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Soluble chitosan–carrageenan polyelectrolyte complexes and their gastroprotective activity
Accepted Manuscript Title: Soluble Chitosan-Carrageenan Polyelectrolyte Complexes and Their Gastroprotective Activity Author: A.V. Volodko V.N. Davydova E.A. Chusovitin I.V. Sorokina M.P. Dolgikh T.G. Tolstikova S.A. Balagan N.G. Galkin I.M. Yermak PII: DOI: Reference:
S0144-8617(13)01070-9 http://dx.doi.org/doi:10.1016/j.carbpol.2013.10.049 CARP 8246
To appear in: Received date: Revised date: Accepted date:
29-8-2013 14-10-2013 14-10-2013
Please cite this article as: Volodko, A. V., Davydova, V. N., Chusovitin, E. A., Sorokina, I. V., Dolgikh, M. P., Tolstikova, T. G., Balagan, S. A., Galkin, N. G., & Yermak, I. M.,Soluble Chitosan-Carrageenan Polyelectrolyte Complexes and Their Gastroprotective Activity, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.10.049 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights
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The soluble polyelectrolyte complexes κ-carrageenan - chitosan were obtained.
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The binding constant of carrageenan with chitosan is 2.11×107 mol-1.
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The thansformation of supramolecular structure of the complexes was shown.
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Gastroprotective activity of complexes depending on their composition was found.
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Corresponding author
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Alexandra V. Volodko,
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G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-East Branch of the
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Russian Academy of Sciences, pr. 100-letya Vladivostoka, 159, Vladivostok 690022,
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Russia, fax: 7(423)2314050, tel.: 7(423)2310719, e-mail: [email protected]
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Soluble Chitosan-Carrageenan Polyelectrolyte Complexes and Their
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Gastroprotective Activity
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A. V. Volodko*a, V. N. Davydovaa, E. A. Chusovitinb, I. V. Sorokinac, M. P. Dolgikhc,
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T. G. Tolstikovac, S. A. Balaganb,d, N. G. Galkinb,d, I. M. Yermaka
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a
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Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok
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690022, Russia
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G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of
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b
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Academy of Sciences, Radio St.5, Vladivostok 690041, Russia
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c
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Russian Academy of Sciences, Lavrentjev Ave. 9, Novosibirsk 630090,Russia
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Institute of Automation and Control Processes of Far Eastern Branch of Russian
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N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the
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Far Eastern Federal University, 8 Suhanova Str., Vladivostok 690950, Russia
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* - corresponding author
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Abstract The soluble polyelectrolyte complexes (PEC) κ-carrageenan (κCG):chitosan was obtained. Binding constant value (2.11×107, mol-1) showed high
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affinity of κ-CG to chitosan. The complex formation of κ-CG:chitosan 1:10 and 10:1
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w/w was shown by centrifugation in a Percoll gradient. Using atomic force
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microscopy we showed that the supramolecular structure of the complexes is
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different from each other and from the macromolecular structure of the initial polysaccharides. The gastroprotective and anti-ulcerogenic effect κ-CG, chitosan and their complexes was investigated on the model of stomach ulcers induced by indometacin in rats. PEC κ-CG:chitosan have gastroprotective properties which depend on their composition. Complex κ-CG:chitosan 1:10 w/w possesses higher gastroprotective activity than the complex 10:1 w/w. These results suggest that the gastroprotective effect of complexes can be associated with their protective layer on
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the surface of the mucous membrane of a stomach, which avoids a direct contact
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with the ulcerogenic agent.
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Keywords: polyelectrolyte complex, chitosan, carrageenan, supramolecular structure, AFM, gastroprotective activity
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1. Introduction 3 Page 3 of 23
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In recent years, polyionic polysaccharides of marine origin have been used in various composite materials. The sulfated polysaccharides of red algae –
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carrageenans (CG), are widely applied due to their unique physical properties,
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structural diversity and biological activity. (Yermak & Khotimchenko, 2003). They are
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composed of alternating 3-linked β-D-galactopyranose and 4-linked α-D-
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galactopyranose or 4-linked 3,6-anhydrogalactose and classified according to the
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number and position of sulfate groups and by the occurrence of 3,6-anhydro bridges
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in α-linked residues. CG is due to its physicochemical properties provides of
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prolonging the necessary concentration of active substance and enhances the
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bioavailability of a drug. PECs, based on CG, have been applied to create systems
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with controlled release of active substances (Van de Velde, Lourenço, Pinheiro, &
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Bakker, 2002). On the basis of CG, micro-encapsulated and tablet forms of drugs
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have been produced (Thommes & Kleinebudde, 2006). It has been shown by
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Schmidt et. al.that CG is effective complexing agent for some tranquilizers and
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antibiotics (Schmidt, Wartewig, & Picker, 2003).
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The combination of physical properties of CG with their strong antiviral,
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antimicrobic and immunomodulatory activities (Yermak & Khotimchenko, 2003;
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Yermak et al., 2012), as well as the ability to form complexes with polycations, allows
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one to create different kinds of bioactive composites. Chitosan, the cationic (1-4)-2-amino-2-deoxy--D-glucan, with degree of
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acetylation typically close to 0.20, is prevalently produced from marine chitin
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(Muzzarelli, 2010; Muzzarelli et al., 2012). Chitosan and its partially depolymerized
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derivatives and oligomers have a wide spectrum of biological activities, and are particularly useful in the area of wound healing, oral delivery, and food industry (AlHilal et al., 2013;Busilacchi et al., 2013; Croisier & Jérôme, 2013; Muzzarelli, 1996; Muzzarelli, 2009; Gamboa, & Leong, 2013). Carrageenan-chitosan PECs can be obtained as films (Yan, Khor, & Lim, 2001), microcapsules (Devi & Maji, 2009; Devi, & Maji, 2010), microspheres (Muzzarelli et al., 2004) and gels (Mitsumata et. al.,
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2003) to be used as drug delivery agents (Li, et al., 2013), cosmeceuticals (Kamel, &
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Abbas, 2013), nano-layered coatings (Pinheiro, et al., 2012), dispersants (Tzoumaki,
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et al., 2013) and beads (Saleem, et al., 2012).
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Obtaining soluble CG:chitosan complexes can expand their application, owing
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to combination of the useful properties of their constituent polysaccharides or
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enhancing biological activity. To study the biological effects of CG:chitosan PECs it is
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necessary to know in detail the complex formation process and supramolecular 4 Page 4 of 23
structure. As it was shown earlier by us, soluble complexes κ-CG:CH may be
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obtained preferentially by mixing the starting components at given ratios. The
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formation process of complexes depended on the CH molecular weight and the
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concentrations and ratios of starting polysaccharides (Volod’ko, Davydova,
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Barabanova, Solov’eva, & Ermak, 2012). This work is devoted to the study of the
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formation and macromolecular organization of κ-CG:chitosan complexes and to the
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investigation of their gastro protective and anti-ulcerogenic activities.
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2. Materials and methods
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2.1. Chitosan
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A sample of chitosan was obtained by alkaline deacetylation of chitin
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(Naberezhnykh, Gorbach, Likhatskaya, Davidova, & Solov’eva, 2008). In this work
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we used sample of chitosan with molecular weight 115 kDa and a degree of N-
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acetylation 6%. The molecular weights of the polycations were determined as
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published earlier (Davydova, Yermak, Gorbach, Drozdov, & Solov'eva, 2000).
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2.2. Isolation of κ-carrageenan
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A sample of κ-CG was isolated by extraction with hot water from C. armatus.
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In this work we used sample of κ-CG with molecular weight 311 kDa. The methods of
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extraction and κ-CG structure determination have been published (Yermak, Kim,
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Titlynov, Isakov, & Solov’eva, 1999).
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2.3. Analytical Methods
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The content of κ-CG was determined by reaction of the polysaccharide sulfate
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group with Taylor’s blue (1,9-dimethylmethylene blue) (Keler & Nowotny, 1986). The optical density was measured at 535 nm on a μ-Quant spectrophotometer (Bio-Tek Instruments Inc., USA). The content of chitosan was determined by reaction of the amino group with trinitrobenzenesulfonic acid (TNBS) (Inman & Dintzins, 1969). The optical density was measured at 410 nm on the μ-Quant spectrophotometer (Bio-Tek Instruments Inc., USA).
2.4. Titration of the tropaeolin 000-II (Orange II) – chitosan complex with κ-CG solution To 80 μl of 0.005 M phosphate buffer (pH 5.0), 40 μl of tropaeolin 000-II
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(sodium salt of 4-(2-hydroxy-1-naphthylazo)benzenesulfonic acid) solution (0.08
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mg/ml) and 20 μl of chitosan solution (100 μg/ml) were added. The mixture was
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incubated for 20 min at 25°C. Then the resulting complex was titrated with solution
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(200 μg/ml) by adding different portions of κ-CG. The absorption was determined with 5 Page 5 of 23
a μ-Quant spectrophotometer (Bio-Tek Instruments Inc., USA) at 483 nm in three
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parallel samples, and the mean arithmetic value was calculated. The value of
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ΔD=Dexp−D0 was calculated, where D0 and Dexp were absorptions of the solutions
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before and after the addition of κ-CG, respectively. The values of ΔDmax and the
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binding constant were determined from the Scatchard dependence in ΔD/Cκ-CG
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versus ΔD coordinates. The chitosan saturation (Q) with κ-CG molecules was
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determined from the ratio ΔD/ΔDmax.
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The number of binding sites on the κ-CG molecule per amino group of
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chitosan was assessed from the saturation curve by plotting a tangent to the point
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that corresponded to the maximal change in the reaction mixture's absorption. Then
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the κ-CG concentration corresponding to the chitosan saturation with the κ-CG was
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determined.
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2.5. Centrifugation in a Percoll Gradient
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Percoll (Sigma, 26 mL, 30%) in NaCl solution (0.15 M) was placed into a 28
an
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mL centrifuge tube. A sample of κ-CG, chitosan or their mixture (2 mL) was layered
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on the Percoll and centrifuged in an angled rotor Heraeus Biofuge Stratos (Germany)
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at 20,000 g for 60 min. After centrifuging, the tube contents were removed through
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the top using a peristaltic pump. Fractions (1.5 mL) were collected. The density of the
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Percoll solution in each fraction was calculated using the refractive index determined
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on a RF-4 refractometer (LOMO). The presence of chitosan and κ-CG in the fractions
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was determined by the reaction of polysaccharide amino group with TNBS (Inman &
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Dintzins, 1969) and by the reaction of sulfate group with Taylor’s blue (Keler &
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Nowotny, 1986), respectively.
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2.6. Atomic Force Microscopy (AFM) The samples were dissolved in distilled de-ionized water at concentrations of
κ-CG and chitosan of 0.1 mg/mL, 0.01 mg/mL; PECs were prepared at the same concentrations at the ratios κ-CG:chitosan 10:1 and 1:10 w/w. Aliquots (12 μl) of each sample in an aqueous solution were deposited onto freshly cleaved mica and dried at 37 °C for 24 h. Morphology of polysaccharide and their complexes was
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studied in air by AFM (Solver P47) in tapping contact mode using a 10 nm radius tip.
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The topographical parameters of the sample macromolecular structure had been
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automatically calculated by a self-developed program “Balagan’s Grain Analysis”.
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The program allows us to separate objects from the substrate and determine
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topographical parameters of each object: the lateral sizes, height, volume, occupied
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area, etc. 6 Page 6 of 23
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The analyzed AFM image is identified with a rectangular two-dimensional matrix where each cell contains a measured height value. At the first stage of the analysis, the image is smoothed and divided into rows and columns. Then based on each column (row), another two columns (rows) are
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calculated: in the first one, the local maxima are eliminated, in the second one – the
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local minima. The calculated four sets of the columns and rows are smoothed out (it
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is possible to choose the smoothing method) and are interpolated. The interpolation
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runs as follows: using the method of least squares, by four points, the closest to the
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interpolated point, a parabola equation is deduced. The interpolated point height is
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determined from the parabola equation. Then from the interpolated columns and
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rows an averaged image is constructed.
At the second stage, the image splits into groups of pixels. To do this, the
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pixels are sorted by descending of their height. Then, starting from the beginning of
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the pixels list, each pixel is coloured (similar to the numbering)by the following rules:
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1. The pixel with coordinates (i1, j1) is adjacent to the pixel (i2, j2), if |i1 - i2 | ≤ 1
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2. If near the pixel there are no coloured pixels, it is assigned a previously unused colour (it will form a new group of pixels); 3. If near a pixel there are coloured pixels, it is assigned a colour of the nearest local maximum.
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and |j1 - j2| ≤ 1;
On the third stage, pixel groups are combined according to their height and
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relative position on the plane. The program does the same actions as on the second
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stage, but it works with pixel groups instead of single pixels.
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On the fourth stage, pixel groups are combined considering pixel groups
relative position in space and their size. In addition, the program examines the spatial distribution of pixels within the group, to reduce the effects of distortion in the case of the objects of interest located on the edge of a terrace. When this step finishes, it is possible to filter pixel groups by size and space position. At the fifth stage, the subtraction of the substrate there occurred. The
substrate relief is determined by analysis of the location of the pixel groups in space. After each object is separated from the substrate, the problem of the object parameters calculation is trivial.
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2.7. Investigation of gastroprotective and antiulcerogenic activity
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The experiments were carried out in three independent series on Vistar female
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rats 200-220 g weighing in accordance with the Guidelines for the Care and Use of 7 Page 7 of 23
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Laboratory Animals. The animals were given standard granulated food and water ad
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libitum. Each group consisted of 6 animals. Model of stomach ulcers. All animals were deprived of food with maintanance
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of free access to water during 24 hours. Then the rats were orally administrated 20
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mg/kg indomethacin (“Fluka BioChemica”, Italy) suspended in water with Tween-80
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(“ICN Biomedicals, Inc.”, USA), and 2 hours later the animals were fed. One day later
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the rats were anesthetized with chloroform and necropsied, their stomachs were
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removed and ulcers of the mucous membrane were counted under a binocular loupe.
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The samples of polysaccharides and their complexes were injected
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intraperitoneally in two modes. When evaluating the gastroprotective effect of κ-CG,
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chitosan and their complexes, the samples injected once 1 hour before indomethacin
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administration. In the study of anti-ulcerogenic action polysaccharides were injected
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during three days prior to the introduction of indomethacin (the last injection was 24
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hours before ulcerogenic agent). Control animals received an equivalent amount of
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water, the reference group was injected with the gastroprotective drug Phosphalugel
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(aluminum phosphate gel, «Astellas Pharma», Netherlands) in the same dose and
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mode as the test polysaccharides. Pauls indexes (PI) were counted in each
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experiment according to the formula: PI=A·B/100, where A – the average number of
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ulcers per animal; B – the number of animals with ulcers in the group (%).
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Antiulcer activity (AUA) was defined as the ratio of PI of the control group
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divided to PI of the treatment group. It is considered that the monitoring agent has
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antiulcer activity on the assumption of AUA 2 units.
PECs κ-CG:chitosan were injected orally at a dose of 5 mg/kg or 0.5
mg/ml/100 g body weight. In this case the dose of the components in the complex were: 0.45 and 4.55 mg/kg. Solutions κ-CG and chitosan were injected at a dose of 4.55 mg/kg. The Phosphalugel was injected at 4.55 mg/kg. The results were analyzed using STATISTICA 6 software. The differences were significant at p<0.05.
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3. Results and discussion
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The method of competitive binding κ-CG with a complex of chitosan/anionic dye was used to determine the binding constants of the polyanion (κ-CG) with a
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polycation (chitosan). The tropaeolin OOO-II anionic dye [sodium 4-(2-hydroxy-1-
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naphthylazo)benzenesulfonate] was used as an anionic dye. The method (Glazunov
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& Gorbach, 1999) is based on the displacement of CG dye from its complex with
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chitosan. The number of seats of binding was calculated. The number of sulfate
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groups of κ-CG associated with one amino group of chitosan was close to
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stoichiometric. The binding constants was calculated using Scatchard and Hill plots
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(table 1). A linear character of the Scatchard plot assumes that no cooperativity
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during the reaction (Nørby, Ottolenghi, & Jensen, 1980) and that one type of binding
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sites involves into this process. This binding constant value (2.11×107, mol-1) showed
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high affinity of κ-CG to chitosan.
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Table 1
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Binding parameters of chitosan with κ-CG
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1.98! 0.31
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Mean value 2.04! 0.26
1.29! 0.13
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Hill’s method
The number of SO42- to 1 NH3+
Quantity chitosan associated with 1 mg κ-CG in saturation point, mg 0.33! 0.03
The method of centrifugation in Percoll gradient was used for direct detection
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Scatchard’s method
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Binding constant, 107
of the complex formation. Figure 1 shows the results of Percoll gradient centrifugation of the complex initial components (Fig. 1a) and its mixtures at the ratio of κ-CG:chitosan 1:10 and 10:1 w/w (Fig. 1b,c).
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Fig. 1. Centrifugation in Percoll gradient: a – initial polysaccharides; b – κ-
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CG:chitosan mixtures 1:10 w/w; c – κ-CG:chitosan 10:1 w/w; 1 – content of amino
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groups chitosan (A474nm); 2 – content of sulfate groups κ-CG (A535nm)
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It can be seen from figure 1 that the curves of κ-CG:chitosan mixtures (Fig. 1b and 1c) differed from those of the initial polysaccharides (Fig. 1a). Under the
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centrifugation conditions chitosan formed a one zone of density and settled in the
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lower part of the gradient whereas κ-CG was separated into two fractions in the
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upper and middle parts of the gradient. The coincidence of the maxima of the elution
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curve for chitosan and κ-CG in their mixture indicated that a κ-CG:chitosan complex
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formed (Fig. 1b,c).
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Two types of complexes with different densities formed irrespective of the
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initial components ratio. In the case of the polyanion high content in the mixture (κ-
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CG:chitosan 10:1 w/w (Fig. 1b)), part of the κ-CG remains unbound, otherwise (κ-
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CG:chitosan 1:10 w/w (Fig. 1c)) part of the chitosan remains free.
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We studied the macromolecular structure of the κ-CG:chitosan complexes, obtained at different ratio, compared with the initial polysaccharides by AFM (Fig. 2).
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Concentrations of the components were the same as in the complexes.
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Fig. 2. AFM topography images: a – chitosan, C=0.01 mg/ml, b – κ-CG, C=0.1
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mg/ml, c - PEC κ-CG:chitosan 10:1 w/w; and a histogram of the meshes area 10 Page 10 of 23
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distribution: d – κ-CG, C=0.1 mg/ml; e – PEC κ-CG:CH 10:1 w/w; on the inserts one
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can see the calculation masks.
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According to the AFM images shown in Fig. 2a, chitosan at a concentration of 0.01 mg/ml produces a homogeneous and very smooth (root-mean-square
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roughness (RMS) – 0.13 nm) film, which is sensitive to AFM probe influences. At the
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scanning of chitosan in tapping contact mode, during recording one of the image
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lines, there was self-excited oscillation of the cantilever. As a result, the probe tip
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impacts the surface for several times with increased force. The crack on the film
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surface was observed during rescanning of the same area.
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The basis of the chemical structure of the κ-CG is a disaccharide repeating unit, which contains the 3,6-anhydrogalactose. It determines the κ-CG ability of
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forming a double helical. As shown on the AFM images (Fig. 2b), κ-CG at
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concentration 0.1 mg/ml forms a three-dimensional network structure with height
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about 2.5 nm that indicates on intermolecular association of the polysaccharide “side-
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by-side” (Funami et. al., 2007). The network is continuous and composed of
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branched fibers. The average wall thickness of the network is (Cκ-CG=0.1 mg/ml) 0.05
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μm. Figure 2d shows a histogram of the meshes area distribution. According to that
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observed for κ-CG unimodal size meshes area distribution, with the maximum at
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about 1600 nm2.
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The macromolecular structure of PEC κ-CG:chitosan 10:1 w/w was different
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from structure of the initial components with the same concentrations of
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polysaccharides, that in the complex. As one can see from Fig. 2, the structure of the
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complex consists of several layers. The bottom layer has a mesh structure similar to the structure of κ-CG, but with considerably smaller wall thickness (0,025 μm). The histogram of the meshes area distribution (Fig. 2e) has a bimodal character with two maxima at about 150 and 300 nm2. This is 10 times smaller than the area corresponding to the peak of the histogram for the κ-CG. The second layer is disposed over the reticlated structure and it is composed of long twisted fibrils. In this
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PEC the amount of the κ-CG was 10 times greater than chitosan, and perhaps the
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two layers correspond to two types of complexes (heavy and light) which were
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observed during the centrifugation of PEC in Percoll gradient (Fig. 1b,c).
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Fig. 3. AFM topography images: a – chitosan, C=0.1 mg/ml, b – κ-CG, C=0.01
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mg/ml, c - PEC κ-CG:chitosan 1:10 w/w
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Increasing of the chitosan concentrations by 10 times results in
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supramolecular structure changes. By the AFM data (Fig. 3a), chitosan composed of
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densely packed spherical particles. The RMS value is 14.3 nm, corresponding to at
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least a level surface, unlike chitosan at a lower concentration (Fig. 2a).
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κ-CG at low concentrations (C=0.01 mg/ml) forms long threadlike structures. Fibers cross over one another, which lead to the increase of the height at the
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crossover points. That may correspond to the formation of the double-structure
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characteristic of κ-CG (Sokolova et al 2013).
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The AFM image of PEC κ-CG:chitosan (1:10 w/w) shows the formation of
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morphologically heterogeneous structure, which is different from those of PEC κ-
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CG:chitosan (10:1 w/w) and from initial polysaccharides. As one can see from Fig. 3,
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sub-layer consist of spherical segments that possibly correspond to free chitosan.
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The heterogeneity observed in the structure is in line with the data obtained by centrifugation, whereby PEC divided in the Percoll gradient into two fractions (Fig. 1c). Also tightly packed formations of complex κ-CG:chitosan 1:10 was observed which probably represent fibrils of κ-CG (average length of 0.5 μm) consisting of segments chitosan.
The observed features of the molecular organization of the PECs κ-
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CG:chitosan reveal that the dependence of the ratio of the initial polysaccharides can
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have a significant impact on their biological effects in the body. We investigated
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gastroprotective and anti-ulcerogenic effect of these complexes (10:1 and 1:10 w/w)
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to test this assumption.
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It was found that the PECs κ-CG:chitosan had various gastroprotective effects
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depending on its composition. PEC κ-CG:chitosan 10:1 w/w reduced ulcers in the
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stomach in two times, and PEC κ-CG:chitosan 1:10 w/w in three times in comparison 12 Page 12 of 23
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with the control (Table 2). Thus when the samples are injected one hour before the
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ulcerogenic impact, the complex with a higher content of κ-CG exhibits a moderate
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activity, while the complex with excess of chitosan has significant gastroprotective
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activity.
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Table 2.
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5.8±1.1
Antiulcer Pauls index activity, units
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Control
The total number of ulcers in the group (6 rats) 35
5.8
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2.3
2.5
0.9
6.4
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Sample
The average number of ulcers per 1 rat
κ-CG:chitosan 2.8±1.0 (р=0.07) 17 10:1 w/w κ-CG:chitosan 1.8±1.0 (р=0.03) 11 1:10 w/w р<0.05 - differences with control are significant
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Comparative gastroprotective activity of PECs κ-CG:chitosan
The further study κ-CG, chitosan and their PEC in comparison with drug
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Phosphalugel showed that the activity of the complex with a high content of
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polycation (κ-CG:chitosan 1:10 w/w) exceeded the activity of the reference drug,
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while the PEC with a high content of polyanion (κ-CG:chitosan 10:1 w/w)
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demonstrated a low gastroprotective effect (Table 3). It is interesting to note the low
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activity of initial polysaccharides (4.55 mg/kg, which corresponds to the maximum
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content of the complexes), as compared with Phosphalugel. This data shows that the
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increase of gastroprotective effect of PEC κ-CG:chitosan 1:10 w/w may not be directly related with gastroprotective properties of chitosan.
Table 3.
Gastroprotective activity κ-CG, chitosan and their complexes in comparison with Phosphalugel Sample
The average number of ulcers per 1 rat
Control κ-CG Chitosan
9.2±1.6 2.8±1.5 (р=0.01) 6.2±1.9 (р=0.24)
The total number of ulcers in the group (6 rats) 55 17 37
Pauls index
Antiulcer activity, units
9.2 1.9 6.2
4.8 1.5 13 Page 13 of 23
κ-CG:chitosan 10:1 w/w κ-CG:chitosan 1:10 w/w
4.2±1.3 (р=0.03)
25
1.3±1.0 (р=0.002) 8
1.0±0.4 6 (р=0.0004) р<0.05 - differences with control are significant
Phosphalugel 1
4.2
2.2
0.4
23.0
0.7
13.1
2
As indicated above, chitosan and κ-CG have various biological activities,
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including antiinflammatory, immunomodulatory and reparative. Such activities may
5
have a modifying effect on the stomach mucosa, increasing its resistance to the
6
effects of pathological factors. An experiment was conducted with a three-day
7
prophylactic injection of κ-CG, chitosan and their PEC to evaluate the possible
8
contribution of non-specific activity of polysaccharides in antiulcer effect. The last
9
injection of the samples was done a day before the reproduction of ulcerative lesions,
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which eliminated the direct contact of the tested samples with indomethacin on the
11
mucosal surface of the stomach. The experiment shows that the introduction of
12
polysaccharides in this mode has not reduced the amount of gastric ulcers induced
13
versus control. There is only reducing the excitability of the gastric mucosa in animals
14
treated with complexes, especially complex κ-CG:chitosan 10:1 w/w, resulting in a
15
reduction of mucosal hyperemia (Table 4). Phosphalugel didn’t exhibit antiulcer
16
activity in these conditions as well.
17
19 20 21
Table 4.
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Anti-ulcerogenic activity κ-CG, chitosan and their PEC at the prophilactic injection to rats with indomethacin-induced stomach ulcer
Sample
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The average number of ulcers per 1 rat
The total number of ulcers in the group (6 rats) 42 44 45
Pauls index
Antiulcer activity, units
Control 7.0±1.2 0.7 κ-CG 7.3±1.4 0.7 1.0 Chitosan 7.5±1.8 0.7 1.0 κ-CG:chitosan 9.2±1.7 55 0.9 0.8 10:1 w/w κ-CG:chitosan 6.7±1.8 40 0.7 1.0 1:10 w/w Phosphalugel 7.2±2.0 43 0.7 1.0 p > 0.05 at all groups - differences with control are unsignificant
23 14 Page 14 of 23
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4. Conclusion
2 3
The soluble PEC κ-CG:chitosan was obtained. Binding constant value showed high affinity of κ-CG to chitosan. The complex formation of κ-CG:chitosan 1:10 and
5
10:1 w/w was proved by centrifugation in a Percoll gradient. Summarizing the results
6
of biological studies, it may be noted that aqueous solutions of PEC κ-CG:chitosan
7
(C=0.5 mg/ml) have gastroprotective activity which depend on their composition.
8
Complex κ-CG:chitosan 1:10 w/w possesses higher gastroprotective activity than the
9
complex κ-CG:chitosan 10:1 w/w. The test polysaccharides and their complexes at
10
preliminary three-days injection do not increase own protective properties of gastric
11
mucosal.
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The differences in the effectiveness of PECs κ-CG:chitosan is likely to be
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associated with features of the supramolecular structure fixed by AFM. We showed
14
that the supramolecular structure of the complexes is different from each other and
15
from the macromolecular structure of the initial polysaccharides. According to this
16
data, κ-CG (C=0.01 mg/ml) forms long threadlike structures, whereas a tenfold
17
increase in the concentration produced mainly coarse three-dimensional networks
18
with a large amount of meshes. Considering that the concentration of κ-CG in
19
aqueous solution of PEC κ-CG:chitosan 1:10 w/w was 0.045 mg/ml, it can be
20
assumed that its high gastroprotective activity is provided by a regular formation of
21
the protective layer which formed due to the optimum conditions of complexation for
22
surface fibrils. These results suggest that the gastroprotective effect of complexes
23
can be associated with the formation of the protective layer on the surface of the
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stomach mucousa, which hinders direct contact with the ulcerogenic agent (indomethacin).
Acknowledgments The work was supported by the Programs for Fundamental Research of the
30
Presidium of the Russian Academy of Science “Molecular-cell biology” and by the
31
Presidium of Far-East Branch of the Russian Academy of Science and a grant № 12-
32
II SD – 05 – 006.
33 34
References
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Sciences, 102 (8), 2644-2654. Mitsumata, T., Suemitsu, Y., Fujii, K., Fujii, T., Taniguchi, T., & Koyama, K.
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salt complex hydrogels. Polymer, 44(23), 7103-7111. Muzzarelli R.A.A. (2010). Chitins and chitosans as immunoadjuvants and non-
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M.G. (2012). A tribute to Henri Braconnot, precursor of the carbohydrate polymers
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Muzzarelli, C., Stanic, V., Gobbi, L., Tosi, G. & Muzzarelli, R.A.A. (2004).
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Muzzarelli, R.A.A. (2009). Chitins and chitosans for the repair of wounded
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Solov’eva, T. F. (2008). Interaction of chitosans and their N-acylated derivatives with
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(2012) k-Carrageenan / chitosan nanolayered coating for controlled release of a model bioactive compound. Innovative Food Science & Emerging Technologies, 16, 227-232.
Saleem, M.A., Kotadia, D.R., Kulkarni, R.V. (2012) Effect of formulation
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Schmidt, A. G., Wartewig, S., & Picker, K. M. (2003). Potential of
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carrageenans to protect drugs from polymorphic transformation. European Journal of
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Sokolova, E. V., Chusovitin, E. A., Barabanova, A. O., Balagan, S. A., Galkin,
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N. G., & Yermak, I. M. (2013).Atomic force microscopy imaging of carrageenans from
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red algae of Gigartinaceae and Tichocarpaceae families. Carbohydrate Polymers,
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93(2), 458-465.
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Thommes, M., & Kleinebudde, P. (2006). Use of κ-carrageenan as alternative pelletisation aid to microcrystalline cellulose in extrusion/spheronisation. I. Influence
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of type and fraction of filler. European Journal of Pharmaceutics and
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Biopharmaceutics, 63(1), 59-67.
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Tzoumaki, M.V., Moschakis, T., Biliaderis, C.G. (2013) Effect of soluble
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polysaccharides addition on rheological properties and microstructure of chitin
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nanocrystal aqueous dispersions. Carbohydrate Polymers, 95 (1), 324-331. Van de Velde, F., Lourenço, N. D., Pinheiro, H. M., & Bakker, M. (2002).
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Carrageenan: A Food‐Grade and Biocompatible Support for Immobilisation
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Techniques. Advanced Synthesis & Catalysis, 344(8), 815-835.
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Volod’ko, A. V., Davydova, V. N., Barabanova, A. O., Solov’eva, T. F & Ermak,. I. M. (2012). Formation of soluble chitosan–carrageenan polyelectrolyte
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complexes. Chemistry of Natural Compounds, 48(3), 353-357.
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Yan, X. L., Khor, E., & Lim, L. Y. (2001). Chitosan‐alginate films prepared with
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chitosans of different molecular weights. Journal of Biomedical Materials Research, 58(4), 358-365.
Yermak, I.M., Barabanova, A.O., Aminin, D.L., .Davydova, B.N., Sokolova,
E.V., Kim Y. H., Shin K. S., & Solov’eva T. F. (2012). Effects of structural peculiarities
23
of carrageenans on their immunomodulatory and anticoagulant activities.
24
Carbohydrate Polymers, 87(1), 713-720.
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Yermak I.M., & Khotimchenko Yu.S. (2003). Chemical properties, biological
26
activities and applications of carrageenan from red algae. In M Fingerman,& R.
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Nagabhushanam (Eds.), Recent advances in marine biotechnology (pp. 207-255).
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New York, USA/London, UK: Plymouth Science Publishers Inc. 18 Page 18 of 23
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Yermak, I. M., Kim, Y. H., Titlynov, E. A., Isakov, V. V., & Solov’eva, T. F.
2
(1999). Chemical structure and gel properties of carrageenans from algae belonging
3
to the Gigartinaceae and Tichocarpaceae, collected from the Russian Pacific coast.
4
Journal of Applied Phycology, 11, 41-48.
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Table 1
2 3
Binding parameters of chitosan with κ-CG Binding constant, 107 Hill’s method 1.98! 0.31
Mean value 2.04! 0.26
The number of SO42- to 1 NH3+ 1.29! 0.13
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Scatchard’s method 2.11! 0.31
Quantity chitosan associated with 1 mg κ-CG in saturation point, mg 0.33! 0.03
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Table 2.
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The total number of ulcers in the group (6 rats)
Pauls index
Antiulcer activity, units
Control
5.8±1.1
35
5.8
-
κ-CG:chitosan 2.8±1.0 (р=0.07) 17 10:1 w/w κ-CG:chitosan 1.8±1.0 (р=0.03) 11 1:10 w/w р<0.05 - differences with control are significant
2.3 0.9
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Sample
The average number of ulcers per 1 rat
2.5 6.4
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Comparative gastroprotective activity of PECs κ-CG:chitosan
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Table 3.
2 3
Gastroprotective activity κ-CG, chitosan and their complexes in comparison with
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Phosphalugel
5
Control 9.2±1.6 55 (р=0.01) 2.8±1.5 17 κ-CG 6.2±1.9 (р=0.24) 37 Chitosan κ-CG:chitosan 4.2±1.3 (р=0.03) 25 10:1 w/w κ-CG:chitosan 1.3±1.0 (р=0.002) 8 1:10 w/w 1.0±0.4 (р=0.0004) 6 Phosphalugel р<0.05 - differences with control are significant
Antiulcer activity, units
9.2 1.9 6.2
4.8 1.5
4.2
2.2
0.4
23.0
0.7
13.1
an
6
Pauls index
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The total number of ulcers in the group (6 rats)
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The average number of ulcers per 1 rat
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Table 4.
2 3
Anti-ulcerogenic activity κ-CG, chitosan and their PEC at the prophilactic injection to rats
4
with indomethacin-induced stomach ulcer The total number of ulcers in the group (6 rats)
Pauls index
1.0 1.0
0.8 1.0 1.0
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5
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Control 7.0±1.2 42 0.7 κ-CG 7.3±1.4 44 0.7 Chitosan 7.5±1.8 45 0.7 κ-CG:chitosan 9.2±1.7 55 0.9 10:1 w/w κ-CG:chitosan 6.7±1.8 40 0.7 1:10 w/w Phosphalugel 7.2±2.0 43 0.7 p > 0.05 at all groups - differences with control are unsignificant
Antiulcer activity, units
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Sample
The average number of ulcers per 1 rat
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S0144-8617(13)01070-9 http://dx.doi.org/doi:10.1016/j.carbpol.2013.10.049 CARP 8246
To appear in: Received date: Revised date: Accepted date:
29-8-2013 14-10-2013 14-10-2013
Please cite this article as: Volodko, A. V., Davydova, V. N., Chusovitin, E. A., Sorokina, I. V., Dolgikh, M. P., Tolstikova, T. G., Balagan, S. A., Galkin, N. G., & Yermak, I. M.,Soluble Chitosan-Carrageenan Polyelectrolyte Complexes and Their Gastroprotective Activity, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.10.049 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Highlights
2
The soluble polyelectrolyte complexes κ-carrageenan - chitosan were obtained.
3
The binding constant of carrageenan with chitosan is 2.11×107 mol-1.
4
The thansformation of supramolecular structure of the complexes was shown.
5
Gastroprotective activity of complexes depending on their composition was found.
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1 Page 1 of 23
Corresponding author
1 2
Alexandra V. Volodko,
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G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-East Branch of the
4
Russian Academy of Sciences, pr. 100-letya Vladivostoka, 159, Vladivostok 690022,
5
Russia, fax: 7(423)2314050, tel.: 7(423)2310719, e-mail: [email protected]
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2 Page 2 of 23
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Soluble Chitosan-Carrageenan Polyelectrolyte Complexes and Their
2
Gastroprotective Activity
3 4
A. V. Volodko*a, V. N. Davydovaa, E. A. Chusovitinb, I. V. Sorokinac, M. P. Dolgikhc,
5
T. G. Tolstikovac, S. A. Balaganb,d, N. G. Galkinb,d, I. M. Yermaka
6 7
a
8
Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok
9
690022, Russia
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G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of
10
b
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Academy of Sciences, Radio St.5, Vladivostok 690041, Russia
12
c
13
Russian Academy of Sciences, Lavrentjev Ave. 9, Novosibirsk 630090,Russia
14
d
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Institute of Automation and Control Processes of Far Eastern Branch of Russian
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N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the
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Far Eastern Federal University, 8 Suhanova Str., Vladivostok 690950, Russia
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* - corresponding author
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Abstract The soluble polyelectrolyte complexes (PEC) κ-carrageenan (κCG):chitosan was obtained. Binding constant value (2.11×107, mol-1) showed high
20
affinity of κ-CG to chitosan. The complex formation of κ-CG:chitosan 1:10 and 10:1
21
w/w was shown by centrifugation in a Percoll gradient. Using atomic force
22
microscopy we showed that the supramolecular structure of the complexes is
24 25 26 27 28 29
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different from each other and from the macromolecular structure of the initial polysaccharides. The gastroprotective and anti-ulcerogenic effect κ-CG, chitosan and their complexes was investigated on the model of stomach ulcers induced by indometacin in rats. PEC κ-CG:chitosan have gastroprotective properties which depend on their composition. Complex κ-CG:chitosan 1:10 w/w possesses higher gastroprotective activity than the complex 10:1 w/w. These results suggest that the gastroprotective effect of complexes can be associated with their protective layer on
30
the surface of the mucous membrane of a stomach, which avoids a direct contact
31
with the ulcerogenic agent.
32 33
Keywords: polyelectrolyte complex, chitosan, carrageenan, supramolecular structure, AFM, gastroprotective activity
34 35
1. Introduction 3 Page 3 of 23
1 2
In recent years, polyionic polysaccharides of marine origin have been used in various composite materials. The sulfated polysaccharides of red algae –
4
carrageenans (CG), are widely applied due to their unique physical properties,
5
structural diversity and biological activity. (Yermak & Khotimchenko, 2003). They are
6
composed of alternating 3-linked β-D-galactopyranose and 4-linked α-D-
7
galactopyranose or 4-linked 3,6-anhydrogalactose and classified according to the
8
number and position of sulfate groups and by the occurrence of 3,6-anhydro bridges
9
in α-linked residues. CG is due to its physicochemical properties provides of
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prolonging the necessary concentration of active substance and enhances the
11
bioavailability of a drug. PECs, based on CG, have been applied to create systems
12
with controlled release of active substances (Van de Velde, Lourenço, Pinheiro, &
13
Bakker, 2002). On the basis of CG, micro-encapsulated and tablet forms of drugs
14
have been produced (Thommes & Kleinebudde, 2006). It has been shown by
15
Schmidt et. al.that CG is effective complexing agent for some tranquilizers and
16
antibiotics (Schmidt, Wartewig, & Picker, 2003).
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The combination of physical properties of CG with their strong antiviral,
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antimicrobic and immunomodulatory activities (Yermak & Khotimchenko, 2003;
19
Yermak et al., 2012), as well as the ability to form complexes with polycations, allows
20
one to create different kinds of bioactive composites. Chitosan, the cationic (1-4)-2-amino-2-deoxy-
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acetylation typically close to 0.20, is prevalently produced from marine chitin
23
(Muzzarelli, 2010; Muzzarelli et al., 2012). Chitosan and its partially depolymerized
24 25 26 27 28 29
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22
derivatives and oligomers have a wide spectrum of biological activities, and are particularly useful in the area of wound healing, oral delivery, and food industry (AlHilal et al., 2013;Busilacchi et al., 2013; Croisier & Jérôme, 2013; Muzzarelli, 1996; Muzzarelli, 2009; Gamboa, & Leong, 2013). Carrageenan-chitosan PECs can be obtained as films (Yan, Khor, & Lim, 2001), microcapsules (Devi & Maji, 2009; Devi, & Maji, 2010), microspheres (Muzzarelli et al., 2004) and gels (Mitsumata et. al.,
30
2003) to be used as drug delivery agents (Li, et al., 2013), cosmeceuticals (Kamel, &
31
Abbas, 2013), nano-layered coatings (Pinheiro, et al., 2012), dispersants (Tzoumaki,
32
et al., 2013) and beads (Saleem, et al., 2012).
33
Obtaining soluble CG:chitosan complexes can expand their application, owing
34
to combination of the useful properties of their constituent polysaccharides or
35
enhancing biological activity. To study the biological effects of CG:chitosan PECs it is
36
necessary to know in detail the complex formation process and supramolecular 4 Page 4 of 23
structure. As it was shown earlier by us, soluble complexes κ-CG:CH may be
2
obtained preferentially by mixing the starting components at given ratios. The
3
formation process of complexes depended on the CH molecular weight and the
4
concentrations and ratios of starting polysaccharides (Volod’ko, Davydova,
5
Barabanova, Solov’eva, & Ermak, 2012). This work is devoted to the study of the
6
formation and macromolecular organization of κ-CG:chitosan complexes and to the
7
investigation of their gastro protective and anti-ulcerogenic activities.
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2. Materials and methods
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2.1. Chitosan
12
A sample of chitosan was obtained by alkaline deacetylation of chitin
13
(Naberezhnykh, Gorbach, Likhatskaya, Davidova, & Solov’eva, 2008). In this work
14
we used sample of chitosan with molecular weight 115 kDa and a degree of N-
15
acetylation 6%. The molecular weights of the polycations were determined as
16
published earlier (Davydova, Yermak, Gorbach, Drozdov, & Solov'eva, 2000).
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2.2. Isolation of κ-carrageenan
18
A sample of κ-CG was isolated by extraction with hot water from C. armatus.
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In this work we used sample of κ-CG with molecular weight 311 kDa. The methods of
20
extraction and κ-CG structure determination have been published (Yermak, Kim,
21
Titlynov, Isakov, & Solov’eva, 1999).
22
2.3. Analytical Methods
23
The content of κ-CG was determined by reaction of the polysaccharide sulfate
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group with Taylor’s blue (1,9-dimethylmethylene blue) (Keler & Nowotny, 1986). The optical density was measured at 535 nm on a μ-Quant spectrophotometer (Bio-Tek Instruments Inc., USA). The content of chitosan was determined by reaction of the amino group with trinitrobenzenesulfonic acid (TNBS) (Inman & Dintzins, 1969). The optical density was measured at 410 nm on the μ-Quant spectrophotometer (Bio-Tek Instruments Inc., USA).
2.4. Titration of the tropaeolin 000-II (Orange II) – chitosan complex with κ-CG solution To 80 μl of 0.005 M phosphate buffer (pH 5.0), 40 μl of tropaeolin 000-II
33
(sodium salt of 4-(2-hydroxy-1-naphthylazo)benzenesulfonic acid) solution (0.08
34
mg/ml) and 20 μl of chitosan solution (100 μg/ml) were added. The mixture was
35
incubated for 20 min at 25°C. Then the resulting complex was titrated with solution
36
(200 μg/ml) by adding different portions of κ-CG. The absorption was determined with 5 Page 5 of 23
a μ-Quant spectrophotometer (Bio-Tek Instruments Inc., USA) at 483 nm in three
2
parallel samples, and the mean arithmetic value was calculated. The value of
3
ΔD=Dexp−D0 was calculated, where D0 and Dexp were absorptions of the solutions
4
before and after the addition of κ-CG, respectively. The values of ΔDmax and the
5
binding constant were determined from the Scatchard dependence in ΔD/Cκ-CG
6
versus ΔD coordinates. The chitosan saturation (Q) with κ-CG molecules was
7
determined from the ratio ΔD/ΔDmax.
ip t
1
The number of binding sites on the κ-CG molecule per amino group of
9
chitosan was assessed from the saturation curve by plotting a tangent to the point
10
that corresponded to the maximal change in the reaction mixture's absorption. Then
11
the κ-CG concentration corresponding to the chitosan saturation with the κ-CG was
12
determined.
us
cr
8
2.5. Centrifugation in a Percoll Gradient
14
Percoll (Sigma, 26 mL, 30%) in NaCl solution (0.15 M) was placed into a 28
an
13
mL centrifuge tube. A sample of κ-CG, chitosan or their mixture (2 mL) was layered
16
on the Percoll and centrifuged in an angled rotor Heraeus Biofuge Stratos (Germany)
17
at 20,000 g for 60 min. After centrifuging, the tube contents were removed through
18
the top using a peristaltic pump. Fractions (1.5 mL) were collected. The density of the
19
Percoll solution in each fraction was calculated using the refractive index determined
20
on a RF-4 refractometer (LOMO). The presence of chitosan and κ-CG in the fractions
21
was determined by the reaction of polysaccharide amino group with TNBS (Inman &
22
Dintzins, 1969) and by the reaction of sulfate group with Taylor’s blue (Keler &
23
Nowotny, 1986), respectively.
25 26 27 28 29
d
te
Ac ce p
24
M
15
2.6. Atomic Force Microscopy (AFM) The samples were dissolved in distilled de-ionized water at concentrations of
κ-CG and chitosan of 0.1 mg/mL, 0.01 mg/mL; PECs were prepared at the same concentrations at the ratios κ-CG:chitosan 10:1 and 1:10 w/w. Aliquots (12 μl) of each sample in an aqueous solution were deposited onto freshly cleaved mica and dried at 37 °C for 24 h. Morphology of polysaccharide and their complexes was
30
studied in air by AFM (Solver P47) in tapping contact mode using a 10 nm radius tip.
31
The topographical parameters of the sample macromolecular structure had been
32
automatically calculated by a self-developed program “Balagan’s Grain Analysis”.
33
The program allows us to separate objects from the substrate and determine
34
topographical parameters of each object: the lateral sizes, height, volume, occupied
35
area, etc. 6 Page 6 of 23
1 2 3
The analyzed AFM image is identified with a rectangular two-dimensional matrix where each cell contains a measured height value. At the first stage of the analysis, the image is smoothed and divided into rows and columns. Then based on each column (row), another two columns (rows) are
5
calculated: in the first one, the local maxima are eliminated, in the second one – the
6
local minima. The calculated four sets of the columns and rows are smoothed out (it
7
is possible to choose the smoothing method) and are interpolated. The interpolation
8
runs as follows: using the method of least squares, by four points, the closest to the
9
interpolated point, a parabola equation is deduced. The interpolated point height is
ip t
4
determined from the parabola equation. Then from the interpolated columns and
11
rows an averaged image is constructed.
At the second stage, the image splits into groups of pixels. To do this, the
us
12
cr
10
pixels are sorted by descending of their height. Then, starting from the beginning of
14
the pixels list, each pixel is coloured (similar to the numbering)by the following rules:
15
1. The pixel with coordinates (i1, j1) is adjacent to the pixel (i2, j2), if |i1 - i2 | ≤ 1
19 20 21
M
18
2. If near the pixel there are no coloured pixels, it is assigned a previously unused colour (it will form a new group of pixels); 3. If near a pixel there are coloured pixels, it is assigned a colour of the nearest local maximum.
d
17
and |j1 - j2| ≤ 1;
On the third stage, pixel groups are combined according to their height and
te
16
an
13
relative position on the plane. The program does the same actions as on the second
23
stage, but it works with pixel groups instead of single pixels.
24 25 26 27 28 29 30 31 32
Ac ce p
22
On the fourth stage, pixel groups are combined considering pixel groups
relative position in space and their size. In addition, the program examines the spatial distribution of pixels within the group, to reduce the effects of distortion in the case of the objects of interest located on the edge of a terrace. When this step finishes, it is possible to filter pixel groups by size and space position. At the fifth stage, the subtraction of the substrate there occurred. The
substrate relief is determined by analysis of the location of the pixel groups in space. After each object is separated from the substrate, the problem of the object parameters calculation is trivial.
33
2.7. Investigation of gastroprotective and antiulcerogenic activity
34
The experiments were carried out in three independent series on Vistar female
35
rats 200-220 g weighing in accordance with the Guidelines for the Care and Use of 7 Page 7 of 23
1
Laboratory Animals. The animals were given standard granulated food and water ad
2
libitum. Each group consisted of 6 animals. Model of stomach ulcers. All animals were deprived of food with maintanance
4
of free access to water during 24 hours. Then the rats were orally administrated 20
5
mg/kg indomethacin (“Fluka BioChemica”, Italy) suspended in water with Tween-80
6
(“ICN Biomedicals, Inc.”, USA), and 2 hours later the animals were fed. One day later
7
the rats were anesthetized with chloroform and necropsied, their stomachs were
8
removed and ulcers of the mucous membrane were counted under a binocular loupe.
ip t
3
The samples of polysaccharides and their complexes were injected
10
intraperitoneally in two modes. When evaluating the gastroprotective effect of κ-CG,
11
chitosan and their complexes, the samples injected once 1 hour before indomethacin
12
administration. In the study of anti-ulcerogenic action polysaccharides were injected
13
during three days prior to the introduction of indomethacin (the last injection was 24
14
hours before ulcerogenic agent). Control animals received an equivalent amount of
15
water, the reference group was injected with the gastroprotective drug Phosphalugel
16
(aluminum phosphate gel, «Astellas Pharma», Netherlands) in the same dose and
17
mode as the test polysaccharides. Pauls indexes (PI) were counted in each
18
experiment according to the formula: PI=A·B/100, where A – the average number of
19
ulcers per animal; B – the number of animals with ulcers in the group (%).
d
M
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cr
9
Antiulcer activity (AUA) was defined as the ratio of PI of the control group
21
divided to PI of the treatment group. It is considered that the monitoring agent has
23 24 25 26 27 28
Ac ce p
22
te
20
antiulcer activity on the assumption of AUA 2 units.
PECs κ-CG:chitosan were injected orally at a dose of 5 mg/kg or 0.5
mg/ml/100 g body weight. In this case the dose of the components in the complex were: 0.45 and 4.55 mg/kg. Solutions κ-CG and chitosan were injected at a dose of 4.55 mg/kg. The Phosphalugel was injected at 4.55 mg/kg. The results were analyzed using STATISTICA 6 software. The differences were significant at p<0.05.
29 30
3. Results and discussion
31
8 Page 8 of 23
1
The method of competitive binding κ-CG with a complex of chitosan/anionic dye was used to determine the binding constants of the polyanion (κ-CG) with a
3
polycation (chitosan). The tropaeolin OOO-II anionic dye [sodium 4-(2-hydroxy-1-
4
naphthylazo)benzenesulfonate] was used as an anionic dye. The method (Glazunov
5
& Gorbach, 1999) is based on the displacement of CG dye from its complex with
6
chitosan. The number of seats of binding was calculated. The number of sulfate
7
groups of κ-CG associated with one amino group of chitosan was close to
8
stoichiometric. The binding constants was calculated using Scatchard and Hill plots
9
(table 1). A linear character of the Scatchard plot assumes that no cooperativity
ip t
2
during the reaction (Nørby, Ottolenghi, & Jensen, 1980) and that one type of binding
11
sites involves into this process. This binding constant value (2.11×107, mol-1) showed
12
high affinity of κ-CG to chitosan.
us
cr
10
13
Table 1
an
14 15 16
Binding parameters of chitosan with κ-CG
19 20 21
1.98! 0.31
d
2.11! 0.31
Mean value 2.04! 0.26
1.29! 0.13
te
18
Hill’s method
The number of SO42- to 1 NH3+
Quantity chitosan associated with 1 mg κ-CG in saturation point, mg 0.33! 0.03
The method of centrifugation in Percoll gradient was used for direct detection
Ac ce p
17
Scatchard’s method
M
Binding constant, 107
of the complex formation. Figure 1 shows the results of Percoll gradient centrifugation of the complex initial components (Fig. 1a) and its mixtures at the ratio of κ-CG:chitosan 1:10 and 10:1 w/w (Fig. 1b,c).
22 9 Page 9 of 23
1
Fig. 1. Centrifugation in Percoll gradient: a – initial polysaccharides; b – κ-
2
CG:chitosan mixtures 1:10 w/w; c – κ-CG:chitosan 10:1 w/w; 1 – content of amino
3
groups chitosan (A474nm); 2 – content of sulfate groups κ-CG (A535nm)
4 5
It can be seen from figure 1 that the curves of κ-CG:chitosan mixtures (Fig. 1b and 1c) differed from those of the initial polysaccharides (Fig. 1a). Under the
7
centrifugation conditions chitosan formed a one zone of density and settled in the
8
lower part of the gradient whereas κ-CG was separated into two fractions in the
9
upper and middle parts of the gradient. The coincidence of the maxima of the elution
10
curve for chitosan and κ-CG in their mixture indicated that a κ-CG:chitosan complex
11
formed (Fig. 1b,c).
cr
Two types of complexes with different densities formed irrespective of the
us
12
ip t
6
initial components ratio. In the case of the polyanion high content in the mixture (κ-
14
CG:chitosan 10:1 w/w (Fig. 1b)), part of the κ-CG remains unbound, otherwise (κ-
15
CG:chitosan 1:10 w/w (Fig. 1c)) part of the chitosan remains free.
16
an
13
We studied the macromolecular structure of the κ-CG:chitosan complexes, obtained at different ratio, compared with the initial polysaccharides by AFM (Fig. 2).
18
Concentrations of the components were the same as in the complexes.
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17
Ac ce p
te
d
19
20 21
Fig. 2. AFM topography images: a – chitosan, C=0.01 mg/ml, b – κ-CG, C=0.1
22
mg/ml, c - PEC κ-CG:chitosan 10:1 w/w; and a histogram of the meshes area 10 Page 10 of 23
1
distribution: d – κ-CG, C=0.1 mg/ml; e – PEC κ-CG:CH 10:1 w/w; on the inserts one
2
can see the calculation masks.
3 4
According to the AFM images shown in Fig. 2a, chitosan at a concentration of 0.01 mg/ml produces a homogeneous and very smooth (root-mean-square
6
roughness (RMS) – 0.13 nm) film, which is sensitive to AFM probe influences. At the
7
scanning of chitosan in tapping contact mode, during recording one of the image
8
lines, there was self-excited oscillation of the cantilever. As a result, the probe tip
9
impacts the surface for several times with increased force. The crack on the film
11
surface was observed during rescanning of the same area.
cr
10
ip t
5
The basis of the chemical structure of the κ-CG is a disaccharide repeating unit, which contains the 3,6-anhydrogalactose. It determines the κ-CG ability of
13
forming a double helical. As shown on the AFM images (Fig. 2b), κ-CG at
14
concentration 0.1 mg/ml forms a three-dimensional network structure with height
15
about 2.5 nm that indicates on intermolecular association of the polysaccharide “side-
16
by-side” (Funami et. al., 2007). The network is continuous and composed of
17
branched fibers. The average wall thickness of the network is (Cκ-CG=0.1 mg/ml) 0.05
18
μm. Figure 2d shows a histogram of the meshes area distribution. According to that
19
observed for κ-CG unimodal size meshes area distribution, with the maximum at
20
about 1600 nm2.
an
M
d
The macromolecular structure of PEC κ-CG:chitosan 10:1 w/w was different
te
21
us
12
from structure of the initial components with the same concentrations of
23
polysaccharides, that in the complex. As one can see from Fig. 2, the structure of the
24 25 26 27 28 29
Ac ce p
22
complex consists of several layers. The bottom layer has a mesh structure similar to the structure of κ-CG, but with considerably smaller wall thickness (0,025 μm). The histogram of the meshes area distribution (Fig. 2e) has a bimodal character with two maxima at about 150 and 300 nm2. This is 10 times smaller than the area corresponding to the peak of the histogram for the κ-CG. The second layer is disposed over the reticlated structure and it is composed of long twisted fibrils. In this
30
PEC the amount of the κ-CG was 10 times greater than chitosan, and perhaps the
31
two layers correspond to two types of complexes (heavy and light) which were
32
observed during the centrifugation of PEC in Percoll gradient (Fig. 1b,c).
33
11 Page 11 of 23
ip t
1
Fig. 3. AFM topography images: a – chitosan, C=0.1 mg/ml, b – κ-CG, C=0.01
3
mg/ml, c - PEC κ-CG:chitosan 1:10 w/w
cr
2
4
Increasing of the chitosan concentrations by 10 times results in
us
5
supramolecular structure changes. By the AFM data (Fig. 3a), chitosan composed of
7
densely packed spherical particles. The RMS value is 14.3 nm, corresponding to at
8
least a level surface, unlike chitosan at a lower concentration (Fig. 2a).
9
an
6
κ-CG at low concentrations (C=0.01 mg/ml) forms long threadlike structures. Fibers cross over one another, which lead to the increase of the height at the
11
crossover points. That may correspond to the formation of the double-structure
12
characteristic of κ-CG (Sokolova et al 2013).
M
10
The AFM image of PEC κ-CG:chitosan (1:10 w/w) shows the formation of
14
morphologically heterogeneous structure, which is different from those of PEC κ-
15
CG:chitosan (10:1 w/w) and from initial polysaccharides. As one can see from Fig. 3,
16
sub-layer consist of spherical segments that possibly correspond to free chitosan.
18 19 20 21 22
te
Ac ce p
17
d
13
The heterogeneity observed in the structure is in line with the data obtained by centrifugation, whereby PEC divided in the Percoll gradient into two fractions (Fig. 1c). Also tightly packed formations of complex κ-CG:chitosan 1:10 was observed which probably represent fibrils of κ-CG (average length of 0.5 μm) consisting of segments chitosan.
The observed features of the molecular organization of the PECs κ-
23
CG:chitosan reveal that the dependence of the ratio of the initial polysaccharides can
24
have a significant impact on their biological effects in the body. We investigated
25
gastroprotective and anti-ulcerogenic effect of these complexes (10:1 and 1:10 w/w)
26
to test this assumption.
27
It was found that the PECs κ-CG:chitosan had various gastroprotective effects
28
depending on its composition. PEC κ-CG:chitosan 10:1 w/w reduced ulcers in the
29
stomach in two times, and PEC κ-CG:chitosan 1:10 w/w in three times in comparison 12 Page 12 of 23
1
with the control (Table 2). Thus when the samples are injected one hour before the
2
ulcerogenic impact, the complex with a higher content of κ-CG exhibits a moderate
3
activity, while the complex with excess of chitosan has significant gastroprotective
4
activity.
5 6
Table 2.
11
5.8±1.1
Antiulcer Pauls index activity, units
cr
Control
The total number of ulcers in the group (6 rats) 35
5.8
-
2.3
2.5
0.9
6.4
us
Sample
The average number of ulcers per 1 rat
κ-CG:chitosan 2.8±1.0 (р=0.07) 17 10:1 w/w κ-CG:chitosan 1.8±1.0 (р=0.03) 11 1:10 w/w р<0.05 - differences with control are significant
an
9 10
Comparative gastroprotective activity of PECs κ-CG:chitosan
The further study κ-CG, chitosan and their PEC in comparison with drug
M
8
ip t
7
Phosphalugel showed that the activity of the complex with a high content of
13
polycation (κ-CG:chitosan 1:10 w/w) exceeded the activity of the reference drug,
14
while the PEC with a high content of polyanion (κ-CG:chitosan 10:1 w/w)
15
demonstrated a low gastroprotective effect (Table 3). It is interesting to note the low
16
activity of initial polysaccharides (4.55 mg/kg, which corresponds to the maximum
17
content of the complexes), as compared with Phosphalugel. This data shows that the
19 20 21 22 23 24
te
Ac ce p
18
d
12
increase of gastroprotective effect of PEC κ-CG:chitosan 1:10 w/w may not be directly related with gastroprotective properties of chitosan.
Table 3.
Gastroprotective activity κ-CG, chitosan and their complexes in comparison with Phosphalugel Sample
The average number of ulcers per 1 rat
Control κ-CG Chitosan
9.2±1.6 2.8±1.5 (р=0.01) 6.2±1.9 (р=0.24)
The total number of ulcers in the group (6 rats) 55 17 37
Pauls index
Antiulcer activity, units
9.2 1.9 6.2
4.8 1.5 13 Page 13 of 23
κ-CG:chitosan 10:1 w/w κ-CG:chitosan 1:10 w/w
4.2±1.3 (р=0.03)
25
1.3±1.0 (р=0.002) 8
1.0±0.4 6 (р=0.0004) р<0.05 - differences with control are significant
Phosphalugel 1
4.2
2.2
0.4
23.0
0.7
13.1
2
As indicated above, chitosan and κ-CG have various biological activities,
ip t
3
including antiinflammatory, immunomodulatory and reparative. Such activities may
5
have a modifying effect on the stomach mucosa, increasing its resistance to the
6
effects of pathological factors. An experiment was conducted with a three-day
7
prophylactic injection of κ-CG, chitosan and their PEC to evaluate the possible
8
contribution of non-specific activity of polysaccharides in antiulcer effect. The last
9
injection of the samples was done a day before the reproduction of ulcerative lesions,
us
cr
4
which eliminated the direct contact of the tested samples with indomethacin on the
11
mucosal surface of the stomach. The experiment shows that the introduction of
12
polysaccharides in this mode has not reduced the amount of gastric ulcers induced
13
versus control. There is only reducing the excitability of the gastric mucosa in animals
14
treated with complexes, especially complex κ-CG:chitosan 10:1 w/w, resulting in a
15
reduction of mucosal hyperemia (Table 4). Phosphalugel didn’t exhibit antiulcer
16
activity in these conditions as well.
17
19 20 21
Table 4.
Ac ce p
18
Anti-ulcerogenic activity κ-CG, chitosan and their PEC at the prophilactic injection to rats with indomethacin-induced stomach ulcer
Sample
22
te
d
M
an
10
The average number of ulcers per 1 rat
The total number of ulcers in the group (6 rats) 42 44 45
Pauls index
Antiulcer activity, units
Control 7.0±1.2 0.7 κ-CG 7.3±1.4 0.7 1.0 Chitosan 7.5±1.8 0.7 1.0 κ-CG:chitosan 9.2±1.7 55 0.9 0.8 10:1 w/w κ-CG:chitosan 6.7±1.8 40 0.7 1.0 1:10 w/w Phosphalugel 7.2±2.0 43 0.7 1.0 p > 0.05 at all groups - differences with control are unsignificant
23 14 Page 14 of 23
1
4. Conclusion
2 3
The soluble PEC κ-CG:chitosan was obtained. Binding constant value showed high affinity of κ-CG to chitosan. The complex formation of κ-CG:chitosan 1:10 and
5
10:1 w/w was proved by centrifugation in a Percoll gradient. Summarizing the results
6
of biological studies, it may be noted that aqueous solutions of PEC κ-CG:chitosan
7
(C=0.5 mg/ml) have gastroprotective activity which depend on their composition.
8
Complex κ-CG:chitosan 1:10 w/w possesses higher gastroprotective activity than the
9
complex κ-CG:chitosan 10:1 w/w. The test polysaccharides and their complexes at
10
preliminary three-days injection do not increase own protective properties of gastric
11
mucosal.
cr
The differences in the effectiveness of PECs κ-CG:chitosan is likely to be
us
12
ip t
4
associated with features of the supramolecular structure fixed by AFM. We showed
14
that the supramolecular structure of the complexes is different from each other and
15
from the macromolecular structure of the initial polysaccharides. According to this
16
data, κ-CG (C=0.01 mg/ml) forms long threadlike structures, whereas a tenfold
17
increase in the concentration produced mainly coarse three-dimensional networks
18
with a large amount of meshes. Considering that the concentration of κ-CG in
19
aqueous solution of PEC κ-CG:chitosan 1:10 w/w was 0.045 mg/ml, it can be
20
assumed that its high gastroprotective activity is provided by a regular formation of
21
the protective layer which formed due to the optimum conditions of complexation for
22
surface fibrils. These results suggest that the gastroprotective effect of complexes
23
can be associated with the formation of the protective layer on the surface of the
25 26 27 28 29
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d
te
Ac ce p
24
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13
stomach mucousa, which hinders direct contact with the ulcerogenic agent (indomethacin).
Acknowledgments The work was supported by the Programs for Fundamental Research of the
30
Presidium of the Russian Academy of Science “Molecular-cell biology” and by the
31
Presidium of Far-East Branch of the Russian Academy of Science and a grant № 12-
32
II SD – 05 – 006.
33 34
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Schmidt, A. G., Wartewig, S., & Picker, K. M. (2003). Potential of
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carrageenans to protect drugs from polymorphic transformation. European Journal of
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Pharmaceutics and Biopharmaceutics, 56(1), 101-110. 17 Page 17 of 23
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Sokolova, E. V., Chusovitin, E. A., Barabanova, A. O., Balagan, S. A., Galkin,
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N. G., & Yermak, I. M. (2013).Atomic force microscopy imaging of carrageenans from
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red algae of Gigartinaceae and Tichocarpaceae families. Carbohydrate Polymers,
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93(2), 458-465.
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Thommes, M., & Kleinebudde, P. (2006). Use of κ-carrageenan as alternative pelletisation aid to microcrystalline cellulose in extrusion/spheronisation. I. Influence
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of type and fraction of filler. European Journal of Pharmaceutics and
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Biopharmaceutics, 63(1), 59-67.
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Tzoumaki, M.V., Moschakis, T., Biliaderis, C.G. (2013) Effect of soluble
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polysaccharides addition on rheological properties and microstructure of chitin
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nanocrystal aqueous dispersions. Carbohydrate Polymers, 95 (1), 324-331. Van de Velde, F., Lourenço, N. D., Pinheiro, H. M., & Bakker, M. (2002).
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cr
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Carrageenan: A Food‐Grade and Biocompatible Support for Immobilisation
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Techniques. Advanced Synthesis & Catalysis, 344(8), 815-835.
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Volod’ko, A. V., Davydova, V. N., Barabanova, A. O., Solov’eva, T. F & Ermak,. I. M. (2012). Formation of soluble chitosan–carrageenan polyelectrolyte
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complexes. Chemistry of Natural Compounds, 48(3), 353-357.
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Yan, X. L., Khor, E., & Lim, L. Y. (2001). Chitosan‐alginate films prepared with
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d
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chitosans of different molecular weights. Journal of Biomedical Materials Research, 58(4), 358-365.
Yermak, I.M., Barabanova, A.O., Aminin, D.L., .Davydova, B.N., Sokolova,
E.V., Kim Y. H., Shin K. S., & Solov’eva T. F. (2012). Effects of structural peculiarities
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of carrageenans on their immunomodulatory and anticoagulant activities.
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Carbohydrate Polymers, 87(1), 713-720.
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Yermak I.M., & Khotimchenko Yu.S. (2003). Chemical properties, biological
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activities and applications of carrageenan from red algae. In M Fingerman,& R.
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Nagabhushanam (Eds.), Recent advances in marine biotechnology (pp. 207-255).
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New York, USA/London, UK: Plymouth Science Publishers Inc. 18 Page 18 of 23
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Yermak, I. M., Kim, Y. H., Titlynov, E. A., Isakov, V. V., & Solov’eva, T. F.
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(1999). Chemical structure and gel properties of carrageenans from algae belonging
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to the Gigartinaceae and Tichocarpaceae, collected from the Russian Pacific coast.
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Journal of Applied Phycology, 11, 41-48.
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Table 1
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Binding parameters of chitosan with κ-CG Binding constant, 107 Hill’s method 1.98! 0.31
Mean value 2.04! 0.26
The number of SO42- to 1 NH3+ 1.29! 0.13
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Scatchard’s method 2.11! 0.31
Quantity chitosan associated with 1 mg κ-CG in saturation point, mg 0.33! 0.03
4
cr
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Table 2.
2
The total number of ulcers in the group (6 rats)
Pauls index
Antiulcer activity, units
Control
5.8±1.1
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5.8
-
κ-CG:chitosan 2.8±1.0 (р=0.07) 17 10:1 w/w κ-CG:chitosan 1.8±1.0 (р=0.03) 11 1:10 w/w р<0.05 - differences with control are significant
2.3 0.9
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Sample
The average number of ulcers per 1 rat
2.5 6.4
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Comparative gastroprotective activity of PECs κ-CG:chitosan
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Table 3.
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Gastroprotective activity κ-CG, chitosan and their complexes in comparison with
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Phosphalugel
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Control 9.2±1.6 55 (р=0.01) 2.8±1.5 17 κ-CG 6.2±1.9 (р=0.24) 37 Chitosan κ-CG:chitosan 4.2±1.3 (р=0.03) 25 10:1 w/w κ-CG:chitosan 1.3±1.0 (р=0.002) 8 1:10 w/w 1.0±0.4 (р=0.0004) 6 Phosphalugel р<0.05 - differences with control are significant
Antiulcer activity, units
9.2 1.9 6.2
4.8 1.5
4.2
2.2
0.4
23.0
0.7
13.1
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Pauls index
ip t
The total number of ulcers in the group (6 rats)
cr
The average number of ulcers per 1 rat
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Sample
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Table 4.
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Anti-ulcerogenic activity κ-CG, chitosan and their PEC at the prophilactic injection to rats
4
with indomethacin-induced stomach ulcer The total number of ulcers in the group (6 rats)
Pauls index
1.0 1.0
0.8 1.0 1.0
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cr
Control 7.0±1.2 42 0.7 κ-CG 7.3±1.4 44 0.7 Chitosan 7.5±1.8 45 0.7 κ-CG:chitosan 9.2±1.7 55 0.9 10:1 w/w κ-CG:chitosan 6.7±1.8 40 0.7 1:10 w/w Phosphalugel 7.2±2.0 43 0.7 p > 0.05 at all groups - differences with control are unsignificant
Antiulcer activity, units
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Sample
The average number of ulcers per 1 rat
6
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7
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23 Page 23 of 23