Tea polyphenol-copper nanomaterial, construction method thereof and antibacterial application thereof

Abstract

The application provides a tea polyphenol-copper nanomaterial and a construction method thereof; catechin compounds are dissolved in deionized water, an alkaline aqueous solution is added to adjust the pH value to 8-9, then a sodium diethyl dithiocarbamate aqueous solution and a copper salt aqueous solution are added respectively, and the tea polyphenol-copper nanomaterial is prepared. And the application in preparing a bacteriostatic agent is provided. The tea polyphenol-copper nanomaterial has the advantages of mild synthesis condition, fast reaction time, simple operation, low cost, strong bacteriostatic capacity, and the half-inhibitory concentration of MRSA and SE is less than 5 muM, and the tea polyphenol-copper nanomaterial can achieve high bacteriostatic effect at a low concentration. And the tea polyphenol-copper nanomaterial has good biological safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibacterial drugs, in particular, to a kind of biological safety that can be used for high-efficiency bacteriostatic tea polyphenol-copper nano material preparation and application. BACKGROUND

[0002] Pathogenic microorganisms reproduce in the host body can cause infectious diseases. Since the 1940s, traditional antibiotics such as penicillin have been on the market, which has greatly reduced the incidence and mortality of infection and has made great contribution to human health. However, due to the irrational use of antibiotics, the types and number of drug-resistant pathogenic bacteria have increased dramatically. Therefore, it is urgent to develop safe, efficient and green new bacteriostatic agents to cope with the current drug-resistant bacteria outbreak crisis.

[0003] Tea polyphenols are natural products of tea plants, mainly including epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin (EC), epicatechin gallate (ECG) and other components. Chinese traditional tea culture for thousands of years and modern medical research show that tea polyphenols have good biological safety and antibacterial activity, but due to its multiple phenolic hydroxyl structure, it is easy to be affected by oxygen, pH and other environmental factors in vitro to cause structural changes, and it is easy to complex with proteins, metal ions and other in vivo to cause activity changes, which greatly limits its biological application. The development of biological nanotechnology in recent years is expected to construct a stable tea polyphenol metal antibacterial material in vitro to solve the problem of inactivation in vitro and in vivo.

[0004] Sodium diethyldithiocarbamate (DEDTC) is the main active metabolite of disulfiram (DSF), a drug for alcoholism. One molecule of disulfiram sulfur can be decomposed into two molecules of DEDTC in vivo, mainly by liver metabolism. At the same time, DEDTC is also a strong chelating agent for metal ions, which can freely pass through the cell membrane after binding with metal, and has more significant biological activity after reacting with copper (Nature, 2017, 552, 1-6; JACS, 2019, 141, 11531-11539). However, DEDTC and copper ions will generate flocculation in water, which is easy to produce precipitation, which is not conducive to clinical application. The existing technology mostly uses emulsification method or core-shell structure to synthesize biological nanomaterials to solve the problem of precipitation, which is complex, harsh conditions, not conducive to environmental protection and industrial production. If tea polyphenols and DEDTC / Cu are used to construct stable nanobiomaterials by a green and efficient method, the problems of tea polyphenol stability and DEDTC / Cu biocompatibility can be solved, which will be conducive to the biological application of tea polyphenols and DEDTC / Cu. Therefore, it is urgent to develop a simple method to construct biological nanomaterials with good biocompatibility and antibacterial activity by tea polyphenols and DEDTC / Cu. SUMMARY

[0005] In order to solve the above technical problems, the present application develops a synthetic simple and efficient safety tea polyphenol-copper nanometer material bacteriostatic agent, and provides its preparation method and application in bacteriostatic agent, which can effectively inhibit the growth of gram-positive bacteria at low concentration, and has good cell safety and blood safety.

[0006] The technical scheme adopted by the present application is:

[0007] A preparation method of tea polyphenol-copper nanometer material, the method is:

[0008] The catechin compound is dissolved in deionized water, an alkaline aqueous solution is added to adjust the pH value to 8-9, then sodium diethyl dithiocarbamate (DEDTC) aqueous solution and copper salt aqueous solution are added respectively to prepare tea polyphenol-copper nanometer material.

[0009] The tea polyphenol is one or more of epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin (EC) and epicatechin gallate (ECG), preferably EGCG 。

[0010] Further, the mass ratio of tea polyphenol, sodium diethyl dithiocarbamate and copper ion is 10-15:2-3:1, preferably 12:2:1 。

[0011] Further, the copper salt is a water-soluble copper salt, preferably copper sulfate.

[0012] The mass concentration of the tea polyphenol is preferably 0.1-10 mmol / L; more preferably 1-5 mmol / L;

[0013] The mass concentration of the sodium diethyl dithiocarbamate is preferably 0.1-5 mmol / L; more preferably 1-3 mmol / L;

[0014] The mass concentration of the copper salt is preferably 0.1-5 mmol / L, more preferably 1-5 mmol / L.

[0015] The average particle size of the tea polyphenol-copper nanometer material prepared by the present application is 3-5 nm.

[0016] The tea polyphenol-copper nanometer material prepared by the present application is a clear and transparent solution state, the nanoparticles are dispersed in the solvent, can be stored stably for more than half a year, without solid-liquid separation, and the physical and chemical properties and biological activity of the tea polyphenol-copper nanometer material do not change obviously.

[0017] The tea polyphenol-copper nanometer material solution can also be freeze-dried to prepare a solid dry powder.

[0018] The tea polyphenol-copper nanomaterial provided by the application has high efficient bacteriostasis capacity and good biological safety, and can be used as a bacteriostatic agent or in combination with other antibacterial agents.

[0019] Specifically, the tea polyphenol-copper nanomaterial can be used for preparing an antibacterial agent for killing pathogenic bacteria, and further, can be used for preparing an antibacterial agent for drug-resistant Staphylococcus aureus or Staphylococcus epidermidis.

[0020] The technical effect of the application is that:

[0021] (1) The tea polyphenol-copper nanomaterial has mild synthesis conditions, fast reaction time, simple operation, low cost, and is conducive to industrialized production.

[0022] (2) The tea polyphenol-copper nanomaterial has strong bacteriostasis capacity, and the half-inhibitory concentration thereof to MRSA and SE is less than 5 μM, and high efficient bacteriostasis effect can be achieved at a low concentration.

[0023] (3) The tea polyphenol-copper nanomaterial has good biological safety, and epigallocatechin gallate in the synthesis substrate is a natural secondary metabolite in tea trees, which is an oxidation product of an anti-alcoholism drug (Antabuse), and copper is also a trace element necessary for human body, so that the bacteriostatic material is non-toxic and harmless to cells, and has biological safety. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The photos of the tea polyphenol-copper nanomaterial and reactants prepared in Example 1 are shown from left to right as EGCG solution, DEDTC solution, CuSO4 solution and product tea polyphenol-copper nanomaterial EDCu.

[0025] Figure 2 The transmission electron microscope (TEM) image of the tea polyphenol-copper nanomaterial prepared in Example 1.

[0026] Figure 3 The particle size distribution graph of the tea polyphenol-copper nanomaterial prepared in Example 1.

[0027] Figure 4 The ultraviolet absorption spectrum graphs of epigallocatechin gallate, sodium diethyldithiocarbamate and tea polyphenol-copper nanomaterial.

[0028] Figure 5 The appearance pictures of the tea polyphenol-copper nanomaterial stored for different time.

[0029] Figure 6 The survival rate column chart of drug-resistant Staphylococcus aureus and Staphylococcus epidermidis treated by tea polyphenol-copper nanomaterials with different concentrations.

[0030] Figure 7The photos of the spread plate of drug-resistant Staphylococcus aureus and Staphylococcus epidermidis treated by tea polyphenol-copper nanomaterials of different concentrations.

[0031] Figure 8 The column chart of the absorbance of the leaked nucleic acid in the culture solution of drug-resistant Staphylococcus aureus and Staphylococcus epidermidis treated by tea polyphenol-copper nanomaterials of different concentrations.

[0032] Figure 9 The column chart of the cell survival rate of human umbilical vein endothelial cells (HUVEC) treated by tea polyphenol-copper nanomaterials of different concentrations.

[0033] Figure 10 The photos of tea polyphenol-copper nanomaterials prepared in Example 5 and raw materials, from left to right, are EGC solution, DEDTC solution, CuSO4 solution and product tea polyphenol-copper nanomaterial EGC-DCu.

[0034] Figure 11 The ultraviolet absorption spectrum of epigallocatechin (EGC), sodium diethyldithiocarbamate (DEDTC) and tea polyphenol-copper nanomaterial EGC-DCu. DETAILED DESCRIPTION

[0035] The present application will be further described below in conjunction with specific examples, and in the following description, many specific details are set forth in order to provide a thorough understanding of the present application, but the scope of protection of the present application is not limited by the specific examples disclosed below.

[0036] Example 1, Preparation and Characterization of Tea Polyphenol-Copper Nanomaterials

[0037] 9.167 mg of epigallocatechin gallate and 1.712 mg of sodium diethyldithiocarbamate were dissolved in 10 mL of deionized water, respectively, to obtain a 2 mM epigallocatechin gallate (EGCG) aqueous solution and a 1 mM sodium diethyldithiocarbamate (DEDTC) aqueous solution, then 1 mM NaOH aqueous solution was added dropwise into 600 μL of the epigallocatechin gallate aqueous solution, and the pH value was tested by pH paper to be between 8 and 9, then 200 μL of 1 mM sodium diethyldithiocarbamate aqueous solution was added, mixed well, and finally 40 μL of 2.5 mM CuSO4 solution was added, mixed well, to obtain tea polyphenol-copper nanomaterials, and the photos under visible light irradiation are shown in Figure 1 from left to right, are EGC solution, DEDTC solution, CuSO4 solution and product tea polyphenol-copper nanomaterial EDCu, and it can be seen that the product is a transparent and clear liquid, which can be stored at 4°C for a long time. The copper element concentration was measured by inductively coupled plasma mass spectrometry (ICP-MS), and the yield was calculated to be 75%.

[0038] The prepared 120 μM (in terms of copper ion concentration) tea polyphenol-copper nano-solution was added dropwise to the ultra-thin carbon film in 5 μL, and the transmission electron microscopy image is shown in Figure 2 The EDCu is uniformly distributed in spherical shape on the ultra-thin carbon film, and has good dispersibility. The particle size distribution diagram is shown in Figure 3 The particle size of the EDCu is about 4 nm. The ultraviolet absorption spectrum of epigallocatechin gallate, sodium diethyldithiocarbamate and EDCu is shown in Figure 4 Compared with the absorption peaks of EGCG (275 nm) and DEDTC (257 and 282 nm), the tea polyphenol-copper nano has obvious absorption peaks at 274 and 436 nm.

[0039] The pictures of EDCu stored at 4℃ for different time are shown in Figure 5 It can be seen that the tea polyphenol-copper nano material has good stability. The longest storage time recorded in the experiment is half a year, and the physical and chemical properties and biological activity of EDCu do not change obviously within half a year.

[0040] Example 2, antibacterial activity of tea polyphenol-copper nano material on drug-resistant Staphylococcus aureus and Staphylococcus epidermidis

[0041] The tea polyphenol-copper nano material EDCu prepared in Example 1 was incubated with methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (SE) in a 96-well plate, and the final concentration was (0, 0.5, 1, 2.5, 5 μM), 0 representing the blank control group. Incubate for about 8 h until the OD 600 of the blank control group reaches about 1, stop incubation, then use the enzyme marker to detect the absorbance (OD 600 ) of the suspension at 600 nm, and evaluate the antibacterial activity of EDCu on MRSA and SE according to the absorbance. The survival rate of MRSA and SE treated with different concentrations of tea EDCu is shown in Figure 6 The tea polyphenol-copper nano material has inhibitory effect on the growth of MRSA and SE, and has obvious concentration dependence. In addition, the MRSA and SE treated with different concentrations of EDCu for 8 h were diluted 10 6 times, and then coated on the agar culture plate for 24 h, as shown in Figure 7 , the EDCu concentrations were 0, 0.5, 1, 2.5, and 5 μM. It can be seen from Figure 7 that the colony forming units of MRSA and SE treated with EDCu are significantly reduced, further proving the antibacterial activity of tea polyphenol-copper nano on MRSA and SE.

[0042] Example 3, antibacterial mechanism of tea polyphenol-copper nano material on MRSA and SE

[0043] The tea polyphenol-copper nano material kills bacteria by destroying the structure of pathogenic bacteria. After the bacterial structure is destroyed, nucleic acid is released from the bacteria into the culture medium, and the amount of leaked nucleic acid in the culture medium can be detected by an enzyme marker to determine the integrity of the bacteria. Different final concentrations of EDCu (0, 0.5, 1, 2.5, 5 μM) were incubated with MRSA and SE in a 96-well plate for 8 h, and then the bacterial suspension was filtered with a 0.22 μm filter membrane. The absorbance value of the filtrate at OD 260 The column chart of the absorbance of the leaked nucleic acid in the culture medium of MRSA and SE treated with different concentrations of EDCu is shown in Figure 8 As the concentration of EDCu increases, the amount of leaked nucleic acid of MRSA and SE increases, indicating that EDCu destroys the wall membrane structure of bacteria and has a concentration-dependent effect.

[0044] Example 4, cell safety evaluation of tea polyphenol-copper nano material

[0045] To evaluate the biological safety of tea polyphenol-copper nano material on human normal cells, human umbilical vein endothelial cells (HUVEC) were selected as the object, and a cell counting kit (CCK-8) was used to determine the survival rate of the cells. First, 2×10 4 HUVEC cells were inoculated into a 96-well plate and cultured at 37°C and 5% CO2 for 24 h, and then tea polyphenol-copper nano material was added to a final concentration of (0, 0.5, 1, 2.5, 5 μM) for 24 h, with 6 parallel groups in each group. After 24 h, the cells were washed three times with serum-free medium, then CCK-8 working solution was added, and the cells were incubated in a cell incubator for 30 min. The absorbance at 450 nm was measured using an enzyme marker. The column chart of the cell survival rate of human umbilical vein endothelial cells (HUVEC) treated with different concentrations of tea polyphenol-copper nano material is shown in Figure 9 Even at a high concentration (5 μM), EDCu has no obvious toxicity to HUVEC cells, indicating that it has good cell safety.

[0046] Further increasing the concentration of tea polyphenol-copper nano material, at a high concentration (7.5 μM), tea polyphenol-copper nano material has no toxicity to HUVEC cells, while at 2.5 μM, tea polyphenol-copper nano material has obvious toxicity to MRSA and SE, and completely inhibits the growth of SE on the plate Figure 7 , indicating that EDCu has good biological safety and can effectively kill pathogenic bacteria at a safe dose.

[0047] Example 5, the preparation method of tea polyphenol-copper nano material has broad spectrum

[0048] Not only EGCG can be used to construct nanomaterials with DEDTC and Cu rapidly, but also the developed method is applicable to other tea polyphenols such as EGC. Prepare 2 mM aqueous solution of epigallocatechin (EGC) and 1 mM aqueous solution of sodium diethyldithiocarbamate (DEDTC), then add 1 mM aqueous solution of NaOH into 500 μL of the EGC solution, test its pH value with pH paper, which is between 8 and 9, then add 200 μL of 1 mM aqueous solution of DEDTC, mix well, finally add 40 μL of 2.5 mM CuCl2 solution, mix well, and obtain tea polyphenol-copper nanomaterials. The photos under visible light irradiation are shown in Figure 10 from left to right are EGC solution, DEDTC solution, CuCl2 solution and product tea polyphenol-copper nanomaterial EGC-DCu, and it can be seen that the product is a transparent and clear liquid, which can be stored at 4°C for a long time.

[0049] The UV absorption spectra of epigallocatechin (EGC), sodium diethyldithiocarbamate (DEDTC) and tea polyphenol-copper nanomaterial EGC-DCu are shown in Figure 11 Compared with the absorption peaks of EGC (264 nm) and DEDTC (257 and 282 nm), the tea polyphenol-copper nanomaterial has obvious absorption peaks at 275 and 434 nm.

Claims

1. A method for constructing tea polyphenol-copper nanomaterials, characterized in that The method is: The epigallocatechin gallate is dissolved in deionized water, a basic aqueous solution is added to adjust the pH value to 8-9, then an aqueous solution of sodium diethyl dithiocarbamate and an aqueous solution of copper salt are added respectively to prepare the tea polyphenol-copper nanomaterial.

2. The method of claim 1, wherein The mass ratio of the epigallocatechin gallate, sodium diethyl dithiocarbamate and copper ion is 10-15:2-3:

1.

3. The method of claim 1, wherein The copper salt is a water-soluble copper salt.

4. The method of claim 3, wherein The copper salt is copper sulfate.

5. The method of claim 1, wherein The mass concentration of the epigallocatechin gallate is 0.1-10 mmol / L; the mass concentration of the sodium diethyl dithiocarbamate is 0.1-5 mmol / L; and the mass concentration of the copper salt is 0.1-5 mmol / L. 6.The tea polyphenol-copper nanomaterial prepared by the method according to any one of claims 1-5. 7.The tea polyphenol-copper nanomaterial according to claim 6 is applied to the preparation of an antibacterial agent for drug-resistant Staphylococcus aureus or Staphylococcus epidermidis.

Images