Materials Science and Engineering C 33 (2013) 72–77
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Bioactive cotton fabrics containing chitosan and biologically active substances extracted from plants G. Mocanu a,⁎, M. Nichifor a, D. Mihai a, L.C. Oproiu b a b
« Petru Poni » Institute of Macromolecular Chemistry 700487 Iasi, Romania National Research & Development Institute for Chemistry & Petrochemistry, Bucharest, Romania
a r t i c l e
i n f o
Article history: Received 14 December 2011 Received in revised form 9 July 2012 Accepted 7 August 2012 Available online 17 August 2012 Keywords: Polysaccharides Chitosan Bioactive textiles Antioxidant activity
a b s t r a c t The paper studies the obtaining of bioactive textiles using chitosan-coated fabrics, in which biologically active substances contained by Viola Tricolor (VT) – an extract of three Viola species (Violaceae) – were immobilized. Chitosan was applied on cotton fabric or on chemically modified cotton (having reactive –CHO or carboxymethyl groups), as tripolyphosphate (TPP) crosslinked fine particles, or by use of glutaraldehyde crosslinking agent. The amount of VT retained on the fabrics was found to depend on the procedure of chitosan application on the cotton. The obtained bioactive textiles are expected to have antioxidant activity due to the biologically active substances from VT; they can be used for obtaining clothes for people with allergies or other skin problems, assuring a controlled release of biomolecules. The study focuses on the in vitro release of VT retained on the fabrics, as well as on its antioxidant activity. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Cellulosic fibers are largely used in medicine as woven or nonwoven fabrics, bandages, wound dressing, scaffolds, sutures, masks, etc.; many commercial medical products have antimicrobial properties. Modern medical fabrics that come in contact with skin have to be biocompatible, non-allergic, non-toxic, bacteriostatic and fungistatic. For example, more environmentally friendly biofunctional textiles have been obtained through loading natural fibers (cotton, bamboo, silk) with resveratrol (extract from Polygonum cuspidatum), a natural polyphenol with antibacterial, anti-fungal, anti-inflammatory and antioxidant activities [1]; skin-friendly textiles for transdermal applications, with antimicrobial and antiallergic effects have been obtained using natural oils as bioactive substances [2]. Antioxidants as L-ascorbic acid and alpha-tocoferol, administered either systemically or topically, can play a good photo-protective role [3]. Wool functionalization with antioxidant gallate esters [4] or phenolic derivatives [5] (caffeic acid) has been reported to result in an improved antioxidant fiber activity, which could have a potential in the development of a protective device against degenerative skin diseases, including those generated by UV radiation. Chitosan, a polysaccharide containing glucosamine and Nacetylglucosamine units present a considerable interest due to its antimicrobial, antitumor and immuno-enhancive effects. These biological activities, in conjunction with its non-toxicity, biodegradability and biocompatibility, recommend the inexpensive chitosan for applications as wastewater treatment, food industry, agriculture, medicine, ⁎ Corresponding author. E-mail address: [email protected] (G. Mocanu). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.08.007
pharmaceuticals and textiles [6]. In the last years, the applications of chitosan in textiles have received great attention in numerous studies [7,8]. The major drawback of chitosan application on textiles consists in its limited durability against repeated laundering. Similarities between the chemical and molecular structures of cellulose and chitosan enable a high affinity between both polymers, the most probable cellulosechitosan intermolecular interactions are based on H-bonds and Van der Waals forces [9]; ionic and/or covalent bonds can be also formed under special conditions and with cellulose treatments. Chitosan has been applied on cotton or cellulose by a number of procedures such as: (i) physical coating of the fabrics with a chitosan solution, followed by drying at 80 °C and 140 °C [10], although some studies revealed that during thermal treatment in air, chemical interactions between the chitosan amino groups with cellulose carbonyl groups occur [11]; (ii) co-crosslinking of chitosan and cotton support with glutaraldehyde [10], or citric acid (with NaH2PO4 as a catalyst) [12], butanetetracarboxylic acid (BTCA) or formaldehyde [13], epichlorohydrine [14]; (iii) introduction of aldehyde groups onto cotton fibers through potassium periodate oxidation of C2– C4 cellulose glucose unit; chitosan is linked on these aldehyde functional groups through the formation of Schiff's base [15]. Through BTCA [16] or citric acid [17] chitosan crosslinking on cotton, the fabrics display improved durability against launderings. Natural or synthetic fibers can be also coated with silver loaded chitosan nanoparticles [18] or chitosan-based polymeric core–shell particles [19]. The extract of heartsease, Viola Tricolor (VT), has been utilized to treat various skin disorders, upper-respiratory problems, rheumatism or as diuretic, due to its anti-inflammatory, expectorant, antioxidant, diuretic properties [20], as well as to its antimicrobial activity [21]. The quantitative analysis of a 10% tincture of air-dried flowering
G. Mocanu et al. / Materials Science and Engineering C 33 (2013) 72–77
aerial parts from three Viola species showed a content of about 2.1% flavonoids, 0.92% polyphenolic carboxylic acids, and 0.09% salicylic acid — a composition that explains its biological activity [22]. Due to its flavonoid and polyphenolic carboxylic acid content, VT has a potential antioxidant activity. The present study aims at preparing bioactive textiles using chitosan for coating of cotton fabrics, as polymeric support for inclusion of biologically active substances. Chitosan has been applied on cotton fabrics or on chemically modified cotton (having aldehyde or carboxymethyl functional groups) either upon crosslinking with glutaraldehyde or as fine particles (after crosslinking with natrium tripolyphosphate). All these cotton–chitosan supports were loaded with VT biologically active substance to obtain bioactive textiles and could be used for obtaining clothes for people with allergies or other skin problems, including degenerative diseases. The in vitro release of retained VT and its antioxidant activity have been studied, to appreciate the performances in application of the obtained devices.
2. Materials and methods Cotton fabric (200 g/m2) was purified by a treatment in 0.5 M NaOH at 95 °C for 90 min to remove non-cellulose compounds and underwent oxidative bleaching by treatment with 0.1 M H2O2 pH=11.2, at 95 °C for 30 min; after each treatment the fibers were washed in distilled water until reaching water conductivity, and then air-dried. Mature cotton fiber contain essentially macromolecules with a high degree of polymerization, about 4000 [23], with a relatively narrow range of molecular weights. Medium weight chitosan (Mw: 190–310 kDa; deacetylation degree of 75–85% [24], 1,1-diphenyl-2-picrylhydrazyl (DPPH), sodium tripolyphosphate (TPP) were purchased form Aldrich, glutaraldehyde 30% aqueous solution, acid orange from Fluka, 3.5 g% Viola Tricolor (VT) solution was kindly supplied by Dr. Anca Toiu-University of Medicine and Pharmacy Iuliu Hatieganu, Faculty of Pharmacy, Cluj Napoca (Romania). 2.1. The chitosan content of the coated cotton fabric was determined by acid orange method [15]. 2.2. In vitro release of the VT retained on the fabrics was studied in solutions simulating a mean sweat composition [25] containing: 0.0613 g/L NaHCO3, 0.36 g/L KCl, 1.056 g/L potassium lactate, and 1.23 g/L NaCl. 0.05 g sample was immersed in 20 mL solution; aliquots were withdrawn at various time intervals and the drug concentration was determined by UV spectrometry at 266 and 336 nm. 2.3. The antioxidant activity of the VT treated fabric was determined through DPPH method [26], and compared with those of ascorbic acid and of the initial VT. Briefly, the samples containing the antioxidant product were immersed in 2 mL of a solution with the same composition as that used for VT release and 1 mL methanolic DPPH solution (0.128 g/L methanol); after 30 min the absorbance at 517 nm was measured. The decreased absorbance of DPPH indicates an increased antioxidant effect. The radical scavenging activity was calculated using the Eq. (1):
Scavenging effect% ¼
A0 −A1 100 A0
73
3. Experimental 3.1. Chemical modification of cotton Several procedures have been used to prepare functionalized cotton, as described below: 3.1.1. Cotton fabric was oxidized with 0.01 M KIO4 at 20 °C for 24 h; the content of the resulting aldehyde groups was determined through a reaction with hydroxylamine hydrochloride (HA), followed by titration with 0.1 N NaOH of the liberated hydrogen chloride [28]. Under the above mentioned conditions, a content of 1.05 meq CHO groups/g sample was obtained. The –CHO groups can react with the –NH2 chitosan groups, forming Schiff bases, assuring a more stable chitosan coating. 3.1.2. Cotton fabric was treated with sodium chloracetate in a basic medium, at 70 °C, in order to introduce reactive carboxymethyl groups. The degree of substitution with carboxymethyl groups determined through conductimetric titrations was of 0.05 meq/g. The presence of weakly acidic COOH groups on cotton will improve chitosan retention through electrostatic interaction forces with NH2 chitosan basic groups.
3.2. Synthesis of chitosan fine particles Fine particles of crosslinked chitosan (with the diameter ranging between 100 and 2500 nm [29]) were prepared as follows [18]: 0.5 g chitosan was dissolved in 100 mL (w/v) of 1% acetic acid solution; its pH was adjusted to 6 with 2 M NaOH solution; then, 0.15 g TPP in 100 mL water was poured dropwise into the chitosan solution, under stirring at 1000 rpm; the mixture was then stirred for additional 15 min more. Finally, the fine particles were separated by centrifugation for 1 h at 4500 rpm. Particle size distribution was determined with a Mastersizer 2000 system (version 5.31, Malvern Instruments England). 3.3. Chitosan application on cotton 3.3.1. Application of chitosan fine particles on cotton fabrics was performed by immersing cotton fabric (dimensions: 2.6/ 7.6 mm) into 1% (w/v) aqueous chitosan particle suspension, and then stirring the mixture for 24 h at room temperature; after that, the fabrics were pressed between 2 microscope slides, to control the pick-up ratio (up to 100% of weight) and dried at 100 °C up to constant weight. 3.3.2. Coating of cotton fabric with glutaraldehyde crosslinked chitosan; 0.5 g chitosan dissolved in 25 mL of a 2% acetic acid solution was mixed with 0.2 mL glutaric dialdehyde and reacted for 1 h at room temperature; weighed cotton fabrics were padded in this solution and left for 1 or 2 h; after that, the fabrics were pressed between 2 microscope slides, to control the pick-up ratio (up to 100% of weight), dried at 90 °C for 3 min and then at 130 °C for 15 min.
ð1Þ
where A0 is the absorbance of a standard DPPH that was prepared in the same conditions, and was not applied on the fabric and A1 is the absorbance of the samples. 2.4. Water uptake of the fabrics was determined through centrifugation at 2000 rpm of a weighted sample, after its swelling in water for 24 h, as reported for ion exchange resins [27].
3.4. Impregnation of fabrics with Viola Tricolor (VT) A dry, pre-weighed fabric (previously chitosan-coated as described above) was immersed for 24 h into 3.5 g% VT solution; the VT retention was determined either from weight increase of the sample, or by UV spectrometry, as a difference from its initial content, based on a calibration curve obtained for UV absorption of VT at 336 nm; the UV spectrum of VT presents two absorption maxima at 266 and 336 nm wavelength that are specific for flavonoids [20].
74
G. Mocanu et al. / Materials Science and Engineering C 33 (2013) 72–77
4. Results and discussion Besides chitosan good bioadhesive property, chitosan delivery system can provide incorporated bioactive agents. Recently, there has been growing interest in delivery systems prepared by ionic gelation between the protonated amino groups of chitosan and anions, such as tripolyphosphate (TPP), because of their very simple and mild preparation conditions, homogeneous particle sizes, and bioadhesive properties. TPP is a non-toxic and multivalent anion that can form crosslinks by ionic interaction between positively charged amino groups of chitosan and multivalent negatively charged TPP molecules [30]. The study aims to investigate the use of chitosan nanoparticles for textile applications as chitosan in nano form is highly active because of very high surface area to volume ratio and is expected to include higher amounts of biologically active substances. Crosslinking with GA of chitosan applied on cotton can improve the durability against repeated laundering; binary aldehyde GA can react not only with NH2 chitosan groups forming Schiff bases, but also with cotton OH groups [10], forming acetal bonds. Unfortunately, GA presents an acute toxicity that has been investigated in many studies [31,32]. The two procedures of cotton coating with TPP crosslinked nanoparticles or GA crosslinked chitosan were used to appreciate their efficiency in obtaining of a more appropriate support for biologically active VT. The size distribution of chitosan fine particles obtained through TPP crosslinking is presented in Fig. 1; a bimodal distribution is observed, with peak maxima at about 1 and 100 μm. The presence of chitosan on cotton fabric was proved by FTIR and SEM spectroscopies. The FTIR spectra of chitosan-coated cotton (Fig. 2) present bands characteristic of the CO amide group around 1650 cm −1 (1652; 1646; 1647 cm −1, respectively) and of the NH amide group at about 1550 cm −1 (1563; 1558; 1559 cm −1, respectively); besides, the presence of these bands in the chitosan spectrum is mentioned in literature [33,34]. Also, bands at around 1420 cm −1 (characteristic for CH amide II [33]) and around 1320 cm −1 (characteristic for –OH, –NH2, –CO groups [34]) appear. The ratio of the bands at about 1320 cm−1 and 1420 cm−1 (the first present in N-acetyl D-glucosamine, the second present in N-acetyl D-glucosamine and glucosamine) was used to calculate the degree of acetylation of chitin and chitosan [34]. The imine –C= N groups (possible formed during GA crosslinking of chitosan or as result of –CHO reaction of periodate oxidized cotton with chitosan) have characteristic bands around 1640 cm −1 [35]–1680 cm −1 [10]; they are difficult to be evidenced due their possible overlapping with the amide characteristic band (1650 cm −1). Spectrum 4 (sample VT-3) presents additional bands around 1100–1200 cm −1, which can be attributed to P = O stretching
Fig. 1. Particles size distribution.
Fig. 2. FTIR spectra of cotton (1) and chitosan coated cotton (2 —sample VT-4; 3 — sample VT-8; 4 — sample VT-3).
[36,18]; the broadening of the band at 1554 cm −1 can be attributed to the electrostatic interactions between amine groups of the chitosan and carboxylic groups of the support [37,38]. The band at 1161 cm−1 can be attributed to the interactions between amine chitosan and phosphate groups, as reported for the chitosan–chondroitinsulfate interpolymeric complex [39]. SEM photos were performed on a TESLA BS 301 apparatus, 100 mag, low vacuum; chitosan-coated cotton shows an uniform coating of the fabrics either with glutaraldehyde crosslinked chitosan (Fig. 3a) or with TPP crosslinked chitosan fine particles (Fig. 3b). The amount of chitosan retained on cotton depends on the coating method used. Cotton fabrics containing functional reactive –CHO or –COOH groups retain higher amounts of TPP crosslinked chitosan fine particles than unmodified cotton (samples 1–3, Table 1). This confirms our initial hypothesis that chitosan particles will be retained on modified cotton both by physical inclusion and by chemical (or electrostatic) bonds. Also, chitosan retention through GA co-crosslinking of the cotton previously modified with reactive functional groups is higher than that obtained for unmodified cotton (samples 4–9, Table 1). In this case, one can assume the formation of chemical bonds between reactive functional groups of cotton and the amino chitosan groups, besides co-crosslinking due to GA. Chitosan is retained in higher amounts on KIO4 oxidized cotton which contains more reactive groups (1.05 meq –CHO reactive groups/g fabric) than on carboxymethyl cotton (0.05 meq –COOH groups/g fabric). The amount of VT retained on the obtained supports depends on the amount of the chitosan coating of the fabrics (Table 1). Chitosan itself retains an important amount of VT, while on the cotton fabrics, it is retained in very small amounts (Table 1). Related to the amount of chitosan (deposed/g cotton), VT is retained in higher amounts on TPP crosslinked chitosan-coated cotton, probably due to a more efficient inclusion into the particle network. Thus, the TPP crosslinked-coated cotton retains the same VT amount as chitosan itself, while GA crosslinked chitosan retains a smaller VT amount than chitosan itself. The samples VT4–VT-6 with approximately the same chitosan content as the corresponding samples VT-7–VT-9 retain more VT because of their less crosslinked structure, obtained through a shorter crosslinking duration, which facilitates the VT diffusion inside the network. The release of the VT retained on the cotton fabrics is presented in Figs. 4 and 5. The differences as to the VT release rate among the obtained supports can be explained by the difference in their hydrophilicity (Fig. 6); a higher release rate is observed for the more hydrophilic cotton coated with TPP crosslinked chitosan (samples VT-1–VT-3, Table 1); the most hydrophilic one – VT-1 – based on unfunctionalized
G. Mocanu et al. / Materials Science and Engineering C 33 (2013) 72–77
75
Fig. 3. SEM photos of the chitosan coated cotton (sample VT-5) (a) and of the cotton treated with TPP crosslinked chitosan fine particles (sample VT-2) (b).
Table 1 Application procedures of chitosan on cotton; chitosan and VT content of the fabrics. Sample
VT-1 VT-2 VT-3 VT-4 VT-5 VT-6 VT-7 VT-8 VT-9 Cotton KIO4 oxidized cotton Carboxy Methylated cotton Chitosan ⁎ ⁎⁎
Procedure
g chitosan/g cotton, calculated from:
TPP crosslinked chitosan deposed on cotton TPP crosslinked chitosan deposed on cotton oxidized with KIO4 TPP crosslinked chitosan deposed on carboxymethylated cotton GA crosslinked chitosan (for 1 h) deposed on cotton GA crosslinked chitosan (for 1 h) deposed on cotton oxidized with KIO4 GA crosslinked chitosan (for 1 h) deposed on carboxymethylated cotton GA crosslinked chitosan (for 2 h) deposed on cotton GA crosslinked chitosan (for 2 h) deposed on cotton oxidized with KIO4 GA crosslinked chitosan (for 2 h) deposed on carboxymethylated cotton –
–
acid orange retention⁎
yield
0.014 0.038 0.029 0.040 0.060 0.052 0.049 0.060 0.059 0 0
0.0163 0.0395 0.0288 0.043 0.062 0.051 0.059 0.065 0.060 –
0 0.217⁎⁎
–
g VT/g (chitosan–cotton system)⁎
g VT/g chitosan
0.0167 0.045 0.037 0.038 0.045 0.043 0.034 0.0357 0.040 b0.01
1.02 1.15 1.27 0.88 0.73 0.84 0.58 0.55 0.67
1.14
Values represent a mean of three measurements that deviated with ±0.5%. Acid orange retention/g chitosan.
cotton presents the highest VT release rate. In the case of the cotton coated with GA crosslinked chitosan (Fig. 5), the samples that have been crosslinked for 2 h (VT-7–VT-9, more crosslinked, less hydrophilic), present a lower release rate than those crosslinked for 1 h (VT-4–VT-6). It should be mentioned that the VT release was studied at a solution/ tested fabric ratio of 20 mL/0.05 g; if the fabric is used for bioactive
Fig. 4. In vitro VT release from VT-1–VT-3 samples, in solutions simulating a mean sweat composition; each value is a mean of three measurements that deviated with ±3%.
dressings, the ratio between sweat and fabrics will be much lower, hence one can appreciate that the VT release rate will be much slower. VT is a plant extract; hence, presumably, some of its components (especially the flavonoids and polyphenolic carboxylic acids) possess antioxidant activity. The main characteristic of an antioxidant is its ability to trap free radicals. Highly reactive free radicals and oxygen species are present in biological systems from a wide variety of sources. These free radicals may oxidize nucleic acids, proteins, lipids or DNA and can initiate degenerative diseases. Antioxidant compounds like phenolic acids, polyphenols and flavonoids scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl and thus inhibit the oxidative mechanisms that lead to degenerative diseases. In order to assess the antioxidant activity of VT, its scavenging effect was studied, comparatively with a known antioxidant, ascorbic acid. The results presented in Fig. 7 show that VT presents an antioxidant effect, but only when it is used in higher concentrations than ascorbic acid. Thus, if a scavenging effect of about 50% is attained for approximately 70 μg ascorbic acid/mL, the same scavenging effect is obtained for about 2 mg VT/mL (a concentration of about 28 times higher); yet, this antioxidant activity can be considered high enough for application domains of bioactive textiles. In vitro studies of the antioxidant activity of wool grafted either with gallate esters [4] or with antioxidant phenolic molecules [5] showed that this novel approach to wool functionalization could have a potential in the development of a protective device against degenerative skin diseases.
76
G. Mocanu et al. / Materials Science and Engineering C 33 (2013) 72–77
Fig. 5. In vitro VT release from VT-4–VT-9 samples, in solutions simulating a mean sweat composition; each value is a mean of three measurements that deviated with ±3%.
In vitro studies on the antioxidant activity of chitosan-coated cotton containing VT were performed; the attempts to determine the antioxidant activity in pure water failed, hence, it may be concluded that the biologically active substances are released only by varying the ionic strength of the environment. The studies were also carried out in solutions simulating a mean sweat composition. Comparatively with the initial VT solution, the chitosan-coated cotton samples containing VT present a lower scavenging effect, probably due to the slower release of the antioxidant substances retained on the fabrics (Figs. 8, 9). The measurements of the free radical scavenging activity of chitosan-coated cotton showed no effect. The active hydroxyl and amino groups in the polymer chains, able to react with free radicals to form stable macroradicals, can be the origin of the scavenging ability; but, according to the reports of Alexandrova et al. [40] the antioxidant activity of chitosan was almost equal to zero. Only at very high concentrations can chitosan scavenge superoxide anions. This may be related to the formation of strong intermolecular and intra‐molecular hydrogen bonds that inhibited the reactivity of hydroxyl and amino groups in the polymer chains [41]. Some authors reported an important antioxidant activity for Low Molecular Weight (LWM) chitosan [42] and LMW and soluble Medium Molecular Weight chitosan with a 98.50% N-deacetylation [43]. Some studies have been demonstrated that molecular weights, as well as the degree of deacetylation, are both potent determinants of action of LMW chitosans [44]. LMW chitosans with a relatively higher degree of deacetylation and lower MW were found to have higher radical scavenging activities on DPPH, hydroxyl and superoxide radicals [45]. High MW chitosans have compact structures, thus making the overall effect of their intra‐molecular hydrogen bonds stronger. The strong effect of intra-molecular hydrogen bonding decreases the reactivity of hydroxyl and amino groups. On the
Fig. 6. Water uptake of VT samples (VT-0 represents uncoated cotton fabric).
Fig. 7. Scavenging effect of VT comparatively with ascorbic acid.
contrary, low molecular weight chitosan has a less compact structure, thus making the overall effect of intra-molecular hydrogen bonding less effective. In our experiments, the scavenging activity of the samples of crosslinked chitosan deposed on cotton, measured at the same concentration as the samples having VT, was almost zero (results are in concordance with the above mentioned literature data [40–43]; hence all antioxidant activity determined was produced by the VT included into the chitosan-coated supports. From the presented data one can see that introduction of reactive groups on cotton through reaction with KIO4 or sodium chloroacetate leads to an increase of the amount of chitosan retained on the cotton and consequently of the VT amount. The use of TPP as crosslinking agent is more advantageous from the viewpoint of lack of the toxicity; also, the TPP-crosslinked chitosan retains higher amounts of VT which is released faster in vitro. GA, crosslinking agent frequently used for chitosan, presents an acute toxicity; chitosan GA crosslinked deposed on cotton (or cotton activated) support retains important amounts of VT, which is released at a smaller rate than TPP crosslinked chitosan. However, the procedure of coating of cotton with GA crosslinked chitosan is less complicated than coating with TPP crosslinked cotton.
5. Conclusions Bioactive textiles were obtained by coating of cotton with chitosan crosslinked with TPP, or GA, followed by loading with biologically active substances contained by VT; the amount of entrapped VT and its release rate can be varied through an appropriate choice of
Fig. 8. Scavenging effect of samples VT-1–VT-3, comparatively with VT.
G. Mocanu et al. / Materials Science and Engineering C 33 (2013) 72–77 [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] Fig. 9. Scavenging effect of samples VT-4–VT-9, comparatively with VT.
the chitosan treated supports. As mentioned, each support presents advantages and disadvantages. The TPP crosslinked cotton, deposed on previously functionalized support retain higher amount of VT (reported to the chitosan content), which is released faster; the GA crosslinked chitosan deposed also on previously functionalized support retains important amounts of VT, which is released gradually. The choice between the presented supports will be performed after subsequent in vivo studies for topical applications. The bioactive textiles obtained could be used for obtaining clothes for people with allergies or other skin problems, due to the specific biological activity of VT, whose antioxidant properties have been proven. The bioactive cotton fabrics are promising innovative biomaterials for medical devices, such as bioactive dressings, wound healing isolation materials and due to their antioxidant properties could have a potential in the development of a protective device against degenerative diseases. References [1] E. Pinho, M. Henriques, R. Oliveira, A. Dias, G. Soares, Fibers Polym. 11 (2) (2010) 271–276. [2] H. Haufe, K. Muschter, J. Siegert, H. Böttcher, J. Sol-Gel Sci. Technol. 45 (2008) 97–101. [3] D. Bonamonte, C. Foti, N. Lionetti, L. Rigano, G. Angelini, Annal. It. Dermat. Allerg. Clin. Speriment. 63 (1) (2009) 10–27. [4] Kh.M. Gaffar Hossain, M. Díaz González, J.M. Dagá Monmany, T. Tzanov, J. Mol. Catal. B: Enzym. 67 (2010) 231–235. [5] S. Jus, V. Kokol, G.M. Guebitza, Enzyme Microb. Technol. 42 (2008) 535–542.
[21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36]
[37] [38] [39] [40] [41] [42] [43] [44] [45]
77
M.N.V. Ravi Kumar, React. Funct. Polym. 46 (2000) 1–27. Y. Gao, R. Cranston, Text. Res. J. 78 (1) (2008) 60–72. D. Knittel, E. Schollmeyer, Lenzinger Berichte. 85 (2006) 124–130. S. Strnad, O. Šauperl, L. Fras-Zemljič, Biopolymers 9 (2010) 181–200. Z. Zhang, L. Chen, J. Ji, Y. Huang, D. Chen, Text. Res. J. 73 (12) (2003) 1103–1106. J. Martinez Urreaga, M.U. de la Orden, Eur. Polym. J. 42 (10) (2006) 2606–2616. D. Alonso, M. Gimeno, R. Olayo, H. Vazquez-Torres, J.D. Sepulveda-Sanchez, K. Shirai, Carbohydr. Polym. 77 (3) (2009) 536–543. K.F. El-tahlawy, M.A. El-bendary, A.G. Elhendawy, S.M. Hudson, Carbohydr. Polym. 60 (4) (2005) 421–430. S.H. Lee, M.J. Kim, H. Park, J. Appl. Polym. Sci. 117 (2) (2010) 623–628. L.F. Zemljic, S. Strand, O. Sauperl, K. Stana-Kelinschek, Text. Res. J. 79 (2009) 219–226. A. Hebeish, F.A. Abdel-Mohdy, M.G. Fouda, Z. Elsaid, S. Essam, G.H. Tammam, E.A. Drees, Carbohydr. Polym. 86 (2011) 1684–1691. X. Fua, Y. Shena, X. Jianga, Y. Yana, Carbohydr. Polym. 85 (2011) 221–227. Ali S. Wazed, S. Rajendran, M. Joshi, Carbohydr. Polym. 83 (2) (2011) 438–446. W. Ye, J.H. Xin, P. Li, K.-L.D. Lee, T.-L. Kwong, J. Appl. Polym. Sci. 102 (2006) 1787–1793. V. Vukics, A. Kery, G.K. Bonn, A. Guttman, Anal. Bioanal. Chem. 390 (2008) 1917–1925. E.W. Banaszczak, W. Bylka, I. Matyawska, O. Goślińska, Z. Muszyński, Fitoterapia 76 (2005) 458–461. A. Toiu, L. Vlase, I. Oniga, M. Tamas, Farmacia LVI (4) (2008) 440–444. Kh.U. Usmanov, T.I. Sushkevitch, J. Polym. Sci. 58 (1962) 1325–1331. Z. Hou, M. Zhang, B. Liu, Q. Yan, F. Yuan, D. Xu, Y. Gao, Food Hydrocol. 26 (2012) 205–211. M.J. Patterson, S.D. Galloway, M.A. Nimmo, Exp. Physiol. 85 (6) (2000) 869–875. N. Ara, H. Nur, Res. J. Med. Sci. 4 (1) (2009) 107–110. K. Pepper, D. Reichenberg, D.K. Hale, J. Chem. Soc. (1952) 3129–3136. D. Bruneel, E. Schacht, Polymer 34 (12) (1993) 2628–2631. C. Buzea, I.I. Pacheco, K. Robbie, Biointerphases 2 (4) (2007) MR17–MR71. A.K. Anal, W.F. Stevens, C. Remunan-Lopez, Int. J. Pharm. 312 (2006) 166–173. L. Sano, A.M. Krueger, P.F. Landrum, Aquat. Toxicol. 71 (2005) 283–296. T. Takigawa, I. Endo, J. Occup. Health 48 (2006) 75–87. E.A. El-Hefian, M.M. Nasef, A.H. Yahaya, E-J. Chem. 9 (3) (2012) 1431–1439. J. Brugnerotto, J. Lizardi, F.M. Goycoolea, W. ArguÈelles-Monal, J. Desbrieres, M. Rinaudo, Polymer 42 (2001) 3569–3580. B. Hoffman, D. Seitz, A. Mencke, A. Kokott, G. Ziegler, J. Mater. Sci. Mater. Med. 20 (7) (2009) 1495–1503. J.R. Azevedo, R.H. Sizilio, M.B. Brito, A.M.B. Costa, M.R. Serafini, A.A.S. Araújo, M.R.V. Santos, A.A.M. Lira, R.S. Nunes, J. Therm. Anal. Calorim. 106 (2011) 685–689. M.H. Lee, M.K. Chun, H.K. Choi, Arch. Pharm. Res. 31 (7) (2008) 932–937. L. Yan, F. Qian, Q. Zhu, Polym. Int. 50 (2001) 1370–1374. G. Kaur, V. Rana, S. Jain, A.K. Tiwary, AAPS Pharm. Sci. Technol. 11 (1) (2010) 36–45. V.A. Alexandrova, G.V. Obukhova, N.S. Domnina, D.A. Topchiev, Macromol. Symp. 144 (1) (1999) 413–422. T. Sun, W. Xie, P. Xu, Macromol. Biosci. 3 (6) (2003) 320–323. T. Yasufuku, M. Anraku, Y. Kondo, T. Hata, J. Hirose, N. Kobayashi, H. Tomida, Pharmaceutics 2 (2010) 245–257. P.J. Chien, F. Sheu, W.T. Huang, M.-S. Su, Food Chem. 102 (2007) 1192–1198. S.K. Kim, N. Rajapakse, Carbohydr. Polym. 62 (2005) 357–368. W. Xie, P. Xu, Q. Liu, Bioorg. Med. Chem. Lett. 11 (2001) 1699–1701.
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Bioactive cotton fabrics containing chitosan and biologically active substances extracted from plants G. Mocanu a,⁎, M. Nichifor a, D. Mihai a, L.C. Oproiu b a b
« Petru Poni » Institute of Macromolecular Chemistry 700487 Iasi, Romania National Research & Development Institute for Chemistry & Petrochemistry, Bucharest, Romania
a r t i c l e
i n f o
Article history: Received 14 December 2011 Received in revised form 9 July 2012 Accepted 7 August 2012 Available online 17 August 2012 Keywords: Polysaccharides Chitosan Bioactive textiles Antioxidant activity
a b s t r a c t The paper studies the obtaining of bioactive textiles using chitosan-coated fabrics, in which biologically active substances contained by Viola Tricolor (VT) – an extract of three Viola species (Violaceae) – were immobilized. Chitosan was applied on cotton fabric or on chemically modified cotton (having reactive –CHO or carboxymethyl groups), as tripolyphosphate (TPP) crosslinked fine particles, or by use of glutaraldehyde crosslinking agent. The amount of VT retained on the fabrics was found to depend on the procedure of chitosan application on the cotton. The obtained bioactive textiles are expected to have antioxidant activity due to the biologically active substances from VT; they can be used for obtaining clothes for people with allergies or other skin problems, assuring a controlled release of biomolecules. The study focuses on the in vitro release of VT retained on the fabrics, as well as on its antioxidant activity. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Cellulosic fibers are largely used in medicine as woven or nonwoven fabrics, bandages, wound dressing, scaffolds, sutures, masks, etc.; many commercial medical products have antimicrobial properties. Modern medical fabrics that come in contact with skin have to be biocompatible, non-allergic, non-toxic, bacteriostatic and fungistatic. For example, more environmentally friendly biofunctional textiles have been obtained through loading natural fibers (cotton, bamboo, silk) with resveratrol (extract from Polygonum cuspidatum), a natural polyphenol with antibacterial, anti-fungal, anti-inflammatory and antioxidant activities [1]; skin-friendly textiles for transdermal applications, with antimicrobial and antiallergic effects have been obtained using natural oils as bioactive substances [2]. Antioxidants as L-ascorbic acid and alpha-tocoferol, administered either systemically or topically, can play a good photo-protective role [3]. Wool functionalization with antioxidant gallate esters [4] or phenolic derivatives [5] (caffeic acid) has been reported to result in an improved antioxidant fiber activity, which could have a potential in the development of a protective device against degenerative skin diseases, including those generated by UV radiation. Chitosan, a polysaccharide containing glucosamine and Nacetylglucosamine units present a considerable interest due to its antimicrobial, antitumor and immuno-enhancive effects. These biological activities, in conjunction with its non-toxicity, biodegradability and biocompatibility, recommend the inexpensive chitosan for applications as wastewater treatment, food industry, agriculture, medicine, ⁎ Corresponding author. E-mail address: [email protected] (G. Mocanu). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.08.007
pharmaceuticals and textiles [6]. In the last years, the applications of chitosan in textiles have received great attention in numerous studies [7,8]. The major drawback of chitosan application on textiles consists in its limited durability against repeated laundering. Similarities between the chemical and molecular structures of cellulose and chitosan enable a high affinity between both polymers, the most probable cellulosechitosan intermolecular interactions are based on H-bonds and Van der Waals forces [9]; ionic and/or covalent bonds can be also formed under special conditions and with cellulose treatments. Chitosan has been applied on cotton or cellulose by a number of procedures such as: (i) physical coating of the fabrics with a chitosan solution, followed by drying at 80 °C and 140 °C [10], although some studies revealed that during thermal treatment in air, chemical interactions between the chitosan amino groups with cellulose carbonyl groups occur [11]; (ii) co-crosslinking of chitosan and cotton support with glutaraldehyde [10], or citric acid (with NaH2PO4 as a catalyst) [12], butanetetracarboxylic acid (BTCA) or formaldehyde [13], epichlorohydrine [14]; (iii) introduction of aldehyde groups onto cotton fibers through potassium periodate oxidation of C2– C4 cellulose glucose unit; chitosan is linked on these aldehyde functional groups through the formation of Schiff's base [15]. Through BTCA [16] or citric acid [17] chitosan crosslinking on cotton, the fabrics display improved durability against launderings. Natural or synthetic fibers can be also coated with silver loaded chitosan nanoparticles [18] or chitosan-based polymeric core–shell particles [19]. The extract of heartsease, Viola Tricolor (VT), has been utilized to treat various skin disorders, upper-respiratory problems, rheumatism or as diuretic, due to its anti-inflammatory, expectorant, antioxidant, diuretic properties [20], as well as to its antimicrobial activity [21]. The quantitative analysis of a 10% tincture of air-dried flowering
G. Mocanu et al. / Materials Science and Engineering C 33 (2013) 72–77
aerial parts from three Viola species showed a content of about 2.1% flavonoids, 0.92% polyphenolic carboxylic acids, and 0.09% salicylic acid — a composition that explains its biological activity [22]. Due to its flavonoid and polyphenolic carboxylic acid content, VT has a potential antioxidant activity. The present study aims at preparing bioactive textiles using chitosan for coating of cotton fabrics, as polymeric support for inclusion of biologically active substances. Chitosan has been applied on cotton fabrics or on chemically modified cotton (having aldehyde or carboxymethyl functional groups) either upon crosslinking with glutaraldehyde or as fine particles (after crosslinking with natrium tripolyphosphate). All these cotton–chitosan supports were loaded with VT biologically active substance to obtain bioactive textiles and could be used for obtaining clothes for people with allergies or other skin problems, including degenerative diseases. The in vitro release of retained VT and its antioxidant activity have been studied, to appreciate the performances in application of the obtained devices.
2. Materials and methods Cotton fabric (200 g/m2) was purified by a treatment in 0.5 M NaOH at 95 °C for 90 min to remove non-cellulose compounds and underwent oxidative bleaching by treatment with 0.1 M H2O2 pH=11.2, at 95 °C for 30 min; after each treatment the fibers were washed in distilled water until reaching water conductivity, and then air-dried. Mature cotton fiber contain essentially macromolecules with a high degree of polymerization, about 4000 [23], with a relatively narrow range of molecular weights. Medium weight chitosan (Mw: 190–310 kDa; deacetylation degree of 75–85% [24], 1,1-diphenyl-2-picrylhydrazyl (DPPH), sodium tripolyphosphate (TPP) were purchased form Aldrich, glutaraldehyde 30% aqueous solution, acid orange from Fluka, 3.5 g% Viola Tricolor (VT) solution was kindly supplied by Dr. Anca Toiu-University of Medicine and Pharmacy Iuliu Hatieganu, Faculty of Pharmacy, Cluj Napoca (Romania). 2.1. The chitosan content of the coated cotton fabric was determined by acid orange method [15]. 2.2. In vitro release of the VT retained on the fabrics was studied in solutions simulating a mean sweat composition [25] containing: 0.0613 g/L NaHCO3, 0.36 g/L KCl, 1.056 g/L potassium lactate, and 1.23 g/L NaCl. 0.05 g sample was immersed in 20 mL solution; aliquots were withdrawn at various time intervals and the drug concentration was determined by UV spectrometry at 266 and 336 nm. 2.3. The antioxidant activity of the VT treated fabric was determined through DPPH method [26], and compared with those of ascorbic acid and of the initial VT. Briefly, the samples containing the antioxidant product were immersed in 2 mL of a solution with the same composition as that used for VT release and 1 mL methanolic DPPH solution (0.128 g/L methanol); after 30 min the absorbance at 517 nm was measured. The decreased absorbance of DPPH indicates an increased antioxidant effect. The radical scavenging activity was calculated using the Eq. (1):
Scavenging effect% ¼
A0 −A1 100 A0
73
3. Experimental 3.1. Chemical modification of cotton Several procedures have been used to prepare functionalized cotton, as described below: 3.1.1. Cotton fabric was oxidized with 0.01 M KIO4 at 20 °C for 24 h; the content of the resulting aldehyde groups was determined through a reaction with hydroxylamine hydrochloride (HA), followed by titration with 0.1 N NaOH of the liberated hydrogen chloride [28]. Under the above mentioned conditions, a content of 1.05 meq CHO groups/g sample was obtained. The –CHO groups can react with the –NH2 chitosan groups, forming Schiff bases, assuring a more stable chitosan coating. 3.1.2. Cotton fabric was treated with sodium chloracetate in a basic medium, at 70 °C, in order to introduce reactive carboxymethyl groups. The degree of substitution with carboxymethyl groups determined through conductimetric titrations was of 0.05 meq/g. The presence of weakly acidic COOH groups on cotton will improve chitosan retention through electrostatic interaction forces with NH2 chitosan basic groups.
3.2. Synthesis of chitosan fine particles Fine particles of crosslinked chitosan (with the diameter ranging between 100 and 2500 nm [29]) were prepared as follows [18]: 0.5 g chitosan was dissolved in 100 mL (w/v) of 1% acetic acid solution; its pH was adjusted to 6 with 2 M NaOH solution; then, 0.15 g TPP in 100 mL water was poured dropwise into the chitosan solution, under stirring at 1000 rpm; the mixture was then stirred for additional 15 min more. Finally, the fine particles were separated by centrifugation for 1 h at 4500 rpm. Particle size distribution was determined with a Mastersizer 2000 system (version 5.31, Malvern Instruments England). 3.3. Chitosan application on cotton 3.3.1. Application of chitosan fine particles on cotton fabrics was performed by immersing cotton fabric (dimensions: 2.6/ 7.6 mm) into 1% (w/v) aqueous chitosan particle suspension, and then stirring the mixture for 24 h at room temperature; after that, the fabrics were pressed between 2 microscope slides, to control the pick-up ratio (up to 100% of weight) and dried at 100 °C up to constant weight. 3.3.2. Coating of cotton fabric with glutaraldehyde crosslinked chitosan; 0.5 g chitosan dissolved in 25 mL of a 2% acetic acid solution was mixed with 0.2 mL glutaric dialdehyde and reacted for 1 h at room temperature; weighed cotton fabrics were padded in this solution and left for 1 or 2 h; after that, the fabrics were pressed between 2 microscope slides, to control the pick-up ratio (up to 100% of weight), dried at 90 °C for 3 min and then at 130 °C for 15 min.
ð1Þ
where A0 is the absorbance of a standard DPPH that was prepared in the same conditions, and was not applied on the fabric and A1 is the absorbance of the samples. 2.4. Water uptake of the fabrics was determined through centrifugation at 2000 rpm of a weighted sample, after its swelling in water for 24 h, as reported for ion exchange resins [27].
3.4. Impregnation of fabrics with Viola Tricolor (VT) A dry, pre-weighed fabric (previously chitosan-coated as described above) was immersed for 24 h into 3.5 g% VT solution; the VT retention was determined either from weight increase of the sample, or by UV spectrometry, as a difference from its initial content, based on a calibration curve obtained for UV absorption of VT at 336 nm; the UV spectrum of VT presents two absorption maxima at 266 and 336 nm wavelength that are specific for flavonoids [20].
74
G. Mocanu et al. / Materials Science and Engineering C 33 (2013) 72–77
4. Results and discussion Besides chitosan good bioadhesive property, chitosan delivery system can provide incorporated bioactive agents. Recently, there has been growing interest in delivery systems prepared by ionic gelation between the protonated amino groups of chitosan and anions, such as tripolyphosphate (TPP), because of their very simple and mild preparation conditions, homogeneous particle sizes, and bioadhesive properties. TPP is a non-toxic and multivalent anion that can form crosslinks by ionic interaction between positively charged amino groups of chitosan and multivalent negatively charged TPP molecules [30]. The study aims to investigate the use of chitosan nanoparticles for textile applications as chitosan in nano form is highly active because of very high surface area to volume ratio and is expected to include higher amounts of biologically active substances. Crosslinking with GA of chitosan applied on cotton can improve the durability against repeated laundering; binary aldehyde GA can react not only with NH2 chitosan groups forming Schiff bases, but also with cotton OH groups [10], forming acetal bonds. Unfortunately, GA presents an acute toxicity that has been investigated in many studies [31,32]. The two procedures of cotton coating with TPP crosslinked nanoparticles or GA crosslinked chitosan were used to appreciate their efficiency in obtaining of a more appropriate support for biologically active VT. The size distribution of chitosan fine particles obtained through TPP crosslinking is presented in Fig. 1; a bimodal distribution is observed, with peak maxima at about 1 and 100 μm. The presence of chitosan on cotton fabric was proved by FTIR and SEM spectroscopies. The FTIR spectra of chitosan-coated cotton (Fig. 2) present bands characteristic of the CO amide group around 1650 cm −1 (1652; 1646; 1647 cm −1, respectively) and of the NH amide group at about 1550 cm −1 (1563; 1558; 1559 cm −1, respectively); besides, the presence of these bands in the chitosan spectrum is mentioned in literature [33,34]. Also, bands at around 1420 cm −1 (characteristic for CH amide II [33]) and around 1320 cm −1 (characteristic for –OH, –NH2, –CO groups [34]) appear. The ratio of the bands at about 1320 cm−1 and 1420 cm−1 (the first present in N-acetyl D-glucosamine, the second present in N-acetyl D-glucosamine and glucosamine) was used to calculate the degree of acetylation of chitin and chitosan [34]. The imine –C= N groups (possible formed during GA crosslinking of chitosan or as result of –CHO reaction of periodate oxidized cotton with chitosan) have characteristic bands around 1640 cm −1 [35]–1680 cm −1 [10]; they are difficult to be evidenced due their possible overlapping with the amide characteristic band (1650 cm −1). Spectrum 4 (sample VT-3) presents additional bands around 1100–1200 cm −1, which can be attributed to P = O stretching
Fig. 1. Particles size distribution.
Fig. 2. FTIR spectra of cotton (1) and chitosan coated cotton (2 —sample VT-4; 3 — sample VT-8; 4 — sample VT-3).
[36,18]; the broadening of the band at 1554 cm −1 can be attributed to the electrostatic interactions between amine groups of the chitosan and carboxylic groups of the support [37,38]. The band at 1161 cm−1 can be attributed to the interactions between amine chitosan and phosphate groups, as reported for the chitosan–chondroitinsulfate interpolymeric complex [39]. SEM photos were performed on a TESLA BS 301 apparatus, 100 mag, low vacuum; chitosan-coated cotton shows an uniform coating of the fabrics either with glutaraldehyde crosslinked chitosan (Fig. 3a) or with TPP crosslinked chitosan fine particles (Fig. 3b). The amount of chitosan retained on cotton depends on the coating method used. Cotton fabrics containing functional reactive –CHO or –COOH groups retain higher amounts of TPP crosslinked chitosan fine particles than unmodified cotton (samples 1–3, Table 1). This confirms our initial hypothesis that chitosan particles will be retained on modified cotton both by physical inclusion and by chemical (or electrostatic) bonds. Also, chitosan retention through GA co-crosslinking of the cotton previously modified with reactive functional groups is higher than that obtained for unmodified cotton (samples 4–9, Table 1). In this case, one can assume the formation of chemical bonds between reactive functional groups of cotton and the amino chitosan groups, besides co-crosslinking due to GA. Chitosan is retained in higher amounts on KIO4 oxidized cotton which contains more reactive groups (1.05 meq –CHO reactive groups/g fabric) than on carboxymethyl cotton (0.05 meq –COOH groups/g fabric). The amount of VT retained on the obtained supports depends on the amount of the chitosan coating of the fabrics (Table 1). Chitosan itself retains an important amount of VT, while on the cotton fabrics, it is retained in very small amounts (Table 1). Related to the amount of chitosan (deposed/g cotton), VT is retained in higher amounts on TPP crosslinked chitosan-coated cotton, probably due to a more efficient inclusion into the particle network. Thus, the TPP crosslinked-coated cotton retains the same VT amount as chitosan itself, while GA crosslinked chitosan retains a smaller VT amount than chitosan itself. The samples VT4–VT-6 with approximately the same chitosan content as the corresponding samples VT-7–VT-9 retain more VT because of their less crosslinked structure, obtained through a shorter crosslinking duration, which facilitates the VT diffusion inside the network. The release of the VT retained on the cotton fabrics is presented in Figs. 4 and 5. The differences as to the VT release rate among the obtained supports can be explained by the difference in their hydrophilicity (Fig. 6); a higher release rate is observed for the more hydrophilic cotton coated with TPP crosslinked chitosan (samples VT-1–VT-3, Table 1); the most hydrophilic one – VT-1 – based on unfunctionalized
G. Mocanu et al. / Materials Science and Engineering C 33 (2013) 72–77
75
Fig. 3. SEM photos of the chitosan coated cotton (sample VT-5) (a) and of the cotton treated with TPP crosslinked chitosan fine particles (sample VT-2) (b).
Table 1 Application procedures of chitosan on cotton; chitosan and VT content of the fabrics. Sample
VT-1 VT-2 VT-3 VT-4 VT-5 VT-6 VT-7 VT-8 VT-9 Cotton KIO4 oxidized cotton Carboxy Methylated cotton Chitosan ⁎ ⁎⁎
Procedure
g chitosan/g cotton, calculated from:
TPP crosslinked chitosan deposed on cotton TPP crosslinked chitosan deposed on cotton oxidized with KIO4 TPP crosslinked chitosan deposed on carboxymethylated cotton GA crosslinked chitosan (for 1 h) deposed on cotton GA crosslinked chitosan (for 1 h) deposed on cotton oxidized with KIO4 GA crosslinked chitosan (for 1 h) deposed on carboxymethylated cotton GA crosslinked chitosan (for 2 h) deposed on cotton GA crosslinked chitosan (for 2 h) deposed on cotton oxidized with KIO4 GA crosslinked chitosan (for 2 h) deposed on carboxymethylated cotton –
–
acid orange retention⁎
yield
0.014 0.038 0.029 0.040 0.060 0.052 0.049 0.060 0.059 0 0
0.0163 0.0395 0.0288 0.043 0.062 0.051 0.059 0.065 0.060 –
0 0.217⁎⁎
–
g VT/g (chitosan–cotton system)⁎
g VT/g chitosan
0.0167 0.045 0.037 0.038 0.045 0.043 0.034 0.0357 0.040 b0.01
1.02 1.15 1.27 0.88 0.73 0.84 0.58 0.55 0.67
1.14
Values represent a mean of three measurements that deviated with ±0.5%. Acid orange retention/g chitosan.
cotton presents the highest VT release rate. In the case of the cotton coated with GA crosslinked chitosan (Fig. 5), the samples that have been crosslinked for 2 h (VT-7–VT-9, more crosslinked, less hydrophilic), present a lower release rate than those crosslinked for 1 h (VT-4–VT-6). It should be mentioned that the VT release was studied at a solution/ tested fabric ratio of 20 mL/0.05 g; if the fabric is used for bioactive
Fig. 4. In vitro VT release from VT-1–VT-3 samples, in solutions simulating a mean sweat composition; each value is a mean of three measurements that deviated with ±3%.
dressings, the ratio between sweat and fabrics will be much lower, hence one can appreciate that the VT release rate will be much slower. VT is a plant extract; hence, presumably, some of its components (especially the flavonoids and polyphenolic carboxylic acids) possess antioxidant activity. The main characteristic of an antioxidant is its ability to trap free radicals. Highly reactive free radicals and oxygen species are present in biological systems from a wide variety of sources. These free radicals may oxidize nucleic acids, proteins, lipids or DNA and can initiate degenerative diseases. Antioxidant compounds like phenolic acids, polyphenols and flavonoids scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl and thus inhibit the oxidative mechanisms that lead to degenerative diseases. In order to assess the antioxidant activity of VT, its scavenging effect was studied, comparatively with a known antioxidant, ascorbic acid. The results presented in Fig. 7 show that VT presents an antioxidant effect, but only when it is used in higher concentrations than ascorbic acid. Thus, if a scavenging effect of about 50% is attained for approximately 70 μg ascorbic acid/mL, the same scavenging effect is obtained for about 2 mg VT/mL (a concentration of about 28 times higher); yet, this antioxidant activity can be considered high enough for application domains of bioactive textiles. In vitro studies of the antioxidant activity of wool grafted either with gallate esters [4] or with antioxidant phenolic molecules [5] showed that this novel approach to wool functionalization could have a potential in the development of a protective device against degenerative skin diseases.
76
G. Mocanu et al. / Materials Science and Engineering C 33 (2013) 72–77
Fig. 5. In vitro VT release from VT-4–VT-9 samples, in solutions simulating a mean sweat composition; each value is a mean of three measurements that deviated with ±3%.
In vitro studies on the antioxidant activity of chitosan-coated cotton containing VT were performed; the attempts to determine the antioxidant activity in pure water failed, hence, it may be concluded that the biologically active substances are released only by varying the ionic strength of the environment. The studies were also carried out in solutions simulating a mean sweat composition. Comparatively with the initial VT solution, the chitosan-coated cotton samples containing VT present a lower scavenging effect, probably due to the slower release of the antioxidant substances retained on the fabrics (Figs. 8, 9). The measurements of the free radical scavenging activity of chitosan-coated cotton showed no effect. The active hydroxyl and amino groups in the polymer chains, able to react with free radicals to form stable macroradicals, can be the origin of the scavenging ability; but, according to the reports of Alexandrova et al. [40] the antioxidant activity of chitosan was almost equal to zero. Only at very high concentrations can chitosan scavenge superoxide anions. This may be related to the formation of strong intermolecular and intra‐molecular hydrogen bonds that inhibited the reactivity of hydroxyl and amino groups in the polymer chains [41]. Some authors reported an important antioxidant activity for Low Molecular Weight (LWM) chitosan [42] and LMW and soluble Medium Molecular Weight chitosan with a 98.50% N-deacetylation [43]. Some studies have been demonstrated that molecular weights, as well as the degree of deacetylation, are both potent determinants of action of LMW chitosans [44]. LMW chitosans with a relatively higher degree of deacetylation and lower MW were found to have higher radical scavenging activities on DPPH, hydroxyl and superoxide radicals [45]. High MW chitosans have compact structures, thus making the overall effect of their intra‐molecular hydrogen bonds stronger. The strong effect of intra-molecular hydrogen bonding decreases the reactivity of hydroxyl and amino groups. On the
Fig. 6. Water uptake of VT samples (VT-0 represents uncoated cotton fabric).
Fig. 7. Scavenging effect of VT comparatively with ascorbic acid.
contrary, low molecular weight chitosan has a less compact structure, thus making the overall effect of intra-molecular hydrogen bonding less effective. In our experiments, the scavenging activity of the samples of crosslinked chitosan deposed on cotton, measured at the same concentration as the samples having VT, was almost zero (results are in concordance with the above mentioned literature data [40–43]; hence all antioxidant activity determined was produced by the VT included into the chitosan-coated supports. From the presented data one can see that introduction of reactive groups on cotton through reaction with KIO4 or sodium chloroacetate leads to an increase of the amount of chitosan retained on the cotton and consequently of the VT amount. The use of TPP as crosslinking agent is more advantageous from the viewpoint of lack of the toxicity; also, the TPP-crosslinked chitosan retains higher amounts of VT which is released faster in vitro. GA, crosslinking agent frequently used for chitosan, presents an acute toxicity; chitosan GA crosslinked deposed on cotton (or cotton activated) support retains important amounts of VT, which is released at a smaller rate than TPP crosslinked chitosan. However, the procedure of coating of cotton with GA crosslinked chitosan is less complicated than coating with TPP crosslinked cotton.
5. Conclusions Bioactive textiles were obtained by coating of cotton with chitosan crosslinked with TPP, or GA, followed by loading with biologically active substances contained by VT; the amount of entrapped VT and its release rate can be varied through an appropriate choice of
Fig. 8. Scavenging effect of samples VT-1–VT-3, comparatively with VT.
G. Mocanu et al. / Materials Science and Engineering C 33 (2013) 72–77 [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] Fig. 9. Scavenging effect of samples VT-4–VT-9, comparatively with VT.
the chitosan treated supports. As mentioned, each support presents advantages and disadvantages. The TPP crosslinked cotton, deposed on previously functionalized support retain higher amount of VT (reported to the chitosan content), which is released faster; the GA crosslinked chitosan deposed also on previously functionalized support retains important amounts of VT, which is released gradually. The choice between the presented supports will be performed after subsequent in vivo studies for topical applications. The bioactive textiles obtained could be used for obtaining clothes for people with allergies or other skin problems, due to the specific biological activity of VT, whose antioxidant properties have been proven. The bioactive cotton fabrics are promising innovative biomaterials for medical devices, such as bioactive dressings, wound healing isolation materials and due to their antioxidant properties could have a potential in the development of a protective device against degenerative diseases. References [1] E. Pinho, M. Henriques, R. Oliveira, A. Dias, G. Soares, Fibers Polym. 11 (2) (2010) 271–276. [2] H. Haufe, K. Muschter, J. Siegert, H. Böttcher, J. Sol-Gel Sci. Technol. 45 (2008) 97–101. [3] D. Bonamonte, C. Foti, N. Lionetti, L. Rigano, G. Angelini, Annal. It. Dermat. Allerg. Clin. Speriment. 63 (1) (2009) 10–27. [4] Kh.M. Gaffar Hossain, M. Díaz González, J.M. Dagá Monmany, T. Tzanov, J. Mol. Catal. B: Enzym. 67 (2010) 231–235. [5] S. Jus, V. Kokol, G.M. Guebitza, Enzyme Microb. Technol. 42 (2008) 535–542.
[21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36]
[37] [38] [39] [40] [41] [42] [43] [44] [45]
77
M.N.V. Ravi Kumar, React. Funct. Polym. 46 (2000) 1–27. Y. Gao, R. Cranston, Text. Res. J. 78 (1) (2008) 60–72. D. Knittel, E. Schollmeyer, Lenzinger Berichte. 85 (2006) 124–130. S. Strnad, O. Šauperl, L. Fras-Zemljič, Biopolymers 9 (2010) 181–200. Z. Zhang, L. Chen, J. Ji, Y. Huang, D. Chen, Text. Res. J. 73 (12) (2003) 1103–1106. J. Martinez Urreaga, M.U. de la Orden, Eur. Polym. J. 42 (10) (2006) 2606–2616. D. Alonso, M. Gimeno, R. Olayo, H. Vazquez-Torres, J.D. Sepulveda-Sanchez, K. Shirai, Carbohydr. Polym. 77 (3) (2009) 536–543. K.F. El-tahlawy, M.A. El-bendary, A.G. Elhendawy, S.M. Hudson, Carbohydr. Polym. 60 (4) (2005) 421–430. S.H. Lee, M.J. Kim, H. Park, J. Appl. Polym. Sci. 117 (2) (2010) 623–628. L.F. Zemljic, S. Strand, O. Sauperl, K. Stana-Kelinschek, Text. Res. J. 79 (2009) 219–226. A. Hebeish, F.A. Abdel-Mohdy, M.G. Fouda, Z. Elsaid, S. Essam, G.H. Tammam, E.A. Drees, Carbohydr. Polym. 86 (2011) 1684–1691. X. Fua, Y. Shena, X. Jianga, Y. Yana, Carbohydr. Polym. 85 (2011) 221–227. Ali S. Wazed, S. Rajendran, M. Joshi, Carbohydr. Polym. 83 (2) (2011) 438–446. W. Ye, J.H. Xin, P. Li, K.-L.D. Lee, T.-L. Kwong, J. Appl. Polym. Sci. 102 (2006) 1787–1793. V. Vukics, A. Kery, G.K. Bonn, A. Guttman, Anal. Bioanal. Chem. 390 (2008) 1917–1925. E.W. Banaszczak, W. Bylka, I. Matyawska, O. Goślińska, Z. Muszyński, Fitoterapia 76 (2005) 458–461. A. Toiu, L. Vlase, I. Oniga, M. Tamas, Farmacia LVI (4) (2008) 440–444. Kh.U. Usmanov, T.I. Sushkevitch, J. Polym. Sci. 58 (1962) 1325–1331. Z. Hou, M. Zhang, B. Liu, Q. Yan, F. Yuan, D. Xu, Y. Gao, Food Hydrocol. 26 (2012) 205–211. M.J. Patterson, S.D. Galloway, M.A. Nimmo, Exp. Physiol. 85 (6) (2000) 869–875. N. Ara, H. Nur, Res. J. Med. Sci. 4 (1) (2009) 107–110. K. Pepper, D. Reichenberg, D.K. Hale, J. Chem. Soc. (1952) 3129–3136. D. Bruneel, E. Schacht, Polymer 34 (12) (1993) 2628–2631. C. Buzea, I.I. Pacheco, K. Robbie, Biointerphases 2 (4) (2007) MR17–MR71. A.K. Anal, W.F. Stevens, C. Remunan-Lopez, Int. J. Pharm. 312 (2006) 166–173. L. Sano, A.M. Krueger, P.F. Landrum, Aquat. Toxicol. 71 (2005) 283–296. T. Takigawa, I. Endo, J. Occup. Health 48 (2006) 75–87. E.A. El-Hefian, M.M. Nasef, A.H. Yahaya, E-J. Chem. 9 (3) (2012) 1431–1439. J. Brugnerotto, J. Lizardi, F.M. Goycoolea, W. ArguÈelles-Monal, J. Desbrieres, M. Rinaudo, Polymer 42 (2001) 3569–3580. B. Hoffman, D. Seitz, A. Mencke, A. Kokott, G. Ziegler, J. Mater. Sci. Mater. Med. 20 (7) (2009) 1495–1503. J.R. Azevedo, R.H. Sizilio, M.B. Brito, A.M.B. Costa, M.R. Serafini, A.A.S. Araújo, M.R.V. Santos, A.A.M. Lira, R.S. Nunes, J. Therm. Anal. Calorim. 106 (2011) 685–689. M.H. Lee, M.K. Chun, H.K. Choi, Arch. Pharm. Res. 31 (7) (2008) 932–937. L. Yan, F. Qian, Q. Zhu, Polym. Int. 50 (2001) 1370–1374. G. Kaur, V. Rana, S. Jain, A.K. Tiwary, AAPS Pharm. Sci. Technol. 11 (1) (2010) 36–45. V.A. Alexandrova, G.V. Obukhova, N.S. Domnina, D.A. Topchiev, Macromol. Symp. 144 (1) (1999) 413–422. T. Sun, W. Xie, P. Xu, Macromol. Biosci. 3 (6) (2003) 320–323. T. Yasufuku, M. Anraku, Y. Kondo, T. Hata, J. Hirose, N. Kobayashi, H. Tomida, Pharmaceutics 2 (2010) 245–257. P.J. Chien, F. Sheu, W.T. Huang, M.-S. Su, Food Chem. 102 (2007) 1192–1198. S.K. Kim, N. Rajapakse, Carbohydr. Polym. 62 (2005) 357–368. W. Xie, P. Xu, Q. Liu, Bioorg. Med. Chem. Lett. 11 (2001) 1699–1701.
