2022 Volume 64 Issue 1 Pages 25-35
Advanced glycation end products (AGEs) influence periodontal disease onset and progression by denaturing proteins in the periodontal tissues and inducing inflammation. Therefore, inhibiting both AGE formation itself and the AGE-induced inflammation would be effective for preventing periodontal disease. This study aimed to find anti-inflammatory ingredients in oral-care products that also exhibit AGE formation inhibitory effect. We evaluated in vitro the AGE formation inhibitory effects of five ingredients with known anti-inflammatory effects in oral-care products: allantoin (ALA), 6-aminohexanoic acid, dipotassium glycyrrhizinate (GKII), lysozyme chloride, and azulene sulfonate sodium hydrate (AZU). Furthermore, in order to assess the implications of the antiglycation effect of these ingredients in periodontal disease, human gingival fibroblasts (HGFs) were treated with AGEs formed in the presence or absence of each ingredient, and the IL-6 expression level in the culture supernatant was analyzed by enzyme-linked immunosorbent assays. Our results showed that ALA, GKII, and AZU inhibited AGE formation in vitro. In addition, compared with bovine serum albumin (BSA) incubated with glyceraldehyde only (i.e. glyceraldehyde-derived AGE), BSA incubated with glyceraldehyde in the presence of each component inhibited IL-6 production from HGFs in vitro, suggesting that these components may prevent gingival inflammation by preventing the formation of AGE. Our in vitro results suggest the possibility that these oral-care product ingredients can help prevent periodontal disease by two distinctive functions-antiglycation and anti-inflammation.
Advanced glycation end products (AGEs) are the final products of the Maillard reaction, a non-enzymatic reaction between reducing sugars and proteins. AGEs are known to accumulate in various organs during physiological aging1-3). Several studies have shown that AGE formation correlates with glycemic control and diabetes affects AGE accumulation4,5). This is expected to be the case in the gingival tissues as well6,7). Furthermore, in gingival tissues, AGEs are present in the epithelium, connective tissue, and gingival sulcus fluid of patients with periodontal disease regardless of diabetes6,8,9), and the AGE content in the gingival sulcus fluid is known to be higher in patients with periodontal disease than in healthy subjects8). These observations suggest a relationship between AGE accumulation in gingival tissues and periodontal disease.
Nonaka et al. and Kido et al. demonstrated that AGEs directly stimulate various gingival cells and promote the production of pro-inflammatory cytokines10,11). In addition, not only do AGEs elicit inflammation by themselves but also exacerbate gingival cell inflammation induced by lipopolysaccharides from Porphyromonas gingivalis, one of the virulence factors related to periodontal disease pathogenesis11-13). Furthermore, Chang PC et al. reported that the administration of aminoguanidine, which inhibits AGE formation, suppresses alveolar bone resorption in non-diabetic rats with experimentally induced periodontal disease14). These findings suggest that AGEs are involved in periodontal disease onset and progression not only in diabetic (diabetes-associated periodontitis) but also in non-diabetic patients.
Chang PC et al. demonstrated that AGEs exacerbate periodontal disease not only by denaturing proteins in the periodontal tissues but also by inducing inflammation e.g., through activating the AGE/RAGE axis pathway14,15). Therefore, in order to inhibit the promoting effect of AGEs on the onset and progression of periodontal disease, it is necessary to prevent both AGE formation itself and AGE-induced inflammation. If we could achieve these two effects with a single active ingredient, we could efficiently control how AGEs affect the gingiva. The purpose of this study was to find anti-inflammatory ingredients in oral-care products that also exhibit an antiglycation effect.
In this study, we evaluated in vitro the AGE formation inhibitory effect of five active ingredients, allantoin (ALA), 6-aminohexanoic acid (AHA), dipotassium glycyrrhizinate (GKII), lysozyme chloride (LYZ), and azulene sulfonate sodium hydrate (AZU) (Table 1), which have been approved in quasi-drug oral-care products by the Ministry of Health, Labor and Welfare in Japan for the prevention of periodontal disease16) and are known to exhibit anti-inflammatory effects17,18). Various mechanisms underlying the anti-inflammatory effects of these five ingredients have been reported. ALA and AZU are reported to exert anti-inflammatory effects by inhibiting the infiltration of inflammatory cells19,20), AHA by antiplasmin21), and GKII by inhibiting the production of leukotrienes and prostaglandins22,23). The mechanism underlying the anti-inflammatory effect of LYZ has not been elucidated in detail24). These five active ingredients were selected because they are water-soluble, which was a condition required for evaluating their inhibitory effect on AGE production, are active ingredients generally used for the prevention of periodontal diseases, and known to have anti-inflammatory effects. In addition to the evaluation of the AGE inhibition property of these five ingredients, in order to assess the implications of their antiglycation effects in periodontal disease, we evaluated how AGEs formed in the presence of each ingredient affect human gingival fibroblasts (HGFs).
Chemical structures and properties of the ingredients
Allantoin (ALA), bovine serum albumin (BSA), and DL-glyceraldehyde were purchased from Merck KGAA (Darmstadt, Germany). Dipotassium glycyrrhizinate (GKII), 6-aminohexanoic acid (AHA), sodium ascorbate (VC), and diethylenetriamine-N, N, N', N'', N''-pentaacetic acid (DTPA) were purchased from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). Lysozyme chloride from Egg White (LYS) and azulene sulfonate sodium hydrate (AZU) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
Methods Antiglycation capacity evaluationThe antiglycation effects of ALA, AHA, GKII, LYZ, and AZU at the concentrations of 6.25, 12.5, and 25.0 mM for glyceraldehyde-induced, fructose-induced, and glyoxal-induced glycation were analyzed using the Collagen Glycation Assay kit: Glyceraldehyde, Collagen Glycation Assay kit: Fructose, and Collagen AGEs Assay Kit, CML-Specific, Glyoxal (Cosmo Bio, Tokyo, Japan) respectively. Dulbecco's phosphate-buffered saline (DPBS) was used as a control in all the assays. Incubation time for glyceraldehyde-, fructose-, and glyoxal-induced glycation were 24 h, 28 d and 24 h, respectively as per manufacturer's instructions. In the assays of the glyceraldehyde- and fructose-induced glycation, the fluorescence intensity (FI) of the glycated collagen was read using Infinite 200 Pro (Tecan Japan, Kanagawa, Japan) with excitation and emission wavelengths of 370 and 440 nm, respectively. The inhibitory rate was calculated as follows: Inhibition (%) = (FI of control − FI of the sample) /FI of control × 100. In the glyoxal-induced glycation assay, the absorbance value (AV) at 450 nm was read using Infinite 200 Pro. The inhibitory rate was calculated as follows: Inhibition (%) = (AV of control − AV of the sample) /AV of control × 100. All assays were performed following the manufacturer's instructions.
Hydroxyl radical scavenging assayThe hydroxyl radical antioxidant capacity (HORAC) of ALA, GKII, and AZU at the concentrations of 6.25, 12.5, and 25.0 mM were determined using the OxiSelect HORAC activity assay kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer's instructions. The HORAC assay is based on the oxidation-mediated quenching of a fluorescent probe by hydroxyl radicals produced by a hydroxyl radical initiator (H2O2) and Fenton reagent. The antioxidant capacity was calculated based on the area under the fluorescent decay curve (Net AUC = AUC (samples) − AUC (distilled water; DW as control) ). VC was used as a positive control.
Glyceraldehyde-derived AGE preparationNon-glycated bovine serum albumin (non-glycated BSA) and glyceraldehyde-derived AGE (glycated BSA) were prepared following the modified method of Takino et al25). Briefly, 25 mg/mL BSA, 5 mM DTPA, and 100 U/mL penicillin-streptomycin (Thermo Fischer Scientific, Waltham, MA, USA) were incubated under sterile conditions with or without 0.1 mol/L DL-glyceraldehyde for 7 d at 37°C. Glycation-inhibited BSA solutions were prepared by adding 12.5 mM of ALA, GKII, or AZU to the above-described solution before incubation (Table 2) followed by the removal of low-molecular-weight reactants including ALA, GKII, AZU, and free DL-glyceraldehyde using a 10-kDa MWCO Amicon Ultra centrifugal filter unit (Merck). Non-glycated BSA, Non-glycated BSA with ALA, BSA with GKII, and BSA with AZU were prepared as negative control solutions by incubating the mixture solutions without DL-glyceraldehyde under the same conditions (Supplementary Table 1).
Reaction mixtures for BSA glycation
HGFs were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in Minimum Essential Medium α (MEM α; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan) and 1% penicillin-streptomycin liquid (Thermo Fischer Scientific) at 37°C in 5% CO2 and 95% humidified air. After reaching a confluence of 80-90%, the cells were detached using 0.05% trypsin-EDTA solution (Thermo Fischer Scientific) in DPBS (Thermo Fischer Scientific) at 37°C for 1 min, and subcultures were formed in culture plates (Sumitomo Bakelite, Tokyo, Japan). HGFs were seeded at 15600 cells/cm2 in the culture plates and pre-cultured in MEM α supplemented with 10% FBS overnight. Pre-cultured HGFs were incubated for 24 h in MEM α without FBS in the presence of various BSA-AGEs prepared as presented in the previous section. After incubation, cell culture supernatants were collected.
IL-6 quantificationThe IL-6 concentration in the cell culture supernatants was evaluated with enzyme-linked immunosorbent assays (ELISA; R&D Systems, Inc, Minneapolis, MN, USA). All assays were performed following the manufacturer's instructions.
Statistical analysisThe data were evaluated for homogeneity of variance among the groups. If the group variance was determined to be homogeneous, Dunnett's multiple comparison test was employed. If the group variance was determined to be heterogeneous, Steel's test was used as indicated in each Figure. The analyses were performed using JMP 14.0.0 software (SAS Institute Inc. Cary, North Carolina, USA). p < 0.05 was considered statistically significant.
To investigate the inhibitory effects of the five anti-inflammatory reagents (ALA, AHA, GKII, LYZ, and AZU) on glycation, we first focused on glyceraldehyde-derived AGEs with particularly strong physiological effects26) that are known to induce inflammation in gingival cells10-12). We evaluated each reagent at the final concentration of 6.25, 12.5, and 25.0 mM. The results showed that ALA at 12.5 mM and 25.0 mM, as well as GKII and AZU at 6.25, 12.5, and 25.0 mM significantly inhibited glyceraldehyde-induced glycation, as compared with the control (12.5 mM ALA; p < 0.01, 25.0 mM ALA; p < 0.001, 6.25-25.0 mM GKII and AZU; p < 0.01) (Fig. 1A). However, AHA and LYZ showed no inhibitory effects (Fig. 1B).
Anti-inflammatory ingredient inhibitory effects on glyceraldehyde-induced glycation
(A) Inhibitory effects of ALA, GKII, and AZU on glyceraldehyde-induced glycation
(B) Inhibitory effects of AHA and LYZ on glyceraldehyde-induced glycation
ALA at 12.5 and 25.0 mM, as well as GKII and AZU at 6.25, 12.5, and 25.0 mM significantly inhibited glycation compared to the control (DPBS) invitro (A), while AHA and LYZ at 6.25 mM, 12.5, and 25.0 mM did not (B). Data are expressed as the mean±SD (n＝4–7). ＊＊p＜0.01, ＊＊＊p＜0.001 compared to control as per Dunnett’s test (A) (B).
We evaluated the inhibitory effects of ALA, GKII, and AZU, which reportedly inhibit glycation by glyceraldehyde, on fructose-induced glycation, which occurs upstream of and affects multiple glycation pathways27). We evaluated each reagent at 6.25, 12.5, and 25.0 mM and found that 12.5 and 25.0 mM GKII and 6.25, 12.5, and 25.0 mM AZU significantly inhibited fructose-induced glycation (p < 0.01). ALA did not inhibit fructose-induced glycation at any of the tested concentrations (Fig. 2A).
ALA, GKII, and AZU inhibitory effects on Fructose and Glyoxal-induced glycation
(A) Inhibitory effects of ALA, GKII, and AZU on Fructose-induced glycation
(B) Inhibitory effects of ALA, GKII, and AZU on Glyoxal-induced glycation
Significantly inhibited fructose-induced glycation could be observed compared to the control (DPBS) invitro in the case of 12.5 and 25.0 mM GKII, as well as 6.25, 12.5, and 25.0 mM AZU. ALA did not inhibit fructose-induced glycation at any of the tested concentrations (A), while ALA at 12.5 and 25.0 mM, as well as GKII and AZU at 6.25, 12.5, and 25.0 mM significantly inhibited glyoxal-induced glycation compared with the control (DPBS) in vitro (B). Data are expressed as the mean±SD (A: n＝4–8, B: n＝3, 4). ＊p＜0.05 compared to control as per Steel’s test (A). ＊＊＊p＜0.001 compared to control as per Dunnett’s test (B).
Last, we evaluated the inhibitory effects of ALA, GKII, and AZU on glyoxal-induced glycation, which resulting in Nε- (carboxymethyl) lysine (CML) that is known to exist in the gingiva6). We evaluated each reagent at 6.25, 12.5, and 25.0 mM. As a result, we found that ALA at 12.5 and 25.0 mM, as well as GKII and AZU at 6.25, 12.5, and 25.0 mM significantly inhibited glyoxal-induced glycation (p < 0.001) (Fig. 2B).
Hydroxyl radical scavenging activity contributes to the antiglycation activity of ALA, GKII, and AZUGlycation and oxidative stress are closely linked28) and reducing sugars promote AGE formation through the formation of hydroxyl radicals29,30). To investigate the mechanism of the ALA, GKII, and AZU antiglycation activity, the hydroxyl radical scavenging activity was examined. The results showed that ALA, GKII, and AZU exhibited significantly higher levels of hydroxy radical scavenging activity compared to the blank sample (DW: control) at 6.25, 12.5, and 25.0 mM (p < 0.001) (Fig. 3).
The hydroxyl radical scavenging activity of ALA, GKII, and AZU
ALA, GKII, and AZU exhibited significant hydroxyl radical scavenging activity compared to the control (DW) at all tested concentrations, 6.25, 12.5, and 25.0 mM. Data are expressed as the mean±SD (n＝4). ＊＊＊p＜0.001 compared to control as per Dunnett’s test.
Next, we evaluated how AGEs affect the pro-inflammatory cytokine production of HGFs. We added glycated BSA to HGFs at 0, 500, 2500, and 5000 μg/mL of total protein concentrations. The IL-6 production of HGFs significantly increased by approximately 3.4-fold on treatment with 5000 μg/mL of glycated BSA compared with that of 0 μg/mL (p < 0.05) (Fig. 4).
Glycated BSA-induced interleukin-6 production by HGFs
IL-6 production by HGFs was significantly increased by the treatment with 5000 μg/mL glycated BSA compared with that with 0 μg/mL. Data are expressed as the mean±SD (n＝3–10). ＊p＜0.05 compared to control as per Steel’s test.
To assess the influence of the ALA, GKII, and AZU antiglycation effect on gingival inflammation, non-glycated BSA, glycated BSA, and glycation-inhibited BSA was added to the HGFs and incubated for 24 h. All BSA solutions were supplemented at the concentration of 5000 μg/mL, which promoted significantly the IL-6 production by HGFs as shown in Fig. 4. Glycated BSA-treated HGFs showed a significant enhancement of IL-6 production compared to those treated with non-glycated BSA (p < 0.001). Glycation-inhibited BSA induced significantly lower IL-6 production in the HGFs compared to glycated BSA (p< 0.001) (Fig. 5).
The effects of ALA, GKII, and AZU antiglycation properties on AGE-induced inflammation by HGFs
Glycated BSA-treated HGFs exhibited significant IL-6 production enhancement compared to those treated with non-glycated BSA. Glycation-inhibited BSA samples induced significantly lower IL-6 production by HGFs compared to the glycated BSA. Data are expressed as the mean±SD (n＝5). ＊＊＊p＜0.001 compared to Glycated BSA as per Dunnett’s test.
In this study, we evaluated the antiglycation effects of five anti-inflammatory ingredients, ALA, AHA, GKII, LYZ, and AZU in oral-care products, in order to identify anti-inflammatory reagents that also exhibit AGE formation inhibitory properties. We discovered that ALA, GKII, and AZU inhibited several glycation pathways in vitro. Moreover, we observed that ALA, GKII, and AZU suppress gingival inflammation by inhibiting AGE formation.
Our results (Fig. 1A, and Fig. 2) showed that the trend of inhibitory effect on glyceraldehyde and fructose induced glycation was similar (AZU > GKII > ALA), whereas that of glyoxal induced glycation inhibition was different (ALA > GKII≒ AZU) (Fig. 1A, and Fig. 2). This might be due to the fact that glyceraldehyde and fructose partly share the same reaction pathways for AGE formation, while glyoxal undergoes an independent pathway27). In contrast, the results (Fig. 1B) showed that AHA and LYZ did not inhibit the glyceraldehyde-derived AGE formation, but rather promoted it. AGEs are known to be formed when free amino groups of proteins undergo glycation with reducing sugars31). In particular, glyceraldehyde is highly reactive and forms cross-links by binding to amino groups27). AHA is an amino acid chemically synthesized from L-lysine21), and LYZ is a protein known to have anti-microbial activity by hydrolyzing bacterial cell walls32). This suggests that certain free amino groups in AHA and LYZ can easily bind to sugars, and they themselves could bind to glyceraldehyde and be glycated. Although AHA and LYZ could potentially suppress collagen glycation by trapping glyceraldehyde themselves, they were excluded from this study because even after washing out the compounds, collagen gel incubated either with AHA or LYZ showed high fluorescence suggesting collagen glycation was not suppressed (Data not shown).
Fig. 3 showed that all the ingredients exhibited hydroxyl radical scavenging ability in the glycation-inhibiting concentration range. In particular, ALA and AZU showed high hydroxyl radical scavenging activity comparable to that of a strong antioxidant, VC, that we chose as a positive control. These results suggest that the hydroxyl radical scavenging effect could contribute to the effector mechanism of each ingredient in inhibiting glycation. Another mechanism known for glycation inhibition is scavenging reactive carbonyl compounds33,34). AZU showed a strong inhibitory effect on glyceraldehyde-induced glycation compared to the glyoxal-induced reaction (Fig. 1A, and Fig. 2B). Glyceraldehyde contains monocarbonyls and glyoxal contains dicarbonyls, suggesting that AZU might specifically inhibit monocarbonyl-induced glycation, i.e., monocarbonyl-trapping ability. In contrast, ALA strongly inhibited glyoxal-induced glycation compared to the glyceraldehyde-induced reaction (Fig. 1A, and Fig. 2B), suggesting that it might exert a specific inhibitory effect on dicarbonyl-induced glycation, i.e., dicarbonyl-trapping ability. These two distinctive possibilities suggested in AZU and ALA could explain the different potentials of compounds in fructose-, glyceraldehyde-, and glyoxal-induced glycation. Although we did not test carbonyl trap properties in this study, further analyses focusing on this aspect would further reveal the mechanisms of antiglycation effects observed in ALA, GKII, and AZU.
Nonaka et al. and Kido et al. reported that AGEs stimulate gingival cells and promote the production of pro-inflammatory cytokines10,11). In this study, we obtained similar results and observed that glyceraldehyde-derived AGEs (glycated BSA, as shown in Table 2) promoted the production of pro-inflammatory cytokine IL-6 by HGFs (Fig. 4). This suggests that AGEs could induce gingival inflammation. The concentration of glycated BSA that promoted IL-6 production by HGFs seemed to be extremely high at 5000 μg/mL and appeared to be very different from the concentration of 500 μg/mL, at which IL-6 production has been observed in previous studies10,11). However, 5000 μg/mL represented the total protein concentration, including not only AGE-BSA but also unreacted BSA. In addition, we also confirmed that the treatment of HGF with 5000 μg/mL of glycated BSA does not show cytotoxicity (Data not shown).
We further evaluated the influence of the ALA, GKII, and AZU antiglycation effect on gingival inflammation, and found that each glycation-inhibited BSAs induced significantly less IL-6 compared to the glycated BSA by HGFs (Fig. 5). ALA, GKII, and AZU are known to have anti-inflammatory effects17,18), but the glycation-inhibited BSA were subjected to ultrafiltration, which removed all compounds other than glycated and intact BSAs, including ALA, GKII, and AZU. The possibility that the residual ALA, GKII, and AZU in the postfiltration sample exerts anti-inflammatory effects on HGF (as shown in Fig. 5) was verified by comparing the IL-6 production by HGF treated with either non-glycated BSA or non-glycated BSA with each ingredient (Supplementary Table 1). The results showed that there was no difference between any of the groups in terms of IL-6 production (Supplementary Fig. 1). This suggests that each ingredients was removed by ultrafiltration and the difference in the amount of glycation-inhibited BSA-induced IL-6 production was due to the antiglycation effect of ALA, GKII, and AZU, and not their anti-inflammatory effects.
In this study, we found three ingredients; ALA, GKII, and AZU used in oral-care products with glycation inhibitory effects, suggesting that these ingredients can help reduce AGE formation and its effects on gingiva i.e., inflammation. Since AGE formation and accumulation occurs day by day, influenced by aging, lifestyles, such as daily diet and systematic dysfunction like diabetes mellitus35,36), middle-aged or elderly people, as well as patients with diabetes mellitus are at the risk of AGE accumulation in gingival tissues and might be subject to AGE-related periodontal disease development. In Japan, the proportion of such middle-aged and elderly people exceeds 50% of the total population37), and the increasing prevalence of periodontal diseases and the increasing medical costs including dental care that accompany aging are also major issues38). Therefore, we believe that periodontal disease prevention focusing on antiglycation effects, which represents particular benefits for the middle-aged and elderly population bears social value.
The findings of this study should be seen in light of certain limitations. The inhibitory effects of ALA, GKII, and AZU on glycation are based on in vitro studies only. It remains unclear whether these ingredients could suppress glycation in vivo. In vivo studies analyzing the antiglycation effect of these ingredients in the periodontal tissue would be required to provide us further insight. Furthermore, clinical trials using these ingredients or toothpaste and mouthwash products containing them would allow us to elucidate how the antiglycation activities affect human periodontal tissue, which would bring us one step closer to the establishment of a novel periodontal disease prevention strategy, antiglycation.
This study aimed to find anti-inflammatory ingredients for oral-care products that also exhibit antiglycation effect to provide a new mechanism for periodontal disease prevention. We observed that ALA, GKII, and AZU, which are known to prevent periodontal disease by their anti-inflammatory effects, also exhibit AGE formation inhibitory effects. Furthermore, we also confirmed that ALA, GKII, and AZU suppress gingival inflammation by inhibiting AGE formation. Since AGEs induce gingival inflammation, potentially related to periodontal disease onset and progression, our results suggest the possibility that ALA, GKII, and AZU are useful in periodontal disease prevention not only for their anti-inflammatory but also for their antiglycation effect.
The authors are grateful for the helpful discussion and suggestions provided by Professor Dr. Shinya Murakami (Osaka University). In addition, we thank Dr. Kota Tsutsumi (Lion Corporation) for their cooperation in conducting this study.
Conflict of Interest: None.
