A plant essential oil composition and its application

Abstract

The present invention discloses a plant essential oil composition and its application. The plant essential oil composition provided by the present invention is composed of essential oil A and essential oil B, wherein essential oil A is selected from rose essential oil or Alpinia oxyphylla essential oil, and essential oil B is selected from Alpinia oxyphylla essential oil or Alpinia officinalis essential oil. The essential oil composition provided by the present invention has a good inhibitory effect on pathogenic bacteria, and its essential oil components have a synergistic antibacterial effect, which greatly reduces the concentration and dosage of the essential oil, and a good antibacterial effect can be achieved by compounding with fewer components. The plant essential oil raw materials used in the present invention are all medicinal and edible plants, which are natural, safe, and non-toxic to the human body. After compounding, not only can the concentration of the essential oil be reduced, but the antibacterial effect will not be affected, the cost will be reduced, and the safety of the essential oil can be improved. Further, it can be used in the preparation of antibacterial drugs to avoid the generation of antibiotic resistance and side effects, and provide a method and idea for preparing more green, natural, safe, low-concentration, and effective antibacterial products.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to a plant essential oil composition and application thereof. Background Art

[0002] Plant essential oils, also known as volatile oils, are oily mixtures extracted from certain plant parts (rhizomes, fruits, seeds, flowers, leaves, etc.) using various extraction methods. They have a distinctive odor and are easily volatile. They are secondary metabolites of plants, and their main components include polyphenols, terpenes, flavonoids, and alkaloids. As natural products, plant essential oils are present in relatively low concentrations, but they are important components in the efficacy of traditional Chinese medicine. Plant essential oils possess a variety of biological activities, such as antibacterial, antioxidant, antiviral, and antitumor activities, and have been widely used in the medical field and food industry. Plant essential oils exhibit excellent antibacterial effects. Compared with chemical antibacterial drugs, plant essential oils are green, non-toxic, natural, and have high nutritional value. Therefore, in recent years, the antibacterial activity of plant essential oils and their application in the pharmaceutical and food industries have attracted considerable attention and attracted extensive research.

[0003] At present, antibacterial drugs or antibacterial agents with high antibacterial effects all contain antibiotic ingredients. Their antibacterial effects are strong, but they are prone to adverse reactions, such as toxic reactions: 1. damage to the nervous system, 2. nephrotoxicity, 3. liver damage, 4. damage to the blood system, allergic reactions or superinfections, and drug resistance. In addition, their costs are high. Since the antibacterial spectrum of a single plant essential oil is relatively narrow and the effect is poor, it is often necessary to use a higher concentration and dosage to achieve a better effect. Therefore, in order to improve the antibacterial effect of plant essential oils, existing research has been conducted to improve the antibacterial effect by adding synergists, or by coordinating multiple antibacterial components or compounding multiple essential oils. For example, the plant essential oil composition containing borneol essential oil, silver-gray chrysanthemum essential oil, and lilac essential oil disclosed in the prior art is compounded with multiple plant essential oils, and it is also necessary to add a water-soluble cationic antibacterial agent to synergistically improve its antibacterial effect, which increases the cost.

[0004] When compounding multiple essential oils, the concentration of the essential oils must be controlled so that it is not too high. If the concentration of the essential oils is too high, not only will the production cost increase significantly, but it may also have an adverse effect on the user's own immune microenvironment, triggering allergic reactions, and even causing skin burns or other irritation reactions, affecting the safety of the essential oils. Therefore, in order to overcome the defects and shortcomings of existing plant essential oils, such as the high concentration required, the potential safety hazards, the need to compound multiple plant essential oils in their essential oil formulations, and the unsatisfactory antibacterial effects, and to avoid the side effects caused by the use of antibiotics, it is necessary to develop more plant essential oil compositions that are highly effective in antibacterial, have low concentrations, few components, and do not require the addition of other synergists, so as to provide methods and ideas for the preparation of more green, natural, safe, and effective antibacterial drugs. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the above-mentioned plant essential oils and provide a plant essential oil composition and application thereof.

[0006] The purpose of the present invention is to provide a plant essential oil composition.

[0007] Another object of the present invention is to provide an application of the plant essential oil composition.

[0008] Another object of the present invention is to provide an antibacterial product.

[0009] Another object of the present invention is to provide an antibacterial drug.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The invention provides a plant essential oil composition, which consists of essential oil A and essential oil B; the essential oil A is selected from rose essential oil or oxyphylla essential oil, and the essential oil B is selected from oxyphylla essential oil or galangal essential oil; and the mass ratio of the essential oil A to the essential oil B is 1:(1-2).

[0012] The present invention uses six medicinal and edible plants—turmeric, galangal, alpinia oxyphylla, tsaoko, kaempferia galanga, and rose—as research subjects. These plants are extracted using steam distillation to obtain six plant essential oils. The antibacterial activity of the six individual essential oils is verified through inhibition zone and MIC tests, showing that the six essential oils have the best antibacterial effect against Staphylococcus aureus, with tsaoko essential oil showing the best antibacterial effect. Further research on compound essential oil combinations using combined drug sensitivity testing yielded plant essential oil compositions with synergistic antibacterial effects: rose essential oil + alpinia oxyphylla essential oil, rose essential oil + galangal essential oil, and alpinia oxyphylla essential oil + galangal essential oil. The three compound essential oils have good antibacterial effects. They inhibit the normal growth and reproduction of bacteria by damaging the bacterial cell membrane structure, causing serious damage to the bacterial morphology and the appearance of pits and cracks on the bacterial surface. They show a strong inhibitory effect on Staphylococcus aureus. The essential oil components have a synergistic antibacterial effect, which can greatly reduce the concentration and dosage of the essential oil. Without affecting the antibacterial effect, the concentration can be reduced by 4-8 times, reducing costs and improving the safety of the use of plant essential oils, providing methods and ideas for the preparation of more green, natural, safe and effective antibacterial drugs.

[0013] At the same time, the plant essential oil raw materials used in the present invention are all plants with medicinal and edible properties. They are natural and safe substances that are non-toxic to the human body and can be used for the preparation of antibacterial drugs, avoiding the drug resistance and side effects of antibiotic antibacterial drugs.

[0014] Turmeric (Curcuma longa L.), also known as Baodingxiang and Huangsiyujin, is a plant of the genus Curcuma in the Zingiberaceae family. The main components of turmeric's volatile oil are zedoaryl and curcumin. As a food-drug congener, turmeric exhibits a variety of pharmacological activities, including anti-tumor, anti-inflammatory, anti-fibrotic, antioxidant, and neuroprotective properties.

[0015] Alpinia officinarum Hance is a plant of the Zingiberaceae family, native to China. Its chemical constituents are primarily classified into three categories: flavonoids, diphenylheptanes, and volatile oils. The volatile oils primarily consist of sesquiterpenes and hemiterpenes, including β-pinene and 1,8-cineole. Alpinia officinarum not only has ornamental value but also medicinal properties, showing efficacy in treating a variety of ailments, including tumors, gastric ulcers, and cardiovascular and cerebrovascular diseases.

[0016] Alpinia oxyphylla Miq., also known as "Zhaijiaozi," is the fruit of the plant Alpinia oxyphylla, part of the Zingiberaceae family. Its main components include sesquiterpenes (nootkatone), flavonoids (chrysin, galangin, and kaempferol), and diarylheptanoids (alpinol). As a traditional Chinese herbal medicine, Alpinia oxyphylla has antibacterial, immunomodulatory, antiviral, anti-inflammatory, and neuroprotective properties.

[0017] Kaempferia galanga L., also known as sand ginger, is a member of the Zingiberaceae family and is cultivated for both seasoning and medicinal purposes. The volatile oil from its rhizomes contains a variety of components, primarily borneol, ethyl cinnamate, and ethyl p-methoxycinnamate. Kaempferia galanga is a spice and natural herbal medicine, with its rhizomes used as both a seasoning and a medicine, exhibiting sedative, analgesic, and antioxidant properties.

[0018] Amomum tsaoko (Crevost & Lem.) is an important edible and medicinal plant in the Zingiberaceae family. It not only removes the fishy odor of food but also adds flavor to it, making it a commonly used spice. Furthermore, it exhibits pharmacological activities such as antibacterial, gastrointestinal regulation, anti-ulcer, and analgesic properties, making it widely used in the food industry and in the treatment of certain diseases.

[0019] Rose (Rosa rugosa Thunb.) is a plant of the genus Rosa in the Rosaceae family. The main components of rose essential oil include citronellol, linalool, and eugenol. Besides its ornamental and nutritional value, rose also boasts medicinal properties, including skin-whitening, anti-depressant, calming, choleretic, emotionally soothing, and sleep-promoting benefits.

[0020] Therefore, the present invention provides the use of the plant essential oil composition in preparing an antibacterial product.

[0021] The invention provides an antibacterial product containing a plant essential oil composition.

[0022] In particular, the present invention shows that due to the synergistic antibacterial effect between essential oils A and B, by reducing the concentration of essential oils A and B in the product by 4-8 times (i.e., reducing it by 1 / 8 or 1 / 4 on the basis of the minimum inhibitory concentration MIC), it also has a significant antibacterial effect, greatly reducing the concentration and dosage of the essential oils. When the composition is rose essential oil and galangal essential oil, the concentrations are ≥1 / 8MIC, ≥1 / 4MIC respectively; when the composition is rose essential oil and galangal essential oil, the concentrations are ≥1 / 8MIC, ≥1 / 4MIC respectively; when the composition is galangal essential oil and galangal essential oil, the concentrations are ≥1 / 4MIC, ≥1 / 8MIC respectively; and according to the actual MIC values ​​of the three pathogens, the minimum use concentration range of essential oils A and B in the specific product is converted, and the effect of the present invention can be achieved within the minimum concentration range.

[0023] Preferably, the concentrations of essential oil A and essential oil B in the product are both ≥0.15 μL / mL.

[0024] Preferably, when the composition in the product is rose essential oil and Alpinia oxyphylla essential oil, their concentrations are ≥0.31 μL / mL and ≥0.15 μL / mL, respectively.

[0025] Preferably, when the composition in the product is rose essential oil and galangal essential oil, their concentrations are ≥0.31 μL / mL and ≥2.5 μL / mL, respectively.

[0026] Preferably, when the composition in the product is Alpinia oxyphylla essential oil and Alpinia officinalis essential oil, their concentrations are ≥0.15 μL / mL and ≥1.25 μL / mL, respectively.

[0027] The present invention provides application of the composition in preparing antibacterial drugs.

[0028] The invention provides an antibacterial drug containing a plant essential oil composition.

[0029] Furthermore, the antibacterial product can inhibit foodborne and infectious pathogenic bacteria and can be used in the development of external-use bactericidal products, such as soaps, toiletries, antibacterial creams and other products.

[0030] Furthermore, the bacteria inhibited are Staphylococcus aureus, Escherichia coli and / or Pseudomonas aeruginosa.

[0031] The present invention has the following beneficial effects:

[0032] The plant essential oil composition provided by the present invention is composed of rose essential oil and oxyphylla essential oil, or rose essential oil and galangal essential oil, or oxyphylla essential oil and galangal essential oil. The components of the essential oil composition have a synergistic antibacterial effect and have a good inhibitory effect on Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. After compounding the two essential oil components, it has a good antibacterial effect. Under the premise of not affecting the antibacterial effect, the concentration and dosage of the essential oil can be greatly reduced, the cost can be reduced, the use safety of the plant essential oil can be improved, and a method and idea for preparing more green, natural, safe and effective antibacterial drugs are provided. At the same time, the plant essential oil raw materials used in the present invention are all plants with medicinal and edible properties, which are natural, safe and have no toxic effects on the human body. They are further used in the preparation of antibacterial drugs, can avoid the drug resistance and side effects of antibiotic antibacterial drugs, and provide more low-concentration, low-component, and no need to add other synergists. Plant essential oil compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of plate spreading after co-cultivation of different compound essential oil compositions are shown.

[0034] Figure 2 Fluorescence images of live / dead bacteria after co-culture (left) and the ratio of live bacteria (right).

[0035] Figure 3 This is a scanning electron micrograph of bacterial morphology after treatment with the compound essential oil composition. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0038] Example 1 Analysis of antibacterial activity of different plant essential oils

[0039] 1. Active ingredient extraction

[0040] Steam distillation was used to extract the medicinal and edible plants: turmeric rhizomes, galangal rhizomes, Alpinia oxyphylla fruit, Kaempferia galanga rhizomes, Amomum villosum fruit, and rose petals. The specific method is as follows: turmeric, galangal, Alpinia oxyphylla fruit, Kaempferia galanga, Amomum villosum fruit, and rose petals were pulverized using a high-speed multifunctional grinder to obtain medicinal powders; 500g of the medicinal powder was weighed and poured into a 10L flask, and 5L of deionized water was added to soak for a period of time. Then, a distillation apparatus was set up for extraction. The extraction was started by heating to boiling and the temperature was adjusted to keep the medicinal solution at a low boiling state for 4 hours. After the distillation was completed, the lower aqueous phase was removed, and the upper essential oil was collected and dried over anhydrous sodium sulfate to obtain turmeric essential oil, galangal essential oil, Alpinia oxyphylla essential oil, Kaempferia galanga essential oil, Amomum villosum essential oil, and rose essential oil. Finally, the obtained essential oils were placed in a 4°C refrigerator for storage.

[0041] 2. Bacteria recovery

[0042] Take Staphylococcus aureus (strain number ATCC6538), Escherichia coli (strain number ATCC25922), and Pseudomonas aeruginosa (strain number ATCC9027) strains stored at -80°C for a long time, pick a small amount of each bacteria with an inoculating loop, inoculate it into sterilized blank LB solid medium by plate streak method, and incubate it in a 37°C incubator for 24 hours until monoclonal colonies appear. Use a sterile pipette tip to pick a monoclonal colony and place it into a 15mL centrifuge tube filled with blank LB liquid medium. Place the 15mL centrifuge tube in a constant temperature shaker for enrichment culture for subsequent experiments.

[0043] 3. Inhibition zone test

[0044] Take the bacterial suspensions of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa after enrichment culture, and dilute the bacterial suspensions with sterile blank LB broth liquid medium to a concentration of 5×10 5 CFU / mL. Then, use a pipette to mix the diluted bacterial solution evenly. Pipette an appropriate amount of the solution into a blank LB broth solid medium and spread it evenly using a disposable sterile applicator. After the medium is air-dried, add a circular filter paper. Finally, pipette 10 μL of essential oil dropwise onto the filter paper. Set up three parallel controls for each essential oil. Incubate the culture dish upside down in a constant temperature incubator for 24 hours. The next day, remove the culture dish and observe for the formation of an inhibition zone. If so, measure the diameter of the inhibition zone. If not, indicate a value of 0. The filter paper used has a diameter of 5.5 mm.

[0045] 4. MIC determination

[0046] The MIC values ​​of six single essential oils, namely turmeric essential oil, galangal essential oil, alpinia oxyphylla essential oil, galangal essential oil, tsaoko essential oil and rose essential oil, against three pathogenic strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were tested by microbroth dilution method. The six essential oils were dissolved in dimethyl sulfoxide to prepare mother liquor, which was then diluted to different concentration gradients as experimental concentrations by the doubling dilution method. The dilution solvent was blank LB broth liquid medium; the three test strains were diluted to 10 6 CFU / mL. Use a pipette to draw 50 μL of gradient concentration essential oil into a 96-well cell culture plate, and add 50 μL of Staphylococcus aureus / Escherichia coli / Pseudomonas aeruginosa bacterial suspension, ensuring that the total volume of each well is 100 μL; set up negative control wells (50 μL essential oil + 50 μL blank LB broth liquid culture medium) and positive control wells (50 μL bacterial solution + 50 μL blank LB broth liquid culture medium), and set up four replicate wells for each experimental gradient concentration as a control. Place the 96-well cell culture plate in a constant temperature incubator and culture for 24 hours, then take it out for observation. The essential oil concentration corresponding to the well where the test bacteria do not grow is the MIC value.

[0047] 5. Antibacterial activity

[0048] The inhibition zone results of six single essential oils with medicinal and edible properties against three pathogenic bacteria, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, are shown in Table 1. As can be seen from the table, compared with Escherichia coli and Pseudomonas aeruginosa, the six edible and medicinal essential oils have the best inhibitory effect on Staphylococcus aureus, and the diameter of the inhibition zone formed by them is larger. Among them, the diameter of the inhibition zone produced by tsaoko essential oil is the largest, reaching 15.60±0.54mm, indicating that tsaoko essential oil has the best inhibitory effect on Staphylococcus aureus; in addition, tsaoko essential oil and rose essential oil have better inhibition on Escherichia coli, and their inhibition zone diameters are 9.77±0.46mm and 9.32±0.38mm respectively, followed by galangal essential oil, turmeric essential oil and kaempferia essential oil, while alpinia oxyphylla essential oil does not produce obvious inhibition zone on Escherichia coli, and turmeric essential oil does not produce inhibition zone on Pseudomonas aeruginosa. Kaempferia essential oil has the largest inhibition zone diameter on Pseudomonas aeruginosa, which is 9.37±0.19mm, followed by alpinia oxyphylla essential oil and rose essential oil. The results show that all six essential oils have a certain inhibitory effect on pathogenic bacteria.

[0049] Table 1 Results of inhibition zone experiments of six single essential oils against three pathogenic bacteria

[0050]

[0051] Table 2 shows the statistical results of the minimum inhibitory concentration (MIC) values ​​of six edible and medicinal essential oils against three pathogenic bacteria: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The results show that turmeric oil, galangal oil, Alpinia oxyphylla oil, kaempferia galanga oil, tsaoko oil, and rose oil have the best inhibitory effects against Staphylococcus aureus, while their inhibitory effects against Escherichia coli and Pseudomonas aeruginosa are relatively poor. The data indicate that among the six edible and medicinal essential oils, Pseudomonas aeruginosa is more sensitive to rose and galangal oils, while less sensitive to kaempferia oil, turmeric oil, Alpinia oxyphylla oil, and tsaoko oils. Combining the inhibition zone test and MIC test results in Table 1, four edible and medicinal essential oils with strong antibacterial activity were screened: galangal oil, Alpinia oxyphylla oil, tsaoko oil, and rose oil.

[0052] Table 2 MIC values ​​of six single essential oils against three pathogens

[0053]

[0054] Example 2 Combined drug sensitivity test of compound essential oil combination

[0055] According to the antibacterial activity verification results of the single essential oils in Example 1, galangal essential oil, alpinia oxyphylla essential oil, tsaoko essential oil and rose essential oil with higher antibacterial activity were selected as alternative components, and Staphylococcus aureus, which was more sensitive to these four essential oils, was used as the test strain to design different compound essential oil combinations through a joint drug sensitivity test.

[0056] According to the MIC results of the four essential oils against Staphylococcus aureus, LB broth liquid medium was used as the dilution solvent to obtain four concentration gradients of essential oil solutions, with the concentration gradient set at 1MIC, 1 / 2MIC, 1 / 4MIC, and 1 / 8MIC. The essential oil solutions of different concentrations were then stored at 4°C for later use. In addition, the Staphylococcus aureus suspension was diluted with sterile blank LB broth liquid medium to a concentration of 10 6 CFU / mL and stored at 4°C for later use.

[0057] Using the checkerboard dilution method, each essential oil (concentration of 1MIC, 1 / 2MIC, 1 / 4MIC, 1 / 8MIC) was combined in pairs on a sterile 96-well cell culture plate, i.e., tsaoko essential oil + rose essential oil, tsaoko essential oil + alpinia oxyphylla essential oil, tsaoko essential oil + galangal essential oil, rose essential oil + alpinia oxyphylla essential oil, rose essential oil + galangal essential oil, alpinia oxyphylla essential oil + galangal essential oil, a total of six combinations. The essential oil concentrations in each well of the 96-well cell culture plate are shown in Table 3.

[0058] Table 3 Schematic diagram of compound essential oil design

[0059]

[0060]

[0061] In a sterile 96-well cell culture plate, 25 μL of each of essential oils A and B with set concentrations were added to the corresponding wells respectively. The two essential oils were mixed evenly, and then 50 μL of diluted Staphylococcus aureus bacterial solution was added, ensuring that the volume of the liquid added to each well was the same. Three replicate wells were set as parallel controls for each different compound concentration. After the sterile 96-well plate was placed in an incubator at 37 °C for 24 hours, it was taken out for observation and result reading. The essential oil concentration corresponding to the well where the test bacteria did not grow was taken as the MIC value. The clarity of the solution in each well was observed, and the antibacterial effect strength and action type of the compound essential oil were determined by calculating the fractional inhibitory concentration index (FIC).

[0062] The FIC calculation formula is as follows:

[0063] FIC = FIC(A) + FIC(B)

[0064] FIC(A) = MIC(A combined test) / MIC(A single test)

[0065] FIC(B) = MIC(B combined test) / MIC(B single test)

[0066] Judgment criteria: If FIC ≤ 0.5, it indicates a synergistic effect between the two components; when 0.5 < FIC ≤ 1, there is an additive effect between the two components; when 1 < FIC ≤ 2, it is an irrelevant effect; when FIC > 2, it is an antagonistic effect, that is, the smaller the FIC index, the better.

[0067] The drug sensitivity test results of the compound essential oils are shown in Table 4. It can be seen that the FIC values of the six essential oil combinations are all less than 1, indicating that their antibacterial action types show an additive effect or a synergistic effect, and there is no irrelevant effect or antagonistic effect. Among them, the FIC values of the combination of tsaoko fruit essential oil with rose essential oil, galangal essential oil, and semen alpiniae oxyphyllae essential oil are all between 0.5 and 1, indicating that there is an additive effect between tsaoko fruit essential oil and the other three essential oils. Compared with rose essential oil, the additive effect between tsaoko fruit essential oil and galangal essential oil and semen alpiniae oxyphyllae essential oil is stronger. The FIC values of the three combinations of rose essential oil + semen alpiniae oxyphyllae essential oil, rose essential oil + galangal essential oil, and semen alpiniae oxyphyllae essential oil + galangal essential oil are all 0.375, showing a synergistic effect. At this time, the concentration ratios of the three compound essential oils are rose essential oil (1 / 8MIC): semen alpiniae oxyphyllae essential oil (1 / 4MIC), rose essential oil (1 / 8MIC): galangal essential oil (1 / 4MIC), and semen alpiniae oxyphyllae essential oil (1 / 4MIC): galangal essential oil (1 / 8MIC). The results show that in the process of the interaction of essential oils, it plays a role of synergistic enhancement, can expand the antibacterial spectrum, and reduce the concentration and dosage of each essential oil.

[0068] Table 4 Combined drug sensitivity test results

[0069]

[0070]

[0071] Example 3 Antibacterial effect of compound essential oil composition

[0072] 1. Antibacterial activity

[0073] The three compound essential oils with synergistic antibacterial effects obtained in Example 2 were named compound essential oil composition 1, whose concentration ratio was rose essential oil (1 / 8 MIC): Alpinia oxyphylla essential oil (1 / 4 MIC), and the specific concentration was 0.3125:0.15625 μL / mL; the compound essential oil composition 2 had a concentration ratio of rose essential oil (1 / 8 MIC): galangal essential oil (1 / 4 MIC), and the specific concentration was 0.3125:2.5 μL / mL; the compound essential oil composition 3 had a concentration ratio of Alpinia oxyphylla essential oil (1 / 4 MIC): galangal essential oil (1 / 8 MIC), and the specific concentration was 0.15625:1.25 μL / mL.

[0074] Take the diluted and 10 6 CFU / mL of Staphylococcus aureus liquid was added, 500 μL of the liquid was added to a 2 mL centrifuge tube, and then the three compound essential oils were added according to the above-set concentrations, 250 μL of each essential oil in the compound essential oil, a total of 500 μL was added to a 2 mL centrifuge tube for co-culture with Staphylococcus aureus, and a blank culture medium (500 μL blank LB liquid medium + 500 μL Staphylococcus aureus liquid) was set as the negative control group, and the 2 mL centrifuge tube was placed in a constant temperature incubator and cultured for 24 hours.

[0075] After 24 hours, the solution in the centrifuge tube was pipetted evenly and an appropriate amount of bacterial solution was taken, diluted 200,000 times with blank LB broth liquid culture medium and 50 μL was pipetted into blank LB broth solid culture medium with a pipette. The solution was evenly spread with a disposable coating stick, and then the culture dish was placed in a constant temperature incubator for 24 hours. Finally, the number of monoclonal colonies in each group of culture dishes was taken out and observed to evaluate the antibacterial ability of the compound essential oil.

[0076] The results are as follows Figure 1 As shown, the negative control group produced more monoclonal colonies after application, while the culture dishes treated with the three essential oil compounds produced significantly fewer monoclonal colonies, indicating that the three essential oil compounds exhibited a strong antibacterial effect, inhibiting the growth and reproduction of Staphylococcus aureus. Furthermore, the order of the number of monoclonal colonies in each group of culture dishes was Compound Essential Oil 1 < Compound Essential Oil 2 < Compound Essential Oil 3. Therefore, among the three essential oil compounds, Compound Essential Oil 1 had the best inhibitory effect on Staphylococcus aureus.

[0077] 2. Live / dead bacteria staining

[0078] The three essential oils were co-cultured with Staphylococcus aureus, and then the Staphylococcus aureus was stained with a live bacteria / dead bacteria double staining kit. The antibacterial ability of the three essential oils was evaluated by calculating the fluorescence intensity. Bacteria with intact membrane structures showed green fluorescence, while bacteria with damaged membrane structures showed red fluorescence. For specific operation methods, refer to the instructions of the live bacteria / dead bacteria double staining kit. Add 1 mL of 10 6 CFU / mL of Staphylococcus aureus was centrifuged at 5000 rpm for 15 minutes at 25°C. The culture medium was discarded and the cells were washed three times with TBST buffer. 500 μL of TBST buffer was then added to resuspend the cells. 500 μL of compound essential oil (250 μL of culture A + 250 μL of essential oil B) was added to the experimental group. A blank culture medium (500 μL blank LB broth liquid culture medium + 500 μL of Staphylococcus aureus culture) was set as the negative control group, and the centrifuge tubes were placed in a constant temperature incubator for co-culture for 24 hours.

[0079] The incubated bacterial suspension was centrifuged at 25°C and 5000 rpm for 15 minutes, washed three times with TBST buffer, and resuspended in 1 mL of TBST buffer. Equal amounts of SYTO 9 and PI were mixed in a centrifuge tube to prepare a dye premix. 3 μL of the dye premix was added to each bacterial suspension, mixed thoroughly, and incubated at room temperature in the dark for 15 minutes. Then, 5 μL of the dyed bacterial suspension was pipetted onto a glass slide and covered with a coverslip. Bacterial fluorescence images were captured using an inverted fluorescence microscope. Finally, the fluorescence images were fitted using Image J, and the fluorescence intensity was calculated using Image Pro Plus. The proportion of live bacteria was analyzed to evaluate the antibacterial effect of the three essential oil compounds.

[0080] The formula for calculating the proportion of live bacteria is:

[0081] Proportion of live bacteria = green fluorescence intensity / (green fluorescence intensity + red fluorescence intensity) × 100%

[0082] The live / dead bacteria staining results after co-culture of the three compound essential oils with Staphylococcus aureus are as follows Figure 2As shown in the figure, green fluorescence represents live bacteria obtained by SYTO 9 staining, while red represents dead bacteria obtained by PI staining. By observing the fluorescence image, it can be found that the green fluorescence of the negative control group is stronger, while the red fluorescence is almost absent; however, the red fluorescence of the Staphylococcus aureus liquid obtained after treatment with the three compound essential oils is stronger, while the green fluorescence is weaker, indicating that most of the bacteria in the liquid are dead bacteria. In addition, using relevant software to fit the fluorescence image and calculate the fluorescence intensity, it was found that the proportion of live bacteria in the negative control group, compound essential oil group 1, compound essential oil group 2, and compound essential oil group 3 were 98%, 72%, 29%, and 22%, respectively, indicating that the three compound essential oils have a strong inhibitory effect on Staphylococcus aureus. Compared with the negative control group, the above results all showed significant differences.

[0083] 3. Effects of compound essential oils on bacterial cell morphology

[0084] Prepare LB broth solid and liquid culture media and sterilize them for later use. Use the plate streak method to dip the activated bacterial solution (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa) into LB broth solid culture medium and culture it in a constant temperature incubator for 24 hours to obtain monoclonal colonies; use a sterilized pipette tip to pick up the monoclonal colonies and place them in LB broth liquid culture medium. Enrich and culture them at 37°C and 130r / min for 24 hours to obtain a high-concentration bacterial suspension. Then use LB broth liquid culture medium to dilute the bacterial suspension to a concentration of 10 6 CFU / mL is reserved.

[0085] 500 μL of bacterial suspension was added to a 2 mL centrifuge tube; each compound essential oil was prepared using LB broth liquid culture medium. Then, 500 μL of each compound essential oil was added to the centrifuge tube containing the bacterial suspension to obtain a compound essential oil treatment group. A negative control group (500 μL blank LB broth liquid culture medium + 500 μL of each bacterial suspension) was set up. The 2 mL centrifuge tubes were placed in a constant temperature incubator and incubated for 24 hours. After incubation, the cells were centrifuged at 25°C, 5000 rpm for 15 minutes and washed three times with phosphate buffered saline (PBS). The cells were then fixed with 2.5% glutaraldehyde solution at room temperature for 2 hours. After fixation, the cells were washed three times with PBS buffer and centrifuged at 25°C, 5000 rpm for 15 minutes. The supernatant was discarded and the cells were dehydrated using a gradient of ethanol (30%, 50%, 70%, 90%, and 100%), with each level left to stand for 15 minutes. Finally, bacterial scanning electron microscopy samples were prepared, and then the obtained samples were freeze-dried and gold-sprayed. The bacterial morphology of each group was observed using a field emission scanning electron microscope to evaluate the effect of compound essential oil treatment on bacterial morphology.

[0086] The results are as follows Figure 3As shown in the figure, the untreated bacteria have a relatively complete and regular morphology. Staphylococcus aureus has a complete spherical structure, while Escherichia coli and Pseudomonas aeruginosa both have a rod-shaped structure. The three bacteria in the negative control group have a relatively uniform size distribution, and the surface is plump and smooth without obvious pits and cracks. However, after treatment with the three compound essential oils, the bacterial morphology is significantly damaged. The spherical structure of Staphylococcus aureus is severely damaged, showing an irregular structure, and the bacteria are completely adhered to each other. The surface of Escherichia coli and Pseudomonas aeruginosa treated with the three compound essential oils is relatively rough, with a small amount of pits and cracks, and due to severe adhesion between the bacteria, the bacteria cannot maintain their complete rod-shaped structure.

[0087] The above results show that the three compound essential oils cause irreversible damage to bacterial morphology, resulting in changes in the physiological morphology of bacterial cells. This is because the three compound essential oils damage the permeability and integrity of the bacterial cell membrane, thereby increasing cell membrane permeability, followed by leakage of bacterial cell contents, which inhibits bacterial growth and reproduction. Therefore, it can be seen that the three compound essential oils have good antibacterial effects. There is a synergistic antibacterial effect between the essential oil components. The combination of two essential oil components has a good antibacterial effect. Without affecting the antibacterial effect, the concentration and dosage of the essential oil can be greatly reduced, reducing costs and improving the safety of plant essential oils. This provides methods and ideas for the preparation of more green, natural, safe and effective antibacterial drugs.

[0088] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An application of a plant essential oil composition in antibacterial treatment, characterized in that: The plant essential oil composition comprises rose essential oil and Alpinia oxyphylla essential oil, or rose essential oil and Alpinia officinalis essential oil, or Alpinia oxyphylla essential oil and Alpinia officinalis essential oil; the mass ratio of the essential oils is 1:(1-2); The bacteria inhibited by the composition are Staphylococcus aureus, Escherichia coli and / or Pseudomonas aeruginosa; When the composition is rose essential oil and Alpinia oxyphylla essential oil, their concentrations are ≥0.31 μL / mL and ≥0.15 μL / mL, respectively; When the composition comprises rose essential oil and galangal essential oil, their concentrations are ≥0.31 μL / mL and ≥2.5 μL / mL, respectively; When the composition comprises Alpinia oxyphylla essential oil and Alpinia officinalis essential oil, their concentrations are ≥0.15 μL / mL and ≥1.25 μL / mL, respectively.

2. Application of a plant essential oil composition in the preparation of an antibacterial product, characterized in that: The plant essential oil composition comprises rose essential oil and Alpinia oxyphylla essential oil, or rose essential oil and Alpinia officinalis essential oil, or Alpinia oxyphylla essential oil and Alpinia officinalis essential oil; the mass ratio of the essential oils is 1:(1-2); The bacteria inhibited by the composition are Staphylococcus aureus, Escherichia coli and / or Pseudomonas aeruginosa; When the composition is rose essential oil and Alpinia oxyphylla essential oil, their concentrations are ≥0.31 μL / mL and ≥0.15 μL / mL, respectively; When the composition comprises rose essential oil and galangal essential oil, their concentrations are ≥0.31 μL / mL and ≥2.5 μL / mL, respectively; When the composition comprises Alpinia oxyphylla essential oil and Alpinia officinalis essential oil, their concentrations are ≥0.15 μL / mL and ≥1.25 μL / mL, respectively.

3. Use of a plant essential oil composition in the preparation of an antibacterial drug, characterized in that: The plant essential oil composition comprises rose essential oil and Alpinia oxyphylla essential oil, or rose essential oil and Alpinia officinalis essential oil, or Alpinia oxyphylla essential oil and Alpinia officinalis essential oil; the mass ratio of the essential oils is 1:(1-2); The bacteria inhibited by the composition are Staphylococcus aureus, Escherichia coli and / or Pseudomonas aeruginosa; When the composition is rose essential oil and Alpinia oxyphylla essential oil, their concentrations are ≥0.31 μL / mL and ≥0.15 μL / mL, respectively; When the composition comprises rose essential oil and galangal essential oil, their concentrations are ≥0.31 μL / mL and ≥2.5 μL / mL, respectively; When the composition comprises Alpinia oxyphylla essential oil and Alpinia officinalis essential oil, their concentrations are ≥0.15 μL / mL and ≥1.25 μL / mL, respectively.

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