Composition as well as preparation method and application thereof in preparation of antibacterial agent

Abstract

The invention relates to the technical field of natural products, and particularly discloses a composition as well as a preparation method and application thereof in preparation of an antibacterial agent. The composition comprises a clausena lansium extract and an olive extract, the clausena lansium extract is extracted by taking clausena lansium peel as a raw material; the olive extract is obtained by extracting olive fruits serving as raw materials. Researches show that the minimum inhibitory concentration of the composition obtained by combining the clausena lansium extract prepared by the method and the olive extract is greatly smaller than that of the single clausena lansium extract or olive extract. Therefore, when the composition disclosed by the invention is used as an antibacterial agent, the use amount of the antibacterial agent can be greatly reduced; the important application value is realized.

Description

Technical Field

[0001] This invention relates to the field of natural product technology, and more specifically to a composition, its preparation method, and its application in the preparation of antibacterial agents. Background Technology

[0002] With increasing public concern for food safety and health, the development of natural preservatives has become a research hotspot in the food industry. While the widespread use of antibiotics has effectively controlled bacterial infections, it has also exacerbated the problem of drug resistance, posing a serious challenge to global public health. In recent years, multidrug-resistant bacteria (such as methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Escherichia coli) have frequently emerged, gradually reducing the effectiveness of conventional antibiotics. According to a report by the World Health Organization (WHO), drug-resistant bacterial infections have become a major global public health crisis, and it is estimated that by 2050, the number of deaths caused by drug-resistant infections may exceed 10 million annually. Therefore, finding new antimicrobial agents to replace or assist antibiotics has become a current research hotspot in the fields of biomedicine and food safety.

[0003] Natural plant-derived antibacterial agents have become a focus of research due to their diverse active ingredients, low toxicity, and broad-spectrum inhibitory effects against various pathogens. Extracts from many traditional medicinal plants have been proven to possess good antibacterial activity and can be used in food preservation, pharmaceuticals, and daily chemical industries. For example, plant components such as tea polyphenols, flavonoids, and phenolic acids have been extensively studied and exhibit good antibacterial properties. However, the antibacterial effects of single plant extracts are often limited, with issues such as high effective concentrations and long durations of action. Therefore, studying the synergistic effects between different plant extracts to improve antibacterial activity, reduce the required concentration, and decrease the risk of bacterial resistance has significant theoretical and practical value.

[0004] In recent years, researchers have gradually discovered that the combined use of multiple plant extracts can enhance their antibacterial effects and reduce the minimum effective concentration (MIC). For example, studies on the synergistic effect of oregano essential oil and cinnamon essential oil have found that their combined use can reduce the MIC by more than 50%. Currently, research on the synergistic antibacterial effects of extracts from wampee, olive, and mandarin orange is still limited, and their optimal ratio and mechanism of action remain unclear. Wampee and olive, as common natural plant resources, are rich in bioactive components and have potential antibacterial effects. Summary of the Invention

[0005] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides a composition, a method for preparing the composition and its application in the preparation of antibacterial agents.

[0006] The technical solution of the present invention is as follows: The present invention first provides a composition comprising a wampee extract and an olive extract; The wampee extract is obtained by extracting wampee peel as raw material; the olive extract is obtained by extracting olive fruit as raw material.

[0007] This invention provides a novel composition. Studies have shown that the composition obtained by combining the wampee extract and olive extract prepared by the method described in this invention has a significantly lower minimum inhibitory concentration (MIC) than either the wampee extract or olive extract alone. The combination of the wampee extract and olive extract prepared by the method described in this invention can produce a synergistic antibacterial effect.

[0008] Preferably, the weight ratio of yellow peel extract to olive extract is 1~5:1~5.

[0009] Most preferably, the weight ratio of yellow peel extract to olive extract is 1:1.

[0010] Preferably, the yellow peel extract is prepared by the following method: After drying the peel of the wampee fruit, it is pulverized to obtain wampee peel powder. Then, an ethanol solution is added, and the mixture is placed in an ultrasonic device for ultrasonic extraction. After the extraction is completed, the extract is collected, concentrated, and dried to obtain the wampee extract.

[0011] Preferably, the olive extract is prepared by the following method: Olive fruits are dried and pulverized to obtain olive fruit powder. Then, an ethanol solution is added, and the mixture is placed in an ultrasonic device for ultrasonic extraction. After extraction, the extract is collected, concentrated, and dried to obtain the olive extract.

[0012] Preferably, the ratio of yellow peel powder or olive fruit powder to ethanol solution is 1g:15~30mL.

[0013] Most preferably, the ratio of yellow peel powder or olive fruit powder to ethanol solution is 1g:20mL.

[0014] Preferably, the ultrasonic extraction time is 20-40 minutes and the extraction temperature is 40-60℃.

[0015] Preferably, the frequency of the ultrasound is 20~40kHz and the power is 500~2000W.

[0016] Preferably, the frequency of the ultrasound is 30kHz and the power is 1000W.

[0017] Preferably, the ethanol solution specifically refers to an aqueous ethanol solution with a volume fraction of 50-95%.

[0018] Most preferably, the ethanol solution specifically refers to an aqueous ethanol solution with a volume fraction of 70%.

[0019] The present invention also provides a method for preparing the above composition, which includes the following steps: mixing yellow peel extract and olive extract evenly to obtain the composition.

[0020] Preferably, the preparation method of the composition further includes the following steps: mixing the yellow peel extract and olive extract evenly and loading them onto a macroporous resin column; first eluting with 2-5 column volumes of 52-54% ethanol aqueous solution to remove impurities; then eluting with 2-5 column volumes of 76-78% ethanol aqueous solution; collecting the eluent eluted with 76-78% ethanol aqueous solution; concentrating and drying to obtain the composition.

[0021] Most preferably, the preparation method of the composition further includes the following steps: mixing the yellow peel extract and the olive extract evenly and loading them onto a macroporous resin column, first eluting with 4 column volumes of 53% ethanol aqueous solution to remove impurities, then eluting with 4 column volumes of 77% ethanol aqueous solution, collecting the eluent eluted with 77% ethanol aqueous solution, concentrating and drying to obtain the composition.

[0022] Preferably, the macroporous resin column is a D101 type macroporous resin column.

[0023] Further research by the inventors revealed that the minimum inhibitory concentration of the composition obtained by mixing the extracts of yellow peel and olive extract and then eluting it under the macroporous resin elution conditions described in this invention is significantly lower than that of the composition obtained by simply mixing the extracts of yellow peel and olive extract.

[0024] The inventors also discovered in their research that the macroporous resin elution conditions described above are crucial; only when the mixture of yellow peel extract and olive extract is further eluted under the macroporous resin elution conditions described above can the minimum inhibitory concentration (MIC) be significantly reduced; however, the MIC of the mixture obtained by further eluting the mixture of yellow peel extract and olive extract under other macroporous resin elution conditions cannot be significantly reduced.

[0025] The present invention also provides the use of the above composition in the preparation of antibacterial agents.

[0026] Preferably, the antibacterial agent specifically refers to an antibacterial agent that has antibacterial activity against Staphylococcus aureus and / or Escherichia coli.

[0027] Beneficial Effects: This invention provides a novel composition. Studies have shown that the composition obtained by combining the wampee extract and olive extract prepared by the method described in this invention has a significantly lower minimum inhibitory concentration (MIC) than either the wampee extract or olive extract alone. Therefore, using the composition described in this invention as an antibacterial agent can significantly reduce the amount of antibacterial agent used; it has significant application value. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments, but the embodiments do not limit the present invention in any way.

[0029] Example 1 Preparation of the composition The composition is obtained by mixing the yellow peel extract and the olive extract at a weight ratio of 1:1.

[0030] The wampee extract is prepared by the following method: dried wampee peel is pulverized to obtain wampee peel powder, then ethanol solution is added, and ultrasonic extraction is performed in an ultrasonic device. After extraction, the extract is collected, concentrated, and dried to obtain the wampee extract. The ratio of wampee peel powder to ethanol solution is 1g:20mL. The ethanol solution specifically refers to a 70% (v / v) aqueous ethanol solution. The ultrasonic extraction time is 30min, the extraction temperature is 50℃, the ultrasonic frequency is 30kHz, and the power is 1000W.

[0031] The olive extract is prepared by the following method: dried olive fruit is pulverized to obtain olive fruit powder, then ethanol solution is added, and ultrasonic extraction is performed in an ultrasonic device. After extraction, the extract is collected, concentrated, and dried to obtain the olive extract. The ratio of olive fruit powder to ethanol solution is 1g:20mL. The ethanol solution specifically refers to a 70% (v / v) aqueous ethanol solution. The ultrasonic extraction time is 30min, the extraction temperature is 50℃, the ultrasonic frequency is 30kHz, and the power is 1000W.

[0032] Example 2 Preparation of the composition The extracts of yellow peel and olive were mixed evenly at a weight ratio of 1:1 and then loaded onto a D101 macroporous resin column. First, impurities were removed by elution with 4 column volumes of 53% ethanol aqueous solution. Then, 4 column volumes of 77% ethanol aqueous solution were used for elution. The eluent obtained from the 77% ethanol aqueous solution was collected, concentrated and dried to obtain the composition.

[0033] The preparation methods for the yellow peel extract and olive extract are the same as in Example 1.

[0034] Comparative Example 1: Preparation of the Composition The extracts of yellow peel and olive were mixed evenly at a weight ratio of 1:1 and then loaded onto a D101 macroporous resin column. First, impurities were removed by elution with 4 column volumes of 5% ethanol aqueous solution. Then, 4 column volumes of 25% ethanol aqueous solution were used for elution. The eluent obtained from the 25% ethanol aqueous solution was collected, concentrated and dried to obtain the composition.

[0035] The preparation methods for the yellow peel extract and olive extract are the same as in Example 1.

[0036] Comparative Example 2: Preparation of the Composition The extracts of yellow peel and olive were mixed evenly at a weight ratio of 1:1 and then loaded onto a D101 macroporous resin column. First, impurities were removed by elution with 4 column volumes of 25% ethanol aqueous solution. Then, 4 column volumes of 55% ethanol aqueous solution were used for elution. The eluent obtained from the 55% ethanol aqueous solution was collected, concentrated and dried to obtain the composition.

[0037] The preparation methods for the yellow peel extract and olive extract are the same as in Example 1.

[0038] Preparation of the composition in Comparative Example 3 The extracts of yellow peel and olive were mixed evenly at a weight ratio of 1:1 and then loaded onto a D101 macroporous resin column. First, impurities were removed by elution with 4 column volumes of 70% ethanol aqueous solution. Then, 4 column volumes of 95% ethanol aqueous solution were used for elution. The eluent obtained from the 95% ethanol aqueous solution was collected, concentrated, and dried to obtain the composition.

[0039] The preparation methods for the yellow peel extract and olive extract are the same as in Example 1.

[0040] Experimental Example 1 In this experiment, Staphylococcus aureus (S. aureus) was ATCC 25923; Escherichia coli (E. coli) was ATCC25922.

[0041] E. coli and S. aureus cultures were taken from glycerol tubes stored at -80 °C and allowed to warm to room temperature for 3 min. LB agar medium was poured into culture plates, and the strain names were labeled at the bottom. Small amounts of bacterial culture were picked up with an inoculation loop and streaked onto LB agar plates, then incubated at 37 °C for 24 h. Colony growth was observed. After streaking twice, a single colony was picked and inoculated into 5 mL of LB liquid medium, and cultured at 37 °C with shaking (180 r / min) for 12 h to obtain the experimental bacterial culture.

[0042] Take 1 mL of overnight culture of E. coli and S. aureus, centrifuge at 6000 r / min for 5 min, discard the supernatant, and resuspend the cells in sterile PBS. Analyze the culture using a UV spectrophotometer (600 nm, OD500). 600 ) Detect the bacterial concentration at OD 600 Bacterial suspensions were aspirated at concentrations of 0.1, 0.5, 0.8, 1.0, and 1.2 and spread on a plate for incubation. After 24 hours of incubation at 37°C, colony counting was performed. The concentration was then adjusted to the 0.5 McFarland standard (approximately 1.5 × 10⁻⁶). 8 CFU / mL). Further dilute to the required concentration for the actual experiment (e.g., 10⁻⁶ CFU / mL). 6 (CFU / mL).

[0043] Sterile 96-well plates were numbered in a laminar flow hood and divided into experimental groups 1-7 and a control group. 100 μL of liquid culture medium was added to each well of the 96-well plate. The sample to be tested was added to the first well of each row at a final concentration of 256 mg / mL. Subsequently, the liquid in the same row was diluted twofold to obtain concentration gradients of 128 mg / mL, 64 mg / mL, 32 mg / mL, 16 mg / mL, 8 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL. 100 μL of 10-well medium was added to each well of the 96-well plate. 6 The test bacterial suspension at CFU / mL was used as a blank control, and the edges of the 96-well plates were sealed. The experiment used the 96-well microdilution method, with a concentration range of 1~256 mg / mL. The wells were sealed with plastic wrap and covered, and incubated at 37℃ for 24 h.

[0044] Among them, experimental groups 1 to 7 tested the yellow peel extract and olive extract prepared according to the method of Example 1, and the compositions prepared in Examples 1 to 2 and Comparative Examples 1 to 3, respectively; the results are shown in Table 1.

[0045] Table 1. Antibacterial test results of the compositions of the present invention Test sample Minimum inhibitory concentration of E. coli Minimum inhibitory concentration of S. aureus Experimental group 1 Yellow peel extract 16 mg / mL 8 mg / mL Experimental group 2 olive extract 8 mg / mL 4 mg / mL Experimental group 3 Example 1 Composition 4 mg / mL 2 mg / mL Experimental group 4 Example 2 Composition 0.5 mg / mL 0.25 mg / mL Experimental group 5 Comparative Example 1 Composition 4 mg / mL 2 mg / mL Experimental group 6 Comparative Example 2 Composition 2 mg / mL 1 mg / mL Experimental group 7 Comparative Example 3 Composition 2 mg / mL 2 mg / mL As shown in Table 1, the minimum inhibitory concentrations (MICs) of experimental group 3 against Staphylococcus aureus and Escherichia coli were significantly lower than those of experimental groups 1 and 2. This indicates that the combination of the extracts prepared by the method described in this invention and the olive extract has a significantly lower MIC than either the extract alone. The combination of the extracts prepared by the method described in this invention and the olive extract can produce a synergistic antibacterial effect.

[0046] As can be seen from the experimental results in Table 1, the minimum inhibitory concentration (MIC) of experimental group 4 against Staphylococcus aureus and Escherichia coli is significantly lower than that of experimental group 3. This indicates that the MIC of the composition obtained by mixing the extract of yellow peel and olive extract and then eluting it under the macroporous resin elution conditions described above is significantly lower than that of the composition obtained by simply mixing the extract of yellow peel and olive extract.

[0047] As can be seen from the experimental results in Table 1, although the minimum inhibitory concentrations (MICs) of Staphylococcus aureus and Escherichia coli in experimental groups 5-7 were partially reduced, the reduction was far less than that in experimental group 4. This indicates that the macroporous resin elution conditions described above in this invention are crucial; only by further eluting the mixture of wampee extract and olive extract under the macroporous resin elution conditions described above in this invention can the MIC of the resulting composition be further significantly reduced. However, the MIC of the mixture obtained by further eluting the mixture of wampee extract and olive extract under other macroporous resin elution conditions cannot be further significantly reduced.

Claims

1. A composition characterized in that, The composition comprises a Clausena lansium extract and an Olea europaea extract. The Clausena lansium extract is extracted from the pericarp of Clausena lansium, and the Olea europaea extract is extracted from the fruit of Olea europaea.

2. The composition of claim 1, wherein, The weight ratio of the Clausena lansium extract to the Olea europaea extract is 1-5:1-5.

3. The composition of claim 1, wherein, The weight ratio of the Clausena lansium extract to the Olea europaea extract is 1:

1.

4. The composition of claim 1, wherein, The Clausena lansium extract is prepared by the following method: The pericarp of Clausena lansium is dried and then crushed to obtain pericarp powder, and then ethanol solution is added and ultrasonic extraction is performed in an ultrasonic device. After the extraction is completed, the extract is obtained, concentrated and dried to obtain the Clausena lansium extract.

5. The composition of claim 1, wherein, The Olea europaea extract is prepared by the following method: The fruit of Olea europaea is dried and then crushed to obtain fruit powder, and then ethanol solution is added and ultrasonic extraction is performed in an ultrasonic device. After the extraction is completed, the extract is obtained, concentrated and dried to obtain the Olea europaea extract.

6. The composition according to claim 4 or 5, characterized in that, The amount ratio of the pericarp powder or the fruit powder to the ethanol solution is 1g:15-30mL. Most preferably, the amount ratio of the pericarp powder or the fruit powder to the ethanol solution is 1g:20mL.

7. The composition according to claim 4 or 5, characterized in that, The ultrasonic extraction is performed for 20-40min at a temperature of 40-60℃.

8. Process for the preparation of a composition according to any one of claims 1 to 7, characterized in that, The composition is prepared by uniformly mixing the Clausena lansium extract and the Olea europaea extract.

9. Use of the composition of any one of claims 1-7 in the preparation of an antibacterial agent.

10. Use according to claim 9, characterized in that, The antibacterial agent is specifically an antibacterial agent having antibacterial effect on Staphylococcus aureus and / or Escherichia coli.