2016 Volume 22 Issue 5 Pages 639-646
Sodium ferric gluconate complex (SFGC) was synthesized by a simple method using D-sodium gluconate (D-SG) and ferric chloride as raw materials, which was found to have chelated iron characteristics by measuring its physicochemical properties. The iron(III) complex was characterized by scanning electron microscopy (SEM), ultraviolet-visible spectrophotometry (UV-Vis), fourier transform infrared (FTIR), X-ray diffraction analysis (XRD) and differential scanning calorimetry (DSC). The results verified that the product was indeed the chelated iron complexes and its iron core was composed of iron oxyhydroxide namely β-FeOOH mineral polymorph. Moreover, antioxidant activity of SFGC was evaluated by the half maximal inhibitory concentration (IC50) against hydroxyl free radical, 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical, nitrite and lipid peroxidation, and their values were 3.65, 8.09, 3.01, 1.90 mg/mL, respectively. These results demonstrate that SFGC is expected to become a good iron supplement with a variety of biological activity or food additive strengthening iron.
Iron deficiency anemia is the most common nutritional deficiency worldwide (Auerbach et al., 2013). Lower hemoglobin and iron deficiency anemia caused by iron deficiency are widely in many countries, especially in developing countries, which caused the serious global nutrition problems (Bartłomiej and Andrzej, 2011; Bereman and Berg, 1989). As the third generation of nutritional iron supplements (Braunschweig et al., 2012), polysaccharide iron(III) complex (PIC) not only have expedient stability and less gastrointestinal irritation, but polysaccharide has more terms of biological activity after it release iron, which are useful components of the body and can be absorbed. Therefore, there is no doubt that PIC are one of the promising oral nutritional iron supplements (Chinese Pharmacopoeia Commission, 2010; Carmona-Jimenez et al., 2015). In recent years, we have seen series of reports that involved using the extracted polysaccharide to prepare PIC, for example, angelica (Eichbaum et al., 2003), limonium (Fu et al., 2014), chitosan (Ghotbi et al., 2009), isomaltooligosaccharide (Goss et al., 2006) and so on.
Gluconic acid, sodium salt is also called D-sodium gluconate (D-SG), which can regulate the body acid-base balance, restore normal nerve action and effectively prevent the occurrence of low sodium syndrome (Jahn et al., 2011; Khawla et al., 2016). D-SG, as a new food additive, has attracted much attention, due to its various advantages such as better stability, chelating property and abundant raw materials. Recently some new preparation techniques and applications emerged and described in details (Kudasheva et al., 2004).
Metallic iron ions in solution were effectively adhered to D-SG as a molecular housing to form stable complexes, the structure of that contain a mineral core composed of iron oxyhydroxide in the β-FeOOH mineral polymorph (Koskenkorva-Frank et al., 2013; Littlewood, 2012). In addition, it is greatly significant in the development of oral iron agents due to its superb stability, no toxic side-effects and easy process operation (Carmona-Jimenez et al., 2015). In this work, a new sodium ferric gluconate complex (SFGC) was prepared by D-SG and ferric chloride with a simple synthesis method. The physicochemical properties and structural characteristics of this SFGC were investigated. Compared the antioxidant activity of D-SG with iron(III) complex, we hoped to get a good iron supplement source with a variety of biological activity or food additive strengthening iron. Furthermore, it also provides the experimental basis for the development of PIC.
Materials D-sodium gluconate (D-SG) was purchased from ChengDu Kelong Chemical Co. Ltd. in Sichuan Province China. 2,2-Diphenyl-1-picryl-hydrazyl (DPPH) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ultrapure water (18.25 MΩ.cm) was used throughout the experiment. All other reagents and chemicals used in this work were of analytical grade.
Preparation of SFGC The 40 mL of 1.18 M Na2CO3 solution was added to another 40 mL solution which contained 10 g D-SG. After that, a solution containing (60 mL) 1.25 M of FeCl3 × 6H2O was added dropwise under continuous stirring. The synthesis of the complex was performed at pH value of 11 (adjusted by the addition of 5 M NaOH). The mixture was heated at 100°C at least 3 h in oil bath, and then filtered and cooled to room temperature. The complex precipitated by 0.2 L ethanol (Li et al., 2012; Liu et al., 2014). After 4 h standing, the ethanol was decanted and the precipitate was centrifuged at 2900×g for 10 min. The supernatant was decanted and the precipitate was redissolved in 0.1 L redistilled water. Dialysis of the solution was used to remove unbound ions (Cl−, Na+, and Fe3+) (Mitsuoka, 2014). Finally, the complex was washed successively from the dialyzed solution by ethanol and acetone, and dried for 3 h in vacuum after decantation. The sodium ferric gluconate complex was obtained as a black powder.
Determination of iron content Atomic absorption spectrometer (Z-5000, Hitachi, Japan) with air-acetylene flame atomization (flame height 7.5 mm, air flow 6.5 L/min, acetylene flow 1.5 L/min), equipped with an HCL lamp for the determination of iron (lamp current 5.0 mA, slit 0.5 nm, wavelength 248.3 nm) was used to detect the iron content of SFGC (Mitic et al., 2011).
Estimation of free iron About 0.1 g of SFGC were dissolved in ultrapure water (10 mL). At the same time, K4[Fe(CN)6] and KSCN solution (0.5 mol/L) were added dropwise to the solution and observed precipitation phenomenon to determine whether there was free iron in solution (Michael et al., 2006), meanwhile, ferric hydroxide colloid was used for reference.
Stability The solutions of SFGC, FeCl3 and FeSO4 (0.01 M, 20 mL) were titrated with 0.01 M NaOH and simultaneously, pH measurements (25°C) were carried out by means of a Hanna PH 213 pH-meter using a Hanna InLab glass electrode. Hydrolysis phenomenon was observed and described in the titration curve (Marshall and Rutherford, 1971).
Reduction Ascorbic acid solutions (10 g/L) with different volumes (1, 2, 3, 4, 5 mL) were added to the 1 mL of 1 mg/mL SFGC solution in a 50 mL volumetric flask, respectively. The solution was mixed with sodium acetate (1 mol/L, 5 mL) and phenanthroline (1 g/L, 3 mL), and the mixture was reduced in a 37°C water bath. The absorbance of the reaction was measured at 510 nm recorded at several time points against the blank (Mao et al., 2015).
Characterization of SFGC The microstructure of SFGC was examined by SEM (Phenom G2 pure, Phenom-World B.V., Netherlands). The powder samples was sprayed on double adhesive tape mounted on aluminium stub. Sample was finally examined at an acceleration voltage of 15 kV under high vacuum (9.0 × 10−5 Pa) and micrograph was recorded (Ma et al., 2010).
The UV-Vis spectra of the D-SG and synthesized Fe(III) complex in water solutions (1 g/L) were recorded on a UV-Vis Spectrophotometer (UV-6100, Mapada Co. Ltd., Shanghai, China). The region 200 – 800 nm was scanned at a resolution of 1 nm at room temperature (Mitsuoka, 2014; Ma et al., 2010). The optical blank solution was redistilled water.
XRD of D-SG and SFGC were recorded on an X-ray diffractometer (X'Pert3 Powder, PANalytical B.V., Netherlands), and the scan was performed under the conditions of 40 kV tube voltage, 20 mA tube current, Cu target, 0.02° scanning step size, 2°/min scanning speed to collect 2° data from 10o to 70° (Pornsunthorntawee et al., 2009).
DSC was carried on a DSC Instrument (200F3, Netzsch, Germany), then D-SG, FeCl3 and SFGC were measured in the atmosphere of flowing oxygen-free nitrogen with a scanning rate of 10°C/min from 30 to 500°C (Phadungath & Metzger, 2011).
Scavenging capacity against hydroxyl free radical The basic theory involved in the Fenton reaction was described in the literature (Pitarresi et al., 2008; Qu et al., 2011). FeSO4 (9 mM, 2 mL), H2O2 (8.8 M, 2 mL) and the SFGC sample solutions of different concentrations (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 mg/mL, 2 mL) were mixed in a 10 mL colorimetric tube. After standing for 5 min, the 2 mL of 9 mM salicylic acid ethanol solution was added and then incubated at 37°C for 0.5 h. The absorbance of the reaction mixture was determined at 510 nm. Scavenging capacity of D-SG and vitamin C (VC) were measured by the same method.
Scavenging capacity of SFGC against hydroxyl free radical was calculated as follows:
where Ao is the absorbance of solution without the sample, and Ax is the absorbance of sample solution. In the case of measurement of scavenging capacity against hydroxyl free radical, the equal volumes of solution without salicylic acid was used as reference for Ao and Ax.
Scavenging capacity against DPPH free radical The basic theory involved in the reaction of DPPH radical and free radical scavenger was followed as description of the previous report (Shilpashree et al., 2015; Shi et al., 2013). DPPH (0.6 mM, 1 mL) and the SFGC sample solutions of different concentrations (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 mg/mL, 3 mL) were mixed in a 10 mL colorimetric tube. After shaking, the mixture was kept in dark at 37°C for 0.5 h. The absorbance of the reaction mixture was determined at 517 nm. Scavenging capacity of SFGC against DPPH free radical was calculated by the Eq. 1. Scavenging capacity of D-SG and VC were measured by the same method.
Scavenging capacity against nitrite The method of N-(1-naphthyl) ethylenediamine dihydrochloride spectrophotometry was followed as description of the previous report (Sun et al., 2006). Citric acid-NaH2PO4 buffer solution (pH 3.0, 1 mL), the SFGC sample solutions of different concentrations (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 mg/mL, 2 mL) and NaNO2 (50 µg/mL, 250 µL) were mixed in a 10 mL colorimetric tube. The mixture was shaken and incubated at 37°C for 0.5 h. The reaction solution was taken out, and then immediately mixed in 4-aminobenzene sulfonic acid solution (0.4%, 1 mL). After standing for 5 min, the 0.5 mL of 0.2% N-(1-naphthyl) ethylenediamine dihydrochloride solution was added and the mixture was then standed at 10 min again. The absorbance of the solution was determined at 538 nm. Scavenging capacity of SFGC against nitrite was calculated by the Eq. 1. Scavenging capacity of D-SG and VC were measured by the same method.
Scavenging capacity against lipid peroxidation Soy lecithin (120 mg) dissolved in 30 mL phosphate buffer (pH 7.4, 0.05 M) was sonicated for 30 min in an ultrasonic generator (KQ5200DE, Ks-csyq, China) until liposome was dispersed completely. Iiposome suspension (1 mL), FeSO4 (5 mM, 2 mL) and the SFGC sample solutions of different concentrations (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 mg/mL, 2 mL) were mixed in a 10 mL colorimetric tube. The mixture was incubated at 37°C for 0.5 h, and shaked every 10 min. The reaction was stopped by the addition of trichloroacetic acid (TCA, 10%, 1 mL) and 2-thiobarbituric acid (TBA, 0.8%, 1 mL), which had been incubated in boiling water bath for 15 min, and then cooled and centrifuged at 4500 rpm for 15 min. The absorbance of the supernatant was determined at 532 nm (Shilpashree et al., 2015; Sun et al., 2006). Scavenging capacity of SFGC against lipid peroxidation was calculated by the Eq. 1. Scavenging capacity of D-SG and VC were measured by the same method.
General properties The synthesized iron complex is odorless, tasteless and brown-black solid powder, but presents a clear red-brown solution once dissolving in water. However, it is not soluble in ethanol, methanol, acetone and other organic reagents. The pH value of sample solution was approximately 6 to 8, and the solution could show Tyndall effect hinting colloidal property (Mitic et al., 2011). The sample solution was added dropwise to the potassium ferrocyanide and potassium thiocyanate solution, but it did not produce precipitation like ferric hydroxide colloid. The results indicated that free iron did not exist nor contaminate in the solution, and the complex had basic stability (Michael et al., 2006).
Stability The bioavailability of iron supplement depended on the dissolving capacity of iron supplement in duodenum (pH6–7), which was the main absorption part of iron (Fu et al., 2014). In order to identify the differences between SFGC and inorganic FeOOH, SFGC solutions, FeCl3 and FeSO4 were titrated with NaOH and the titration results were shown in table 1. For the SFGC solution, the precipitate was not observed and always remained clear solution in a range of pH 5–13. However, for the FeCl3 solution, the precipitate was initially and generously observed in pH 3.03 and 6.70. As for FeSO4 solution, this pH of start turbid was 3.34, and the pH of a large number of turbid was 6.76. These results implied that FeCl3 and FeSO4 were turned into iron hydroxide which were not absorbed and used by organism under physiological pH condition according to the study in the previous researches (Phadungath and Metzger, 2011). But the complex SFGC was fully soluble under physiological conditions, and it was neither hydrolyzed nor precipitated with high stability and better bioavailability.
Reduction Iron is mainly absorbed by duodenum and upper jejunum mucosa, and only the dissolved irons could be absorbed. Ascorbic acid not only made the iron release from the SFGC, but also made trivalent iron reduce to divalent iron (Qu et al., 2011). Fig. 1 shows the impact of the dissolution and reduction rate of iron in the SFGC by the amount of ascorbic acid added. With the ascorbic acid added, the dissolution and reduction rate of iron gradually increased, and finally tended towards stability in 4 h. According to this result we can conclude that the SFGC had better capability of the dissolution and reduction and the bioavailability, and had not significant irritation of alimentary canal because the solution did not contain Fe3+. These results are in agreement with others studies which reported that iron(III) complexs were formed mainly by isomaltooligosaccharide, chitosan and inonotus obliquus polysaccharide (Mao et al., 2014; Pitarresi et al., 2008).
A-T curves of the reaction system between SFGC and VC
Microstructure The SEM images of SFGC shown in Fig. 2 revealed that the shapes of SFGC were different, and the surface of SFGC was flatter and smoother, which showed small and uniform white flake like structures embedded all over the complex surface when it was observed with the magnification.
SEM images of SFGC: 1000×(a), 100×(b)
UV-Vis spectrum analysis The UV-Vis spectrums of SFGC and D-SG were presented in Fig. 3. The main absorption wavelength of SFGC in the ultraviolet spectra is 221 nm (Fig. 3.a), whereas the main absorption wavelength of D-SG is 214 nm (Fig. 3.b). The absorption intensity of SFGC was significantly higher than D-SG in ultraviolet region, since electron transfer transition band of ligand (D-SG) to the core of iron ion caused the strong absorption in the ultraviolet region. Nevertheless, the SFGC complex had weak absorption in the visible region, and it was because of electronic transition from a lower d-orbit to a higher d-orbit in center ion produced d-d transition bands. In general, the results showed that D-SG and iron reacted successfully and formed to the SFGC complex.
UV-Vis spectrums of SFGC (a) and D-SG (b)
FTIR spectrum analysis The FTIR spectrums of D-SG and SFGC shown in Fig. 4 were found to be substantially similar, since the major component of the complex skeleton was D-SG. There were stretching vibration absorption peak of O-H (about 3420 cm−1) and C-H (about 2940 cm−1), in addition, there was the deformation vibration absorption peak of H-O-H (about 1640 – 1400 cm−1) in both Fig. 4 (a, b). Therefore, iron ions did not make a significant change in the structure of D-SG. However, the peak shape of the stretching vibration absorption band of C-O (1084 cm−1) and fingerprint region (900~400 cm−1) had some change in Fig. 4 (a). There was the characteristic peak of structure at 820, 650 and 534 cm−1 in the spectrum of SFGC, which were attributed to the characteristic absorption peak of β-FeOOH (both in 900 – 420 cm−1) according to the study in the previous researches (Tomida et al., 2009; Tang et al., 2013). These results explained that D-SG and iron were synthesized to a new substance (SFGC) and the iron core of complex was polymerized β-FeOOH structure.
FTIR spectrums of SFGC (a) and D-SG (b)
X-ray spectrum analysis The XRD spectrums of SFGC and D-SG were shown in Fig. 5, which was some significant difference in the two spectra. D-SG had many weak diffraction peaks, however, SFGC had no sharp crystallization peak. The result suggested that SFGC had poor crystallinity and was amorphous shape, which was obviously different from the raw material.
X-ray spectrums of SFGC (a) and D-SG (b)
DSC thermogram The DSC curves of SFGC, D-SG and FeCl3 with different temperature were shown in Fig. 6. It was observed that it had a clear endothermic peak of D-SG at 214.9°C presumably due to the decomposition of D-SG, and FeCl3 had only one obvious exothermic peak appeared at about 65°C. However, there were two new exothermic peaks appearing at about 270 and 320°C for SFGC, which was different from the formers. The results indicated that the complex was a new substance (SFGC), and the peak shape of exothermic peak was better, which reflected the complex SFGC had a high thermal stability (Wan, 2013).
DSC curves of SFGC (a), D-SG (b) and FeCl3 (c)
Scavenging capacity against hydroxyl free radical Hydroxyl free radical is considered to be the most active and toxic free radical, which can directly damage a variety of biofilms and lead to all kinds of diseases (Wang et al., 2015). Through calculating the scavenging capacity (Eq. 1) of SFGC, D-SG and VC against hydroxyl free radical, it was found that all of them have scavenging activity. The ·OH scavenging capacity of VC (Y=1.64933+30.47371X, R2=0.9953) was stronger than SFGC (Y = 1.24667 + 15.51143X,R2=0.9850) and D-SG (Y=7.98821+5.91857X, R2=0.9731), and scavenging capacity of SFGC was slightly stronger than D-SG. The activity boosted with the increase of concentration (X), and there was linear relationship in a certain concentration range. This result was similar to the one obtained by Mao et al. (2015) (Pitarresi et al., 2008) (isomaltooligosaccharide iron complex (IIC)). D-SG molecule containing hydroxyl groups and a carboxyl group as the electron donor, which reacted to achieve the effect of free radical scavenging with hydroxyl free radical. Spatial structure was changed after D-SG and Fe(III) ion reacting, which caused a synergistic effect of ligand moiety on exposed active group, promoted the role of hydroxyl radical and made the scavenging capacity increase.
Scavenging capacity against DPPH free radical DPPH is also called 1,1-Diphenyl-2-picrylhydrazyl radical, which is a very stable free radical of nitrogen center, and has been widely used for quantitative determination of the antioxidant capacity of biological samples and food (Wang et al., 2008). The scavenging capacity (Eq. 1) of SFGC, D-SG and VC against DPPH free radical showed that all of them had scavenging activity. The DPPH scavenging capacity of VC (Y=40.27179+1.30976X, R2=0.9321) was stronger than SFGC (Y=23.4053+3.28762X, R2=0.9659) and D-SG (Y=7.95679+4.75602X, R2=0.9795), and scavenging capacity of SFGC was slightly stronger than D-SG. The activity boosted with the increase of concentration (X), and there was linear relationship in a certain concentration range. Ma et al. (2010) (Wei and Nan, 2011) reported that at a concentration of 0.5 mg/mL and more, the scavenging rate of Flammulina velutipes polysaccharide and its chelate with iron on DPPH radicals there was also a linear relationship. D-SG was paired with single-electron of DPPH free radical, which brought in the scavenging effect. After iron was coordinated, the synergistic effect of iron and D-SG made the scavenging capacity against DPPH free radical increase.
Scavenging capacity against nitrite The content of nitrosamine is little in natural food, but nitrogenous substances, such as primary amine, secondary amine, amino acid, phospholipid and so on, are widely present in food. They can be converted to nitrosamine with nitrite under certain conditions, which is a strong carcinogen to humans and animals (Wang et al., 2006). The scavenging capacity (Eq. 1) of SFGC, D-SG and VC against nitrite showed that all of them had scavenging activity. The scavenging capacity of VC (Y=47.47571+11.89524X, R2=0.9005) was stronger than SFGC (Y=20.33893+9.86381X, R2=0.9306) and D-SG (Y=12.66786+3.63595X, R2=0.9751), and scavenging capacity of SFGC was stronger than D-SG. The activity boosted with the increase of concentration (X), and there was linear relationship in a certain concentration range. These results are in agreement with the study of soluble soybean polysaccharide iron complex of Wan Z. (2013) (Sun et al., 2006). Indeed, as compared to polysaccharide, polysaccharide iron complex led to the higher proportion of high-scavenging capacity. D-SG and sodium nitrite were reacted by oxidation-reduction reaction to reduce the content of nitrite, and the scavenging capacity against sodium nitrite was promoted after coordinating. Iron can both get electron and lose electron and can produce a synergistic effect with D-SG.
Scavenging capacity against lipid peroxidation Intermediate product of lipid peroxidation-radical can cause atherosclerosis, get protein molecule together, seriously damage biofilms, damage biomacromolecule and cause aging (Zhang et al., 2012). The calculated scavenging capacity (Eq. 1) of SFGC, D-SG and VC against lipid peroxidation showed that all of them had scavenging activity. The scavenging capacity of VC (Y=40.78286+8.77262X, R2=0.9325) and SFGC (Y=34.542+8.12X, R2=0.9272) was stronger than D-SG (Y=9.3053+0.6942X, R2=0.9001), and scavenging capacity of VC was slightly stronger than SFGC. The activity boosted with the increase of concentration (X), and there was linear relationship in a certain concentration range. The hydroxyl and carboxyl groups in the D-SG molecule could not be reacted with the decomposed product of lipid peroxidation-malondialdehyde (MDA), but lipid peroxidation could be inhibited and lipid antioxidant activity was significantly increasing after the iron coordinating. It may be the electronic effect of iron weakening the carbonyl activity of MDA or iron reacted with MDA preventing the condensation of MDA and TBA. These results are in agreement with others studies which reported that iron(III) complexs are formed mainly by isomaltooligosaccharide (Pitarresi et al., 2008) and soluble soybean polysaccharide (Sun et al., 2006).
The half maximal inhibitory concentration (IC50) values of SFGC against hydroxyl free radical, DPPH free radical, nitrite and lipid peroxidation (3.65, 8.09, 3.01, 1.90 mg/mL) were shown in Fig. 7. Scavenging capacity of SFGC against four substances arrayed in descending order: lipid peroxidation > nitrite > hydroxyl free radical > DPPH free radical. Compared with D-SG (7.10, 8.84, 10.27, 58.62 mg/mL), the capacity of SFGC to scavenge hydroxyl free radical, DPPH free radical, nitrite and lipid peroxidation were risen by 48.59%, 8.48%, 70.69% and 96.76%, respectively. By data for antioxidant activity performed on SFGC have been reported in the literature.
Comparison on IC50 values of SFGC
SFGC was synthesized using D-SG and ferric chloride as raw materials based on physicochemical properties, which has been proved to have characteristic properties like chelated iron. SFGC was shown to have higher stability and better stripping reduction capability by hydrolysis and reduction experiments. Therefore, SFGC had the basic properties of ideal oral iron agents. SFGC was characterized by the complex structure, and the iron core of complex was polymerized β-FeOOH structure. The results of the antioxidant activity test showed that scavenging capacity of SFGC against hydroxyl free radical, DPPH free radical, nitrite and lipid peroxidation were significantly higher than that of D-SG, but were lower than that of VC. These results demonstrate that SFGC is expected to become a good iron supplement source with a variety of biological activity or food additive strengthening iron. The present study establishes the scientific basis for the research and development of iron supplement of SFGC, which possesses higher practical value and provides new ideas for the development of PIC.
Acknowledgments This work has been supported by Chongqing Engineering Research Center for Pharmaceutical Process and Quality Control Capacity Building Project (CSTC2012gg-yyjsb10002-33) and Southwest University Dr. Fund Projects (SWU110056, SWU110057).