The complex world of polysaccharides edited by Desiree Nedra Karunaratne

 

The Complex World of Polysaccharides 

606 

2. Experimental 

2.1. Chemicals 

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/high-

performance liquid chromatography grade and used without further purification; ultrapure 

water was supplied by a Milli-Q system. 

2.2. Spectroscopic measurements 

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 

of chitosan were conducted in these conditions. 

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 

performed at room temperature. 

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 

concentration of chitosan in acid aqueous solution). 

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 

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The Chitosan as Dietary Fiber: An in vitro Comparative  

Study of Interactions with Drug and Nutritional Substances  607 

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. 

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 hydrogen-

bonding 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). 

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