Pectin from apples with a 70~75% degree of esterification was purchased from Sigma-Aldrich (St. Louis, USA). Xanthan gum, hydroxypropyl methylcellulose (HPMC), chitosan from crab shells, sodium alginate from brown algae, RP, and α,α-diphenyl-β-picrylhydrazyl (DPPH) were also purchased from Sigma-Aldrich (St. Louis, USA).

The anti-oxidative effects of pectin and other polysaccharides were evaluated using the DPPH assay described by Yamaguchi, with some modifications [16]. An aqueous solution (0.5 mg/ml) of pectin or other polysaccharides were prepared by dissolving them in water. Chitosan does not dissolve in pure water, and instead was dissolved in a solution of 1% acetate (0.5 mg/ml). A DPPH solution (0.1 ml) was mixed with 0.03 ml of the samples prepared. The mixtures were well shaken and incubated for 30 min at room temperature, and the absorbance of the solution was measured at 517 nm against a solvent (control) using an xMark microplate absorbance spectrophotometer (Bio-Rad, USA). The DPPH radical scavenging effect was calculated by the equation below.

To observe a linear relationship between the concentration and anti-oxidative effects of pectin, 0.1 ml of DPPH solution was mixed with 0.03 ml of varying concentrations of pectin, which were then analyzed as described above.

Concentrations of 0.1 v/v% and 0.01 v/v% of RP were prepared by dissolving the indicated amounts of RP in ethanol, after which 5 ml of the RP solutions were mixed with 5 ml of varying concentrations of pectin. After 5 min of vortexing, the mixtures were incubated at 40℃. Every 2 hours for a total of 8 hours, 200 µl of the samples were taken and the levels of intact RP in pectin hydrogels were analyzed at 325 nm using an xMark microplate absorbance spectrophotometer (Bio-Rad, USA).

All reported data are mean±SD. Statistical significance was checked by Student's t-test one-way ANOVA followed by Student's t-test or Dunnett's test and p<0.05 was considered to be significantly different unless otherwise indicated.

Prior to studying the stabilizing effect of pectin on RP, the anti-oxidative activity of pectin and other polysaccharides were measured by DPPH assay, which is a widely used antioxidant assay based on the decrease in the absorbance of a radical-containing solution. DPPH is a stable free radical which has an unpaired electron at nitrogen atom and it exhibits purple color with a maximum absorption at 517 nm. When DPPH reacts with anti-oxidants, it is reduced to DPPH-H and as a result of that absorbance value decreases. Measuring the scavenging effect of anti-oxidants is based on the decrease in the absorbance of a DPPH solution according to the reaction below. In this way, the anti-oxidative activity of certain samples can be obtained by measuring DPPH radical scavenging effect.

Among the polysaccharides that we analyzed, as shown in Fig. 2, pectin exhibited considerable anti-oxidative properties, with an approximately 5-fold greater DPPH radical scavenging activity compared with the other polysaccharides. There was a statistical difference between the anti-oxidative effects of pectin and the other polysaccharides tested (p<0.05). Phenolic compounds and flavonoids are known to have anti-oxidative effects due to their abundance of hydroxyl groups having hydrogen- or electron-donating properties [17,18]. As shown in Fig. 1, pectin is primarily a polymer of α-(1-4)-linked D-galacturonic acid with hydroxyl groups at the 2' and 3' carbon atom positions [19]. Xanthan gum is a hetero polysaccharide consisting of repeated pentasaccharide units with numerous hydroxyl groups [20]. HPMC is a cellulose derivative polymer with methyl and hydroxypropyl groups, but does not contain free hydroxyl groups that can interact with DPPH free radicals [21]. Chitosan is a linear copolymer of β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine containing one hydroxyl group for each glucosamine unit [22]. Lastly, sodium alginate is a sodium salt of alginic acid that contains two hydroxyl groups at the 2' and 3' carbon atom positions [23]. Except for HPMC, all of the polysaccharides we examined have hydroxyl groups that may produce an anti-oxidative effect, and thus similar anti-oxidative effects were expected. However, pectin exhibited a considerably higher anti-oxidative activity compared with the other polysaccharides. The reason for this may be attributed to the low viscosity of pectin compared to the other polysaccharides (data not shown). Specifically, the higher viscosity of the sample polysaccharides may have prevented appropriate interaction between the hydroxyl groups present in the polysaccharides and DPPH free radicals. Thus, hydroxyl-group containing polysaccharides may demonstrate good anti-oxidative activity when their viscosities are not overly high.

As shown in Fig. 3, the DPPH radical scavenging effect of pectin increased gradually with increasing concentrations of pectin. This result may stem from the increased density of hydroxyl groups generating the anti-oxidative effect.

Two different concentrations of RP concentrations were evaluated to determine if there were concentration-dependent effects. First, the remaining amount of RP as a function of time was determined in the absence of pectin. We found that the levels of RP greatly decreased from 100% to 39% and 61% at RP concentrations of 0.01 and 0.1%, respectively, during a 2 hour storage period at 40℃ (Fig. 4). These results implied that the degradation of RP in ethanol was dependent upon the concentration of the substrate. The reason for the greater degradation of the lower concentration of RP may have been due to the ease with which the outer environment was able to break down RP. Next, upon addition of pectin at various concentrations from 0.1~0.5 mg/ml, the stability of RP was relatively enhanced for both lower and higher concentrations of RP. Especially, addition of pectin at a lower concentration of RP (0.01% RP) significantly increased its stability at all concentrations of pectin tested (p<0.05). On the other hand, the ability of pectin to stabilize RP was drastically reduced at higher concentration of RP (0.1% RP). The reason for this result is not clear, but was consistent with our preliminary examination of the viscosities of the lower and higher RP solutions. Specifically, the lower concentration of RP was less viscous than the higher concentration RP, and thus a more effective interaction with pectin could be expected.

RP stability in ethanol and in the presence of various concentrations of pectin was monitored as a function of time (Fig. 5). The remaining amounts of RP decreased considerably for an initial 2-hour period, after which the rate of decrease slowed. As shown in Figs. 4 and 5, the stabilizing effect of pectin was greater at lower RP concentration despite the fact that the absolute levels of RP in the test solutions were greater for the more highly concentrated RP solution. We are currently performing additional experiments to clarify the mechanism behind the effect of substrate concentration on the stability of RP.

In the present study, we clearly showed that pectin exhibits a considerably stronger anti-oxidative activity compared with the other polysaccharides we examined. The anti-oxidative effects of pectin are considered to be due to the hydroxyl groups present in its structure. The anti-oxidant effects of pectin increased in a dose-dependent manner, which was likely due to the increased amount of hydroxyl groups. The stability of RP was considerably improved in the presence of pectin, because of its anti-oxidative effects and also the increased amount of pectin, which led to enhanced stabilization of RP. The stability of RP was comparatively affected by the concentration of RP itself. From the results obtained in this study, we suggest that pectin may be a promising component for cosmeceutical bases where stabilization of RP is necessary, which may prevent the need for costly stabilization techniques when RP is incorporated into cosmetic products.