Raw HA solution isolated from the soil was purchased from Honlih Ltd. Company (Taiwan). HA was precipitated by adding HCl and the supernatant discarded. The sediment was then dissolved in H₂O and neutralized with NaOH. After removing suspended particle, the filtrate was lyophilized as standard HA sample. HA standard solution used in this study was prepared by dissolving HA in H₂O. The UV-visible absorption spectrum of HA solutions showed a maximum over the range 190–240 nm. A wavelength of 230 nm was selected to ensure sufficient precision for relatively concentrated HA solutions (30 µg/mL). Measurements for doubling dilutions from stock HA solution at 32 µg/mL were performed in a quartz cell of 1 cm path length at a wavelength of 230 nm by a Hitachi Model U-2000 spectrophotometer. The calibration curve (r² = 0.9989) was applied.

The adsorption analysis conducted in this study was similar to that in previous publications [40,41] and is described below. One half gram of cellulose, diethylaminoethyl cellulose (DEAE-cellulose), carboxymethyl cellulose (CM-cellulose), chitin, chitosan or activated charcoal was added to 50 mL of a 0.1 M HCl solution containing HA at a concentration of 40 µg/mL. These solutions of pH 1.0 were shaken at 37 °C for 1 h or the desired period as experimental design in a thermostatically controlled shaker bath and then 70 mL of 0.2 M Na₂HPO₄ solution was added by titration within 5 min to maintain a final pH value of 7.1 to 7.4. When sampling, 1 mL of solution was applied and the insoluble matter was removed by a bench-top centrifuge at 10,000 × g for 10 min. The supernatant was collected and the concentration of HA was measured by a spectrophotometer of U-2000 from Hitachi.

Chitosans with different DD values were prepared from chitin with DD value of 25.0% by alkaline deacetylation as described before. And that is, the deacetylation reaction was performed in 10-fold volume of concentrated NaOH under nitrogen to avoid oxidation. The reaction time was 2 h for chitosans with similar molecular weights [42].

The DD value of chitosan was measured by the first derivative UV-spectrophotometry method (1DUVS) with NAG as the calibration curve [43]. Briefly, the absorption spectra of chitosan in various acetic acid solutions were scanned within 190–250 nm. The zero crossing point (ZCP) was obtained by superimposing 1DUVS of 10, 20, and 30 mM of acetic acid solutions at 203 nm. The DD values of the chitosan samples were determined by the formula:

                                DD＝ 100 − [A/(W − 204A)/161 + A] × 100                                         (1)

where A is the amount of NAG determined and W is the mass of chitosan sample used [43].

The procedure for the adsorption experiments was the same as that described previously in this study except that a fixed amount (0.1 g) of DEAE-cellulose, chitin, chitosan or activated charcoal was used. The aqueous solutions with different HA concentrations were placed in a 0.5 L flask and shaken at 37 °C in a thermostatically controlled shaker bath. After the pH of the solution had been achieved neutrality over a 1-hour period, the residual concentration of HA was measured with standard curve by spectrophotometry.

Chitosan is a linear polymer of predominantly glucosamine units and behaves as a polyelectrolyte at acidic pH values. Because of its ability to form ionic bonds at low pH values, chitosan can bind to different types of anions, such as bile acids or free fatty acids [44]. In the present study, 0.1 M HCl solution was used to imitate gastric acid in the stomach based on a previous study [40]; the pH value of the solution was then raised to 7.1–7.4 to simulate the environment of the upper intestinal tract. Activated charcoal, a well-known adsorbent of organic substances by van der Waal’s forces, was used as a positive control. In their chemical structure, chitosan and cellulose are very similar. In acid condition, only the chitosan is protonized. Therefore, cellulose, cellulose derivatives with charged molecules, DEAE-cellulose of positive charge and CM-cellulose of negative charge, were also investigated. As shown in (Figure 1A), DEAE-cellulose, activated charcoal, chitin and chitosan adsorbed HA effectively. DEAE-cellulose, a cellulose derivative with positive charges, adsorbed HA dramatically, while cellulose and CM-cellulose, both bearing negative charges, did not adsorb HA. This finding suggests that chitosan adsorbs HA by ionic interaction or van der Waal’s forces.

The adsorption rate was monitored (Figure 1B). The result showed that the adsorption capacity of chitosan was similar at different incubation times (1, 60 and 300 min) at neutral pH values, which were not obviously exchanged during the incubation time. These results support the finding of Popa et al. [45]. In their study, chitosan and the polyphenols formed a complex, and only 5% of polyphenol was released from the complex after 2 h in a weakly alkaline environment (pH 7.8). Further, the release rate of polyphenols from chitosan-polyphenol complexes was low at neutral pH values, and nearly zero after 90 min.

Adsorption isotherms are important to describe how adsorbates will interact with adsorbent. Therefore, these isotherms are critical for optimizing the use of chitosan as an adsorbent [42]. The adsorption isotherms for DEAE-cellulose, chitin, chitosan and activated charcoal were shown in Figure 2. Where q_e is the amount of HA adsorbed per gram of adsorbent and C_e is the concentration of HA remaining in the solution. The data show that chitosan possesses a much higher adsorption capacity than the other three adsorbents.

It is necessary to analyze the adsorption process by theoretical or empirical equation. The most widely used equation is the Langmuir equation [42], which is represented as:

                                                θ = q_e/q_∞ = K C_e (1 + K C_e)                                                 (2)

where θ is the fractional coverage, q_∞ is the amount of HA adsorbed. Per gram of adsorbent with 100 percent coverage and K is the Langmuir constant.

The essential characteristics of the Langmuir equation can be expressed in terms of the separation factor, RL, which is defined as:

                                                R_L = 1/(1 + K C₀)                                                               (3)

where C₀ is the highest initial HA concentration (mg/L).

The value of R_L indicates that the shape of the isotherm is either unfavorable (R_L > 1), linear (R_L = 1), favorable (0 < R_L < 1) or irreversible (R_L = 0). The parameters in the Langmuir equation and separation factors calculated for chitosan, DEAE-cellulose, chitin and activated charcoal are listed in Table 1. Our results indicate that approximately 3.3 grams of HA was adsorbed by 1 gram of chitosan. The adsorption capacity of chitosan was much greater than those of chitin, DEAE-cellulose and activated charcoal. This suggests that the interaction between chitosan and HA is predominantly due to ionic bonding, much like that between chitosan and polyphenolic compounds, as has been reported previously [45].

To confirm the ionic interaction between chitosan and HA, chitosans with different DD values were prepared. The reaction time was controlled for chitosans with similar molecular weights [42]. The DD values for chitosan prepared in 40% NaOH at 110 °C, 50% NaOH at 110 °C and 50% NaOH at 135 °C were calculated to be 72.8%, 82.0% and 84.7%, respectively, by the 1DUVS method. Figure 3 shows the adsorption isotherms for chitosans with different DD values. The adsorption capacity of chitosan on HA increased with increasing DD values.

According to the results obtained in this study, we suggest that chitosans with a high DD value readily dissolve in the stomach because of the presence of gastric acid. Therefore, their surface area is maximal under these conditions. The dissolved chitosan mixes with HA to form chitosan-HA complexes and subsequently an insoluble gel form in the small intestine. Chitosan’s binding to HA is supported by the positive charges on glucosamine units. This is why chitin and DEAE-cellulose, both with a less positive charge and less surface area, adsorbed less HA than chitosan. These results also interpret the low adsorption capacity of complex beads prepared from activated clay and chitosan, which were prepared by Chang et al. [46]. The maximum amount of HA adsorbed was 149 mg per gram of complex bead, which was insoluble in acid condition and possessed less surface area.

Chitosan is a versatile functional food for obesity prevention, hypercholesterolemia inhibition and immune stimulation [38,39,47]. The adsorption of hazardous materials is also conducted in this study. Adsorption of humic acid in the presence of sun-flower seed oil has been performed in another experiment (data not shown). It suggested that adsorption effect and fat entrapping of chitosan could occur synergistically due to the different mechanism. It was reported that chitosan formed a thin layer on the surface of fat droplets to prevent digestion by enzymes [38,40]. As a result, there is enough surface area of chitosan to expose for adsorption of humic acid effectively in the presence of a lipid source. In vitro simulation of gastrointestinal condition was conducted using HCl and Na₂HPO₄ in this study. However, we have conducted adsorption analysis on HA in this study. This study may not completely imitate the real in vivo conditions owing to the complicated dietary composition in the gastrointestinal tract. It is worth conducting further investigation to evaluate the BFD prevention effect of chitosan.