N. V. R. (2000). A review of chitin and chitosan applications. Reactive and Functional Polymers, 46(1), 1–27. Kuroda, M., Uchida, S., Watanabe, K., & Mimaki, K. (2009). Chromones from the tubers of
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Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Synthesis, characterization and antioxidant activity of chitosan-chromone derivatives
T
⁎
Cahit Demetgül , Neslihan Beyazit Mustafa Kemal University, Chemistry Department, Faculty of Arts and Sciences, 31060, Hatay, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords: Chitosan derivatives Chromone Terephthalaldehyde Antioxidant activity
In this study, a new chromone-functionalized chitosan Schiff base and its cross-linked derivative were synthesized and characterized by FT-IR, UV–vis, 13C CP/MAS solid-state NMR, TGA, XRD-powder and SEM measurements and elemental analysis data. Degrees of substitution (DS) were determined from the elemental analysis by using the C/N ratios. The in vitro antioxidant activity of high molecular chitosan and its chromone derivatives was evaluated as radical scavengers against 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH%). The results showed that both of the chitosan-chromone derivatives have good antioxidant potential which might be due to the phenolic group introduced after chemical modification of chitosan with a chromone derivative. Chromone-chitosan Schiff base (CSCH) had a better ability to scavenging DPPH radical (IC50, 0.88 mg/mL) than that of its cross-linked derivative (CSCH-TP) obtained by using terephthalaldehyde (IC50, 1.32 mg/mL).
1. Introduction
fungal (Grindlay and Reynolds, 1986; Jovanovic, Steenken, Tosic, Marjanovic, & Simic, 1994), anti-cancer (Martens and Mithöfer, 2005), anti-HIV (Zhou, Shi, & Lee, 2010), anti-ulcers (Parmer, Tariq, & Ageel, 1987), antiinflammatory (Gabor, 1986), wound healing (Maho & Yoshiyuki, 2010), and anti-oxidant (Kuroda, Uchida, Watanabe, & Mimaki, 2009). It was reported that the chemical modification of chitosan molecule with a chromone derivative led to an enhancement in the biological activities such as antimicrobial (Kumar & Koh, 2012), fungicidal and insecticidal (El Badawy, 2008). But there is no any previous report on the antioxidant activity of a chitosan-chromone derivative. In this study, a new chitosan-chromone Schiff base (CSCH) and its crosslinked derivative (CSCH-TP) were synthesized by the condensation reaction of 6-formyl-7-hydroxy-5-methoxy-2-methylbenzopyran-4-one (CH) with chitosan (CS) and terephthalaldehyde (TP) was used as a crosslinking agent. Their structural characterization was carried out by means of UV–vis, FT-IR, 13C NMR spectroscopy, XRD Diffraction, TGA, SEM imaging techniques and elemental analyses. Furthermore, the antioxidant activities of the obtained materials were evaluated by using DPPH scavenging assay.
Chitosan (CS) is the second-most abundant biopolymer derived from the N-deacetylation of chitin. The design and synthesis of chitosanbased materials has been attracted by researcher groups due to their unique biological and physicochemical properties such as biocompatibility, biodegradability and film-forming ability. Various novel chitosan derivatives have been obtained by chemical modifications of reactive hydroxyl and amino/acetamido groups attached to the macromolecule backbone (Kumar, 2000; Jiao et al., 2011; Muzzarelli, 1977). For instance, Schiff bases that can be synthesized by condensation of amino groups of chitosan with aldehydes or ketones have been received extensive interests for their expanded biofunctional properties such as antibacterial, antitumor and antioxidant activities (Elshaarawy, Refaee, & El-Sawi, 2016; Tamer et al., 2017). Chitosan and especially self-assembled chitosan materials have therefore become of great interest as new functional biomaterials with significant potential in numerous fields (Guo et al., 2015; Jiao et al., 2015; Zhao et al., 2015). In numerous studies, chitosan was reported as a potential antioxidant agent (Kim & Thomas, 2007; Vinsova & Vavrikova, 2011; Kaya et al., 2014). Natural compounds isolated from plants possess biological activities can be used to improve the antioxidant effect of chitosan (Pasanphan & Chirachanchai, 2008). Chromone and its derivatives are an important class of heterocycles, mainly present in natural products and exhibit a wide range of pharmacological properties such as anti-bacterial, anti-
⁎
2. Materials and methods 2.1. Materials Chitosan with high molecular weight (> 75% deactylated) was
Corresponding author. E-mail address: [email protected] (C. Demetgül).
https://doi.org/10.1016/j.carbpol.2017.11.074 Received 2 October 2017; Received in revised form 15 November 2017; Accepted 20 November 2017 Available online 24 November 2017 0144-8617/ © 2017 Elsevier Ltd. All rights reserved.
Carbohydrate Polymers 181 (2018) 812–817
C. Demetgül, N. Beyazit
summarized in Scheme 1.
purchased from Sigma-Aldrich. Glacial acetic acid, terephthalaldehyde (TP), methanol, ethanol and the organic solvents were purchased from Merck Co. The anti-oxidant reagent 1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich. All reagents were of analytical grade and were as received without further purification. Elemental analyses (C, H and N) were performed using a Thermo Scientific Flash 2000 CHNS/O Analyzer. The FT-IR and UV–vis spectra were recorded on a Perkin Elmer Spectrum Two with U-ATR FTIR spectrometer and on a OPTIZENα UV–vis Spectrometer, respectively. 0.02 g of sample was dissolved in 25 mL of acetic acid 1%. The electronic absorbance of samples in a quartz cell scanned from 230 to 500 nm was recorded. 13C NMR spectra were obtained by a Bruker Superconducting FT/NMR Spectrometer Avance TM 300 MHz WB with CP/MAS technique (cross-polarization, magic-angle-spinning). The Xray diffraction patterns of samples were recorded at room temperature on a using a Rigaku System RadB X-Ray Diffractometer, using monochromated Cu Kα radiation in the range 2–40° (2θ), at 25 °C. The surface morphology of chitosan and derivatives were examined by using a JEOL JSM 5500 scanning electron microscope (SEM) at 10 kV. The thermogravimetry analyses (TGA) were carried out in nitrogen atmosphere using METTLER TOLEDO, heated from 30 °C to 1000 °C at a heating rate of 10 °C min−1.
2.3. Antioxidant activity 2.3.1. DPPH scavenging activity CSCH or CSCH-TP (2 mL in 1.0% (v/v) glacial acetic acid solution) was added to a methanolic solution of DPPH (2.0 × 10−4 M, 2.0 mL) and 5 mL of methanol. The mixture was shaken for 10 s and left to stand at room temperature for 30 min. The scavenging activity of CSCH or CSCH-TP on DPPH% was determined by the absorbance at 517 nm. The percentage of scavenging activity was calculated by using the following Eq. (1): % scavenging activity = (Abscontrol−Abssample)/Abscontrol × 100
(1)
where Abscontrol is the absorbance of the control (without the test sample) and Abssample is the absorbance of the sample (with the test sample). 3. Results and discussion 3.1. Elemental analysis The elemental analysis data of chitosan and its chromone derivatives (CSCH and CSCH-TP) are listed in Table 1. Carbon to nitrogen (C/ N) ratios of CS, CSCH and CSCH-TP were found to be 5.57, 8.41 and 10.62%, respectively. The increase in the C/N ratios of chitosan-chromone derivatives can be explained by the chemical modification of chitosan with a nitrogen-free chromone analog. From Table 1, the degree of substitution (DS) of the chitosan derivatives to −NH2 group on chitosan was calculated by following Eq. (2), modified with base in the model of Inukai et al. (Inukai, Chinen, Matsuda, Kaida, & Yasuda, 1998):
2.2. Preparation of chitosan-chromone derivatives, CSCH and CSCH-TP The chromone derivative (6-formyl-7-hydroxy-5-methoxy-2-methylbenzopyran-4-one, CH) was obtained by the oxidation of visnagin, according to the procedure described before by our group (Beyazit, Çatıkkaş, Bayraktar, & Demetgöl, 2016; Gönaydın & Beyazit, 2004). For the preparation of CSCH, 0.5 g of powder chitosan was dissolved in 25 mL of 1.0% (v/v) glacial acetic acid. The mixture was vigorously stirred under the reflux condition until the chitosan was completely dissolved. 0.2 g of chromone derivative in 50 mL methanol solution was added drop wise into the chitosan solution and the mixture was stirred under reflux condition for 4–5 h. The yellow color product was filtered off, washed with ethanol and dried in vacuo. For the preparation of CSCH-TP, 0.3 g of CSCH was dissolved in 40 mL of 1.0% (v/v) glacial acetic acid and 0.3 g of terephthalaldehyde (TP) in 25 mL ethanol solution were added drop wise into the CSCH solution. The mixture was stirred under reflux condition for 4–5 h. The yellow color product was filtered off, washed with ethanol and dried in vacuo. The synthesis route of chitosan-chromone derivatives are
DS =
(C / N ) m − (C / N ) o n
(2)
Where (C/N)m is the C/N of the chitosan derivative, (C/N)o is the C/N of the original chitosan, n is the number of carbon introduced after chitosan modification. The DS values obtained were about 0.33 and 0.50 for CSCH and CSCH-TP, respectively. 3.2. UV–vis spectra The electronic spectra of chitosan and its derivatives were measured
Scheme 1. The synthesis of chitosan-chromone Schiff base (CSCH) and its cross-linked derivative (CSCH-TP).
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results suggest that chitosan-chromone derivatives have less crystalline and more amorphous structure than the pure chitosan. Also the XRD spectrum of cross-linked chitosan derivative (CSHCH-TP) showed a weaker and broader crystalline peak as compare to that of CSCH. It is known that the crystalline structure of chitosan is mainly stabilized by intermolecular and intramolecular hydrogen bonding (Wang, Du, Fan, Liu, & Hu, 2005). The intramolecular hydrogen bonding of chitosan is formed between amino group at C-2 and hydroxyl group at C-3. The decreasing of crystallinity can be explained by deformation of some of the inter-molecular and intra-molecular hydrogen bonding because of the steric hindrance after Schiff base formation (Franca, Lins, Freitas, & Straatsma, 2008; Wan, Creber, Peppley, & Bui, 2004).
Table 1 Elemental analyses results of CS, CSCH and CSCH-TP. Compound
Element (%)
Chitosan (CS) CSCH CSCH-TP
C
H
N
C/N
45.34 50.66 55.22
7.03 6.10 5.68
8.23 6.01 5.20
5.57 8.41 10.62
in the range of 230–500 nm as shown in Fig. S1. It is well known that a maximum absorption band was observed at 235 nm corresponding to the n-π* transition and there is not any transition observed between 300 and 800 nm in the electronic spectrum of chitosan (Baran & Menteş, 2016; Tamer et al., 2016). On the other hand in the electronic spectra of CSCH and CSCH-TP, two new significant bands were observed at 260 and 305 nm assigned to π-π* transition of aromatic rings and n-π* transition of azomethine groups, respectively. The results revealed that a condensation reaction occurred between chitosan and chromone derivative to form an imine bond.
3.5. Thermal analysis (TGA) The thermal gravimetric analysis (TGA) of chitosan was previously reported in the literature. Chitosan exhibits two stage thermal decomposition process: the first one (T < 100 °C) is commonly associated with the loss of physisorbed water molecules, whereas the second stage occuring at ca. 300 °C corresponds to the decomposition of chitosan backbone (Corazzari et al., 2015. According to the TGA thermograms (Fig. S5 and S6), the main decomposition process of CSCH and CSCH-TP takes place at ca. 230 °C, lower than the decomposition stage of original chitosan backbone. The results suggested that chitosan derivatives are less thermally stable than the chitosan. The thermal instability of chitosan-chromone derivatives might be due to the substitution of free primary amino groups with chromone rings which is breaking the interand intra-molecular hydrogen bonding and lowering the crystallinity of chitosan (Demetgül, 2012).
3.3. FT-IR spectra FT-IR spectra of pure chitosan and its derivatives are illustrated in Fig. S2, S3 and S4. The spectrum of chitosan shows basic characteristic peaks at 3355 cm−1 (overlapped stretching vibrations between OeH and NeH groups), 2920 and 2871 cm−1 (CeH stretch), 1640 and 1589cm−1 (amide II band due to CeO and NeH stretching, respectively), 1063 and 1026 cm−1 (skeletal vibration involving CeO stretch) (Demetgül & Serin, 2008; Kumar, Koh, Kim, Gupta, & Dutta, 2012). In the FT-IR spectra of CSCH and CSCH-TP, the peak at 3355 cm−1 shifted to 3365 and 3381 cm−1, respectively and broadened due to the OeH absorption of chromone ring. The new peaks were observed in the FT-IR spectra of chitosan-chromone derivatives. The peaks observed at 3071 and 3087 cm−1 was attributed to the aromatic CeH stretch for CSCH and CSCH-TP, respectively. Furthermore the strong new peaks were also observed at 1630 and 1639 cm−1 assigned to the characteristic absorbance of azomethine (C]N) group, confirming the formation of Schiff base.
3.6.
13
C NMR analysis
It was previously reported that the typical 13C CP-MAS solid-state NMR chemical shift values of pure chitosan were observed at 23 ppm (eCH3e), 58 ppm (C2), 61 ppm (C6), 76 ppm (C5,C3), 85 ppm (C4), 106 ppm (C1), 174 ppm (C]O) [Carbohydrate Polymers Volume 173, 1 October 2017, Pages 714–720]. As compared with chitosan, new peaks were observed at 20 ppm (chromone eCH3e), 103 and 110 ppm (overlapped signals between C-1 of chitosan and chromone ring carbons), 165 ppm (overlapped signals between azomethine carbons and chromone carbons adjacent to oxygen atoms) in the spectrum of CSCH and CSCH-TP (Figs. 2 and 3). The intensity of the signal at 174 ppm was strengthened which was ascribed to carbonyl carbons of both chitosan and chromone scaffolds. When compared to CSCH, the spectrum of CSCH-TP exhibits new peaks at 129 and 137 ppm attributed to the aromatic carbons of terephthalaldehyde, confirming the formation of crosslinked Chitosan derivative.
3.4. X-Ray diffraction (XRD) study The XRD spectra of pure chitosan (CS), CSCH and CSCH-TP are given in Fig. 1. X-Ray Diffraction study of CS exhibits two sharp crystalline peaks at 2θ = 10° and 2θ = 20° in agreement with the previous reports (Baran, Açıksöz, & Menteş, 2016). In the XRD patterns of CSCH and CSCH-TP, the intensity of peaks decreased and broadening of the peaks was observed when compared with the free chitosan. These
3.7. Scanning electron microscopy (SEM) The SEM images of chitosan and chitosan-chromone derivatives (CSCH and CSCH-TP) are represented in Fig. 4. The difference of structural morphology between chitosan and its chromone derivatives was also supported by their SEM images. While the pure chitosan had a nonporous and smooth membranous phase surface, the SEM image of CSCH showed a polymorphic porous structure. On the other hand, crosslinked chitosan derivative, CSCH-TP, exhibited a more regular smooth surface compared to the surface of CSCH. 3.8. DPPH radical scavenging activity Fig. 5 shows the scavenging activity of chitosan and its chromone derivatives for the DPPH radical. The scavenging activity of chitosanchromone derivatives was enhanced compared with that of original chitosan. The IC50 value for CSCH was 0.88 mg/mL, which was slightly higher than the 1.32 mg/mL for CSCH-TP.
Fig. 1. XRD Diffractograms of chitosan and its derivatives.
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C. Demetgül, N. Beyazit Fig. 2.
13
C CP-MAS NMR Spectrum of CSCH.
from the plants (Sousa, Guebitz, & Kokol, 2009). Chromone and its derivatives are an important class of oxygencontaining natural heterocyclic compounds and they have been known as antioxidant agents (Keri, Budagumpi, Pai, & Balakrishna, 2014). In the literature, there are few studies in which chitosan was modified with a chromone derivative (Huang et al., 2013; El Badawy, 2008; Kumar and Koh, 2012). As far as we know, the antioxidant activity of a
In numerous studies, original chitosan was investigated as a potential antioxidant agent. The antioxidant activity of chitosan has been associated with its strong hydrogen-donating ability and it was reported that a low molecular weight and a higher concentration have a positive effect on the activity (Vinsova & Vavrikova, 2011). There have been many studies which intended to improve its biological activities by chemical modification of chitosan with natural compounds obtained
Fig. 3.
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13
C CP-MAS NMR Spectrum of CSCH-TP.
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Fig. 4. SEM images of CS (a), CSCH (b) and CSCH-TP (c).
enhancement in the antioxidant activity was observed after the functionalization of chitosan with a chromone which is an important scaffold in medicinal chemistry. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.2017.11.074. References Baran, T., & Menteş, A. (2016). Polymeric material prepared from Schiff base based on Ocarboxymethyl chitosan and its Cu(II) and Pd(II) complexes. Journal of Molecular Structure, 1115, 220–227. Baran, T., Açıksöz, E., & Menteş, A. (2016). Highly efficient, quick and green synthesis of biarlys with chitosan supported catalyst using microwave irradiation in the absence of solvent. Carbohydrate Polymers, 142, 189–198. Beyazit, N., Çatıkkaş, B., Bayraktar, Ş., & Demetgül, C. (2016). Synthesis, characterization and catecholase-like activity of new Schiff base metal complexes derived from visnagin: theoretical and experimental study. Journal of Molecular Structure, 1119, 124–132. Corazzari, I., Nistico, R., Turci, F., Faga, M. G., Franzoso, F., Tabasso, S., et al. (2015). Advanced physico-chemical characterization of chitosan by means of TGA coupled on-line with FTIR and GCMS: Thermal degradation and water adsorption capacity. Polymer Degradation and Stability, 112, 1–9. Demetgül, C., & Serin, S. (2008). Synthesis and characterization of a new vic-dioxime derivative of chitosan and its transition metal complexes. Carbohydrate Polymers, 72, 506–512. Demetgül, C. (2012). Synthesis of the ketimine of chitosan and 4, 6-diacetylresorcinol, and study of the catalaselike activity of its copper chelate. Carbohydrate Polymers, 89, 354–361. El Badawy, M. (2008). Chemical modification of chitosan: synthesis and biological activity of new heterocyclic chitosan derivatives. Polymer International, 57, 254–261. Elshaarawy, R. F. M., Refaee, A. A., & El-Sawi, E. A. (2016). Pharmacological performance of novel poly-(ionic liquid)-grafted chitosan-N-salicylidene Schiff bases and their complexes. Carbohydrate Polymers, 146, 376–387. Franca, E. F., Lins, R. D., Freitas, L. C. G., & Straatsma, T. P. (2008). Characterization of chitin and chitosan molecular structure in aqueous solution. Journal of Chemical Theory and Computation, 4(12), 2141–2149. Günaydın, K., & Beyazit, N. (2004). The chemical investigations on the ripe fruits of Ammi visnaga (Lam.) lamarck growing in Turkey. Natural Product Research, 18(2), 169–175. Gabor, M. (1986). Anti-inflammatory and anti-allergic properties of flavonoids. Progress in Clinical and Biological Research, 213, 471–480. Grindlay, D., & Reynolds, T. (1986). The aloe vera phenomenon: A review of the properties and modern uses of the leaf parenchyma gel. Journal of Ethnopharmacology, 16, 117–151.
Fig. 5. DPPH radical scavenging activity of chitosan and its chromone derivatives.
chitosan-chromone derivative has never been investigated before. In our study, DPPH radical scavenging activity assays showed that a considerable amount of increase in the antioxidant activity was observed for chitosan-chromone derivatives (CSCH and CSCH-TP) as compared with chitosan alone. This might be due to the phenolic group of chromone ring condensed with chitosan to afford the corresponding Schiff base. However, after the crosslinking with terephthalaldehyde, the activity was decreased slightly which might be related to the reduced active amino group content of chitosan backbone. 4. Conclusion In the present work, a new chemically modified chitosan with a chromone ring and its cross-linked derivative were synthesized and characterized by elemental analysis data and various spectroscopic methods. SEM, TGA and XRD studies demonstrated that chitosanchromone derivatives (CSCH and CSCH-TP) have polymorphic, porous surface, low thermal stability and low cristallinity as compared with original chitosan. Chitosan alone has been reported to have antioxidant activity and in order to improve its free radical scavenging activity, it has been modified in several ways. In this study, the effect of chitosan modification with chromone moiety on its antioxidant activity was evaluated by using the DPPH radical scavenging assay. As a result, an 816
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