Disinfection performance and synthesis conditions of the EGCG–Cu complex Open Access

Tea polyphenols serve as natural antimicrobial agents, displaying evident inhibitory effects on waterborne pathogenic bacteria including coliforms, Staphylococcus aureus, Proteus and Shigella flexneri (Feng et al. 2023; Zhang et al. 2023b). Numerous studies indicate that tea polyphenols possess antioxidant, anti-tumor, anti-hyperlipidemia and potent broad-spectrum antibacterial properties (Malik et al. 2003; Wen et al. 2023). Additionally, recent investigations have demonstrated that polyphenols and catechins can effectively suppress the proliferation of antibiotic resistance genes in aquatic environments (Yu et al. 2023). As such, tea polyphenols with their economic, eco-friendly and environmental friendly attributes have been proposed as novel disinfectants for drinking water (Cuimin et al. 2016). Catechin, constituting over 70% of total tea polyphenols, harbors epicatechin gallate (EGCG) a main ingredient with substantial bacteriostatic function (Yang et al. 2003, 2014). EGCG is acknowledged for its various therapeutic properties, including antibacterial, anticancer, antihypertensive, anticoagulant and antiulcer effects (Azam et al. 2004; Ganeshpurkar & Saluja 2020; Liczbiński & Bukowska 2022).

Nature always provides rich inspiration for the design of functional materials (Li et al. 2022; Zhang et al. 2023a). Polyphenols can affect cellular behavior due to their inherent functional characteristics such as antioxidant and photothermal behavior (Cao et al. 2022). In recent years, alongside evaluations of EGCG impact on organic substances, studies focused on the physiological ramifications between EGCG and metal ions. The structure of flavonoid is of a high degree of super-delocalization, and its expansive π conjugate structure renders it an ideal spatial configuration for metal ion chelation. When catechins interact with metal ions, complexation or redox reactions transpire, leading to alterations in their chemical structure. These transformations equip catechins with additional physiological and biochemical properties. The multi-metal complex plays a significant role in biological activities or contributes to new biological properties of catechins. Research indicates that Mn (II)–EGCG can push up EGCG and inhibit α-glucosidase, thereby reducing the risk of type 2 diabetes mellitus (Li et al. 2023). Fe³⁺, as a catalytic medium in the Ni²⁺-EGCG network, exhibits significant bactericidal effects and the ability to eradicate pathogenic biofilms (Chen et al. 2021). The interaction between EGCG and copper ions is primarily responsible for cytomembrane damage in prostate cancer cells (Yu et al. 2007). Yoshioka et al. (2001) utilized spin trapping to identify the formation of hydroxyl radicals in the complexation system of EGC, EGCG, EC and copper ions, revealing that EGCG forms a complex with Cu²⁺, which made the system divalent copper reduced to monovalent copper. This monovalent copper can react with both oxygen and hydrogen peroxide present in the solution, leading to the creation of highly reactive free radicals. These radicals demonstrate strong oxidizing capacity and are capable of inducing the DNA molecules’ degradation through oxidative processes (Sun et al. 2011). Kumamoto et al. (2001) investigated the antioxidant activities of 13 metal ions after complexing with EGCG and discovered that catechins can be oxidized automatically in the presence of copper ions, while the complex of EGCG and Cu²⁺ exhibits enhanced antioxidant capacities under acidic conditions. Similarly, Hayakawa et al. (2004) found significant differences in the antioxidant capacities of EGCG and EGCG in the presence of Cu²⁺. This disparity to the complexation ability of EGCG with metal ions forms a relatively stable complex and inhibits reactive oxygen species production. Holloway et al. (2015) observed that the heat-treated complex of catechins and Cu²⁺ demonstrated superior bactericidal efficacy than the non-heated complex. Therefore, this study selected Cu²⁺ as the central ion and EGCG as the ligand, employing the hydrothermal synthesis to investigate the bactericidal effects of synthesized substances on Escherichia coli, an indicator bacterium in drinking water, aiming to pave the path for the potential application of tea polyphenols and EGCG in the field of drinking water disinfection.

The EGCG utilized in this experiment was purchased from Nanjing Guang-run Biological Products Co., Ltd, No. GR0799, with a molecular weight of 458.4 and purity over 98%, and was white-like dry powder. The strain of E. coli was from China Industrial Microbial Culture Collection Management Center, No. 10004. Copper sulfate, sodium bicarbonate, boric acid buffer, potassium bromide (spectrum pure), ethanol, sodium chloride and nutrient agar were also used for this experiment.

The reactant ratio between EGCG and Cu²⁺ was examined by the molar ratio method (Liu et al. 2021). EGCG in 11.5 mg (0.025 mmol) was dissolved in 25 mL of distilled water and then 0.05 mol/L of borax solution and 0.2 mol/L of boric acid solution were prepared. Borate buffer with pH of 7.6 was composed of 1.5 mL of borax solution and 8.5 mL of boric acid solution. About 0.5 mL of EGCG stock solution was diluted to 10 mL of borate buffer, and 3 mL of the diluted solution was transferred to the quartz tube. Subsequently, the full spectrum of ultraviolet radiation was determined using a scanning interval of 190–600nm, the ABS scanning mode was selected, and the scanning step was set at 1 nm, the EGCG solution was scanned with a UV spectrophotometer at full wavelength, and 0.0025 mol/L CuSO₄ solution 20 μL, full band UV scanning was performed after each addition.

Infrared spectroscopy was carried out by the KBr pellet method (Ridgway & Aulton 1974). EGCG–Cu complex powder of 1–2 mg and spectral pure KBr of 200 mg were mixed evenly and made into a translucent sample by the tablet press, which was scanned by an infrared spectrometer with the air collected as the background, and the data were processed by OMNIC spectrum software.

According to the Standard examination methods for drinking water – Microbiological parameters (GB/T 5750.12-2023), the bacterial total count was determined by the plate counting method after serial dilution.

To probe the impact of reaction pH on complexation, the molar ratio of the reactants was maintained at 1:1. The pH of the solution was altered to 4.0, 5.0, 6.0, 7.0 and 8.0 with sodium hydrogen carbonate, and the reaction mixture was stirred for 30 min at room temperature by a magnetic stirrer.

Controlling the molar ratio of reactants at 1:1 and maintaining a reaction pH of 7, the effect of reaction temperature on complex formation was investigated at 30, 40, 50, 60 and 70 °C with magnetic stirring for 30 min. The results are shown as follows.

The molar ratio of the limiting reagent was 1:1, and the reaction pH was 7. The reaction was carried out under 40 °C with magnetic stirring for 10, 20, 30, 60, 120 and 240 min, and the effect of reaction time on the complexation was explored with the following results.

The molar ratio of the reactant was n_(Cu2+):n_(EGCG) = 1:2, 1:1, 2:1, 3:1 and 4:1, the reaction pH was kept at 7, and the reaction was stirred for 60 min at 40 °C. The results were presented as follows.

To synthesize the necessary complex, a hydrothermal method was employed; 0.5 mmol of EGCG was dissolved in 30 mL of distilled water. Then, 1 mmol of copper sulfate was prepared as a solution and gradually added. After mixing thoroughly, the pH level was adjusted to 7 by 1 mol/L of sodium bicarbonate. The mixture was stirred by a constant temperature magnetic stirrer for 1 h at 40 °C to ensure the complete complex formation. The resulting EGCG–Cu was washed multiple times with a 1:1 ethanol–water solution and finally dried through vacuum freeze-drying overnight.

As can be seen from Figure 7, the molar ratio of EGCG to Cu²⁺ in the cuvette was constantly changed. The waveform of the ultraviolet (UV) scan curve of the measured solution in boric acid changed significantly, and its characteristic peak position at 300 nm began to shift backwards, eventually stabilizing at a wavelength of approximately 320 nm. This indicated that the complexation reaction of the EGCG in solution gradually occurred with the addition of Cu²⁺, generating a complex with a specific structure, resulting in a red shift of the absorption wavelength of the characteristic peak by 20 nm.

Upon the formation of a metal complex with copper ions, the UV spectrum of EGCG underwent significant changes, as shown in Figure 9. The UV absorption of EGCG was observed in the B band of the benzene ring in the A and B rings. In addition to E band absorption near 200 nm, there was a characteristic peak at 275 nm in the UV region that did not change with solubility. The characteristic peak at 275 nm showed a significant shift due to complexation, as a portion of the phenolic hydroxyl groups reacted with the metal ions to form coordination bonds. The lone pairs of electrons on phenolic hydroxyl groups and the pi-electron system of the benzene ring formed p–π conjugation, which was strengthened by the coordination bonds, leading to a shift toward a longer wavelength (Jing 2011). Yue (2016) explored the UV spectra of the dye lignin and copper ion complex and found that the characteristic peak of the dye lignin increased from 336 to 423 nm after complexation. This was attributed to the formation of coordination bonds between copper ions and the phenolic hydroxyl groups of dye lignin. The complexed molecule was planar, and the conjugated system was enlarged, resulting in an increase in absorption intensity and a redshift of the characteristic peak. This demonstrates that structural changes at the molecular level can be inferred from changes in the characteristic peak. The formation of the complex between EGCG and copper ions indeed caused a change in the overall structure of EGCG, confirming the success of the complexation reaction.

The effects of EGCG–Cu complex and EGCG on the growth of E. coli at the same concentration were compared. The results were as follows.

The Cu²⁺ concentration measured in water continuously increases with time, indicating that Cu²⁺ in the complex is continuously released into the aqueous solution. Within the first 24 h, the release of Cu²⁺ increases rapidly from 0 to 14 mg/L. Subsequently, a rapid increase of Cu²⁺ concentration becomes slower, especially after 100 h. So, EGCG–Cu can maintain its bactericidal effect within a relatively long time.

In the blank group, a few bacteria deaths occurred at 48 h, indicating that E. coli entered the decline phase as the nutrients in the water were depleted. When the Cu²⁺ concentration was 0.125 mg/L, the antibacterial rate increased with the contact time. At 24 h of contact time, the antibacterial rate reached its maximum, at around 41%, and it almost did not increase further after 24 h, indicating that E. coli is highly sensitive to Cu²⁺. When the Cu²⁺ concentration increased to 0.5 mg/L, the antibacterial rate still showed a positive correlation with time, reaching a peak of 84% at 24 h. As the Cu²⁺ concentration increased, the bactericidal effect on E. coli became more pronounced, and this effect became more evident with time. At 4 mg/L of Cu²⁺, the antibacterial rate reached nearly 100% at 18 h. At 20 mg/L, the antibacterial rate had already reached over 80% at 2 h, and it achieved a 100% antibacterial effect after 6 h of contact. Overall, both the Cu²⁺ concentration and the contact time are positively correlated with the antibacterial rate. The higher the Cu²⁺ concentration and the longer the contact time, the more pronounced the inhibitory effect on E. coli.

The blank group bacteria multiplied significantly and caused the solution to be turbid. The EGCG group was clear but visibly tea red, as EGCG easily undergoes oxidation and polymerization reactions, forming colored quinone polymers. The EGCG–Cu group is clear without turbidity or odor, indicating that the E. coli in the solution is completely inhibited and never grown within 14 days. The black precipitate is the complex, which proves that the complex can exist in the solution for a long time, and even a small amount of dissolved complex can achieve a considerable disinfection effect. Furthermore, due to the chelation between the phenolic hydroxyl groups in the complex and copper ions, it does not undergo electron-loss reactions without colored quinones like EGCG. Therefore, the chromaticity of the solution treated with EGCG–Cu is less than 15, which is very favorable for its application as a disinfectant.

Utilizing complex yield as the determinant factor, the optimal conditions for complexation were established in this study. The complexation ratio between EGCG and Cu²⁺ was investigated by employing the molar ratio method. The examination of characteristic peaks and functional group variations in EGCG and the complex was carried out by UV and infrared spectroscopy. In addition, the antibacterial effect of the complex was evaluated through its influence on a bacterial suspension of E. coli. The following conclusions were drawn:

• (1)

  The optimum experimental conditions for the complexation reaction were determined by experiments: the pH of the complexation reaction was 7; the reaction temperature was 40 °C; the reaction time was 60 min and the ratio of the reactants was 1:2. The results showed that the amount of complex formation was the highest under this condition.

• (2)

  The molar ratio of the complexation of EGCG and Cu²⁺ was explored by the mole ratio method. The absorbance value of the new substance's characteristic peak was used to fit the line, revealing that the complexation ratio of EGCG and Cu²⁺ was 1:2. UV characteristic peak displacement proved that the benzene ring structure of EGCG has changed, resulting in the production of a new substance. The absorption peak changes of various functional groups on the infrared spectrum indicated that the complexation reaction occur on the phenolic hydroxyl group of EGCG's benzene ring.

• (3)

  The relationship between the released Cu²⁺ and the disinfection performance of the EGCG–Cu complex was studied by the ICP spectrometer. The results showed that Cu²⁺ in the EGCG–Cu complex is continuously released into the aqueous solution, which can maintain a long-term bactericidal effect.

• (4)

  Through testing the bactericidal activity of E. coli, copper ion complexation modification of EGCG was shown to significantly enhance its antibacterial properties, and the chromaticity of the EGCG–Cu complex antibacterial solution is less than that of EGCG, which is very favorable for its application as a disinfectant.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.