Dental caries is a common microbial infectious disease, which involves the hard tissue of tooth. Streptococcus mutans is one of the dominating and resident germs of oral, also is the dominating germ factor of the occurrence of caries is discussed by Loesche1. Streptococcus mutans has the capacity of growth, adherence and acidogenicity at tooth surfaces and lead to further demineralization during the formative process of dental plaque2. Therefore, there has been increasing attention focused on inhibitory effect of S. mutans to discover new compounds. The use of natural products and their preparations to treat diseases has been extensively studied based on their biological activities for various purposes worldwide have received increased attention in recent years following the described3-5. The researchers have discovered some natural plants such as Mangnolia officinalis, Galla chinesis and Bauhinia championii (Benth.) Benth, etc., rich in polyphenols have exhibited significant anticaries activity as discussed6,7.
Pomegranate contains substantial amounts of polyphenol compounds such as ellagic acid, ellagic tannins and gallic acid and the highest content is in Pericarpium Granati as discussed by Li et al.8. The separation and purification of polyphenols from Pericarpium Granati technology used SP-700, D-141 and HZ-818 resins as described by Zhang et al.9, Zhu et al.10 and Tang et al.11. However, SP-700, D-141 and HZ-818 resins were expensive and not easy to purchase. Moreover, previous studies showed that polyphenols from Pericarpium Granati have a variety of biological activity effects such as anti-tumor, antioxidant, bactericidal, anti-inflammatory12-14. However, it has not been reported the effects on S. mutans. In this study, a crude extract was prepared from Pericarpium Granati, the appropriate resin was selected for separation and purification of polyphenols by static adsorption/desorption experiment. The optimal adsorption/desorption parameters were determined by dynamic curve. The objective was to develop more practical technology for providing high-purity polyphenols from Pericarpium Granati crude extract using macroporous resins, further to evaluate the anticariogenic activities against S. mutans in vitro, which is provided scientific evidence for further development and enhanced the application of polyphenols from Pericarpium Granati on caries prevention.
MATERIALS AND METHODS
Materials and reagents: The sample of Pericarpium Granati was purchased from Liuzhou, China, identified by Professor Guangwei Huang (Liuzhou LMZ Co., Ltd., China). Gallic acid standard (98%) (Kuer Chem. Co., Ltd., China). Streptococcus mutans strain (ATCC 25175) (GIMCC, China). The TPY liquid medium (Guangdong HKM Co., Ltd., China).
Total phenolic content: The total phenolic content was determined using the Folin-Ciocalteu assay described by Dorman et al.15 with slight modification. The 2.0 mL of sample solution was mixed with 4.0 mL of Folin-Ciocalteu reagent and 10.0 mL of 20% sodium carbonate solution and then diluted to 50 mL with pure water. The absorbance at 760 nm was measured using a UV-visible spectrophotometer (UV-2000, Shanghai Analytical Instrument Overall Factory, Shanghai, China) after 2 h of incubation at 30°C. Gallic acid was used to generate a calibration curve at concentrations of 20-120 μg mL1, the linear regression equation was y = 0.0067x+0.0102 and R2 = 0.9987.
Preparation of crude extract from Pericarpium Granati: The powder of Pericarpium Granati (40 g) was extracted with 800 mL 60% ethanol-aqueous solution at 65°C for 100 min as discussed by Jiang et al.16. The filtrates were collected and concentrated to a crude extract with a rotary evaporator at 65°C.
Adsorbents: Five kinds of tested macroporous resins including D101, AB-8, HPD-100, HPD-450 and HPD-600 were cheap and easy to obtain commercially, purchased from Donghong Chemical Co., Ltd. (Shandong, China) and their unique physical properties are listed in Table 1.
The pretreatment of macroporous resins following the described by Wang et al.17 with slight modification. The resins were soaked in 3 Bed Volumes (BV) of 95% ethanol-aqueous overnight, washed successively with ethanol and pure water until the eluent was clear. In succession, soaked in same amount 1 M NaOH and HCl for 6 h, washed with pure water until the eluent was neutral18. Then the resins were dried at 65°C for 24 h, subsequently placed in a desiccator for later use.
Static adsorption, desorption and kinetics experiments: For the purpose to investigate the adsorption/desorption properties of five kinds of tested resins for polyphenols, the static adsorption/desorption and theirs kinetics experiments were performed as follows19: Dry resins 2.0 g and 20 mL of sample solution at concentration of 10.0 mg mL1 were put into a 50 mL glass-stoppered conical flask and then flask was consecutively shaken for 8 h with 120 rpm at 25°C, the static adsorption capacity and adsorption ratio were determined at adsorption equilibrium.
|Table 1:|| Physical properties of the tested resins
During the adsorption process, the adsorption kinetics was studied by monitoring the content of polyphenols at equal time intervals. After completion of the adsorption process, filtered and washed the resins with pure water and then the separated resins were put into another 50 mL glass-stoppered conical flask. Subsequently, 20 mL of 60% ethanol-aqueous solution was added and the flask was consecutively shaken for 8 h with 120 rpm at 25°C, the static desorption capacity and desorption ratio were determined at desorption equilibrium. During the desorption process, desorption kinetics was studied by measuring the concentration of polyphenols at equal time intervals in the desorption solution. The optimal resin was selected on the basis of theirs adsorption/desorption properties which were calculated in term of the following equations.
Adsorption capacity (mg g1):
Adsorption ratio (%):
Desorption ratio (%):
where, C0, C1 and C2 are the primary and various adsorption/desorption stage concentrations (μg mL1) of polyphenols, respectively, V1 and V2 are the volume of the sample and ethanol-aqueous solution (mL), respectively and W1 is the weight of dried resin (g).
Dynamic adsorption/desorption experiments
Optimization of the feeding parameter: A series of feeding parameters, including concentration, flow rate and volume were optimized. The general procedure, tested resins were wet-packed with 20 mL (a bed volume, 1 BV). After feeding, the eluent was collected and the concentration of polyphenols or adsorption ratio was determined according to the calibration curve20,21. The sample solutions at concentration 2.0, 2.4, 2.8, 3.2, 3.6, 4.0 mg mL1 flowed slowly through the column at rate of 3.0 BV h1, respectively. The eluent was collected and the optimal feeding concentration was determined according to the dynamic adsorption ratio curve. On the other hand, the feeding flow rate experiment as follows. The sample solution at concentration of 3.2 mg mL1 flowed through the column with the rate of 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 BV h1, respectively. The optimal feeding flow rate was determined according to the dynamic adsorption ratio curve. At last, the sample solutions at concentration of 3.2 mg mL1 flowed through the column at 3.0 BV h1. The eluent was collected every 20 mL (1 BV) and the optimal feeding volume was determined according to the leakage point.
Optimization of the eluent parameter: A series of eluent parameters, including concentration, flow rate and volume were optimized. The general procedure, test resins were wet-packed with 20 mL (a bed volume, 1 BV), the adsorption-saturation was reached according to the adsorption parameters obtained, eluted the column with solution. The elution effluent was collected and the concentration of polyphenols or desorption ratio was determined according to the calibration curve. The optimal eluent concentration was investigated using a series of ethanol-aqueous solutions (0, 20, 40, 60, 80 and 100% (v/v), respectively) eluted the column at 3.0 BV h1. The optimal eluent concentration was determined according to the dynamic desorption curve. The optimal eluent flow rate was evaluated by comparing the 60% ethanol-aqueous solution and the rate was set at 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 BV h1, respectively. The optimal eluent flow rate was determined according to the dynamic desorption curve. The optimal desorption eluent volume was investigated using 60% ethanol-aqueous solution eluted the column at rate of 2.0 BV h1, every 10 mL (0.5 BV) of the eluent was collected, the optimal eluent volume was determined according to the dynamic concentration curve as discussed by Liu et al.22.
|Table 2:|| Static adsorption/desorption properties of the tested resins
Anti-Streptococcus mutans activity experiments
Effect on growth of Streptococcus mutans: Minimal Inhibitory Concentration (MIC) was investigated by agar disc diffusion method as described by Khurram et al.23. A series of solutions at concentration from 50.0-1.56 mg mL1 were done in TPY liquid medium, S. mutans (0.9×108 CFU mL1, 0.1 mL) were put into each culture tube containing previous TPY liquid medium (2.0 mL), anaerobic incubation for 48 h at 37°C. The MIC value according to no visible S. mutans produced growth with visual inspection.
Effect on adherence of Streptococcus mutans: According to the MIC value, glass surface adherence assay was investigated at concentrations of MIC and below according to methods previously described24,25 with slight modifications, respectively. The S. mutans (0.9×108 CFU mL1, 0.1 mL) were pipetted into a glass burette by angle of 30°, which contained 10 mL of TPY liquid medium (different concentrations tested polyphenols and 1% sucrose), anaerobic incubation for 48 h at 37°C, supernatants were removed and washed three times with pure water. The adhered bacteria were resuspended into 5% NaOH solution and then the supernatant was removed by centrifuging, suspended in normal sodium and measured the OD value at 540 nm.
Effect on acidogenicity of Streptococcus mutans: A pH drop program was employed to evaluate the effect on acidogenicity of S. mutans as described by Joycharat26. Streptococcus mutans (0.9×108 CFU mL1, 0.1 mL) was grown in 10 mL TPY liquid medium which containing 1% sucrose and tested polyphenols at concentrations of MIC and below, respectively. The initial pH was adjusted to 7.4 and anaerobic incubation for 48 h at 37°C, measured terminal pH of supernatants and calculated pH reduction.
Statistical analysis: All of the experiments were performed in triplicate and the results were expressed as Mean±SD. Analysis of variance was performed by ANOVA test and a statistically significant (p<0.05) or very significant (p<0.01) was considered.
RESULTS AND DISCUSSION
Static adsorption/desorption experiments
Static adsorption/desorption properties of resins: The different properties of five kinds of tested resins for polyphenols were obtained as shown in Table 2. These resins can adsorb polyphenols through physical adsorption and the results were shown the adsorption capacity and adsorption ratio for polyphenols that HPD-100 and HPD-450 are higher than others at adsorption equilibrium because they have larger surface area or suitable polarity, which may provide the higher adsorption capacities for polyphenols. The slightly low adsorption capacity of AB-8 resin may be explained by its smaller surface area than that of D101 and HPD-100.
On the other hand, the desorption ratio were slightly higher than the adsorption ratio for all the resins. Polarity has played an important role for desorption capacity, generally a higher polarity imply a weaker capacity of desorption. Therefore, desorption capacity of HPD-600 was significantly lower compared with HPD-100, which had the highest desorption capacity. However, HPD-450 exhibited the second highest desorption capacity, may be due to the fact that HPD-450 have larger surface area than that of AB-8.
Static adsorption kinetic curve: The adsorption kinetic curve of polyphenols on five kinds of tested resins was screened. The results were shown in Fig. 1, the adsorption ratio was fast increases and then it gradually increased and finally slowed down until adsorption equilibrium. For HPD-450 resin, the adsorption capacity increases rapidly within 1 h, then almost reached adsorption equilibrium at 3 h, but HPD-100 resin reached adsorption equilibrium at 5 h. The reason is HPD-450 resin has suitable polarity to attract polyphenols and faster rate to reach adsorption equilibrium than HPD-100. However, the adsorption for HPD-600 resin reached to equilibrium at 3 h too, but the adsorption capacity far below HPD-450.
Static desorption kinetic curve: The effects of desorption time on the desorption rate of five kinds of tested resins were shown in Fig. 2. The kinetic desorption properties for polyphenols showed a similar tendency that desorption ratio of polyphenols increases accordingly. At the initial 1 h, the desorption process of all resins increased rapidly, however, the rate of adsorption and diffusion get to equal with the concentration increases, desorption ratio did not change obviously after 2 h, showed the desorption equilibrium almost achieved.
||Static adsorption kinetic curve of the tested resins
|Fig. 2:|| Static desorption kinetic curve of the tested resins
Dynamic adsorption/desorption experiments: The HPD-100 and HPD-450 exhibited better adsorption/desorption capacity than those of others by static experiments. Although, HPD-450 resin had slightly lower desorption capacity than that of HPD-100, its adsorption ratio was slightly higher and has a shorter time to reach adsorption equilibrium compared with HPD-100. Therefore, HPD-450 resin was selected for further dynamic investigation with comprehensive consideration of adsorption/desorption properties.
Dynamic adsorption curve of feeding concentration: The process of adsorption includes adsorption and diffusion equilibrium between polyphenols and the resin. The adsorption reaches the breakthrough point when polyphenols starts to leak from the resin. Figure 3 shows the dynamic effects of different feeding concentration on adsorption. The adsorption ratio rapidly increased when the sample solution concentration from 2.0-3.2 mg mL1 where is the highest adsorption ratio. After that, the adsorption ratio decrease gradually with the sample solution concentration increased. A high concentration of polyphenols causes to insufficient exchanged between polyphenols and resin and harm to the adsorption capacity. Therefore, the feeding concentration at 3.2 mg mL1 was finally selected for further experiments.
Dynamic adsorption curve of feeding flow rate: Figure 4 shows as the adsorption ratio rapidly decreased as the feeding flow rate from 3.0-6.0 BV h1. It may be due to incomplete adsorption of polyphenols, since the flow rate of sample solution was faster. However, in the flow rate interval of 1.0-3.0 BV h1, in regard to the adsorption ratio, there was no significant difference at flow rate. It means the rate of change has little effect on the absorption ratio at low rate. Considering that a lower flow rate in general to affect efficiency, 3.0 BV h1 was selected to further experiment as an optimal feeding flow rate.
||Dynamic adsorption curve of feeding concentration
|Fig. 4:|| Dynamic adsorption curve of feeding flow rate
Dynamic adsorption curve of feeding volume: The leakage point generally refers to the concentration of eluent reaches 5.0% of that of initial concentration. The leakage point curve experiments at the feeding flow rate of 3.0 BV h1 is shown in Fig. 5. In the initial process, polyphenols in the sample solutions was almost completely adsorbed over the first 6.0 BV and the concentration of polyphenols closed to zero. However, the leakage process started gradually increased as the elution volume increased from 6.0-8.0 BV. The leakage process increased rapidly after volume of more than 9.0 BV and the maximum loading sample volume of the resin column was 9.0 BV, which the concentration of polyphenols in the effluent solutions reached the leakage point. At this stage, the adsorption capacity of the resin approaches saturation, it can be considered that there would no more accommodate polyphenols.
Dynamic desorption curve of ethanol-aqueous concentration: The dynamic desorption curve is used to display the profile of desorption process and obtained in terms of the concentration and the flow rate of eluent solution. Furthermore, eluent solution volume were used to perform desorption ratio experiments as well. Figure 6 shows, few desorption at the 0 and 20% at ethanol-aqueous concentration. However, the desorption ratio increased sharply, while the ethanol-aqueous concentration from 20-40%. Finally, the desorption ratio reached a peak at the 60%. After that, desorption ratio decrease gradually with the ethanol-aqueous concentration increased. Polyphenols from Pericarpium Granati exhibited certain polarity, the desorption ratio was highest while the ethanol-aqueous concentration closes to similar polarity.
||Curve of adsorption leak of polyphenols
|Fig. 6:|| Dynamic desorption curve of ethanol-aqueous concentration
|Fig. 7:|| Dynamic desorption curve of eluent flow rate
Therefore, 60% ethanol-aqueous displayed the highest desorption ratio compared with that of others and as the optimal concentration to through the column.
Dynamic desorption curve of eluent flow rate: Figure 7 shows, in the flow rate interval of 1.0-2.0 BV h1, in regard to the desorption ratio, there was no significant difference at flow rate.
||Dynamic desorption curve of eluent volume
|Table 3:|| Effect on growth of Streptococcus mutans
|+: Bacterial growth and -: No bacterial growth
|Table 4:|| Effect on adherence of Streptococcus mutans
||Effect on acidogenicity of Streptococcus mutans
However, there have a slowly decreased of desorption ratio during the eluent flow rate from 2.0-6.0 BV h1. High flow rate reduced the time of adsorption-desorption exchange between the eluent and HPD-450 resin is harmful to desorption for polyphenols from resin. Take into account that lower eluent flow rate in general to affect efficiency, the eluent flow rate of 2.0 BV h1 was selected for further experiments.
Dynamic desorption curve of eluent volume: The effects of eluent volume on desorption ratio were investigated with the aim of decreasing the solution consumption and making more efficient. The results were shown in Fig. 8. Polyphenols concentration in eluent solutions was increased rapidly with the eluent volume increased and reached a peak at the 40 mL (2.0 BV). After that, polyphenols concentration decrease gradually with the eluent volume increased. The concentration of polyphenols in the eluent closes to zero when the volume of elution was more than 70 mL (3.5 BV). It can be considered that there was no more polyphenols desorped by eluent solutions. Therefore, an eluent volume of 3.5 BV was selected as optimal parameter.
Purification of polyphenols on HPD-450 resin: The optimal parameters were as follows based on the dynamic experiments: The 9.0 BV of feeding solution at concentration of 3.2 mg mL1 through the column at 3.0 BV h1, then for desorption 3.5 BV 60% ethanol-aqueous at 2.0 BV h1 eluted. According to the optimal parameters, the content of polyphenols was increased from 34.9±1.6% to 71.1±3.1%, with a recovery yield of 85.5±3.7%, the results was consistent with the described by Zhang et al.9, Zhu et al.10 and Tang et al.11. However, HPD-450 resin has the advantage of cheap and easy to obtain commercially compared with SP-700, D-141 and HZ-818 resins that it worth to further study its industry applications.
Antibacterial activity against Streptococcus mutans: The antibacterial activities against S. mutans were evaluated using disc diffusion method and effects on growth, adherence and acidogenicity were investigated. The results of the antibacterial activity are given in Table 3-5.
Effect on growth of Streptococcus mutans: Table 3 shows, the MIC value of crude extract is 12.5 mg mL1, but the MIC value of purification of polyphenols is 6.25 mg mL1 compared with 3.13-100.00 mg mL1 as previously described by Jin et al.6 Jiang et al.7, the antibacterial activity on growth of S. mutans is outstanding. The purification of polyphenols has higher polyphenols content displayed higher inhibition ability for growth of S. mutans. The results illustrate polyphenols from Pericarpium Granati is one of the anticaries material basis therefore worthy of further study, which is consistent with previous research by Jin et al.6 and Jiang et al.7.
Effect on adherence of Streptococcus mutans: Table 4 shows that the effect on adherence of S. mutans. The results indicated that there were very significant effect on adherence of S. mutans when the concentration is greater than or equal to 3.13 mg mL1 (p<0.01) compared with 0.78-12.5 mg mL1 according to the described by Jin et al.6 Jiang et al.7 and He et al.27, the inhibition ability is moderate. The S. mutans adhesion to the tooth surface, resulting in the formation of dental plaque, it is an important part of the formative of dental caries. Since the polyphenols from Pericarpium Granati has significant inhibitory effect on the adhesive function of S. mutans that it might be used in the area of dental caries prevention and treatment in the future.
Effect on acidogenicity of Streptococcus mutans: The effect on acidogenicity of S. mutans was given in Table 5. The authors found that polyphenols from Pericarpium Granati exhibited obviously inhibitory effects compared with control group when the concentration of purification of polyphenols is 1.56 mg mL1 (p<0.05). There are very significantly inhibitory effects showed while the concentration of polyphenols is greater than 1.56 mg mL1 (p<0.01). The results also indicated polyphenols from Pericarpium Granati have excellent effects of inhibition acidogenicity of S. mutans compared with 0.78-12.5 mg mL1 following the described by Jin et al.6 Jiang et al.7 and Wang et al.28. The acidogenicity of S. mutans is one of the most important indicators for dental caries, pH drop cause demineralization of tooth and development of the dental plaque. Due to the significantly inhibit acidogenicity of S. mutans, polyphenols from Pericarpium Granati were expected to become a good candidate for further development as an oral care agent for preventing dental caries.
In this study, HPD-450 was to select as the optimal resin of separation and purification of polyphenols from Pericarpium Granati and high-purity polyphenols was successfully separated and purified from the Pericarpium Granati extract according to the optimal parameters. The experiment of anti-S. mutans activity showed the purification of polyphenols exhibited significantly inhibited activity of growth, adherence and acidogenicity. It illustrated that polyphenols is the one of anticaries material basis of Pericarpium Granati extract. Therefore, further studies are worth to perform and it is expected to become a good candidate for further development as an oral care agent for preventing dental caries.
This study was financially supported by Postdoctoral Research Fund (2013) from Liuzhou LMZ Co., Ltd.