Carbohydrate Polymers 60 (2005) 3338 www.elsevier.

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Preparation of high antimicrobial activity thiourea chitosanAgC complex

Center for Applied Radiation and Materials, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, P.O. Box 800-204, 2019 Jia LuoRoad, Shanghai 201800, China b Department of Food Science, Xiangtan University, Xiangtan, Hunan 410115, China Received 25 October 2004; accepted 11 November 2004 Available online 19 December 2004

Abstract Thiourea chitosan was prepared by the reaction of chitosan with ammonium thiocyanate in ethanol. Thiourea chitosan was characterized by FT-IR, 13C NMR, and elemental analysis; XPS conrmed that in thiourea chitosanAgC complex, S atoms coordinated to silver ions and were the major electron donors; O atoms also coordinated to silver ions and they were another electron donors next to S atoms. N atoms did not take part in coordination. Thiourea chitosanAgC complex overcomes the instability of AgC. Antimicrobial activities of the complex was evaluated against six species of bacteria and molds. The complex showed a wide spectrum of antimicrobial activities, whose MIC values against bacteria were 20 times lower than those of chitosan, 100 times lower than those of sodium diacetate and 200 times lower than those of sodium benzoate, respectively; the complex has a better antibacterial activity than antifungal activity. q 2004 Elsevier Ltd. All rights reserved.
Keywords: Chitosan; Thiourea chitosan; Antimicrobial activity; Silver iron; Complex

1. Introduction Chitosan is a natural nontoxic biopolymer derived by the deacetylation of chitin. Chitosan and its derivatives have attracted considerable interest due to their antimicrobial and antifungal activities (Jung et al., 1999). The antimicrobial activity of chitosan depend on several factors such as the kind of chitosan (deacetylation degree, molecular weight) used, the pH of the medium, the temperature, etc. The mechanism of the antimicrobial activity has not been fully elucidated yet, but several hypotheses have been postulated. The most feasible hypothesis is a change in cell permeability due to interactions between the polycationic chitosan and the electronegative charges on the cell surfaces. This interaction leads to the leakage of intracellular electrolytes and proteinaceous constituents (Chen, Liau, & Tsai, 1998; Feng et al., 2000; Papineau, Hoover, Knorr, & Farkas,

* Corresponding author. Tel./fax: C86 215 955 8905. E-mail address: [email protected] (G. Wu). 0144-8617/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbpol.2004.11.020

1991; Sudarshan, Hoover, & Knorr, 1992; Young & Kohle, 1982). Other mechanisms are the interaction of diffused hydrolysis products with microbial DNA, which leads to the inhibition of the mRNA and protein synthesis (Hadwiger et al., 1986) and the chelation of metals, spore elements and essential nutrients (Cuero, Osuji, & Washington, 1991). Silver or silver ions have long been known to have powerful antibacterial activity. Silver is widely applied in some medical elds for its high antimicrobial activity and low concentration (Kang, Jung, & Jeong, 2000). The complex of chitosan with AgC exhibits antibacterial activity (Zhan, Xiong, Liu, & Xie, 2002). However, AgC including the adsorptive Ag(I) by chitosan is instable, and can be easily reduced as Ag, which limits its medical application. According to hard and soft acid theory, thiourea compounds are very efcient at chelating noble metals and widely used for extracting noble metals including silver, gold, etc. (Chanda & Rempel, 1990). In order to introduce thiourea group into chitosan main chain, thiourea grafting chitosan using glutaraldehyde

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as cross-linking agent was prepared (Guibal, Vincent, & Navarro Mendoza, 2000a; Guibal et al., 2001). However, the obtained thiourea grafting chitosan can not dissolve into any solvent, which restricts its use as antimicrobial. A kind of soluble chitosan modied by thiourea is desired. In this paper, thiourea chitosan (TU-CTS) was prepared by the reaction of citosan with ammonium thiocyanate, which was easy to be dissolved into 1% acetic acid solution. Thiourea chitosan and AgC formed a stable thiourea chitosanAgC(TU-CTS-AgC) complex, which remarkably enhanced the antimicrobial activity of chitosan as well as the stability of AgC.

2. Experimental 2.1. Materials Chitosan with deacetylation degree of 90% was purchased from Yuhuan Organisms Co. Ltd. (Zhejiang Province, China). Beef extract, potato starch, glucose and peptone were purchased from Shanghai Chemical Agent Co. (Shanghai, China). The microorganisms tested were provided by Xiangtan University in China. The other reagents were of analytical grade and used without further purication. 2.2. Characterizations of thiourea chitosan and thiourea chitosanAgC C NMR spectra were performed on a Bruker (AVACE) AV-500 spectrometer in D2O/HCl and D2O/CF3COOD, respectively; IR spectra were recorded on Nicolet-170 SX FT-IR spectrophotometer in KBr discs; S% was measured in a SC-132 sulfur meter (LECO), while C%, N% and H% were measured by Elemental Analyzer-MOD 1106 (Carlo Erba Strumentazione); AgC content was measured by a Hitachi 180-80 atomic absorption spectrometry. X-ray photoelectron and X-ray excited Auger spectra were obtained with a Physical Electronics PHI 5700 spectrometer with using nonmonochromated Mg Ka radiation (hnZ1253.6 eV) and operating at 15 kV and 20 mA; the vacuum in the analysis chamber was better than 10K9 mbar. Binding energies were corrected using the binding energy values for C 1 s of adventitious carbon xed at 284.8 eV. 2.3. Preparation of thiourea chitosan and thiourea chitosanAgC A mixture of 16.1 g (0.1 mol) chitosan powder, 15.2 g (0.2 mol) ammonium thiocyanate and 150 ml ethanol in a three-neck ask with amagenetic stirring was reuxed for 12 h. After cooling down to room temperature,
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the precipitate was collected by ltration and repeatedly washed with ethanol, then was dissolved in 500 ml of 1% (v/v) acetic acid solution. By adding 10% (w/v) NaOH solution into the solution and ltrating, the precipitate was collected and successfully washed with water and nally dried to give 16.5 g of thiourea chitosan. 0.3 g of thiourea chitosan was dissolved in 30 ml of acetic acid (1%, v/v), followed by addition of 25 ml of AgNO3 solution (0.01 g AgNO3) into the solution by stirring. After the mixture was stirred for 3 h at room temperature, the precipitate obtained by adding 200 ml acetone into the mixture was collected by ltration, successively washed with 95% ethanol, and dried to give thiourea chitosanAgC complex. The sorption amount of AgC in thiourea chitosanAgC complex was 1.44 mg/g as determined by atomic absorption spectrometry. 2.4. Evaluation of antimicrobial activity in vitro The agar plate method was used to determine the minimum inhibition concentration (MIC) of chitosan, sodium diacetate, sodium benzoate and thiourea chitosan AgC complexe. The 1% (w/v) solutions of chitosan, sodium diacetate, sodium benzoate and thiourea chitosan AgC were prepared in 1% (v/v) acetic acid. Duplicate two-fold serial dilutions of each sample were added to nutrient broth (beef extract 5 g, peptone10 g to 1000 ml distilled water, pH 7.0) for the nal concentration of 0.1, 0.05, 0.025, 0.0025, 0.00125, and 0.00025%; 0.1%(v/v) of acetic acid was used as a control instead of samples. Then they were autoclaved at 121 8C for 25 min. The culture of each bacterium was diluted by sterile distilled water to 105106 CFU/ml. A loop of each suspension was inoculated on nutrient medium with the sample or the control added. After inoculation, the plates were incubated at 37 8C for 72 h, and the colonies were counted and the MIC values were obtained. The MIC was considered to be the lowest concentration that completely inhibits against bacteria comparing with the control, disregarding a single colony or a faint haze caused by the inoculum (Speciale, et al., 2002). Following the procedure described above, slight modications were made in order to obtain the MIC values of chitosan, sodium diacetate, sodium benzoate and thiourea chitosanAgC against molds. Potato starch glucose media (potato starch 10 g, glucose 20, agar 20 g and pH value in nature) was used for culturing molds. The culture of each mold spores was diluted by sterile distilled water to 105 CFU/ml. A loop of each suspension was inoculated on nutrient medium with the sample or the control added. The inoculated plates were incubated at 30 8C for 10 days, and the colonies were counted and the MIC values were obtained.

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Scheme 1. Synthesis of thiourea chitosan.

Table 1 Elemental analysis results of CTS and TU-CTS Sample Mole ration of glucosamine unit to ammonium thiocyanate 1:1 1:1.5 1:2 1:2.5 Elemental analysis (%) S 0 0.4 0.5 0.5 0.6 C 38.70 38.07 37.81 37.75 37.52 N 8.51 8.46 8.35 8.27 7.85 H 6.53 6.45 6.38 6.35 6.28

CTS TSC-CTS I TSC-CTS II TSC-CTS III TSC-CTS IV

3. Results and discussion 3.1. Characterization of thiourea chitosan and thiourea chitosanAgC A traditional method of introducing thiourea group into chitosan is carried out using glutaraldehyde as cross-linking reagent (Guibal et al., 2000a; Guibal et al., 2001). The obtained product is an insoluble solid and not used for bacteriostasis. Here we used a different approach to introduce thiourea group into chitosan. It is well known that when temperature is above 70 8C, part of ammonium thiocyanate converts into its isomeric compound, thiourea. Our strategy is that ammonium thiocyanate and chitosan are together heated to form chitosan thiocyanate and ammonia; similarly, chitosan thiocyanate is heated to convert into thiourea chitosan. The preparation of thiourea chitosan is illustrated in Scheme 1. As can be seen from Table 1, with increasing the mole ratio of glucosamine unit to ammonium thiocyanate, sulfur content in thiourea chitosan slightly increased. The sulfur content in thiourea chitosan is less than 0.6%. It indicates that only a small amount of chitosan thiocyanate is converted into thiourea chitosan and the yield is low. Although chitosan thiocyanate is similar to ammonium thiocyanate, the reaction activity of chitosan thiocyanate is lower than ammonium thiocyanate upon heating. Further work is needed to investigate for this difference. However, a small quantity of thiourea chitosan could chelate with a few of silver ions which is and enough to remarkably enhance the antimicrobial activity of chitosan. Structure changes of thiourea chitosan and thiourea chitosanAgC were conrmed by FT-IR spectra (Fig. 1).

The FT-IR spectra of thiourea chitosan shows new peaks at 1535.1 cmK1, which is assigned to the characteristic absorbance peak of thiourea groups (Padhye, 1985; Rao & Kasturi, 1964); In addition, the signal from amide I at 1646.7.9 cmK1 (n(CZO)) and 1600.2 cmK1 (d(NH2)) (Fernandez Cervera et al., 2004) disappeared and was replaced by a new peak at 1631.5 cmK1 (d(NH2)) and its intensity became weak as result of the amino group of chitosan being substituted. These results suggest thiourea group are part of chitosan. Comparing with thiourea chitosan, in the FT-IR spectra of thiourea chitosanAgC, a new peak at 825.4 cmK1 appeared, which was assigned to the stretching vibration of SAgC (Lobana, Bhargava, Sharma, & Kumar, 2003).

Fig. 1. FT-IR spectra of chitosan, thiourea chitosan and thiourea chitosanAgC.

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S. Chen et al. / Carbohydrate Polymers 60 (2005) 3338

Fig. 2(a) and (b) show the 13C NMR spectra of chitosan and thiourea chitosan in D2O/CF3COOD and D2O/HCl, respectively. It can be seen that the signals of 13C NMR spectra in Fig. 2(a) are stronger and clearer than in Fig. 2(b),

since chitosan and thiourea chitosan have better solubility in D2O/CF3COOD than in D2O/HCl. Whether in D2O/CF3COOD or D2O/HCl, the 13C-NMR chemical shifts for chitosan at 58.3(C2), 62.5(C6),

Fig. 2. (a) 13C NMR spectra of chitosan and thiourea chitosan in D2O/CF3COOD. (b) 13C NMR spectra of chitosan and thiourea chitosan in D2O/HCl.

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Fig. 3. Ag 3d, S 2p, O 1s and N 1s XPS spectra.

72.5(C3), 77.2(C5), 79.1(C4) and 100.0(C1) (ppm) were detected (Dung, Milas, Rinaudo, & Desbriers, 1994; Rinaudo, Desbrieres, le Dung, Thuy Binh, & Dong, 2001). In contrast to chitosan, the signals for thiourea chitosan upshifted and appeared at 57.1(C2), 61.6(C6), 71.4(C3), 76.1(C5), 78.6(C4) and 98.9(C1) (ppm). It was noticeable that the weaker signals for substituted C2 remarkably downshifted and appeared at 66.5 ppm (C2 0 ). Hence, it could be concluded that a few of C2 amino groups were substituted by thiourea groups because the stronger signals for unsubstituted C2 at 57.1 ppm still were observed. Comparing Fig. 2(a) with Fig. 2(b), the weaker signals at 163.3 ppm in Fig. 2(b) was assigned to the carbons of thiourea groups (Klimova, Klimova, Martinez Garcia, Mendez Stivalet, & Ruiz Ramirez, 2001), which were superimposed by the stronger signals for C]O in CF3COOD and could not be identied in Fig. 2(a). The 13CNMR spectrum suggests C2 amino group of chitosan was substituted by thiourea group. The evidences of elemental analysis, FTIR, and 13 C-NMR obviously support the amino groups of chitosan were partly converted into thiourea groups. As well known, XPS is a very useful tool for detecting the interaction between builders in complex. Fig. 3 shows the Ag 3d, S 2p, O 1s and N 1s XPS spectra.

Ag3d electron spectra for AgNO3 presented two signal peaks with the binding energy (BE) of 374.3 and 380.2 eV. After AgC chelating with thiourea chitosan, the peaks signicantly shifted to 372.3 and 378.4 eV, respectively; this result indicates that the electron density at the Ag atoms is higher than that of the free ligand; the S 2p electron spectra of the complex show the BE value is signicantly shifted to 167.1 from 164.9 eV, indicating that the electron density at the S atoms is greatly less than that of the free ligand. This implies the coordination of S atoms to silver ions and S atoms are the major electron donors; similarly, the electron density at the O atoms is lower than that of the free ligand, which is conrmed by the BE shifting from 535.7 eV to 536.7 eV in the O 1s electron spectra of the complex; O atoms are another electron donors next to S atoms. The BE value of N1s is close to that of the free ligand, indicating no coordination of N atom. The thiourea chitosan chelating with AgC could be described based on hardsoft-acidbase (HSAB) theory: AgC acting as a soft acid is the acceptor of electrons given by S and O atoms in thiourea chitosan acting as a soft base. 3.2. Antimicrobial activities The antimicrobial activities of chitosan, thiourea chitosanAgC complex and sodium diacetate sodium benzoate are shown in Table 2.

Table 2 Antibacterial activity of CTS, TU-CTS-AgC, sodium diacetate and sodium benzoate Antibacterial Microorganisms Staphylococcus aureus Eschercihia Coli Bacillus subtilis Aspergillus avus Mucor kacilliformis Paccilomgces uarioti CTS(w/v, %) TU-CTS-AgC (TU-CTS-AgC%, w/v) 0.05 0.05 0.05 0.5 0.5 0.5 0.0025 0.0025 0.0025 0.05 0.05 0.05 (AgC%, w/v) 0.000012 0.000012 0.000012 0.00024 0.00024 0.00024 Sodium diacetate (w/v, %) 0.25 0.25 0.25 0.5 0.5 0.5 Sodium benzoate (w/v,%) 0.5 0.5 0.5 0.5 0.5 0.5

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S. Chen et al. / Carbohydrate Polymers 60 (2005) 3338 Cuero, R. G., Osuji, G., & Washington, A. (1991). N-carboxymethyl chitosan inhibition of aatoxin production: role of zinc. Biotechnological Letters, 13, 441444. Dung, P., Milas, M., Rinaudo, M., & Desbriers, J. (1994). Water soluble derivatives obtained by controlled chemical modications of chitosan. Carbohydrate Polymers, 24, 209214. Feng, Q. L., Wu, J., Chen, G. Q., Cui, F. Z., Kim, T. N., & Kim, J. O. (2000). A mechanistic study of the antibacterial effect of silver ions on E. coli and Staphylococcus aureus. Journal of Biomedical Material Research, 52, 662668. Fernandez Cervera, M., Heinamaki, J., Rasanen, M., Maunu, S. L., Karjalainen, M., Nieto Acosta, O. M., Iraizoz Colarte, A., & Yliruusi, J. (2004). Solid-state characterization of chitosans derived from lobster chitin. Carbohydrate Polymers , 18. Guibal, E., Vincent, T., & Navarro Mendoza, R. (2000a). Synthesis and characterization of a thiourea-derivative of chitosan for platinumrecovery. Journal of Applied Polymer Science, 75(1), 119134. Guibal, E., von Offenberg Sweeney, N., Zikan, M. C., Vincent, T., & Tobin, J. M. (2001). Competitive sorption of platinum and palladium on chitosanderivatives. International Journal of Biological Macromolecules, 28, 401408. Hadwiger, L. A., Kendra, D. F., Fristensky, B. W., & Wagoner, W. (1986). Chitosan both activates genes in plants and inhibits RNA synthesis in fungi. In R. A. A. Muzzarelli, C. Jeuniaux, & G. W. Gooday (Eds.), Chitin in nature and technology (pp. 209214). New York: Plenum Press, 209214. Jung, B. O., Lee, Y. M., Kim, J. J., Choi, Y. J., Jung, K. J., & Chung, S. J. (1999). The antimicrobial effect of water soluble chitosan. Journal of Korean Industrial Engineering Chemistry, 10, 660665. Kang, H. Y., Jung, M. J., & Jeong, Y. K. (2000). Antibacterial activity and the stability of an AgC solution made using metallic silver. Korean Journal of Biotechnology and Bioengineering, 15, 521524. Klimova, T., Klimova, E. I., Martinez Garcia, M., Mendez Stivalet, J. M., & Ruiz Ramirez, L. (2001). The reactions of semicarbazide and thiourea with ferrocenyl-substituted, a, b-enones. Journal of Organometallic Chemistry, 633, 137142. Lobana, T. S., Bhargava, G., Sharma, V., & Kumar, M. (2003). Thiosemicarbazonates of silver(I): synthesis, spectroscopy and reactivity towards triphenylphosphine. Indian Journal Of Chemistry, 42a, 309312. Padhye, S. (1985). Transition metal complexes of semicarbazones and thiosemicarbazones. Coordination Chemical Review, 63, 127135. Papineau, A. M., Hoover, D. G., Knorr, D., & Farkas, D. F. (1991). Antimicrobial effect of water-soluble chitosans with high hydrostatic pressure. Food Biotechnology, 5, 4557. Rao, C. N. R., & Kasturi, T. R. (1964). Contribution to the infared spectra of organosulphur compounds. Canadian Journal of Chemistry, 42, 3642. Rinaudo, M., Desbrieres, J., le Dung, P., Thuy Binh, P., & Dong, N. T. (2001). NMR investigation of chitosan derivatives formed by the reaction of chitosan with levulinic acid. Carbohydrate Polymers, 46, 339348. Speciale, A., Musumeci, R., Blandino, G., Milazzo, I., Caccamo, F., & Nicoletti, G. (2002). Minimal inhibitory concentrations and time-kill determination of morioxacin against aerobic and anaerobic isolates. International Journal of Antimicrobial Agents, 19, 111118. Sudarshan, N. R., Hoover, D. G., & Knorr, D. (1992). Antibacterial action of chitosan. Food Biotechnology, 6, 257272. Young, D. H., Kohle, H., & Kauss, H. (1982). Effect of chitosan on membrane permeability of suspension cultured Glycine max and Phaseolus vulgaris cells. Plant Physiology, 70, 14491454. Zhan, X. J., Xiong, Y. Z., Liu, Z., & Xie, D. Z. (2002). Synthesis of silver carboxymethyl chitosan and its experimental study on its bacteriostasis. China Journal of Biochemistry Pharmacology, 22(3), 142144.

It can be seen that thiourea chitosanAgC complex shows a wide spectrum of antimicrobial activities against all of the microorganisms tested. It is also noticed that the complex has good antibacterial activity and antifungal activity. Generally, the complexes has better antibacterial activity than antifungal activity. Although only a few silver ions are loaded on thiourea chitosan, compared with chitosan, sodium diacetate and sodium benzoate, thiourea chitosanAgC has much better antimicrobial activities, whose MIC values against bacteria were 20 times lower than chitosan, 100 times lower than sodium diacetate and 200 times lower than sodium benzoate, respectively. The chelation AgC signicantly enhanced the antimicrobial activities of chitosan. Generally, the inhibitory effect of silver on bacteria can be explained by the reaction of AgC with proteins by combing the SH groups of enzymes, which leads to the inactivation of the proteins. Another inhibition mechanism of silver ions on microorganism is believed that silver ions affect DNA molecules by losing their replication abilities. (Feng, et al., 2000).

4. Conclusion Thiourea chitosan was prepared by the reaction of chitosan with ammonium thiocyanate in ethanol. Thiourea chitosan was characterized by FT-IR, 13C NMR, and elemental analysis; XPS conrmed that in thiourea chitosanAgC complex, S atoms coordinated to silver ions and were the major electron donors; O atoms were another electron donors next to S atoms. Thiourea chitosan AgC complex improved the instability of AgC. In vitro antimicrobial activities of the complex were evaluated against six species of bacteria and molds. The complex showed a wide spectrum of effective antimicrobial activities. Its MIC values much lower than those of chitosan, sodium diacetate and sodium benzoate. The complex has better antibacterial activity than antifungal activity.

Acknowledgements This work was nancially supported by "Hundred Talents" project of the Chinese Academy of Sciences.

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