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Preparation and characterization of chitosan-stearate complexes and in vitro evaluation on the adsorption of deoxycholic acid salt.


Chilosan(CS), which is nontoxic and biodegradable (1-3), is a naturally occurring copolymer of glucosamine and N-aeetyl-glucosamine produced by the deacelylation of chitin (4). Because of the unique polycalionic nature, CS has received considerable attention for being commercially applied in biomedical, food, and chemical industries (5), (6), especially the superb performance in lowering plasma cholesterol and lipid which has been verified in vitro (7), (8) and in vivo (9-11).

A number of studies have demonstrated that CS samples can influence bile salts metabolism determining the digestion of lipids in the small intestine. Nevertheless, the binding capacity of CS for bile salts decreases sharply in the alkaline solution systems. Muzzarelli et al. have mentioned that clarifying the bile salt collection ability of highly charged (i.e., of a very high degree of deacelylation. DD) and hydrophobically modified CSs is crucial (12). Previous studies have shown that higher-molecular-weight CSs bind bile sails heller, while DD seems to exert no effect on the bile salt-binding capacity (13).

Chitosan-stearate complexes (CSC) are prepared by reacting CS with stearic acid which do not elevate the risk of coronary heart disease (11). Introducing hydrophobic molecular groups may indicate a cause of bile sail sequestration by CSC. Moreover. Furda has suggested that CS-fatty acid complexes bound additional lipids after being digested, including natural triglycerides, falty and bile acids, and cholesterol and other sterols. Therefore, the complexes can be used as food additives or pharmaceuticals to reduce the absorption of lipids and cholesterol (14).

However, the relationship between the physico-chemical properties of CS-fatty acid complexes preparations and their potential interaction with bile acids has seldom been referred, although the information is critical in the production and utilization of such hypolipidemic complexes. Thus, this study aims to optimize the preparation procedure of CSC and test the physico-chemical properties.



High molecular weight CS with 90% DD was obtained from Nantong Shuanglin Biotechnology (Jiangsu. China). Stearic acid, sodium stearale, anhydrous diethyl ether. NaOH and HCl were supplied by Sinophartn Chemical Reagent (Shanghai, China),

Synthesis of CSC

In this experiment, CSCs were prepared by a modified method of Lin et al. (15). The interaction was designed in accordance with the proportion of the carboxyl group of stearic acid to the free amino group of CS. The mole ratios of n([COO.sup.-])/n([NH.sup.3.sub.+]) were 0.4, 0.6, 0.8, 1.0. and 1.2. Briefly. CS was dissolved in a 2.5 wt% HCl aqueous solution to form a 5 wt% CS solution. Sodium stearate was dispersed into deionized water at 80[degrees]C to reach different concentrations. Then 200 mL of the sodium stearate solution was added to 1000 mL of the CS solution, which was then stirred at 250 rpm and 80[degrees]C for 2 h. The resulting precipitate was filtered, washed with deionized water, and then dried in a vacuum freeze-dryer. Part of the sediment was further purified by extraction with anhydrous diethyl ether in a Soxhlel apparatus and then dried to get rid of the excessive ether.

Determination of the Amount of Combined Stearic Acid

In order to distinguish that the adsorption of CS to stearic acid by electrostatic reaction from that by other protocols. anhydrous diethyl ether was used to wash the precipitate to remove the stearic acid adsorbed by CS via nonelectrostatic reaction. Thus, the nitrogen values of the samples which were obtained utilizing the Kjeldahl method for nitrogen determination (16) can be used to calculate the amount of combined stearic acid. The ratio of combined stearic acid (R) can be calculated by Eq. I.


Where N, is the nitrogen values of CS and [N.sub.2] is that of CSC. M is the molecular weight of stearic acid. 166 is the average molecular weight of the CS residues. R is defined as the amount of carboxylic acid combined to each unit of CS sugar residues. When CS is adsorbed to stearic acid via electrostatic reaction, R ranges from 0 to the DD of CS. The R value exceeding the DD of CS suggests that CS adsorbed to stearic acid with other patterns in addition to electrostatic reaction.

FTIR Spectroscopy

FTIR spectra were obtained using a Nicolel Nexus 470 instrument (Nicolel Instrument, Thermo Company. Madison, USA). All samples were prepared into KBr pellets by blending [Tilde]2 mg of the powdered polymer and scanned against a blank KBr pellet background ranging between 4000 and 400 [cm.sup.-1] with the spectral resolution of 4.0 [cm.sup.-1]. The spectra were analyzed by using the OMNIC 4.0 software.

X-Ray Diffraction

X-ray diffraction (XRD) analyses were carried out using Cu K[varies] or [proportional] radiation at 40 kV and 40 mA on a Bruker AXS D8 Advance diffractometer (Germany). The XRD scans were recorded from 5 to 80[degrees] 2[theta] with 0.02[degrees] of step-width and 3 s of counting time.

Thermogravimetric Analysis

Thermogravimetric analysis (TGA) was performed on a Mettler Toledo TGA/SDTA851 Thermogravimeter (Mettler Toledo Corp., Zurich. Switzerland). STARe software (version 9.01) was used to analyze the thermal stability of the samples. In this work, the mass of each sample was about 5 mg. The samples were heated from 30 to 350[degrees]C at the rate of 20[degrees]C/min under [N.sub.2] at 30 mL/ min during the analysts.

In Vitro Evaluation on the Adsorption of Deoxycholic Acid Salt

The in vitro binding to bile salt was tested according to the method [I7] with a slight modification. The bile salt binding capacity was determined by colorimetry. A color reaction was triggered by a 1% (v/v) aqueous solution of furfural. Deoxycholic acid salt (1 mg/mL) was selected for analysis. The analytical sample of sodium deoxycholate was dissolved in pH 7.6 phosphate buffer to form the deoxycholic acid salt solution. Totally, 0.1 g of the sample was added to a 25 mL colorimetrie tube and digested in 6 mL of 0.01 M hydrochloric acid (HCl) for 1 h in a 37[degrees]C shaker bath. After this acidic incubation simulating gastric digestion, the sample pH was adjusted to 7.6 with 6 mL of sodium hydroxide (NaOH). To each test sample was added 12 mL of sodium deoxycholate solution. Phosphate buffer (pH 7.6) was added to individual substrate blanks without sodium deoxycholate. Then the tubes were incubated for 2 h in a 37[degrees]C shaker bath again. The mixtures were transferred to 50 mL centrifuge tubes and centrifuged at 12.000 rpm for 20 min.

We targeted to determine the concentration of bile salt in the supernatant after being incubated at 37[degrees]C and pH 7.6. The supernatant (1 mL) was transferred into a second set of labeled tubes, mixed with 1mL 1% (v/v) furfural solution, and ice-bathed for 5 min. Then 10 mL of 70% sulphuric acid was added, and the resulting solution was mixed well and heated in a 70[degrees]C water bath for 10 min. Finally the mixtures were moved quickly to ice bath and left still for 2 min, and the absorbance was thereafter measured using a Metertek SP-830 spectrophotometer at the wavelength of 510 nm. The bile salt concentrations were corrected based on the mean recoveries of the mixture after subtracting the individual substrale blanks.

The bile-salt-binding capacity was described by Eq. 2.

The bile salt - binding capacity(%)


Where [m(BS).sub.supernatant] is the amount of permeated (unbound) bile salt in the supernatant of the intestine model (mg), and [m(BS)] is the starting amount of bile salt inside the digested chyme of the model (mg).

Statistical Analysis

The results were expressed as mean values [+ or -]SD. The data obtained were subjected to analysis of variance (ANOVA). P Less than 0.05 was considered statistically significantly different.


Preparation of CSC

The floccules were smaller and weakly aggregated at low stearic acid concentration, while they were prone to accumulation with increasing concentration. Finally. CSCs were obtained as beige powders after the floccules were washed with anhydrous diethyl ether and then dried.

As shown in Fig. 1, the R value (B) of the total CS absorbed to stearic acid was appreciably higher than that (C) after the floccules were washed with anhydrous diethyl ether and the ratio (A) of n([COO.sup.-])/n([NH.sub.3.sup.+]). The results indicate that CS might be adsorbed to stearic acid via different mechanisms, including electrostatic interaction and physical adsorption-entrapment. Furthermore, less aggregates formed between CS and lowly concentrated stearic acid, leading to the remaining of free CS molecules.

Maximal CS was adsorbed to stearic acid (R [approximately equal to] 1.90) when the ratio of n([COO.sup.-])/n([NH.sub.3.sup.+]) was about 0.6. whereas least CS was adsorbed via electrostatic interaction (R [approximately equal to] 0.11).

It has previously been suggested that adding CS into a solution containing negatively charged emulsions can reverse surface charge from negative to positive, indicating that cationic CS molecules are adsorbed to the surfaces of the anionic surfactant-coated droplets which may subsequently reduce absorption (18), (19). Accordingly. CS bound to stearic acid with charge neutralization and yielded CSC with decreasing free amine groups ([NH.sub.3.sup.+]) of CS molecules, thereby entrapping less stearic acid.

FTIR Spectroscopy

CSCs were synthesized through the electrostatic attraction between the carboxylic groups of stearic acid and the amino groups of CS. which was confirmed by the FTIR spectra of CS and CSC shown in Fig. 2. The basic characteristic peaks of CS are discerned at: 3400 [cm.sup.-1] (N-H and 0-H stretch). and 1597 [cm.sup.-1] (N-H bend).

The weaker characteristic absorption of CSC at around 3400 [cm.sup.-1] than that of CS can be attributed to the formation of complex molecules in the presence of [NH.sub.3.sup.+]. The vibrational band at about 1600 [cm.sup.-1] corresponding to primary amino groups disappeared, giving rise to two new bands in the vicinity of the absorption peak. This behavior reflects the interaction between the amino and carboxylic groups. The absorption peaks at 2917.02 and 2845.62 [cm.sup.-1] can be assigned to the saturated alkyl. Thus these results confirm that the complexes were prepared by the electrostatic interaction between the carboxylic groups of stearic acid and the amino groups of CS. The process may occur following:

Chitosan-[NH.sub.2] + HOOC-R [right arrow] or [vector] Chitosan-[NH.sub.3.sup.+] ...[.sup.-COO-R]

X-Ray Diffraction

It has previously been suggested that CS has at least six crystalline polymorphs, including "tendon," "annealed." "1-2," "L-2," "form I," and "form II" (20). The XRD patterns of CS and its complexes are shown in Fig. 3. The XRD patterns of the CS used herein exhibit two characteristic peaks at the 2[theta] of [Tilde]11[degrees] and 20[degrees]. which are characteristic "L-2" polymorph (21). However. the peak at 11[degrees] (2[theta]) in the XRD patterns of the CSC disappeared. which indicates that CS and stearic acid interacted. resulting in a new crystal structure.


The thermal properties of CS and the complexes were characterized by TGA and DTG. Some characteristic parameters of the thermodegradation were determined according to the thermogravimetric curves. Figure 4 exhibits the TGA curves of CS and CSC comprising two stages. The DTG curve (Fig. 5) of CS shows a peak at [Tilde]65[degrees]C during the first stage of degradation that is related to water loss. Considering that some free amine groups of CS had been occupied during electrostatic interaction, CSC still possessed a smaller number of amine groups available to interact with water. Therefore, some water molecules that were originally bound to amine groups were bound to hydroxyl groups, which thus increased the peak temperature of CSC.

The second thermal degradation process occurred from 240 to 320[degrees]C. The degradation temperature ([T.sub.max] of CS peaked at about 305[degrees]C. However. the [T.sub.max], of CSC decreased, inferring that CSC was less thermally stable than CS. The results might be ascribed to the disruption of crystalline structure. Moreover, the elevated R value of CSC can be attributed to the decreased [T.sub.max], which indicates that CSC was prone to less thermally stable in case that amine groups bound more stearic acid via electrostatic attraction. It is well-acknowledged that slightly changed crystalline structure can significantly alter the corresponding thermal stability (21), (22). Therefore, the variation of peak position is expected to reflect the physical and molecular changes upon the electrostatic attraction between amine and carbonyl groups.

Adsorption of Deoxycholic Acid Suit

The hypolipidemic and hypocholesterolemic activities of CS and other digestion-resistant polysaccharides are associated with their binding capacities against bile acids (23), (24). In vitro assay has been widely accepted to estimate the potential bile acid binding capacity of polysaccharides including CS. ready-to-eat breakfast cereals, and other dietary fibers (7), (25).

As we known, the DD and molecular weight of a CS determine the chemical and physiological properties. Previous studies have shown that the bile salt-binding capacity was substantially affected by the viscosity-average molecular weight, and the CS sample with highest molecular weight bound bile salts most effectively. In contrast, DD hardly influenced the bile-salt-binding capacity (13). Therefore, the adsorption capacities of high-molecular-weight CS and its stearale toward deoxycholic acid sah were investigated in this study.

Influence of pH on the bile salt-binding capacity of CS and Its Complex. CSC and CS were of different adsorption properties. The pH variations may evidently influence the biological functionality of CS because ils solubility and electrical characteristics are pH dependent. The bile salt-binding capacities of CSC and CS also differed with pH variations. The pKa of the CS amino groups is around 6.3 (19) and that of deoxycholic acid is about 5.3 (5). Actually, electrostatic interactions only took place by ionic bonds when the amino groups of CS and deoxycholic acid were highly cationized and the anionized, respectively. However, the ionization of CS amine groups was significantly hindered in slightly alkaline solutions, which decreased the electrostatic attraction between CS molecules and deoxycholic acid sait.

As shown in Fig, 6, the binding capacity of CS for deoxycholic acid salt was higher at pH 6.0 than that at pH 7.0 or pH 5.0. Moreover, the capacity for bind bile salt at pH 5.0 was higher than that at pH 7.0 probably due to the compromise of electrostatic interactions that the free amino groups of CS combined [H.sup.+] in the solution, leading to high degree of cationization and low anionization degree of deoxycholic acid simultaneously.

On the other hand, sodium deoxycholate is one of the bio-surfactants in the gastrointestinal tract dominating the digestion and absorption of fat (26). The curve plotting the binding capacity of CSC changed similarly. CSC bound more deoxycholic acid salt than CS at the identical pH, which can be attributed to the increased hydrophobicity after CS combined the long carbon chain of stearic acid.

Adsorption of Deoxycholic Acid Salt to CSC at pH 7.6. The correlation between the bile salt-binding capacity and the hydrophobic characteristics of complexes was also determined in this section. As illustrated in Fig. 7. the CSCs with higher R values, which are more hydrophobic. are more subject to being adsorbed to bile salt. Aboul 42% of deoxycholic acid salt was adsorbed at low R value ([approximately equal to]0.11 ). which was raised with increasing R value. Most bile salt was adsorbed when R reached maximum ([approximately equal to]0.3), which may be ascribed to the augmented hydrophobicity that allowed the complexes to pack more deoxycholic acid salt. The binding of bile acid salts by CS stearate reduced the emulsion of fats in food, which thus inhibited the absorption of fats.


This study reports that CSCs were synthesized via the electrostatic interaction between the carboxylic groups of stearic acid and the amino groups of CS. CS underwent crystalline structure change and thermal stability decrease upon yielding CSC. Additionally, CSC bound bile salt better than CS in the test utilizing an in vitro model simply simulating the digestive system. The CSC containing more hydrophobic long carbon chain of stearic acid had higher bile-salt-binding capacity. The results herein suggest that the materials may be industrialized as food additives or pharmaceuticals to reduce the absorption of lipids and cholesterol.


CSC    chitosan-stearate complex

CS     chitosan

DD     deacetylation degree


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Yiyu Xu, (1) Yanshun Xu, (1) Jiali Zhang, (2) Wenshui Xia (1)

(1) State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiang nan University, Wuxi, Jiangsu 214122, China

(2) Biosynthesis and Biomaterials Laboratory; School of Medicine and Pharmaceutics, Jiangnan University, Wuxi, Jiangsu 214122, China

Correspondence to: Wenshui Xia; e-mail:

Contract grant sponsor: Science and Technology Plan Project of Guangdong Province; contract grant numbers; 2010B090400467; contract grant sponsor: Fund Project for Transformation of Scientific and Technological Achievements of Jiangsu Province; contract grant number: BA2009082.

DOI 10.1002/pen.23592

Published online in Wiley Online Library (

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Author:Xu, Yiyu; Xu, Yanshun; Zhang, Jiali; Xia, Wenshui
Publication:Polymer Engineering and Science
Article Type:Report
Date:Mar 1, 2014
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