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Study of the interactions between polyphenolic compounds and chitosan
Reactive & Functional Polymers 45 (2000) 35–43 www.elsevier.com / locate / react
Study of the interactions between polyphenolic compounds and chitosan Marcel-Ionel Popa*, Nicolae Aelenei, Valentin I. Popa, Daniela Andrei Technical University ‘ Gh Asachi’, Faculty of Industrial Chemistry, D. Mangeron 71, Iasi, RO 6600 Iasi, Romania Received 4 January 1999; received in revised form 7 January 2000; accepted 12 January 2000
Abstract This paper discusses the interaction between chitosan and the polyphenols separated from spruce wood bark. The chitosan and the polyphenols formed a complex and the release of the polyphenols occurred only in an alkaline medium (pH . 9) in a two-step process. Analysis of the release of the polyphenols showed that in the initial stage the diffusion occurs according to the Fick’s law, while in the last stage, the process develops according to a zero-order kinetics. 2000 Elsevier Science B.V. All rights reserved. Keywords: Polyphenols; Chitosan; Controlled release
1. Introduction The polyphenols represent one of the main classes of plants’ secondary metabolites. They are characterized by a special complexity and structures which differ from one plant to another. They play a special part in the growth and reproduction processes. They are distinguished by the following general features: (1) water solubility — although when pure some plant polyphenols may be difficult to dissolve in water, in the natural state polyphenol–polyphenol interactions usually ensure some minimal solubility in aqueous media; (2) molecular masses — natural polyphenols encompass a large molecular mass *Corresponding author. E-mail address: [email protected] (M.-I. Popa).
range from 500 to 3000–4000; (3) structure and polyphenolic character-polyphenols, per 1000 relative molecular mass, possess some 12–16 phenolic groups and 5–7 aromatic rings; (4) intermolecular complexation, besides giving the usual phenolic reactions they have the ability to precipitate some alkaloids, polysaccharides, gelatin and other protein from solution. These complexation reactions are not only of intrinsic scientific interest as studies in molecular recognition and possible biological function, but they could have important and wide-ranging practical applications [1]. Considering their function in metabolic processes, as well as some important in vitro manifested properties of polyphenols, much recent research has focused on their separation and structural characterization, as well as on their possible applications [2–4]. If one re-
1381-5148 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S1381-5148( 00 )00009-2
36
M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
searches their utilization as antioxidant agents [5] or plant growth regulators [6,7], than the study of polyphenols’ interaction with some complexing agents has a special significance. This would permit the development of some isolation techniques for polyphenols from extraction media, and some controlled release systems. This paper studies the manner in which the polyphenols separated from spruce wood bark interact with chitosan, and also the stability of the complex thus obtained under different pH conditions. 2. Experimental The studied polyphenols were extracted from spruce wood bark, with an 1% ammonia solution, by a technique described elsewhere [4]. The chitosan with a relative molecular mass of 150 000 and a 12% acetylation degree was ´ obtained from the Departement de Genie Chimique of the University of Sherbrooke, Canada. The interaction between the two reactants was studied on an 1% aqueous solution of polyphenols and an 1% solution of chitosan. The dissolution of the chitosan involved the stirring of the solid chitosan in a 1 M hydrochloric acid solution, followed by the neutralization of the clear solution thus obtained with a 1 M NaOH solution, up to a pH of 5.7. Afterwards, the solution of polyphenols was added to the chitosan, the systems being stirred continuously, at room temperature, for 4 h. After the reaction, the precipitate formed was separated through centrifugation at 3500 rpm, for 10 min. The unreacted polyphenols were removed by repeated washings with distilled water. Finally, the product was washed with acetone and dried in vacuum, at room temperature.
basic and in a weakly acid medium, the study of the kinetics was realised through spectophotometry. The studied polyphenols are soluble only in a strongly acid medium. The visible absorption spectrum of the aqueous solutions of polyphenols shows a maximum over the 350nm domain, regardless of the pH value, starting from a pH of 7.0. A wavelength of 450 nm has been selected in order to assure a sufficient precision of the method for solutions with relatively high concentrations (0.1 g / dl). The experimental data necessary for drawing the calibration curve were obtained from the study of ten aqueous solutions with concentrations between 0 and 0.1 g / dl, prepared by diluting a standard solution of 0.1 g / dl concentration. Measurements were made in 1-cm thick glass cell, at a wavelength of 450 nm. The calibration curve is plotted in Fig. 1. Similar calibration curves were recorded for buffer solutions with pH 6.5, 7.8 and 9.8. In all situations the experimental points were placed on the same straight line, with an error below 1%. For the subsequent calculations we utilised the average calibration curve. The equation of the curve was obtained by the least-square method A 5 6.8882c
2.1. Analysis of polyphenols Due to the intensely brown-reddish colour of the aqueous solutions of polyphenols, both in a
Fig. 1. Calibration curve for polyphenols in aqueous solution.
M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
where A is the solution’s absorbancy and c is the concentration. In all subsequent kinetic studies, the concentrations of the solutions, expressed in mg / ml, were calculated according to the relation: c 5 A / 0.6882.
2.2. Release of polyphenols from the complex The study of polyphenols’ release from the chitosan–polyphenols complex was carried out by an elution technique in aqueous medium of different pH values, under continuous stirring, at 258C. Preliminary qualitative determinations indicated that, in an acid medium, no release occurs. Therefore only the release in a basic medium, using buffer solutions with 7.8, 8.9, 9.9 and 11.9 pH values was studied. The sample, appearing as a fine powder, was introduced into the eluent and stirred continuously for 300 min. Now and then, samples were extracted from the solution and their absorbances measured. Afterwards, the samples were re-introduced into the release medium. In all experiments, the weight of the complex sample was 0.1 g, for an eluent volume of 50 ml. The total amount of polyphenols present in the samples was determined by measuring the solution’s absorbency 48 h after the initiation of release and checking its value 78 h later. No modification of the eluent’s concentration was noticed after 78 h in any sample. The maximum quantity of polyphenols released, at pH.8.5, after 72 h, was: m f 5 82.2 mg, which represents 82.2% of the polyphenols retained in the complex.
3. Results and discussion Chitosan is a linear polymer of mainly anhydroglucosamine and behaves as a linear polyelectrolyte at acidic pH [8]. As chitosan is a polymer, it has a higher positive charge density, one charge per glucosamine unit.
37
The efficacy of polyphenols as complexing agents derives principally from their relative molecular mass and size, from the many phenolic groups in the same molecule and associated aromatic nuclei. The process of complexation between polyphenols and chitosan may be reversible or irreversible [1]. Reversible complexation of polyphenols may be considered as a two-stage process, in the first of which the polyphenols and chitosan, by the development of non-covalent forces, are in equilibrium with the soluble complex. As the position of this equilibrium changes then, as a second stage, these soluble complexes may well aggregate and precipitate from solution. The whole process is however usually reversible and under suitable conditions the precipitated complex may be redissolved. Polyphenols 1 chitosan 1 H 2 O A [Polyphenols] n ? [Chitosan] m 1 H 2 O Precipitation A [Polyphenols] a ? [Chitosan] b This mechanism is confirmed by our results, since the curve’s aspect shows that the release of the polyphenols from the complex occurs at a higher rate with increasing pH. The general aspect of polyphenols’ release from the complex is presented in Fig. 2, which plots graphically the fraction from the total amount of polyphenols, released as a function of time. In a weakly alkaline medium, (pH 7.8) the release occurs to a reduced extent, a release degree of about 5% from the amount of polyphenols in the sample being attained after 120 min, this value remaining constant up to 300 min. The total amount released at pH 7.8, after 96 h, was about 6%, which perhaps represents
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M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
Fig. 2. Kinetics of polyphenols’ release from chitosan–polyphenols complex, as a function of time, at various pH values.
only the polyphenols physically included in the complex. With increasing pH both the release rate in the initial period (the first 90 min) and the released amount of polyphenols increases. The estimation of the release rate at certain times has been calculated with the relation 1 Dm 1 m n 11 2 m n r 5 ] ]] 5 ] ]]]] m s Dt m s t n 11 2 t n
cesses and practically independent of pH is installed. For pH values of 7.8 the release shows the same stages, characteristic for the initial 0–90 min and the main 90–300 min periods. The only exception is that, in this case, the release ends after 90 min and, at longer times, the release rate is practically equal to zero.
(2)
where r is the relation rate at average time t 5 (t n 1 t n 11 ) / 2 and m s is the sample’s weight. The moment at which release rate is evaluated with Eq. (2) representing an average value: t 5 (t n 1 t n 11 ) / 2, Dm is the polyphenols released over a Dt time interval. The release rate as a function of time is plotted in Fig. 3. Apart from the sample eluted at pH 7.8, which exhibits a very reduced release rate — close to zero nearly on the whole period — all the samples showed a strong decrease of the release rate in the initial period, differentiated as function of pH. After 120 min a regime of constant rate, characteristic for zero order pro-
3.1. Kinetic aspects of polyphenol release from the complex The process of transport of a bioactive agent through a polymer or through controlled release systems may be described with Fick’s diffusion law. Nevertheless, numerous exceptions from such a mechanism occur. That happens because of the specific features of the transport medium, the device’s geometrical elements, the diffusing agent molecular sizes — medium and, respectively, diffusing — eluent interactions. The solutions of Fick’s equation depends on them to a large extent. A review of the mathematical models applied in the study of controlled release
M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
39
Fig. 3. Variation of polyphenols’ release rate in time, as a function of pH.
has been recently presented by Narasimhan and Peppas [9]. In the case of drugs dispersed on non-porous polymeric matrices, the mathematical model — assuming a pseudo-stationary state — was introduced by Higuchi [10], and developed by Paul and McSpaden [11], who obtained a dependence between the weight of released drug and the time as follows 2c s A ] D m t 5 ]] ]t (3) erfj * p
œ
where c s is the solubility of the drug in the polymer; A is the surface of the polymer film’s cross section; D is the coefficient of the drug’s diffusion through the polymer; m t is the weight of drug released at moment t; erfj * is an error function of the j * 5 x * /(Dt)1 / 2 . When the drug or the bioactive agent is dissolved in vitrous polymers or must penetrate through non-porous thin membranes (with a thickness of d ), a simplified solution of Fick’s law may be written ]] m 16Dt ]t 5 ]] (4) mf pd 2
œ
where m t is the weight of drug released at moment t; m f is the weight of drug at t 5 ` (assuming that m f is the drug mass initially present in the sample, which is released completely after a sufficiently long time); D5 diffusion coefficient; d 5thickness of the film. An equation of the (4) type is applicable only for low diffusion times, more exactly for the situation in which up to 60% of the total amount of drug (m t /m f , 0.6) is released. For the same type of system, but longer release times, or when m t /m f . 0.6, the solution of Fick’s law is
S D
m p 2 Dt 8 ]t 5 1 2 ]2 exp ]] mf d p
(5)
The confirmation of the mechanism involves checking the linearity of the experimental data in the coordinates ln (1 2 m t /m f ) 2 t, according to the equation
S D
mt 8 p 2D ln 1 2 ] 5 ln ]2 1 ]] t mf p d2
S
D
(6)
If the drug is dispersed into a porous polymeric matrix, the kinetic equation is [12]
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M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
]]]]]] m t 5 AœDeff Css2Cd 2 e Csdt
(7)
where e is the void fraction in the porous material: Cd is the concentration of the drug in the polymer film, Cs is the drug solubility in the polymer and Deff is the effective diffusion coefficient. Another procedure relatively simple but attractive — for practical reasons — involves the description of the controlled release with the ‘power law’, according to which the relative amount released is expressed as a function of the release time m ]t 5 kt n mf
(8)
Korsmeyer and Peppas [13] stated first such an equation in the case of controlled release. A detailed analysis on the application of Eq. (8) to the study of swelling or rigid systems and also to various geometries of the release system was made by Ritger and Peppas [14]. Usually, according to the linearized equation ln (m t /m f ) 5 ln k 1 nln t
(9)
the experimental data are expressed in logarithmic co-ordinates: ln (m t /m f ) 2 ln t, the value of exponent n being evaluated from the slope of the straight line thus obtained. For the acceptance of linear regression in the case of the data under analysis, the confidence interval should be at least 95% (r 2 . 0.95). If the value of the n obtained is 0.5, the mechanism of drug transport is Fickian and the drug release rate is time-dependent. If 0.5 , n , 1, the transport is anomalous and the release rate is once again time- dependent. If n51.0, the transport is of the second type and the release rate is time-independent or in other words, zero-order release is obtained. Our studies on the behaviour of the new release system obtained involved the checking of both mathematical models described above. The results obtained in the process of polyphenol release from the chitosan–polyphenols complex, in a moderate basic medium, are given
as examples. The used sample was a raw powder, and the parameters of the release process were the following: weight of sample 0.1 g; volume of eluent 50 ml; pH 9.9; temperature 258C, intensive stirring. The control of the release was monitored spectrophotometrically, at a wavelength of 450 nm, the experimental results obtained with this sample being presented in Fig. 4. Two domains may be distinguished on the release curve, as follows: an initial period (0–60 min), over which variation is non-linear, having a decreasing slope, and a main period (90–720 min) over which variation is linear. In order to check whether the release occurs through a Fick type diffusion, which is characteristic to non porous polymers, the experimental data have been represented as both m t /m f 2 t 1 / 2 (Fig. 5) and ln (1 2 m t /m f ) 2 t (Fig. 6). Fig. 5 shows that the model is suitable for the system used here, the confidence interval for the initial period (m t /m f , 0.6) being of 99.4%, which indicates a good linearity between the variables taken into study. It is surprising that, in the given coordinates, a linear dependence is also obtained for the main release period. This may be due to the fact that the tested model is particularly adequate for the transport through thin films, while in our case the substance presents itself as a powder. Fig. 6 shows the validity of the model used. Indeed, the equation is adequate for release ratios over 0.6, which, in our situation, corresponds to release times longer than 60 min. Within the 60–270 min domain, the experimental data form a straight line, the confidence interval being 99.7%. Eq. (6), applied to the initial period is not satisfactory, the curve thus obtained showing important deviations from linearity (in this case, the confidence interval being below 95%). If Eq. (8) is used for processing the data regarding polyphenol release from the chitosan– polyphenols complex, at pH 9.9, it will show that, in the initial moments (60–90 min), release occurs through a mechanism of unidirectional
M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
41
Fig. 4. Relative amount of polyphenols released as a function of time, at pH 9.9.
Fig. 5. Fick’s law mechanism checking for elution at pH 9.9.
diffusion. Indeed, as plotted graphically in Fig. 7, in ln m t 2 ln t coordinates, a straight line having the slope very close to 0.5 was obtained, and also a very good confidence interval. The second time interval (60–270 min) is
characterized by a zero-order kinetics, for which in Eq. (8) n51, and thus the equation becomes m ]t 5 kt mf
(10)
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M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
Fig. 6. Graphical checking of Eq. (6).
4. Conclusions
Fig. 7. Graphical checking of Eq. (9).
As shown Fig. 8, the obtained linearity is very good, which justifies the assertion that the release of polyphenols from chitosan is governed, with the exception of the initial period, by zero order kinetics — most suitable for systems of controlled release.
An ionic complex was obtained between chitosan and the polyphenols extracted from spruce wood bark. The process of polyphenol release from the chitosan—polyphenols complex was studied and some conclusions were drawn. Release does not occur in an acid medium, the complex being insoluble, while polyphenol retention is achieved through strong chemical bonds. In a weakly alkaline conditions (pH 7.8), only about 6% of the total amount of polyphenols is released from the complex, which probably represents the compound dissolved physically in the polymer. The amount of released polyphenols and the release rate in the initial stage increase with the increase of the pH of the elution medium. At an alkaline pH, two distinct stages may be observed in the release process, namely: an initial stage (0–60 min), during which the release rate decreases in time — the decrease being more significant at the higher pH — and a
M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
43
Fig. 8. Graphical checking of a zero order kinetics.
main release stage (60–300 min) during which the release rate is constant and independent on pH. The study of the release mechanism showed that, in the initial stage, release occurs through a Fick diffusion mechanism, while, in the main period, the release occurs according to a zeroorder kinetics. Acknowledgements The study is part of a cooperation program established between Technical University of Iasi-Romania and University of SherbrookeCanada. References [1] E. Haslam, Polyphenol complexation in polyphenolic phenomena, in: A. Scalbert (Ed.), Polyphenolic Phenomena, INRA, Paris, 1993, pp. 23–33.
[2] J.B. Harbone, New naturally occurring plant polyphenols, in: A. Scalbert (Ed.), Polyphenolic Phenomena, INRA, Paris, 1993, pp. 19–22. [3] V.I. Popa, C. Beleca, Cellulose Chem. Technol. 28 (1994) 613–620. [4] V.I. Popa, C. Beleca, M. Popa, R. Bodirlau, I. Bara, E. Truta, Bull. Soc. Brot. Ser. 2, (Portugal) 67 (1966) 245–255. [5] L. Barolary, F. Xi, I. Norris, J. Wood Chem. Technol. 17 (1997) 73–90. [6] B. Kosikova, E. Slavikova, Drevarsky Vyokum 1–2 (1994) 33–38. [7] V.I. Popa, C. Beleca, M. Popa, R. Bodarlan, P. Vidrascu, Rev. Roum. Chim. 40 (1995) 691–698. [8] D.N.S. Hon, Chitin and chitosan: medical application, in: S. Dumitriu (Ed.), Polysaccharides in Medicinal Applications, Marcel Dekker, New York, 1966, pp. 631–650. [9] B. Narasimhan, N.A. Peppas, Controlled drug delivery, Kinam Park, in: ACS Professional Reference Book, A, Chemical Society, Washington, DC, 1997, pp. 529–557. [10] T. Higuchi, J. Pharm. Sci 50 (1961) 874. [11] D.R. Paul, S.K. Mc Spaden, J. Membr. Sci 1 (1976) 33. [12] T. Higuchi, J. Pharm. Sci. 52 (1963) 1145. [13] R.W. Korsmayer, N.A. Peppas, in: S.Z. Mansdorf, T.J. Roseman (Eds.), Controlled Release Delivery Systems, Marcel Dekker, New York, 1983, pp. 77–90. [14] P.L. Ritger, N.A. Peppas, J. Control. Rel. 5 (1987) 23–36.
Study of the interactions between polyphenolic compounds and chitosan Marcel-Ionel Popa*, Nicolae Aelenei, Valentin I. Popa, Daniela Andrei Technical University ‘ Gh Asachi’, Faculty of Industrial Chemistry, D. Mangeron 71, Iasi, RO 6600 Iasi, Romania Received 4 January 1999; received in revised form 7 January 2000; accepted 12 January 2000
Abstract This paper discusses the interaction between chitosan and the polyphenols separated from spruce wood bark. The chitosan and the polyphenols formed a complex and the release of the polyphenols occurred only in an alkaline medium (pH . 9) in a two-step process. Analysis of the release of the polyphenols showed that in the initial stage the diffusion occurs according to the Fick’s law, while in the last stage, the process develops according to a zero-order kinetics. 2000 Elsevier Science B.V. All rights reserved. Keywords: Polyphenols; Chitosan; Controlled release
1. Introduction The polyphenols represent one of the main classes of plants’ secondary metabolites. They are characterized by a special complexity and structures which differ from one plant to another. They play a special part in the growth and reproduction processes. They are distinguished by the following general features: (1) water solubility — although when pure some plant polyphenols may be difficult to dissolve in water, in the natural state polyphenol–polyphenol interactions usually ensure some minimal solubility in aqueous media; (2) molecular masses — natural polyphenols encompass a large molecular mass *Corresponding author. E-mail address: [email protected] (M.-I. Popa).
range from 500 to 3000–4000; (3) structure and polyphenolic character-polyphenols, per 1000 relative molecular mass, possess some 12–16 phenolic groups and 5–7 aromatic rings; (4) intermolecular complexation, besides giving the usual phenolic reactions they have the ability to precipitate some alkaloids, polysaccharides, gelatin and other protein from solution. These complexation reactions are not only of intrinsic scientific interest as studies in molecular recognition and possible biological function, but they could have important and wide-ranging practical applications [1]. Considering their function in metabolic processes, as well as some important in vitro manifested properties of polyphenols, much recent research has focused on their separation and structural characterization, as well as on their possible applications [2–4]. If one re-
1381-5148 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S1381-5148( 00 )00009-2
36
M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
searches their utilization as antioxidant agents [5] or plant growth regulators [6,7], than the study of polyphenols’ interaction with some complexing agents has a special significance. This would permit the development of some isolation techniques for polyphenols from extraction media, and some controlled release systems. This paper studies the manner in which the polyphenols separated from spruce wood bark interact with chitosan, and also the stability of the complex thus obtained under different pH conditions. 2. Experimental The studied polyphenols were extracted from spruce wood bark, with an 1% ammonia solution, by a technique described elsewhere [4]. The chitosan with a relative molecular mass of 150 000 and a 12% acetylation degree was ´ obtained from the Departement de Genie Chimique of the University of Sherbrooke, Canada. The interaction between the two reactants was studied on an 1% aqueous solution of polyphenols and an 1% solution of chitosan. The dissolution of the chitosan involved the stirring of the solid chitosan in a 1 M hydrochloric acid solution, followed by the neutralization of the clear solution thus obtained with a 1 M NaOH solution, up to a pH of 5.7. Afterwards, the solution of polyphenols was added to the chitosan, the systems being stirred continuously, at room temperature, for 4 h. After the reaction, the precipitate formed was separated through centrifugation at 3500 rpm, for 10 min. The unreacted polyphenols were removed by repeated washings with distilled water. Finally, the product was washed with acetone and dried in vacuum, at room temperature.
basic and in a weakly acid medium, the study of the kinetics was realised through spectophotometry. The studied polyphenols are soluble only in a strongly acid medium. The visible absorption spectrum of the aqueous solutions of polyphenols shows a maximum over the 350nm domain, regardless of the pH value, starting from a pH of 7.0. A wavelength of 450 nm has been selected in order to assure a sufficient precision of the method for solutions with relatively high concentrations (0.1 g / dl). The experimental data necessary for drawing the calibration curve were obtained from the study of ten aqueous solutions with concentrations between 0 and 0.1 g / dl, prepared by diluting a standard solution of 0.1 g / dl concentration. Measurements were made in 1-cm thick glass cell, at a wavelength of 450 nm. The calibration curve is plotted in Fig. 1. Similar calibration curves were recorded for buffer solutions with pH 6.5, 7.8 and 9.8. In all situations the experimental points were placed on the same straight line, with an error below 1%. For the subsequent calculations we utilised the average calibration curve. The equation of the curve was obtained by the least-square method A 5 6.8882c
2.1. Analysis of polyphenols Due to the intensely brown-reddish colour of the aqueous solutions of polyphenols, both in a
Fig. 1. Calibration curve for polyphenols in aqueous solution.
M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
where A is the solution’s absorbancy and c is the concentration. In all subsequent kinetic studies, the concentrations of the solutions, expressed in mg / ml, were calculated according to the relation: c 5 A / 0.6882.
2.2. Release of polyphenols from the complex The study of polyphenols’ release from the chitosan–polyphenols complex was carried out by an elution technique in aqueous medium of different pH values, under continuous stirring, at 258C. Preliminary qualitative determinations indicated that, in an acid medium, no release occurs. Therefore only the release in a basic medium, using buffer solutions with 7.8, 8.9, 9.9 and 11.9 pH values was studied. The sample, appearing as a fine powder, was introduced into the eluent and stirred continuously for 300 min. Now and then, samples were extracted from the solution and their absorbances measured. Afterwards, the samples were re-introduced into the release medium. In all experiments, the weight of the complex sample was 0.1 g, for an eluent volume of 50 ml. The total amount of polyphenols present in the samples was determined by measuring the solution’s absorbency 48 h after the initiation of release and checking its value 78 h later. No modification of the eluent’s concentration was noticed after 78 h in any sample. The maximum quantity of polyphenols released, at pH.8.5, after 72 h, was: m f 5 82.2 mg, which represents 82.2% of the polyphenols retained in the complex.
3. Results and discussion Chitosan is a linear polymer of mainly anhydroglucosamine and behaves as a linear polyelectrolyte at acidic pH [8]. As chitosan is a polymer, it has a higher positive charge density, one charge per glucosamine unit.
37
The efficacy of polyphenols as complexing agents derives principally from their relative molecular mass and size, from the many phenolic groups in the same molecule and associated aromatic nuclei. The process of complexation between polyphenols and chitosan may be reversible or irreversible [1]. Reversible complexation of polyphenols may be considered as a two-stage process, in the first of which the polyphenols and chitosan, by the development of non-covalent forces, are in equilibrium with the soluble complex. As the position of this equilibrium changes then, as a second stage, these soluble complexes may well aggregate and precipitate from solution. The whole process is however usually reversible and under suitable conditions the precipitated complex may be redissolved. Polyphenols 1 chitosan 1 H 2 O A [Polyphenols] n ? [Chitosan] m 1 H 2 O Precipitation A [Polyphenols] a ? [Chitosan] b This mechanism is confirmed by our results, since the curve’s aspect shows that the release of the polyphenols from the complex occurs at a higher rate with increasing pH. The general aspect of polyphenols’ release from the complex is presented in Fig. 2, which plots graphically the fraction from the total amount of polyphenols, released as a function of time. In a weakly alkaline medium, (pH 7.8) the release occurs to a reduced extent, a release degree of about 5% from the amount of polyphenols in the sample being attained after 120 min, this value remaining constant up to 300 min. The total amount released at pH 7.8, after 96 h, was about 6%, which perhaps represents
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M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
Fig. 2. Kinetics of polyphenols’ release from chitosan–polyphenols complex, as a function of time, at various pH values.
only the polyphenols physically included in the complex. With increasing pH both the release rate in the initial period (the first 90 min) and the released amount of polyphenols increases. The estimation of the release rate at certain times has been calculated with the relation 1 Dm 1 m n 11 2 m n r 5 ] ]] 5 ] ]]]] m s Dt m s t n 11 2 t n
cesses and practically independent of pH is installed. For pH values of 7.8 the release shows the same stages, characteristic for the initial 0–90 min and the main 90–300 min periods. The only exception is that, in this case, the release ends after 90 min and, at longer times, the release rate is practically equal to zero.
(2)
where r is the relation rate at average time t 5 (t n 1 t n 11 ) / 2 and m s is the sample’s weight. The moment at which release rate is evaluated with Eq. (2) representing an average value: t 5 (t n 1 t n 11 ) / 2, Dm is the polyphenols released over a Dt time interval. The release rate as a function of time is plotted in Fig. 3. Apart from the sample eluted at pH 7.8, which exhibits a very reduced release rate — close to zero nearly on the whole period — all the samples showed a strong decrease of the release rate in the initial period, differentiated as function of pH. After 120 min a regime of constant rate, characteristic for zero order pro-
3.1. Kinetic aspects of polyphenol release from the complex The process of transport of a bioactive agent through a polymer or through controlled release systems may be described with Fick’s diffusion law. Nevertheless, numerous exceptions from such a mechanism occur. That happens because of the specific features of the transport medium, the device’s geometrical elements, the diffusing agent molecular sizes — medium and, respectively, diffusing — eluent interactions. The solutions of Fick’s equation depends on them to a large extent. A review of the mathematical models applied in the study of controlled release
M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
39
Fig. 3. Variation of polyphenols’ release rate in time, as a function of pH.
has been recently presented by Narasimhan and Peppas [9]. In the case of drugs dispersed on non-porous polymeric matrices, the mathematical model — assuming a pseudo-stationary state — was introduced by Higuchi [10], and developed by Paul and McSpaden [11], who obtained a dependence between the weight of released drug and the time as follows 2c s A ] D m t 5 ]] ]t (3) erfj * p
œ
where c s is the solubility of the drug in the polymer; A is the surface of the polymer film’s cross section; D is the coefficient of the drug’s diffusion through the polymer; m t is the weight of drug released at moment t; erfj * is an error function of the j * 5 x * /(Dt)1 / 2 . When the drug or the bioactive agent is dissolved in vitrous polymers or must penetrate through non-porous thin membranes (with a thickness of d ), a simplified solution of Fick’s law may be written ]] m 16Dt ]t 5 ]] (4) mf pd 2
œ
where m t is the weight of drug released at moment t; m f is the weight of drug at t 5 ` (assuming that m f is the drug mass initially present in the sample, which is released completely after a sufficiently long time); D5 diffusion coefficient; d 5thickness of the film. An equation of the (4) type is applicable only for low diffusion times, more exactly for the situation in which up to 60% of the total amount of drug (m t /m f , 0.6) is released. For the same type of system, but longer release times, or when m t /m f . 0.6, the solution of Fick’s law is
S D
m p 2 Dt 8 ]t 5 1 2 ]2 exp ]] mf d p
(5)
The confirmation of the mechanism involves checking the linearity of the experimental data in the coordinates ln (1 2 m t /m f ) 2 t, according to the equation
S D
mt 8 p 2D ln 1 2 ] 5 ln ]2 1 ]] t mf p d2
S
D
(6)
If the drug is dispersed into a porous polymeric matrix, the kinetic equation is [12]
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M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
]]]]]] m t 5 AœDeff Css2Cd 2 e Csdt
(7)
where e is the void fraction in the porous material: Cd is the concentration of the drug in the polymer film, Cs is the drug solubility in the polymer and Deff is the effective diffusion coefficient. Another procedure relatively simple but attractive — for practical reasons — involves the description of the controlled release with the ‘power law’, according to which the relative amount released is expressed as a function of the release time m ]t 5 kt n mf
(8)
Korsmeyer and Peppas [13] stated first such an equation in the case of controlled release. A detailed analysis on the application of Eq. (8) to the study of swelling or rigid systems and also to various geometries of the release system was made by Ritger and Peppas [14]. Usually, according to the linearized equation ln (m t /m f ) 5 ln k 1 nln t
(9)
the experimental data are expressed in logarithmic co-ordinates: ln (m t /m f ) 2 ln t, the value of exponent n being evaluated from the slope of the straight line thus obtained. For the acceptance of linear regression in the case of the data under analysis, the confidence interval should be at least 95% (r 2 . 0.95). If the value of the n obtained is 0.5, the mechanism of drug transport is Fickian and the drug release rate is time-dependent. If 0.5 , n , 1, the transport is anomalous and the release rate is once again time- dependent. If n51.0, the transport is of the second type and the release rate is time-independent or in other words, zero-order release is obtained. Our studies on the behaviour of the new release system obtained involved the checking of both mathematical models described above. The results obtained in the process of polyphenol release from the chitosan–polyphenols complex, in a moderate basic medium, are given
as examples. The used sample was a raw powder, and the parameters of the release process were the following: weight of sample 0.1 g; volume of eluent 50 ml; pH 9.9; temperature 258C, intensive stirring. The control of the release was monitored spectrophotometrically, at a wavelength of 450 nm, the experimental results obtained with this sample being presented in Fig. 4. Two domains may be distinguished on the release curve, as follows: an initial period (0–60 min), over which variation is non-linear, having a decreasing slope, and a main period (90–720 min) over which variation is linear. In order to check whether the release occurs through a Fick type diffusion, which is characteristic to non porous polymers, the experimental data have been represented as both m t /m f 2 t 1 / 2 (Fig. 5) and ln (1 2 m t /m f ) 2 t (Fig. 6). Fig. 5 shows that the model is suitable for the system used here, the confidence interval for the initial period (m t /m f , 0.6) being of 99.4%, which indicates a good linearity between the variables taken into study. It is surprising that, in the given coordinates, a linear dependence is also obtained for the main release period. This may be due to the fact that the tested model is particularly adequate for the transport through thin films, while in our case the substance presents itself as a powder. Fig. 6 shows the validity of the model used. Indeed, the equation is adequate for release ratios over 0.6, which, in our situation, corresponds to release times longer than 60 min. Within the 60–270 min domain, the experimental data form a straight line, the confidence interval being 99.7%. Eq. (6), applied to the initial period is not satisfactory, the curve thus obtained showing important deviations from linearity (in this case, the confidence interval being below 95%). If Eq. (8) is used for processing the data regarding polyphenol release from the chitosan– polyphenols complex, at pH 9.9, it will show that, in the initial moments (60–90 min), release occurs through a mechanism of unidirectional
M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
41
Fig. 4. Relative amount of polyphenols released as a function of time, at pH 9.9.
Fig. 5. Fick’s law mechanism checking for elution at pH 9.9.
diffusion. Indeed, as plotted graphically in Fig. 7, in ln m t 2 ln t coordinates, a straight line having the slope very close to 0.5 was obtained, and also a very good confidence interval. The second time interval (60–270 min) is
characterized by a zero-order kinetics, for which in Eq. (8) n51, and thus the equation becomes m ]t 5 kt mf
(10)
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M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
Fig. 6. Graphical checking of Eq. (6).
4. Conclusions
Fig. 7. Graphical checking of Eq. (9).
As shown Fig. 8, the obtained linearity is very good, which justifies the assertion that the release of polyphenols from chitosan is governed, with the exception of the initial period, by zero order kinetics — most suitable for systems of controlled release.
An ionic complex was obtained between chitosan and the polyphenols extracted from spruce wood bark. The process of polyphenol release from the chitosan—polyphenols complex was studied and some conclusions were drawn. Release does not occur in an acid medium, the complex being insoluble, while polyphenol retention is achieved through strong chemical bonds. In a weakly alkaline conditions (pH 7.8), only about 6% of the total amount of polyphenols is released from the complex, which probably represents the compound dissolved physically in the polymer. The amount of released polyphenols and the release rate in the initial stage increase with the increase of the pH of the elution medium. At an alkaline pH, two distinct stages may be observed in the release process, namely: an initial stage (0–60 min), during which the release rate decreases in time — the decrease being more significant at the higher pH — and a
M.-I. Popa et al. / Reactive & Functional Polymers 45 (2000) 35 – 43
43
Fig. 8. Graphical checking of a zero order kinetics.
main release stage (60–300 min) during which the release rate is constant and independent on pH. The study of the release mechanism showed that, in the initial stage, release occurs through a Fick diffusion mechanism, while, in the main period, the release occurs according to a zeroorder kinetics. Acknowledgements The study is part of a cooperation program established between Technical University of Iasi-Romania and University of SherbrookeCanada. References [1] E. Haslam, Polyphenol complexation in polyphenolic phenomena, in: A. Scalbert (Ed.), Polyphenolic Phenomena, INRA, Paris, 1993, pp. 23–33.
[2] J.B. Harbone, New naturally occurring plant polyphenols, in: A. Scalbert (Ed.), Polyphenolic Phenomena, INRA, Paris, 1993, pp. 19–22. [3] V.I. Popa, C. Beleca, Cellulose Chem. Technol. 28 (1994) 613–620. [4] V.I. Popa, C. Beleca, M. Popa, R. Bodirlau, I. Bara, E. Truta, Bull. Soc. Brot. Ser. 2, (Portugal) 67 (1966) 245–255. [5] L. Barolary, F. Xi, I. Norris, J. Wood Chem. Technol. 17 (1997) 73–90. [6] B. Kosikova, E. Slavikova, Drevarsky Vyokum 1–2 (1994) 33–38. [7] V.I. Popa, C. Beleca, M. Popa, R. Bodarlan, P. Vidrascu, Rev. Roum. Chim. 40 (1995) 691–698. [8] D.N.S. Hon, Chitin and chitosan: medical application, in: S. Dumitriu (Ed.), Polysaccharides in Medicinal Applications, Marcel Dekker, New York, 1966, pp. 631–650. [9] B. Narasimhan, N.A. Peppas, Controlled drug delivery, Kinam Park, in: ACS Professional Reference Book, A, Chemical Society, Washington, DC, 1997, pp. 529–557. [10] T. Higuchi, J. Pharm. Sci 50 (1961) 874. [11] D.R. Paul, S.K. Mc Spaden, J. Membr. Sci 1 (1976) 33. [12] T. Higuchi, J. Pharm. Sci. 52 (1963) 1145. [13] R.W. Korsmayer, N.A. Peppas, in: S.Z. Mansdorf, T.J. Roseman (Eds.), Controlled Release Delivery Systems, Marcel Dekker, New York, 1983, pp. 77–90. [14] P.L. Ritger, N.A. Peppas, J. Control. Rel. 5 (1987) 23–36.