(−)-Epigallocatechin gallate (EGCG), is the main catechin found in green tea [1
]. Many studies have reported its potential health benefits, including anti-inflammatory [2
], and antioxidant actions [4
] as well as the prevention of cancer and cardiovascular diseases [5
] in cells, animal models, and even humans. EGCG is also known to have antimicrobial effects against both Gram-negative and Gram-positive bacteria by damaging their cell membranes, which results in the downregulation of enzymes involved in biosynthesis and the prevention of bacterial adherence to their host cell [8
]. Thus, EGCG can decreases the production of toxic bacterial metabolites [8
]. Based on its proven health benefits, EGCG appears to have a substantial potential for use in medical applications.
However, EGCG is seldom used in the medical field, possibly due to its instability. In solution, EGCG donates hydrogen atoms, leading to the generation of superoxide and oxidized products. Subsequently, this causes the formation of theasinensin A and compound P2 as the major dimers, which may be the reason for the change in color of EGCG aqueous solution to brown [9
]. Aqueous EGCG solution is highly unstable, as EGCG can easily degrade through oxidation and epimerization [10
]. The rates of these two reactions are affected by many factors, such as the presence of oxygen or sunlight, as well as changes of temperature, pH and ionic strength [9
]. In air-saturated buffer, EGCG undergoes auto-oxidation, forming H2
], while under low oxygen partial pressure, generated by extensive flushing with nitrogen gas, EGCG has shown to be stable, with 95% remaining after 6 h [15
]. As for the pH level, the acidic environments enhance the stability of EGCG, but neutral and alkaline pH level cause auto-oxidation on the B-ring and produce theasinensin A and product P2 [17
]. In addition, previous studies have reported that following exposure to a solar simulator emission, EGCG almost completely degrades within an hour [12
]. The activity of EGCG is also dependent on the salt concentration of ambient medium, the degradation EGCG increases with rising ionic strength of the solvent. In addition, temperature is a significant parameter governing EGCG stability and the mode of its degradation [19
]. Several works have suggested that EGCG undergoes degradation via oxidation at temperatures below 50 °C, while epimerization was more commonly occurs at higher temperatures [19
]. Through epimerization, EGCG transforms to its trans
-epimer (−)-gallocatechin gallate (GCG) [14
], while epimerization of EGCG should not significantly alter its biological effects [25
The stability of EGCG is affected by many factors, limiting its application in many cases; however, we hope to apply EGCG to periodontitis treatment. Periodontitis is one of the major oral inflammatory diseases, encountered in routine dentistry practice [28
]. The imperative factor behind the pathogenesis of periodontal infections is the accumulation of dental plaque, which results in gingival inflammation [29
]. EGCG has already been used in some clinical experiments as a mouthwash [30
] and a local drug route [32
], which showed that it has a healing effect on periodontal tissue. As ultrasonic dental scalers are widely used in the treatment of periodontitis, they are a potential delivery route for EGCG. However, before using this approach, it is necessary to investigate the stability of EGCG under ultrasound conditions to determine whether the stability of EGCG is affected by ultrasound itself, or whether it is influenced by sunlight or oxygen.
This is the first study to investigate whether ultrasound had an effect on the stability of EGCG. In this study, an ultrasonic dental scaler was used to atomize the EGCG aqueous solution, so EGCG may have extreme contact with air and sunlight over a short time period. Without oxygen, EGCG in aqueous solution was rather stable, even when it was atomized into a mist, which demonstrates that ultrasonic vibration itself does not make EGCG degrade. However, when the EGCG aqueous solution was atomized into air, EGCG in solution appeared less stable than under the storage conditions. The EGCG aqueous solution in the control group in this part of the study was exposed to oxygen and sunlight in the short-term, and it was stable, while the degradation of atomized EGCG aqueous solution with oxygen was significantly higher. These results demonstrate that ultrasound may accelerate the degradation of EGCG because of the extreme contact with oxygen and sunlight, rather than because of the ultrasonic vibration. In addition, the high concentration of EGCG (2.00 mg/mL) was still stable even under ultrasonic conditions. Thus, the use of EGCG aqueous solution through an ultrasonic dental scaler to treat periodontal infection may be feasible, because EGCG would not obviously decrease even when subjected to the ultrasonic conditions. Meanwhile, EGCG aqueous solution could actually reach the infected area and exercise its antimicrobial capacity through the work tip of the ultrasonic dental scaler, providing a new method for dentists to control the periodontal infection and may potentially improving treatment outcomes.
On the other hand, previous studies have proven that sunlight and oxygen affected the stability of EGCG [12
], but we are the first to compare the strength of these two factors’ effects on EGCG in aqueous solution. Both parts of the study showed that, whether exposed to sunlight or not, the degradation level of EGCG did not change a lot, while EGCG decreased much more following exposure to oxygen. The Pearson correlation coefficients also confirmed that oxygen has a more pronounced effect on the stability of EGCG than sunlight.
In addition, the present study also has demonstrated that the stability of EGCG is concentration-dependent in water at room temperature, and the higher the initial concentration of EGCG is, the more stable it will be. This result is accordance with the previous studies, which showed that the initial concentration of EGCG in aqueous solution is an important factor for stability—a clear difference was shown, between the µM and mM range [16
]. However, the reason for this phenomenon is not clear et al. showed that the rates of oxidation and epimerization reactions increased at the low concentrations of EGCG aqueous solution. As more water was present in these solutions, the pH, molecular mobility, and dissolved oxygen levels were greater, while at high concentrations of EGCG aqueous solution, the pH, molecular mobility, and dissolved oxygen levels decreased, so the rates of the two reactions were inhibited [36
]. Thus, we propose that the oxidation and epimerization contributed to this phenomenon. Further study is needed to better explain the differences in the reaction mechanisms between the high concentration and low concentration solutions of EGCG.
However, the previous studies suggested that 1% EGCG exposed to a solar simulator emission would degrade almost 70% in an hour [12
], while the 2.00 mg/mL EGCG aqueous solution in the present study was rather stable. This may relate to the formulation of EGCG. In those studies, the formulation of EGCG was an emulsion, while aqueous solution was used in the present study. In addition, the illumination in those studies was much higher than that in this study. On the other hand, the aim of those studies was to investigate the photodegradation of EGCG, but the effect of oxygen should be considered. As for the mechanism by which sunlight affects EGCG, the previous study suggested that the mechanism of EGCG photodegradation is different from the decomposition pathways involved in the catechin chemical instability [12
]. The photochemical process was described in a previous review: The primary photochemical reaction is directly due to the absorption of a photon [37
]. In other words, if there is a certain overlap between the absorption spectrum of the molecule and the incident radiation, radiation may provide energy to the molecule, which may be the reason why sunlight could accelerate the degradation process of EGCG. Thus, sunlight and oxygen both affect the stability of EGCG, and EGCG should be kept out of air and sunlight. Although EGCG is less stable in aqueous solution than in solid form, EGCG must be in water to be used with the ultrasonic dental scaler, which was why we investigated the stability of the EGCG aqueous solution in the present study.
Asah et al. [38
] and Hirasaw et al. [39
] reported that 0.50 mg/mL and 1.00 mg/mL EGCG solutions are the minimum inhibitory concentrations, respectively. However, Sang et al. [16
] showed that both 0.50 mg/mL and 1.00 mg/mL EGCG aqueous solution were unstable, but 3.20 mg/mL EGCG aqueous solution is stable, so we aimed to find the minimum stable concentration. We found that the 2.00 mg/mL EGCG aqueous solution remained stable for two days, and we also aimed to cover the range of concentrations used in most animal studies in accordance with the study of Sang et al. [16
]. Thus, the other four low concentrations were performed in the present study.
Interestingly, EGCG still degrades even if under conditions without sunlight and air, which suggests that EGCG degradation occurs regardless of auto-oxidation. This phenomenon may be explained by epimerization of EGCG to GCG. In the study of Sang et al. [16
], when the auto-oxidation of EGCG was prevented, epimerization of EGCG to GCG became appreciable. GCG was the major product formed from EGCG at high concentrations, whereas with low concentrations of EGCG, EGCG dimers were formed mainly. The rates of these two reactions are affected by the level of oxygen, the concentration of EGCG, and the level of antioxidants [16
]. However, the degradation products were not identified in the present study, and the further study was needed.
Although the EGCG aqueous solution was strictly kept out of sunlight or air in the present study, there were still some limitations. Regarding the isolation from oxygen, though the cap was tightened immediately, there was still some oxygen dissolved in the water. In addition, while EGCG was being measured in HPLC, sunlight was kept out, but it was impossible to keep air out of the HPLC machine. However, the present study showed that short time of air exposure had almost no impact on the stability of EGCG, which rules out the possibility that the existence of air could have interfered with the results during the HPLC analysis. On the other hand, when the EGCG aqueous solution was exposed to sunlight, sunlight may have influenced the temperature change. However, the whole study was performed in the laboratory, where the room temperature indoors was maintained at 25–28 °C, so the influence of sunlight on temperature was slight.
4. Materials and Methods
(−)-Epigallocatechin gallate (EGCG, 98% purity) was purchased from Yuanye Biotechnology Co. Ltd. (Shanghai, China). Mmembrane syringe filters (0.22 μm) were purchased from Dingguo Changsheng Biotechnology Co. Ltd. (Beijing, China). An ultrasonic dental scaler was from Guilin zhuomuniao Medical Devices Co. Ltd. (Guilin, China). A TA8120 digital luxmeter was purchased from Tasi Electronic Co. Ltd. (Suzhou, China). A MZ-2CNT diaphragm vacuum pump was from BRAND Trading Co. Ltd. (Shanghai, China) was used.
4.2. High Performance Liquid Chromatography (HPLC) Analysis
Analytical conditions. Prior to the analysis, all samples were filtered by 0.22 μm membrane syringe filters. Chromatographic analysis was performed on a Waters 2695 Series (Waters Technologies Shanghai Limited, Shanghai, China) LC system containing a quaternary pump, an online degasser, an autosampler, and a thermostatic column compartment set at 30 °C. Chromatographic separation was conducted on a Waters XSelect HSS T3 C18 column (4.6 mm × 250 mm, 5 μm). A gradient method was employed using mobile phase A consisting of distilled H2
O, acetonitrile, acetic acid, and EDTA (888/90/20/2, v
) and phase B consisting of distilled H2
O, acetonitrile, acetic acid, and EDTA (178/800/20/2, v
). The initial ratio is 100% A (10 min). Subsequently, the solvent composition was changed to 68/32 (A/B 15 min) and 100% A (10 min). The flow rate was 1 mL/min, and the injection volume was 10 μL. The UV detection wavelength was 278 nm [40
Selectivity and Specificity. For the chromatographic method, developing a separation involves demonstrating specificity, which is the ability of the method to accurately measure the analyte response in the presence of all interferences. EGCG aqueous solution samples were prepared according to the procedures described above and analyzed.
Linearity. The calibration curve of HPLC method was evaluated by analyzing a series of standard EGCG aqueous solution samples at concentration from 0.005 to 2.5 mg/mL. The determination coefficient (R2) was calculated by means of the least-square analysis. The lower limit of quantification (LLOQ) is the lowest concentration of EGCG in a sample that can be determined with acceptable precision and accuracy.
Precision and Accuracy. Intra-day/inter-day accuracies and precisions of the method were evaluated by analyzing samples at 2 concentration levels in 4 replicates during a single day and in duplicate over 2 consecutive days. The precision was represented by relative standard deviation (RSD). The accuracies were determined by calculating the percentage of measured concentrations to the nominal concentrations of samples.
4.3. Stability of EGCG Aqueous Solution under Different Storage Conditions
The EGCG aqueous solution was prepared with distilled H2O. In order to prevent sunlight and air entering the solution, we used glass bottles wrapped up with tinfoil. After adding the pre-weighed EGCG power into the bottle, it was diluted with distilled H2O to full of bottle, and then the cap was tightened immediately. A series concentrations (0.05, 0.10, 0.15, 2.00 mg/mL) of EGCG aqueous solutions were prepared at room temperature (25–28 °C), with each concentration was divided into four conditions as follows: (A) exposed to neither sunlight nor air (tightened the cap of bottle and wrapped the bottle with tinfoil), (B) exposed to sunlight but not air (tightened the cap but did not wrap the bottle), (C) not exposed to sunlight but air (opened the cap and wrapped the bottle with tinfoil), and (D) exposed to sunlight and air (opened the cap but did not wrap the bottle). The diameter of the bottle mouth was about 1 cm, and the illumination intensity was about 300–500 lux. Each concentration of EGCG solution was stored under conditions A to D, respectively, for 6 h, 12 h, 24 h, 36 h, and 48 h, and the residual concentration of EGCG was measured with HPLC. The whole experiment was performed in the laboratory at room temperature (25–28 °C).
4.4. Stability of EGCG Aqueous Solution Atomized by the Ultrasonic Dental Scaler
A series of concentrations (0.05, 0.10, 0.15, 2.00 mg/mL) of EGCG aqueous solutions were prepared described in Section 2.3
. The vibration frequency of the ultrasonic work tip was 42 kHz. Though the ultrasonic dental scaler, the EGCG solution was atomized into a mist. For the test group, each concentration of EGCG solution was atomized in a large glass bottle under the four conditions which were similar to Section 2.3
: (a) exposed to neither air nor sunlight (inserted the tip into the vacuous bottle created by vacuum pump, and wrapped the bottle with tinfoil), (b) exposed to sunlight but not air (inserted the tip into the vacuous bottle created by vacuum pump, but did not wrap the bottle with tinfoil), (c) not exposed to sunlight but air (wrapped the bottle with tinfoil, but the bottle was not vacuous), (d) exposed to air and sunlight (the bottle was not wrapped with tinfoil, and the bottle was not vacuous), and the atomized solution was collected. The experiments were performed in the order of low to high concentrations, and there was a minimum period of 5 min between concentrations in which the next concentration of EGCG solution was run though the handpiece to wash out any excess of the last concentration of EGCG solution in the system.
For the control group, the EGCG aqueous solution was exposed to air and sunlight for the same amount of time as the test group in culture dishes, so that the EGCG solution was able to form a thin layer and have full contact with sunlight and air. The diameters of culture dishes were 10 cm.
4.5. Statistical Analysis
A group t-test was used to compare the significance of the differences between the groups of residual EGCG in EGCG aqueous solutions atomized by ultrasound, and a p-value < 0.05 was accepted as significant. Data are expressed as the mean value ± standard deviation (mean ± SD) (n = 3).