Degradation of organic solvent-saturated chitin hydrogel by plasma in liquid

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1 Degradation of organic solvent-saturated chitin hydrogel by plasma in liquid Thanadon Kaewyoo a,b, Ratana Rujiravanit a,b,c* and Nagahiro Saito d a) The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok, Thailand b) NU-PPC Plasma Chemical Technology Laboratory, Chulalongkorn University, Bangkok, Thailand c) Center of Excellence on Petrochemical and Materials Technology, Chulalongkorn University, Bangkok, Thailand d) Department of Materials, Physics and Energy Engineering, Graduate School of Engineering, Nagoya University, Nagoya, Japan Keywords: β-chitin, degradation, chitoloigosaccharides, solution plasma processing ABSTRACT A discharge of plasma in liquid, so called solution plasma, was used to reduce molecular weight of chitin hydrogel dispersed in a dilute salt solution. In this study, chitin was prepared from squid pen. It is known that chitin has intense hydrogen bonding interaction in its structure, leading to its high crystallinity and low solubility. To overcome this obstacle, chitin in the form of hydrogel was prepared by dissolving chitin in CaCl 2 - saturated methanol, followed by precipitation of chitin solution in water. The remaining hydrogen bonding interaction in chitin hydrogel was further disrupted by submersion of chitin hydrogel in various organic solvents, i.e. DMSO, DMS and ethanol. It was found that DMF-saturated chitin hydrogel had the lowest crystallinity and could undergo degradation by solution plasma in a dilute KCl solution. The effects of KCl concentration and plasma treatment time on molecular weight reduction of DMF-saturated chitin hydrogel were investigated. It was found that not only molecular weight of the degraded chitin but also the yield of water-soluble product of degraded chitin was influenced by KCl concentration and plasma treatment time. It might be concluded that solution plasma could be applied to produce water-soluble chitooligosaccharide from DMF-saturated chitin hydrogel dispersed in KCl solution. Moreover, anti-cancer activity against lung cancer cells of the watersoluble chitooligosaccharides was evaluated. *ratana.r@chula.ac.th INTRODUCTION In the present day, cancer is the major cause of death of people around the world. It has been reported that in the year 2015, 8.8 million people died because of the cancer. (World Health Organization) Nowadays, many researchers are still working on developing new medicines for curing cancers. Not only synthetic compounds but also natural products have been investigated for their anti-cancer activities against various types of cancers. Some potential anti-cancer drugs are derived from polysaccharides. (Zhao et al., 2010) It has been reported that oligomers of chitin and of its derivative, chitosan, that are also known as chitooligosaccharides (COS), exhibited antitumor and anticancer activities. (Karagozlu et al., 2015) Chitin is a polysaccharide consisting of two monomeric units, i.e. D-glucosamine and N-acetyl-D-glucosamine units, that are linked together by β-(1,4)-glycosidic linkage. Chitin is a structural constituent in crustaceans such as shrimp, crab and squid. Chitin that is derived from shrimp and crab shells has random coil structure and is defined as alphachitin and chitin that is derived from squid pen has beta-sheet structure and is defined as on Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 1

2 beta-chitin. Since beta-chitin has less inter-molecular hydrogen bonding interaction than alpha-chitin, beta-chitin is more susceptible to swelling and degradation reaction than alpha-chitin. (Rinaudo, 2006) To render beta-chitin more susceptible to degradation reaction, beta-chitin in the form of hydrogel can be prepared by dissolving beta-chitin in CaCl 2 -saturated methanol to obtain chitin solution, followed by the addition of chitin solution into water to precipitate the polymer as chitin hydrogel. (Tamura et al., 2006) In this study, the crystallinity of chitin in organic solvent are investigated before the degradation process. Typically, chitooligosaccharides (COS) has molecular weight in the range of 1 kda to 6 kda. (Šimůnek et al.,2012). COS can be normally prepared by various types of degradation reactions of chitin, such as chemical degradation, enzymatic degradation and physical degradation. (Qin et al., 2002). Although chemical degradation by using a concentrate acid is a low-cost process operating with a fast reaction to produce COS (Kazami et al.,2015), there is a risk on exposure to the corrosive, harmful chemical and a careful acid waste management is required to prevent sequential environmental problems. Although COS with a specific molecular weight can be produced under a mild condition using enzyme, the enzymatic degradation is a high cost process and spends relative long reaction time to generate COS. Recently, physical degradation methods to produce COS have received more attention due to the fast reaction rate and its potential for mass production. Physical degradation could be induced by providing energy impact to affect chain scission of polymers. Several forms of energy impact have been used to degrade polymers. In the literature, there has been reported on using plasma discharge in liquid or solution plasma to degrade chitosan and alginate. (Watthanaphanit et al., 2013) Along with the discharge of plasma, several forms of highly active species including positively charged particles, negatively charged particles and free electron are generated in a solution that can further induced bond cleavage in a polymer chain. In this study, chitin prepared from squid pen was used to produce chitooligosaccharides by applying solution plasma treatment. To facilitate degradation reaction of chitin, amorphous chitin in the form of chitin hydrogel was firstly prepared by dissolving chitin powder in CaCl 2 -saturated methanol to obtain chitin solution. By the addition of chitin solution into water, chitin hydrogel was formed. After the removal of CaCl 2 by dialysis, chitin hydrogel was subject to solvent exchange process in order to further disrupt the remaining crystalline region in chitin hydrogen and to obtain organic solvent-saturated chitin hydrogel. Organic solvents used in this study were DMF, DMSO and ethanol. DMF-saturated chitin hydrogel was found to have the lowest crystallinity and had been used for the production of COS by solution plasma treatment. DMF-saturated chitin hydrogel was dispersed in a dilute KCl solution before undergoing plasma treatment. The effects of KCl concentration and plasma treatment time on the reduction of molecular weight of chitin and production yield of COS were investigated. Biological activity of COS in the aspect of anti-cancer activity against lung cancer was examined in comparison with the normal cells. EXPERIMENTAL A. Materials on Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 2

3 Squid pens (Loligo duvauceli) was provided by Marine Bio Resources Co., Ltd. (Thailand). Anhydrous sodium hydroxide pellets (NaOH, analytical grade, Univar), Hydrochloric acid (HCl, analytical grade, RCI Lab Scan), Calcium Chloride dihydrate (CaCl 2.2H 2 O, analytical grade, Univar), Methanol (CH 3 OH, analytical grade, RCI Lab Scan), Acetone (C 3 H 6 O, commercial grade, RCI Lab Scan), Potassium Chloride (KCl, analytical grade, Univar), Dimethylformamide (DMF, analytical grade, QRëC Quality Reagent Chemical), Dimethyl sulfoxide (DMSO, analytical grade, RCI Lab Scan), Absolute ethanol (C 2 H 5 OH, analytical grade, RCI Lab Scan) B. Chitin preparation Squid pens were cut into small pieces (1-2 centimetres, approximately). And then 200 g. of squid pens were demineralized by soaking in 3 litres of 1 M hydrochloric acid solution, followed by occasional stirring at room temperature for 3 days. Hydrochloric acid solution was changed every 24 hr (repeated for 3 times). The demineralized squid pens were deproteinized by boiling at 80 C in 4 % (w/v) of sodium hydroxide solution with stirring for 4 hr. The ratio of sodium hydroxide solution to the demineralized squid pens was10:1. After deproteinization, the obtained chitin was dried at 60 C for 2 days. C. Chitin hydrogel preparation Calcium solvent was prepared by heating calcium chloride dihydrate (CaCl 2.2H 2 O) and methanol under reflux at 70 C for 4 hr. 225 g of CaCl 2.2H 2 O was added into300 ml of methanol. The mixture was heated with continuous stirring for 4 hr until a homogeneous solution of calcium solvent was obtained. Chitin powder was then slowly added into the calcium solvent. The suspension was heated at 70 C with continuous stirring for 4 hr and chitin solution was obtained. After that chitin hydrogel was formed by the addition of chitin solution into an excess amount of water. Calcium chloride in chitin hydrogel was then removed by dialysis against water for 3 days with a change of fresh water 4 times per day. D. Organic solvent-saturated chitin preparation Chitin hydrogel was centrifuged at rpm for 30 min in order to remove excess water from chitin hydrogel. The precipitate of chitin hydrogel was then collected and added into different organic solvents, i.e. DMSO, DMF and ethanol. The ratio of organic solvent to chitin hydrogel was 10: 1 by weight. In case of DMSO and DMF, the suspensions of chitin in organic solvents were heated at 110 C for 40 minutes in order to completely remove the remaining water in chitin gel. The chitin gel suspension was centrifuged at rpm for 30 minutes. After that the supernatant was drained off from chitin gel and fresh organic solvents were added to chitin gel. The solvent exchange process was repeated for 3 times to obtain organic solvent-saturated chitin gel. E. Characterization of organic solvent-saturated chitins on Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 3

4 Crystallinity of oven-dried organic solvent-saturated chitin were determined by X- ray diffraction (XRD) and the chemical structure of oven-dried chitin hydrogel and ovendried organic solvent-saturated chitin were investigated by FT-IR F. Degradation of organic solvent-saturated chitin via solution plasma The organic solvent-saturated chitin gel was dispersed in 0.16 % potassium chloride solution. After that the organic solvent-saturated chitin gel suspended in the dilute KCl solution was subject to solution plasma treatment in order to accomplish degradation. The frequency, voltage and pulse width for solution plasma treatment were15.0 khz, 2.24 kv and 2 μs, respectively. Solution plasma treatment times were 240, 360, 480, 600 and 720 minutes. G. Characterization of degraded chitin Weight-average molecular weight and polydispersity index (PDI) of degraded chitin was investigated by gel permeation chromatography (GPC) and chemical structure of degraded chitin was investigated by 1 H-NMR. RESULTS AND DISCUSSION A. Characterization of organic-saturated chitin Crystallinity of the oven-dried organic solvent-saturated chitin was investigated by X-ray diffraction (XRD) spectrophotometer. According to the XRD spectra shown in Figure 1 a., XRD spectra of the oven-dried ethanol-saturated chitin gel and the oven-dried chitin hydrogel showed the sharp two peaks at 2 = 9 degree and 2 = 19 degree, indicating the presence of crystalline structure of chitin in the samples. It might be explained that by doing solvent exchange from water to ethanol, ethanol did not disrupt the crystalline region of chitin gel and existed in the amorphous region of chitin gel. After the ethanol-saturated chitin gel were dryied, ethanol was removed from the chitin gel and, as a consequence, there was no much change in crystalline structure of the chitin samples. In case of DMFsaturated and DMSO-saturated chitin, the peaks at 2 = 19 were slightly broaden when compared with that of chitin hydrogel. It might be implied that DMF-saturated and DMSOsaturated chitin had lower crystallinity than the original chitin hydrogel, indicating the disruption of hydrogen bonding interaction within the crystalline region of chitin gel samples by DMSO and DMF. FT-IR spectra of chitin hydrogel and organic solvent-saturated chitin gel are shown in Figure 1 (b).the characteristic peaks of chitin at 1550, 1650 and 3100 cm -1 were observed for all samples. These peaks refer to functional groups and chemical bonding in chitin. First, the peak at 1650 cm -1 refers to the amide I or C=O in the structure of acetmido group of chitin. Second, the peaks at 1550 and 3100 cm -1 refer to N-H bonding. They might be concluded that did not make a change to the chemical structure of chitin. on Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 4

5 a b) 9 19 Ethanol/Chitin hydrogel Chitin hydrogel DMF/Chitin hydrogel DMSO/Chitin hydrogel Figure 1. a.) XRD spectra of oven-dried organic solvent-saturated chitin b.) FT-IR spectra of oven-dried organic solvent-saturated chitin. B. Characterization of degraded chitin Degradation of organic solvent-saturated chitin gel by solution plasma treatment was investigated. From XRD results, DMSO-saturated and DMF-saturated chitin gel had less crystallinity than ethanol-saturated chitin gel and original chitin hydrogel. However, for degradation of DMSO-saturated chitin gel by solution plasma treatment in dilute KCl solution, the discharge of plasma became unstable after 2 hr of plasma treatment time. Accordingly, DMF-saturated chitin gel was selected for the further study on degradation by solution plasma treatment. a) b) Figure 2 a.) Production yield of water-soluble degraded chitin b.) Weight-average molecular weight of water-soluble degraded chitin The weight-average molecular weight of water-soluble degraded chitin was determined by gel permeation chromatography (GPC). The GPC result is shown in Figure 2 (b). The weight-average molecular weight of water-soluble degraded chitin was in the range of Da Or equivalent to degree of polymerization of 5 7 monomeric on Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 5

6 units. It was found that the weight-average molecular weight slightly increased with the increasing of solution plasma treatment time. It might be explained that at the beginning of plasma treatment chitin existing in the amorphous region was firstly degraded. At the prolonged plasma treatment time, chitin in the crystalline region in chitin gel was gradually degraded and dissolved into the solution. Under this phenomenon, the portion of higher molecular weight of water-soluble chitin became larger with the longer plasma treatment time. The XRD spectra of freeze-dried chitin gel remaining after degradation as a function of plasma treatment time are shown in Figure 3 (a). It was found that the crystallinity of chitin samples decreased with the increasing of solution plasma treatment time, indicating the degradation of chitin existing in the crystalline region by the action of active species generated the discharge of plasma. According to Figure 2 (a), the maximum production yield of water-soluble chitin was 45% on dry weight basis of chitin in the DMF-saturated chitin gel. a.) b.) DOH NH OH6 H1 OH3 H2-H6 H GlcNAc Figure 3 a.) XRD spectra of water-insoluble degraded chitin gel after plasma treatment as a function of plasma treatment time. b.) 1 H-NMR of water-soluble degraded chitin. 1 H-NMR of water-soluble degraded chitin is shown in Figure 3 (b). The characteristic peaks of chitin were observed at the chemical shift of 1.95, 5.73, 5.99, 6.09 and 8.28 ppm referring to hydrogen of methyl group in GlcNAc unit, OH3, H1, OH6 and NH, respectively and the range of ppm corresponding to H2-H6 in pyranose ring. It was confirmed by the 1 H-NMR analysis that the water-soluble degraded chitin obtained by solution plasma treatment was chitin/chitosan. Degree of deacetylation of degraded on Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 6

7 chitin was calculated by using integral intensity of H GluNAc and H2-H6 (Hirai et al., 1991) and it was equal to %. CONCLUSIONS Chitin hydrogel, an amorphous form of chitin, can be prepared via the dissolution of chitin powder in CaCl 2 -saturated methanol, followed by precipitation of chitin hydrogel after the addition of chitin solution into a large amount of water. Water in chitin hydrogel was replaced by different organic solvents, i.e. DMSO, DMF and ethanol, in order to disrupt the remaining crystalline region and facilitate the degradation of chitin gel by solution plasma treatment. DMF was found to be the most effective organic solvent in terms of plasma stability and the lowest crystallinity of chitin gel. The appreciable production yield of water-soluble chitin having molecular weight of approximately 1,500 Da was around 45% on the dry weight basis of chitin gel obtained at the plasma treatment time of 720 min. It could be concluded that solution plasma is an effective tool for production of water-soluble chitin from degradation of amorphous chitin gel saturated in DMF. ACKNOWLEDGEMENTS This thesis work was financially supported by the Government Budget for the year 2016 and the NU-PPC Plasma Chemical Technology Laboratory, Chulalongkorn University. REFERENCES Hirai, A., Odani, H., and Nakajima, A. (1991). Polymer Bulletin, 26(1), Jang, M., Kong, B., Jeong, Y., Lee, C., H., & Nah, J.. (2004). Journal of Polymer Science Part A: Polymer Chemistry, 42 (14), Karagozlu, M.Z. and Kim, S.,K. (2015) Handbook of Anticancer Drugs from Marine Origin. pp , Springer. Kazami, N., Sakaguchi, M., Mizutani, D., Masuda, T., Wakita, S., Oyama, F., Kawakita, M., and Sugahara, Y. (2015). Carbohydrate Polymers, 132, Qin, C., Dua, Y., Xiao, L., Li, Z., and Gao, X. (2002). International Journal of Biological Macromolecules, 31, Rinaudo, M. (2006). Progress in Polymer Science, 31 (7), Šimůnek, J., Brandysová, V., and Koppová, I. (2012) Folia microbiologica, 57 (4), Tamura, H., Nagahama, H., and Tokura, S. (2006) Cellulose, 13, Watthanaphanit, A. and Saito, N. (2013). Polymer degradation and stability, 98(5), Zhao, L., Chen, Y., Ren, S., Han, Y., and Cheng, H. (2010). Carbohydr Res, 345(5), on Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 7