Green Preparation Method and Application of Litsea cubeba Essential Oil for Nano Silver

Abstract

This invention discloses the use of Litsea cubeba essential oil in the preparation of nano-silver, and establishes a corresponding preparation method. The method involves using emulsified Litsea cubeba essential oil as a reducing agent and stabilizer, and promoting the formation of Litsea cubeba essential oil-nano-silver through ultrasound and heating. This method is green, environmentally friendly, safe, effective, and low-cost. The process does not require the addition of additional chemical reagents as stabilizers, and the generated nano-silver has a strong bactericidal effect against drug-resistant bacteria. Therefore, the Litsea cubeba essential oil-nano-silver of this invention can alleviate the problem of drug-resistant bacterial infections to a certain extent, has the potential to replace antibiotics, and can be developed into a novel antibacterial agent, opening up new avenues for the application of Litsea cubeba essential oil.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation technology and its application, and in particular relates to a method for green preparation of nano-silver from Litsea cubeba essential oil and its application. Background Technology

[0002] A major challenge in the pharmaceutical and biomedical fields is the development of antibiotic resistance in pathogens, which poses a serious threat to human and animal health. Methicillin-resistant Staphylococcus aureus (MRSA) is a prime example, referring to Staphylococcus aureus carrying the mec A gene or with a MIC value ≥4 μg / mL for oxacillin. It has become one of the most widespread pathogens in clinical tissue infections, causing pneumonia, wound infections, mastitis, myocarditis, endocarditis, osteomyelitis, and other diseases, seriously threatening public health. Multidrug-resistant (MDR) Escherichia coli has been listed as one of the major challenges facing humans and animals worldwide. Infection can lead to enteritis, cystitis, peritonitis, diarrhea, and other diseases. In severe cases, it can invade the bloodstream, causing sepsis and bacteremia, endangering life. It has become a thorny public health problem, urgently requiring the development of novel antimicrobial agents to replace antibiotics in combating drug-resistant bacterial infections.

[0003] Nanosilver, as a novel antibacterial agent with broad-spectrum antibacterial activity and low susceptibility to drug resistance, is urgently sought to replace ineffective antibiotics in combating infections caused by multidrug-resistant bacteria. Nanosilver exhibits a maximum absorption peak at 400-450 nm; theoretically, a larger peak indicates a higher nanosilver content, and a smaller wavelength corresponding to the maximum absorption peak indicates a smaller nanosilver particle size. Green synthesis of nanosilver is an economical, environmentally friendly, and efficient method. Currently, extracts from various plant parts, such as bark, leaves, fruits, stems, and seeds, have been successfully used in the synthesis of nanosilver. Nanosilver synthesized using green methods exhibits better bioactivity than that synthesized using other methods because the bioactive components in the plant extracts act as capping agents and stabilizers, encapsulating the nanosilver particles. Therefore, rapid, high-concentration green synthesis is of great significance for the increasingly demanding requirements of nanotechnology.

[0004] Essential oils are mixtures of volatile organic compounds from plants. Their rich bioactive components may become the main active ingredients in the synthesis of nano-silver. For example, alcohols, esters, ethers, aldehydes, ketones, lactones, phenols and phenolic ethers can act as surface-active molecules to stabilize nano-silver particles. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method and application for the green preparation of nano-silver from Litsea cubeba essential oil, which is efficient, convenient and non-toxic. The obtained Litsea cubeba essential oil-nano-silver has strong bactericidal activity against MDR Escherichia coli and MRSA and is not prone to drug resistance.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] Litsea cubeba essential oil is used to prepare nano-silver.

[0008] A green method for preparing nano-silver from Litsea cubeba essential oil involves using emulsified Litsea cubeba essential oil as a reducing agent and stabilizer, and promoting the formation of Litsea cubeba essential oil-nano-silver through ultrasound and heating.

[0009] The above-mentioned green method for preparing nano-silver from Litsea cubeba essential oil involves mixing Litsea cubeba essential oil, Tween 80, and anhydrous ethanol to prepare Litsea cubeba essential oil nanoemulsion; adjusting the pH of the Litsea cubeba essential oil nanoemulsion to 9 with sodium hydroxide solution; mixing the Litsea cubeba essential oil nanoemulsion with silver nitrate solution and reacting it under ultrasonic conditions to obtain Litsea cubeba essential oil-nano-silver solution.

[0010] The concentration of Litsea cubeba essential oil nanoemulsion is 0.1 g / 10 mL; the volume ratio of Litsea cubeba essential oil, Tween 80, and anhydrous ethanol is 1:4:2, forming a clear and transparent nanoemulsion that is miscible with aqueous solution, facilitating subsequent reduction reactions; the concentration of sodium hydroxide solution is 0.1 mol / L, and the concentration of silver nitrate solution is 0.1 mol / L; the volume ratio of Litsea cubeba essential oil nanoemulsion to silver nitrate solution is 1:1-5:1.

[0011] The volume ratio of Litsea cubeba essential oil nanoemulsion to silver nitrate solution was 3:1, resulting in the smallest silver nanoparticle size. Transmission electron microscopy showed that the particle size was 5-15 nm, and the dispersibility was good.

[0012] The Litsea cubeba essential oil-nano silver solution was collected and precipitated by high-speed centrifugation and then freeze-dried to obtain Litsea cubeba essential oil-nano silver particles.

[0013] During the reaction, the ultrasonic power was 240W, 100%, the reaction temperature was 60℃, and the reaction time was 6h; the centrifugation time was 90min, and the rotation speed was 12000rpm.

[0014] The nano-silver prepared above.

[0015] The above-mentioned application of nano-silver in the preparation of antibacterial drugs.

[0016] The antibacterial drugs are those that target MDR E. coli or MRSA.

[0017] Addressing the problems existing in traditional nano-silver preparation methods, and leveraging the natural origin and high bioactivity of Litsea cubeba essential oil, the inventors have for the first time used Litsea cubeba essential oil to prepare nano-silver and established a corresponding preparation method. This method uses emulsified Litsea cubeba essential oil as a reducing agent and stabilizer, promoting the formation of Litsea cubeba essential oil-nano-silver through ultrasound and heating. This method is green, environmentally friendly, safe, effective, and low-cost. The process requires no additional chemical reagents as stabilizers, and the resulting nano-silver exhibits strong bactericidal effects against drug-resistant bacteria.

[0018] Compared with the prior art, the innovations and advantages of this invention are as follows:

[0019] 1. This invention is the first to utilize Litsea cubeba essential oil to prepare nano-silver using a green synthesis method. The main components of Litsea cubeba essential oil include α-citral and β-citral, which have strong reducing properties and the potential to reduce silver ions to nano-silver. The inventors used its rich bioactive components as stabilizers and reducing agents to obtain Litsea cubeba essential oil-nano-silver with small particle size, strong stability, and good dispersibility.

[0020] 2. This invention uses ultrasound and heating to promote the synthesis of nano-silver. The preparation process is simple, safe, mild, low-cost, and environmentally friendly. The entire preparation process does not require the introduction of additional chemical reagents as stabilizers and complexing agents, avoiding the use of toxic chemicals and reducing the cytotoxicity and environmental toxicity of nano-silver.

[0021] 3. The Litsea cubeba essential oil-nano silver prepared in this invention is less prone to drug resistance and exhibits strong bactericidal activity against MDR Escherichia coli and MRSA. Its MIC and MBC against MDR Escherichia coli are 25 μg / mL and 50 μg / mL, respectively, while its MIC and MBC against MRSA are 50 μg / mL and 100 μg / mL, respectively. Time-killing curve results show that the Litsea cubeba essential oil-nano silver with MIC and MBC can kill all MDR Escherichia coli at 60 min and 30 min, respectively, and kill all MRSA at 4 h and 6 h, respectively. Therefore, the Litsea cubeba essential oil-nano silver of this invention can alleviate the problem of drug-resistant bacterial infections to a certain extent, has the potential to replace antibiotics, and can be developed into a novel antibacterial agent.

[0022] 4. The green synthesis method of nano-silver of this invention opens up new avenues for the application of Litsea cubeba essential oil. Attached Figure Description

[0023] Figure 1 The UV-Vis absorption spectra of Litsea cubeba essential oil-nano silver synthesized in different proportions in Example 2 are shown.

[0024] Figure 2 This is a transmission electron microscope image of Litsea cubeba essential oil-nano silver in Example 2. In the image, A is 200 nm and B is 50 nm.

[0025] Figure 3 This is the amplification result of the mecA resistance gene of MRSA in Example 3. In the figure: M is the marker and 1 is the mecA resistance gene.

[0026] Figure 4 The graph shows the bactericidal effect of Litsea cubeba essential oil and nano-silver in Example 5. In the graph, A is the bactericidal curve for MDR Escherichia coli, and B is the bactericidal curve for MRSA. Detailed Implementation

[0027] Example 1: Preparation of Litsea cubeba essential oil-nano silver

[0028] (1) Accurately weigh 0.1g of Litsea cubeba essential oil (Jiangxi Xuesong Natural Medicinal Oil Co., Ltd., 1kg), 0.4g of Tween 80 and 0.2g of anhydrous ethanol (mass ratio of 1:4:2), add 10mL of deionized water and mix well until the solution is clear and transparent to obtain Litsea cubeba essential oil nanoemulsion.

[0029] (2) The pH of the Litsea cubeba essential oil nanoemulsion was adjusted to 9 using 0.1 mol / L sodium hydroxide;

[0030] (3) Mix Litsea cubeba essential oil nanoemulsion and 0.1 mol / L silver nitrate in ratios of 1:1, 2:1, 3:1, 4:1 and 5:1 respectively, and immediately place them in an ultrasonic instrument (power of 240W, 100%, temperature of 60℃) for 6 hours to obtain Litsea cubeba essential oil-nano silver solution.

[0031] (4) Centrifuge the Litsea cubeba essential oil-nano silver solution after the reaction at high speed. The centrifugation conditions are 12000 rpm and 90 minutes. The precipitate is dried in a freeze dryer to obtain Litsea cubeba essential oil-nano silver dried particles and stored at room temperature.

[0032] After the reaction, the solution color immediately changed from colorless to reddish-brown, indicating that Litsea cubeba essential oil has strong reducing properties.

[0033] Example 2: Physical characterization of Litsea cubeba essential oil-nano silver

[0034] (1) The synthesized Litsea cubeba essential oil-nano silver solution was observed under a UV-Vis spectrophotometer, showing its absorption peak at 400-450 nm. A larger peak value indicates a higher content of Litsea cubeba essential oil-nano silver; a smaller wavelength corresponding to the maximum absorption peak indicates a smaller particle size of Litsea cubeba essential oil-nano silver. The synthesis ratio conditions that resulted in the highest concentration of Litsea cubeba essential oil-nano silver were selected, and further experiments were conducted. Figure 1 As shown, the Litsea cubeba essential oil-nano silver in a ratio of 3:1 has the maximum absorption peak at 423nm. Therefore, Litsea cubeba essential oil-nano silver prepared under this ratio condition was selected for Examples 3-5.

[0035] (2) The shape, particle size, and dispersibility of Litsea cubeba essential oil-silver nanoparticles were observed under a transmission electron microscope. For example... Figure 2 As shown, the Litsea cubeba essential oil-nano silver particles are spherical with a particle size range of 5-15 nm and good dispersibility.

[0036] Examples 1-2 show that the optimal reaction ratio of Litsea cubeba essential oil to nano-silver is Litsea cubeba essential oil: silver nitrate = 3:1. Under this ratio, the reaction is fast and simple due to the promoting effects of pH = 9, ultrasound (240W, 100%), and 60℃. The synthesized Litsea cubeba essential oil-nano-silver has a maximum absorption peak at 423nm, a particle size of 5-15nm, and is uniformly dispersed.

[0037] Example 3: Screening study of MDR Escherichia coli and MRSA

[0038] (1) The resistance of *E. coli* to nine antibiotics—rifampin, tetracycline, meropenem, colistin, fosfomycin, penicillin, amikacin, ampicillin, and chloramphenicol—was analyzed according to CLSI (Clinical and Laboratory Standards Institute 2020). The bacteria were cultured in LB broth to the logarithmic growth phase, and the bacterial suspension was diluted to 10⁻¹⁰ with MHB broth. 5 The MIC was determined by adding 50 μL of antibiotic and 50 μL of bacterial culture to a 96-well plate and mixing thoroughly. The mixture was then serially diluted 2-fold in MHB medium and incubated at 37°C for 18 hours. The lowest concentration of antibiotic at which no bacterial growth was observed was the MIC. The results are shown in Table 1.

[0039] Table 1. Results of drug resistance of drug-resistant Escherichia coli to multiple antibiotics

[0040]

[0041] Table 1 shows that *Escherichia coli* is resistant to eight antibiotics: rifampin, tetracycline, colistin, fosfomycin, penicillin, amikacin, ampicillin, and chloramphenicol. These include rifamycins, tetracyclines, polymyxins, fosfomycins, β-lactams, and aminoglycosides. Strains resistant or insensitive to at least three different types of antibiotics can be defined as MDR bacteria. Therefore, this *E. coli* strain is an MDR *E. coli* strain.

[0042] (2) MRSA was determined by PCR amplification of the mecA resistance gene, and the results are as follows: Figure 3 As shown, the mecA resistance gene was amplified in the DNA of MRSA, indicating that this Staphylococcus aureus is MRSA.

[0043] Example 4: MIC and MBC tests of Litsea cubeba essential oil-nano silver against MDR Escherichia coli and MRSA.

[0044] (1) MIC test was performed on 96-well plates. The bacterial suspension was diluted to 10 μL with MHB broth. 5 CFU / mL, 50 μL of bacterial culture was added to each well of a 96-well microplate. Subsequently, different concentrations of nano-silver were added to each well. The 96-well plates were incubated at 37°C for 18-24 h. The lowest concentration of Litsea cubeba essential oil-nano-silver that showed no bacterial growth was the MIC. The minimum inhibitory concentrations of Litsea cubeba essential oil-nano-silver against MDR Escherichia coli and MRSA were 25 μg / mL and 50 μg / mL, respectively.

[0045] (2) The solution with no bacterial growth was inoculated onto LB agar medium and incubated at 37°C for 12-18 h. The minimum concentration of Litsea cubeba essential oil-nano silver without bacterial growth was the MBC. The MBC of nano silver against MDR Escherichia coli and MRSA were 50 μg / mL and 100 μg / mL, respectively.

[0046] Example 5: Bactericidal curve test of Litsea cubeba essential oil-nano silver against drug-resistant Escherichia coli and MRSA.

[0047] Drug-resistant E. coli and MRSA were cultured in LB broth for 10 hours. 7 CFU / mL, nano-silver was added to each tube, and the concentration was adjusted to MIC and MBC, respectively. The tubes were incubated at 37℃. The control group consisted of LB broth tubes with an equal volume of sterile physiological saline. After incubation for 0-120 min from each tube, 100 μL of the mixture was extracted, diluted to different concentrations, and then added to LB agar, evenly distributed using a spreader. Based on bacterial counts between 30-300, the bacterial concentrations after treatment with Litsea cubeba essential oil-nano-silver and physiological saline were calculated. MIC Litsea cubeba essential oil-nano-silver killed bacteria at 1 h and 6 h, respectively, while MBC nano-silver killed bacteria at 30 min and 4 h, respectively. These results are consistent with scientific evidence regarding bacterial structure. Gram-positive bacteria have thicker cell walls than Gram-negative bacteria, making them more resistant to environmental factors.

[0048] Examples 3-5 show that Litsea cubeba essential oil-nano silver exhibits strong bactericidal effects against MDR Escherichia coli and MRSA.

[0049] In summary, Examples 1-5 demonstrate that a green, efficient, convenient, and economical method for preparing Litsea cubeba essential oil-nano silver has been developed. This method eliminates the need for additional chemical reagents as stabilizers, thus reducing the cytotoxicity and environmental toxicity of nano silver. Litsea cubeba essential oil-nano silver exhibits strong bactericidal activity against common clinical drug-resistant bacteria (MDR Escherichia coli, MRSA), and has the potential to replace antibiotics in combating drug-resistant bacterial infections. It is expected to be developed into a novel bactericidal agent to replace antibiotics that are prone to developing resistance.

Claims

1. Litsea cubeba essential oil is used to prepare nano-silver; characterized in that... The preparation was carried out as follows: Litsea cubeba essential oil, Tween 80, and anhydrous ethanol were mixed to prepare Litsea cubeba essential oil nanoemulsion; the pH of the Litsea cubeba essential oil nanoemulsion was adjusted to 9 with sodium hydroxide solution; the Litsea cubeba essential oil nanoemulsion was mixed with silver nitrate solution and reacted under ultrasonic conditions to obtain Litsea cubeba essential oil-silver nanoemulsion solution; the concentration of the Litsea cubeba essential oil nanoemulsion was 0.1 g / 10 mL; the volume ratio of Litsea cubeba essential oil, Tween 80, and anhydrous ethanol was 1:4:2; the concentration of sodium hydroxide solution was 0.1 mol / L, the concentration of silver nitrate solution was 0.1 mol / L; and the volume ratio of Litsea cubeba essential oil nanoemulsion to silver nitrate solution was 1:1-5:

1.

2. A method for the green preparation of nano-silver from Litsea cubeba essential oil, characterized in that: Litsea cubeba essential oil, Tween 80, and anhydrous ethanol were mixed to prepare a Litsea cubeba essential oil nanoemulsion. The pH of the Litsea cubeba essential oil nanoemulsion was adjusted to 9 with sodium hydroxide solution. The Litsea cubeba essential oil nanoemulsion was mixed with silver nitrate solution and reacted under ultrasonic conditions to obtain a Litsea cubeba essential oil-nano silver solution. The concentration of the Litsea cubeba essential oil nanoemulsion was 0.1 g / 10 mL. The volume ratio of Litsea cubeba essential oil, Tween 80, and anhydrous ethanol was 1:4:

2. The concentration of sodium hydroxide solution was 0.1 mol / L, and the concentration of silver nitrate solution was 0.1 mol / L. The volume ratio of Litsea cubeba essential oil nanoemulsion to silver nitrate solution was 1:1-5:

1.

3. The method for green preparation of nano-silver from Litsea cubeba essential oil according to claim 2, characterized in that: The volume ratio of the Litsea cubeba essential oil nanoemulsion to the silver nitrate solution is 3:

1.

4. The method for green preparation of nano-silver from Litsea cubeba essential oil according to claim 3, characterized in that: The Litsea cubeba essential oil-nano silver solution was collected and precipitated by high-speed centrifugation and then freeze-dried to obtain Litsea cubeba essential oil-nano silver particles.

5. The method for green preparation of nano-silver from Litsea cubeba essential oil according to claim 4, characterized in that: In the reaction, the ultrasonic power was 240W, 100%, the reaction temperature was 60℃, and the reaction time was 6h; the centrifugation time was 90min, and the rotation speed was 12000rpm.

6. The nanosilver prepared according to claim 1 or 2.

7. The application of the nano-silver according to claim 6 in the preparation of antibacterial drugs: the antibacterial drug is an anti-MDR Escherichia coli or anti-MRSA drug.

Images