Plant essential oil composition and application thereof

Abstract

The invention discloses a plant essential oil composition and application thereof. The plant essential oil composition provided by the invention consists of essential oil A and essential oil B, the essential oil A is selected from rose essential oil or fructus alpiniae oxyphyllae essential oil, and the essential oil B is selected from fructus alpiniae oxyphyllae essential oil or rhizoma galangae essential oil. The essential oil composition provided by the invention has a relatively good inhibition effect on pathogenic bacteria, and every two essential oil components have a synergistic antibacterial effect, so that the concentration and the dosage of the essential oil are greatly reduced, and a relatively good antibacterial effect can be achieved by compounding relatively few components. The adopted plant essential oil raw materials are all medicinal and edible plants, are natural, safe and non-toxic to a human body, after compounding, the concentration of the essential oil can be reduced, the antibacterial effect cannot be affected, the cost is reduced, the use safety of the essential oil is improved, and the essential oil is further used for preparing antibacterial drugs and has a good application prospect. The generation of drug resistance and side effects of antibiotic antibacterial drugs can be avoided, and a method and a thought are provided 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 plant volatile oils, are oily mixtures extracted from certain parts of plants (rhizomes, fruits, seeds, flowers, leaves, etc.) using different extraction methods. They have a unique smell and are easily volatile. They are secondary metabolites of plants. Their main components include polyphenols, terpenes, flavonoids, alkaloids, etc. Plant essential oils are natural products with low content, but they are important components for Chinese medicine to exert its efficacy. Plant essential oils have 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 show good antibacterial effects. Compared with chemical antibacterial drugs, plant essential oils have the advantages of being green, non-toxic, natural, and having high nutritional value. Therefore, in recent years, the antibacterial activity of plant essential oils and their application in the fields of medicine and food industry have attracted much attention and have attracted extensive research.

[0003] At present, antibacterial drugs or antibacterial agents with high antibacterial effects all contain antibiotic ingredients, which have strong antibacterial effects, but 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, and their cost is relatively high. Due to the narrow antibacterial spectrum and poor effect of a single plant essential oil, 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 for use, 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, trigger allergic reactions, and even cause 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 high concentrations, potential safety hazards, the need to compound multiple plant essential oils in their essential oil formulations, and 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 can effectively inhibit bacteria, have low concentrations, few components, and do not require the addition of other synergists, so as to provide methods and ideas for preparing 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 an essential oil A and an 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, namely, turmeric, galangal, alpinia oxyphylla, tsaoko, kaempferia galanga and rose, as research objects, extracts them by steam distillation to obtain six plant essential oils, verifies the antibacterial activity of the six single essential oils by antibacterial zone test and MIC test, and shows that the six medicinal and edible essential oils have the best antibacterial effect on Staphylococcus aureus, among which tsaoko essential oil has the best antibacterial effect. Further, the combination of compound essential oils is studied by joint drug sensitivity test, and the plant essential oil composition with synergistic antibacterial effect is obtained: 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 causing 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 oils. 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 that are both medicinal and edible, are natural and safe substances, are non-toxic to the human body, can be used for the preparation of antibacterial drugs, and avoid the drug resistance and side effects of antibiotics.

[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 volatile oil are aromatic turmeric flavonoids and curcumone. As one of the medicinal and edible substances, turmeric has a variety of pharmacological activities, such as anti-tumor, anti-inflammatory, anti-fibrosis, antioxidant, and neuroprotective.

[0015] Alpinia officinarum Hance is a plant of the Zingiberaceae family, native to China. The chemical components of Alpinia officinarum Hance are mainly divided into three categories: flavonoids, diphenylheptanes and volatile oil components, among which the volatile oil components mainly include sesquiterpenes and hemiterpenes, including β-pinene, 1,8-cineole, etc. Alpinia officinarum Hance not only has ornamental value, but also has medicinal value. It has certain therapeutic effects on many diseases, such as tumors, gastric ulcers, and cardiovascular and cerebrovascular diseases.

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

[0017] Kaempferia galanga L., also known as sand ginger, is a plant of the genus Kaempferia in the ginger family. It is often cultivated for seasoning or medicinal purposes. The volatile oil of Kaempferia rhizome contains a variety of ingredients, mainly borneol, ethyl cinnamate, ethyl p-methoxycinnamate, etc. Kaempferia is a spice and natural Chinese herbal medicine. Its rhizome can be used as a seasoning or as a medicine. It has sedative, analgesic and antioxidant effects.

[0018] Tsaoko (Amomum tsaoko Crevost & Lem.) is an important edible and medicinal plant of the Zingiberaceae family. It can not only remove the fishy smell of food, but also add flavor to food. It is a daily spice. In addition, it has pharmacological activities such as antibacterial, gastrointestinal function regulation, anti-gastric ulcer, and analgesia, and can be widely used in the food industry and the treatment of certain diseases.

[0019] Rose (Rosa rugosa Thunb.) is a plant of the genus Rosa in the family Rosaceae. The main components of rose essential oil are citronellol, linalool and eugenol. In addition to its ornamental and nutritional value, rose also has high medicinal value, with whitening, anti-depression, calming nerves and gallbladder, calming emotions, and promoting sleep.

[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 comprising a plant essential oil composition.

[0022] In particular, the present invention shows that due to the synergistic antibacterial effect between essential oil A and essential oil B, by reducing the concentration of essential oil A and essential oil B in the product by 4-8 times (i.e., reducing 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 oil. 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 oil A and essential oil B in the specific product is calculated, 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 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 the two essential oil components are compounded, they have a good antibacterial effect, and 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, and the use safety of the plant essential oil can be improved, so as to provide methods and ideas for preparing more green, natural, safe and effective antibacterial drugs. At the same time, the plant essential oil raw materials used in the present invention are all plants with the same origin of medicine and food, which are natural, safe, and have no toxic effects on the human body. They are further used for the preparation of antibacterial drugs, and can avoid the drug resistance and side effects of antibiotic antibacterial drugs, and provide more low-concentration, few components, 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.

[0034] Figure 2 Fluorescence staining 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 the bacterial morphology after treatment with the compound essential oil composition. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. 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, the reagents and materials used in the following examples are commercially available.

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

[0039] 1. Extraction of active ingredients

[0040] The medicinal and edible plants: turmeric rhizomes, galangal rhizomes, yi xiao ren fruits, kaempferia rhizomes, tsaoko fruits and rose petals were extracted by steam distillation. The specific method is as follows: turmeric, galangal, yi xiao ren, kaempferia, tsaoko and rose were crushed by a high-speed multifunctional grinder to obtain drug powder; 500g of drug powder was weighed and poured into a 10L flask, 5L of deionized water was added and soaked for a period of time, and then a distillation device was set up for extraction, and the timing was started when it was heated to boiling. The temperature was adjusted to keep the drug 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 with anhydrous sodium sulfate to obtain turmeric essential oil, galangal essential oil, yi xiao ren essential oil, kaempferia essential oil, tsaoko essential oil and rose essential oil, respectively. Finally, the obtained essential oil was placed in a 4℃ 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 for a long time at -80°C, pick a small amount of each bacteria with an inoculation loop, inoculate it into a sterilized blank LB solid culture medium by streaking, and place it in a 37°C incubator for 24 hours until a monoclonal colony appears. Use a sterilized pipette tip to pick a monoclonal colony and place it in a 15mL centrifuge tube filled with blank LB liquid culture medium, and place the 15mL centrifuge tube in a constant temperature shaker for enrichment culture for subsequent experiments.

[0043] 3. Inhibition zone test

[0044] Take the enriched cultured Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa bacterial suspensions and dilute them 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, draw an appropriate amount of bacterial solution into the blank LB broth solid culture medium, and evenly apply the bacterial solution with a disposable sterile applicator; after the culture medium is blown dry, add a circular filter paper. Finally, draw 10μL of essential oil and drop it on the filter paper. Set three parallel controls for each essential oil, and place the culture dish upside down in a constant temperature incubator for 24 hours. Take out the culture dish the next day and observe whether there is an inhibition zone. If so, measure the diameter of the inhibition zone. If not, use the value 0 to represent it. The diameter of the filter paper used is 5.5mm.

[0045] 4. MIC determination

[0046] The MIC values ​​of six single essential oils, namely, turmeric essential oil, galangal essential oil, yizhiren essential oil, kaempferia 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 into different concentration gradients as experimental concentrations by multiple dilution method, and the dilution solvent was blank LB broth liquid culture medium; and the three tested strains were diluted to 10 6 CFU / mL. Use a pipette to draw 50μL of gradient concentration essential oil into a 96-well cell culture plate, and add 50μL of Staphylococcus aureus / Escherichia coli / Pseudomonas aeruginosa bacterial suspension to ensure that the total volume of each well is 100μL; set up negative control wells (50μL essential oil + 50μL blank LB broth liquid culture medium) and positive control wells (50μL bacterial solution + 50μL 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 it for 24 hours, then take it out for observation, and take the essential oil concentration corresponding to the well where the test bacteria do not grow as the MIC value.

[0047] 5. Antibacterial activity

[0048] The inhibition zone results of six kinds of 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 Amomum villosum essential oil is the largest, reaching 15.60±0.54mm, indicating that Amomum villosum essential oil has the best inhibitory effect on Staphylococcus aureus; in addition, Amomum villosum essential oil and rose essential oil have better inhibition on Escherichia coli, and the diameters of the inhibition zones are 9.77±0.46mm and 9.32±0.38mm, respectively, followed by Alpinia galanga essential oil, turmeric essential oil and Kaempferia galanga 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 galanga 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 the six essential oils have a certain inhibitory effect on pathogenic bacteria.

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

[0050]

[0051] The results of the minimum inhibitory concentration (MIC) values ​​of the six edible-medicine homologous single essential oils against three pathogenic bacteria, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, are shown in Table 2, showing that the six edible-medicine homologous essential oils, namely turmeric essential oil, galangal essential oil, schizonepeta alpiniae essential oil, kaempferia galanga essential oil, tsaoko essential oil and rose essential oil, have the best inhibitory effect on Staphylococcus aureus, while the inhibitory effect on Escherichia coli and Pseudomonas aeruginosa is relatively poor. The data show that among the above six edible-medicine homologous essential oils, Pseudomonas aeruginosa is more sensitive to rose essential oil and galangal essential oil, while it is less sensitive to kaempferia essential oil, turmeric essential oil, schizonepeta alpiniae essential oil and tsaoko essential oil. Combined with the results of the inhibition zone test and MIC test in Table 1, four edible-medicine homologous essential oils with strong antibacterial activity were screened, namely galangal essential oil, schizonepeta alpiniae essential oil, tsaoko essential oil and rose essential 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, alkyl 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 combinations of compound essential oils 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 dilute the essential oil solutions with four concentration gradients, and the concentration gradient was set to 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 chessboard dilution method, the essential oils (concentrations of 1MIC, 1 / 2MIC, 1 / 4MIC, and 1 / 8MIC) were combined in pairs on a sterile 96-well cell culture plate, namely, six combinations of tsaoko essential oil + rose essential oil, tsaoko essential oil + alkyl oxyphylla essential oil, tsaoko essential oil + galangal essential oil, rose essential oil + alkyl oxyphylla essential oil, rose essential oil + galangal essential oil, and alkyl oxyphylla essential oil + galangal essential oil. 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, add 25 μL of essential oils A and B with set concentrations to the corresponding wells respectively. Mix the two essential oils, and then add 50 μL of diluted Staphylococcus aureus bacterial solution, ensuring that the volume of the liquid added to each well is the same. Set three replicate wells as parallel controls for each different compound concentration. After placing the sterile 96-well plate in an incubator at 37 °C for 24 hours, take it out, observe, and read the results. The essential oil concentration corresponding to the well where the test bacteria do not grow is the MIC value. Observe the clarity of the solution in each well and determine the antibacterial effect strength and action type of the compound essential oil 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 additive or synergistic effects, and there are no irrelevant or antagonistic effects. 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 / 8 MIC): semen alpiniae oxyphyllae essential oil (1 / 4 MIC), rose essential oil (1 / 8 MIC): galangal essential oil (1 / 4 MIC), and semen alpiniae oxyphyllae essential oil (1 / 4 MIC): galangal essential oil (1 / 8 MIC). The results show that in the process of the interaction of essential oils, it plays a role in synergistic enhancement, can expand the antibacterial spectrum, and reduce the concentration and usage amount 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 effect obtained in Example 2 were named as compound essential oil composition 1, whose concentration ratio was rose essential oil (1 / 8MIC): Alpinia oxyphylla essential oil (1 / 4MIC), and the specific concentration was 0.3125:0.15625 μL / mL; the compound essential oil composition 2, whose concentration ratio was rose essential oil (1 / 8MIC): Alpinia galanga essential oil (1 / 4MIC), and the specific concentration was 0.3125:2.5 μL / mL; the compound essential oil composition 3, whose concentration ratio was Alpinia oxyphylla essential oil (1 / 4MIC): Alpinia galanga essential oil (1 / 8MIC), and the specific concentration was 0.15625:1.25 μL / mL.

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

[0075] After 24 hours, blow the solution in the centrifuge tube evenly and take an appropriate amount of bacterial solution, dilute it 200,000 times with blank LB broth liquid culture medium and use a pipette to draw 50 μL into blank LB broth solid culture medium, use a disposable coating stick to spread the solution evenly, then place the culture dish in a constant temperature incubator and culture for 24 hours, finally take out and observe the number of monoclonal colonies in each group of culture dishes 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 coating, while the monoclonal colonies in the culture dishes treated with the three compound essential oils were significantly reduced, indicating that the above three compound essential oils showed a strong antibacterial effect and could inhibit the growth and reproduction of Staphylococcus aureus. In addition, the number of monoclonal colonies in each group of culture dishes was ranked as compound essential oil 1 < compound essential oil 2 < compound essential oil 3, so among the above three compound essential oils, 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 liquid was centrifuged at 25°C and 5000rpm for 15min, the culture medium was discarded and washed three times with TBST buffer, and then 500μL TBST buffer was added to resuspend the liquid. 500μL compound essential oil (250μL bacterial liquid A + 250μL essential oil B) was added to the experimental group. A blank culture medium (500μL blank LB broth liquid culture medium + 500μL Staphylococcus aureus liquid) was set as the negative control group, and the centrifuge tube was placed in a constant temperature incubator for co-culture for 24 hours.

[0079] The incubated bacterial solution was centrifuged at 25°C and 5000rpm for 15min, washed three times with TBST buffer, and then resuspended in 1mL TBST buffer. Equal amounts of SYTO 9 and PI were mixed in a centrifuge tube to obtain a dye premix, and 3μL of the dye premix was added to each group of bacterial suspensions. After mixing, the mixture was incubated at room temperature in the dark for 15min, and then 5μL of the dyed bacterial solution was pipetted with a pipette and dropped onto a glass slide, covered with a coverslip, and the bacterial fluorescence image was taken with an inverted fluorescence microscope. Finally, Image J was used to fit the fluorescence image and Image Pro Plus was used to calculate the fluorescence intensity. The proportion of live bacteria was analyzed to evaluate the antibacterial effect of the three compound essential oils.

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

[0081] Live bacteria ratio = green fluorescence intensity / (green fluorescence intensity+red fluorescence intensity)×100%

[0082] The live / dead bacteria staining results after co-culture of three kinds of compound essential oils with Staphylococcus aureus are as follows Figure 2As shown in the figure, the green fluorescence represents live bacteria obtained by SYTO 9 staining, while the red fluorescence represents dead bacteria obtained by PI staining. By observing the fluorescence graph, it can be found that the green fluorescence of the negative control group is strong, 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 strong, and the green fluorescence is weak, indicating that most of the bacteria in the liquid are dead bacteria. In addition, by fitting the fluorescence images and calculating the fluorescence intensity using relevant software, 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 for sterilization. Use the plate streak method to dip the activated bacterial solution (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa) into LB broth solid culture medium, place it in a constant temperature incubator and culture it for 24 hours to obtain monoclonal colonies; use a sterilized pipette tip to pick up 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, and 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 with LB broth liquid culture medium, and 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, and a negative control group (500 μL blank LB broth liquid culture medium + 500 μL of each bacterial suspension) was set up, and the 2 mL centrifuge tube was placed in a constant temperature incubator for incubation for 24 hours. After the incubation, centrifuge at 25 ° C, 5000 rpm for 15 minutes, and washed three times with phosphate buffered saline (PBS); then fix the bacteria at room temperature for 2 hours with 2.5% glutaraldehyde solution. After fixation, wash three times with PBS buffer, centrifuge at 25 ° C, 5000 rpm for 15 minutes, discard the supernatant, and dehydrate the bacteria with gradient ethanol (30%, 50%, 70%, 90%, 100%), and let each level 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, the untreated bacteria have a relatively complete and regular morphology, Staphylococcus aureus has a complete spherical structure, while Escherichia coli and Pseudomonas aeruginosa 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 more obviously damaged. The spherical structure of Staphylococcus aureus is severely damaged, showing an irregular structure, and the bacteria are completely adhered to each other. The surfaces of Escherichia coli and Pseudomonas aeruginosa treated with the three compound essential oils are relatively rough, with a small amount of pits and cracks, and the bacteria cannot maintain a complete rod-shaped structure due to severe adhesion between the bacteria.

[0087] The above results show that the three compound essential oils cause irreversible damage to the bacterial morphology, causing 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, which leads to an increase in the permeability of the cell membrane, followed by the leakage of the bacterial cell contents, which inhibits the growth and reproduction of bacteria. It can be seen that the three compound essential oils have good antibacterial effects, and there is a synergistic antibacterial effect between the essential oil components. The compounding of two essential oil components has a good antibacterial effect. Under the premise of not affecting the antibacterial effect, it can greatly reduce the concentration and dosage of essential oils, reduce costs, and improve 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.

[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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A plant essential oil composition, characterized in that It is composed of A essential oil and B essential oil; The A essential oil is selected from rose essential oil or Alpinia oxyphylla essential oil, and the B essential oil is selected from Alpinia oxyphylla essential oil or Alpinia officinalis essential oil; the mass ratio of the A essential oil to the B essential oil is 1:(1-2).

2. Use of the composition according to claim 1 in the preparation of antibacterial products.

3. An antibacterial product, characterized in that: The invention comprises the composition according to claim 1.

4. The product according to claim 3, characterized in that: The concentrations of essential oil A and essential oil B in the product are both ≥0.15 μL / mL.

5. The product according to claim 4, characterized in that: 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.

6. The product according to claim 4, characterized in that: 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.

7. The product according to claim 4, characterized in that: 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.

8. Use of the composition according to claim 1 in the preparation of antibacterial drugs.

9. An antibacterial drug, characterized in that: The invention comprises the composition according to claim 1.

10. The use according to claim 2 or 8, characterized in that: The bacteria inhibited by its products or drugs are Staphylococcus aureus, Escherichia coli and / or Pseudomonas aeruginosa.

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