**3. Results and discussion**

Absorption spectra and chemical structures of chemicals are shown in Figure 2. Spectral data in Figure 2.a shows that vitamin B12 absorb significantly within 425–600 nm range, as described in the literature (Zheng & Lu, 1997; British Pharmacopoeia, 1998) and the present work chose the absorption peak at ~550 nm to assess the spectral behavior. Similar graphs have been obtained for vitamin B2 and fluoxetine. Figure 2.b shows that the absorption maximum for the vitamin B2 occurs at ~440 nm, data consistent with the literature ((United States Pharmacopeia, 2007). Figure 2.c. shows the absorption spectrum of fluoxetine consistent with the literature (Fregonezi-Nery et al., 2008) that exhibits two absorption maxima at 270 and 275 nm. The last one maximum was chosen to monitor the spectral behavior of fluoxetine.

606 The Complex World of Polysaccharides

water was supplied by a Milli-Q system.

of chitosan were conducted in these conditions.

concentration of chitosan in acid aqueous solution).

**2.2. Spectroscopic measurements** 

performed at room temperature.

**3. Results and discussion** 

Chitosan and fluoxetine hydrochloride were purchased from Aldrich Chemical Co. (St. Louis, MO, USA), and vitamin B2 (riboflavin, 96%) and vitamin B12 (cyanocobalamin,, USP Grade) from Vetec Co. (Duque de Caxias, RJ, Brazil).and Merck Co. (Darmstadt, Hessen, Federal Republic of *German), respectively.* Other chemicals were ultraviolet/highperformance liquid chromatography grade and used without further purification; ultrapure

Previous studies have shown that the best conditions to solubilize chitosan are: chitosan 1% (w/v) dissolved in aqueous solution of glacial acetic acid 1% (v/v) under stirring (Signini & Campana Filho, 1999; Rodrigues, 2005; Rodrigues et al., 2008), so measurements in presence

Chitosan has no fluorescence emission or absorption in the experimental conditions. The absorption spectra of chemicals (fluoxetine, B2 and B12) were measured using quartz cuvettes with 1 cm of optical pathway. The fluorescence measurements were performed at excit= 275 nm and emis= 305 nm for vitamin B12 (Li and Chen, 2000); at excit= 440 nm and emis= 305 nm for vitamin B2 (United States Pharmacopeia, 2007; Association of Official Analytical Chemists, 2005); and at excit= 230 nm and emis= 290 nm for fluoxetine

(United States Pharmacopeia, 2007; Association of Official Analytical Chemists, 2005).

Absorbance measurements were taken at maximum of the absorption spectra and

Initially, variations of both fluorescence and absorption spectra of the chemicals (fluoxetine, B12 and B2) were taken as a function of their concentration in acid aqueous solution and after in different concentrations of chitosan in aqueous acid solution, at the same range of chemicals concentration. The variation in the spectra of the chemicals (fluoxetine, vitamins B2 and B12) was also studied by keeping fixed the concentration of vitamin and varying the

Absorption spectra and chemical structures of chemicals are shown in Figure 2. Spectral data in Figure 2.a shows that vitamin B12 absorb significantly within 425–600 nm range, as described in the literature (Zheng & Lu, 1997; British Pharmacopoeia, 1998) and the present work chose the absorption peak at ~550 nm to assess the spectral behavior. Similar graphs have been obtained for vitamin B2 and fluoxetine. Figure 2.b shows that the absorption maximum for the vitamin B2 occurs at ~440 nm, data consistent with the literature ((United States Pharmacopeia, 2007). Figure 2.c. shows the absorption spectrum of fluoxetine

**2. Experimental** 

**2.1. Chemicals** 

The chitosan-chemicals (fluoxetine, vitamins B2 and B12) interaction have been studied in aqueous acid solution by the monitoring the fluorescence and UV-visible spectra of chemicals, each monitored separately.

In three cases, the increase in concentration of chemical causes an increase in both absorption and fluorescence intensities due to the increase of species that absorb and emit light and this increases is linear profile always indicating that self-aggregation processes are not occurring in this concentration range (data not shown).

Subsequently, chemicals (fluoxetine, vitamins B2 and B12) were studied in the absence and the presence of chitosan, at concentrations 0.050 g.L-1, 0.60 g.L-1 and 1.0 g.L-1 of polysaccharide, keeping fixed the chemicals concentration (8.5x10-5 mol.L-1). With the increase of chitosan concentration both fluorescence and absorption intensities of chemicals are increased.

Figures 3, 4 and 5 show the behavior of fluorescence intensities to fluoxetine, vitamin B12 and B2, respectively. In all graphics, fluorescence intensities significantly increase when chitosan concentration goes from zero to 1.0 g.L-1. This is a common behavior of fluorescent molecules when they migrate from the solution for environment of different polarity (Kalyanasundaram, 1987) and is due to the influence of microenvironment formed by chitosan on the photophysics of the chemical that is changed due to spatial hindrance that it suffers and due to loss of part of rotational freedom of substituent groups, (Kalyanasundaram & Thomas, 1977; Valeur, 2001). Then, with increase concentration of chitosan, the microenvironment becomes more rigid and the lifetime of the chemicals (fluoxetine, vitamins B2 and B12) in the excited states are living longer (Kalyanasundaram, 1987). However, fluorescence intensities of vitamin B12 and fluoxetine show similar increase rate while vitamin B2 is markedly lower. The increase of fluorescence intensities with the polysaccharide concentration has been observed also to vitamin in pharmaceutical formulations containing dextran (Alda et al., 1996).

Absorption intensities of chemicals (fluoxetine, vitamins B2 and B12) increase with the chitosan concentration similarly to the of fluorescence intensities, Figure 6. The reason for this behavior is the increased stiffness of environment generated by chitosan chains. However, in this case, intensities show the following increasing order: vitamin B2, fluoxetine and vitamin B12.

In chemical structure of all chemicals (fluoxetine, vitamins B2 and B12) there are rings with double bonds and polar groups that can interact strongly with the similar groups of chitosan. There are also OH groups in the molecular structure of chitosan favor hydrogenbonding type interactions with polar groups of chemicals. These interactions can influence the absorption and the emission processes of radiation of molecules reducing the rotational degrees of freedom of the molecule (Ramamurthy, 1991).

In general way, results demonstrated that three chemicals (fluoxetine, vitamins B2 and B12) are transferred to microenvironment generated by weakly acidic solution of chitosan but in different proportions, due to the structural feature and solubility of each. Table 1 describes the relative increase of the fluorescence and absorption intensities of the chemicals when chitosan concentration ranges from zero to 1.0 g.L-1.

Both vitamins belong to the class of hydro soluble vitamins (Sun et al., 2007), while the fluoxetine drug is slightly soluble in water (Darwish, 2005). The low solubility promotes some molecules of fluoxetine to migrate from aqueous environment to the more rigid environment generated by chitosan (the higher the concentration) causing a proportionately greater increase of absorbance and fluorescence intensities. However, the fluorescence intensities of vitamin B12 also increase in the same proportion and the absorbance intensities in proportion even higher, despite the hydro soluble nature of this vitamin.

**Figure 2.** Absorption spectra and chemical structures of vitamin B12 (A), vitamin B2 (B) and fluoxetine (C).

These results demonstrate that the transfer process of chemicals from aqueous environment to the chitosan aggregates is influenced by solubility of the molecules in water and/or by the molecular structure. Particularly, the structure molecular of vitamin B12 seems to have an interesting effect in this case. Vitamin B12 belong to the cobalamins, a class of octahedral Co(III) complexes which contain a planar framework called a corrin, with the metal center coordinated in the equatorial position by the four corrin nitrogens. Her energies of excited states are sensitive to the nature of the ligands of center coordinated and are influenced by water content of the surrounding environment (Solheim et al., 2011). These characteristics may be the reason for the more significant increase of the spectral properties of the vitamin B12, with increasing concentration of chitosan (lower water content), compared with the vitamin B2.

608 The Complex World of Polysaccharides

chitosan concentration ranges from zero to 1.0 g.L-1.

In general way, results demonstrated that three chemicals (fluoxetine, vitamins B2 and B12) are transferred to microenvironment generated by weakly acidic solution of chitosan but in different proportions, due to the structural feature and solubility of each. Table 1 describes the relative increase of the fluorescence and absorption intensities of the chemicals when

Both vitamins belong to the class of hydro soluble vitamins (Sun et al., 2007), while the fluoxetine drug is slightly soluble in water (Darwish, 2005). The low solubility promotes some molecules of fluoxetine to migrate from aqueous environment to the more rigid environment generated by chitosan (the higher the concentration) causing a proportionately greater increase of absorbance and fluorescence intensities. However, the fluorescence intensities of vitamin B12 also increase in the same proportion and the absorbance intensities in proportion even higher, despite the hydro soluble nature of this vitamin.

**Figure 2.** Absorption spectra and chemical structures of vitamin B12 (A), vitamin B2 (B)

and fluoxetine (C).

In fact, some molecules of fluoxetine or vitamins B2 or B12, are transferred to microenvironment generated by weakly acidic solution of chitosan. Among the three, the vitamin B2 is transferred in a smaller proportion. However, for all of them is expected that its loss in the diet, caused by administration of chitosan, is not so significant.

From our observations, possible risks to the patient should be considered when prolonged treatment with chitosan is prescribed and perhaps extra care should be taken when chitosan and fluoxetine are prescribed together in slimming diets. In the case of vitamins, essential for many physiological functions, there must be some precautions to minimize the impacts generated by this therapeutic, as the replacement of nutrients in the diet of patient.

**Figure 3.** Fluorescence spectra of fluoxetine in acid aqueous solution of chitosan. Chitosan concentrations: 0.00; 0.050; 0.60 and 1.0 (from the base to the top).

**Figure 4.** Fluorescence spectra of vitamin B12 in acid aqueous solution of chitosan. Chitosan concentrations: 0.00; 0.050; 0.60 and 1.0 (from the base to the top).

**Figure 5.** Fluorescence spectra of vitamin B2 in acid aqueous solution of chitosan. Chitosan concentrations: 0.00; 0.050; 0.60 and 1.0 (from the base to the top).

**Figure 6.** Behavior of absorption intensities of fluoxetine, vitamin B12 and B2.


**Table 1.** Relative increase on the fluorescence (I) and absorption (ABS) intensities of the fluoxetine, vitamins B2 and B12 when chitosan concentration ranges from zero to 1.0 g.L-1.

This paper seeks to warn to possible problems connected with the excessive loss of vitamins and other nutrients by the body during prolonged treatment with chitosan, as well as due the concomitant use of chitosan and fluoxetine.

## **4. Conclusions**

610 The Complex World of Polysaccharides

**Figure 4.** Fluorescence spectra of vitamin B12 in acid aqueous solution of chitosan. Chitosan

**Figure 5.** Fluorescence spectra of vitamin B2 in acid aqueous solution of chitosan. Chitosan

concentrations: 0.00; 0.050; 0.60 and 1.0 (from the base to the top).

concentrations: 0.00; 0.050; 0.60 and 1.0 (from the base to the top).

Innumerous studies have described the formation of aggregates in naturals (Kim et al., 2000; Pelletier et al., 2000; Zhbankov et al., 2003), as chitosan ((Rodrigues, 2005; Hennen, 2005; Rodrigues et al., 2008) and synthetic (Kalyanasundaram, 1987; Neumann & Rodrigues, 1994; Neumann et al., 1995; Gomes et al., 2006; Gomes et al., 2007; Sur, 2010) polymers solutions and these molecular structures occur due to intra- and intermolecular interactions.

Chitosan is a polysaccharide precursor of materials suitable to release and/or dissolve drugs into the human body (Hennen, 2005), among other uses. However, the study of spectral properties of chemicals, fluoxetine, vitamins B2 and B12, demonstrated that the microenvironment generated by weakly acidic solution of chitosan also is able to sequester some B2, B12 and fluoxetine molecules, despite the hydro soluble nature of vitamins.

The results described in the current work demonstrated the wide range of possibilities in the studies of interaction between chitosan, used in diets as anti-obesity supplement, and molecules that are present in the human body, as well as with other drugs. Moreover, our work demonstrates the need for more studies on the subject as a means of providing information on the use of chitosan in diets as anti-obesity supplement.
