A plant essential oil composition with antibacterial activity and its preparation method and application

Abstract

The invention discloses a plant essential oil composition with antibacterial activity and a preparation method thereof. The plant essential oil composition is composed of a clove light oil component and a cinnamon heavy oil component in a volume ratio of 3.3:1 to 9.5:1. The invention finds through a large number of experimental studies that the combination of undistilled and refined clove essential oil and cinnamon essential oil has a poor antibacterial effect and has no obvious synergistic effect, while the combination of the clove light oil component and the cinnamon heavy oil component obtained by distillation and refining in a volume ratio of 3.3:1, 8:1 or 9.5:1 has a good synergistic antibacterial effect on Corynebacterium fasciatum, Enterococcus faecium and Escherichia coli, respectively, and can be used to prepare antibacterial drugs.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine, and in particular, relates to a plant essential oil composition with antibacterial activity and a preparation method and application thereof. Background Art

[0002] Plant essential oils mainly refer to volatile components from plant flowers, leaves, stems, seeds, fruits, roots or skins. They have antibacterial, antioxidant, anti-tumor and other biological activities, and have good application prospects in the pharmaceutical and food industries.

[0003] Clove is the dried flower bud of Eugenia caryophyllata Thunb., a plant of the Myrtaceae family, and is used in the fields of medicine, spices, food preservatives, etc. Volatile oil is one of its main active ingredients, with biological activities such as antiviral, antibacterial, anticancer, antioxidant and anti-inflammatory.

[0004] Cinnamon is the dried bark of Cinnamomum cassia Presl, a plant of the Lauraceae family. It contains a large amount of volatile oil and has a fragrant smell, and is often used as a spice. At the same time, volatile oil, as one of its main active ingredients, has biological activities such as antibacterial, insecticidal, and anti-cancer.

[0005] The clove-cinnamon drug pair can enhance the warming-yang and expelling-cold effects of both, but there is currently no report on the antibacterial application of the combination of clove light oil component and cinnamon heavy oil component. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to screen out the best volume ratio of clove light oil component and cinnamon heavy oil component through a large number of experiments for combination to achieve synergistic antibacterial application.

[0007] Technical solution: In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0008] The invention discloses a plant essential oil composition with antibacterial activity, which consists of a clove light oil component and a cinnamon heavy oil component.

[0009] As a preferred embodiment, in the above-mentioned plant essential oil composition with antibacterial activity, the volume ratio of the clove light oil component to the cinnamon heavy oil component is 3.3:1 to 9.5:1.

[0010] As a more preferred embodiment, in the above-mentioned plant essential oil composition with antibacterial activity, the volume ratio of the clove light oil component to the cinnamon heavy oil component is 3.3:1, 4:1, 8:1 or 9.5:1.

[0011] As a particularly preferred embodiment, in the above-mentioned plant essential oil composition with antibacterial activity, the volume ratio of the clove light oil component to the cinnamon heavy oil component is 3.3:1, 8:1 or 9.5:1.

[0012] The plant essential oil composition with antibacterial activity of the present invention, wherein the clove light oil component is prepared by the following method:

[0013] Clove essential oil extraction: Clove medicinal powder is soaked in water, extracted by steam distillation, centrifuged, essential oil is collected, and stored away from light;

[0014] Clove essential oil refining: Clove essential oil is loaded into a molecular distillation feed bottle, liquid nitrogen is loaded into a cold trap, the refrigeration device is turned on, distillation is performed, and clove light oil components are collected;

[0015] The cinnamon heavy oil component is prepared by the following method:

[0016] Extraction of cinnamon essential oil: cinnamon powder, add water, soak, extract by steam distillation, centrifuge, collect essential oil, and store in dark place;

[0017] Cinnamon essential oil refining: cinnamon essential oil is put into a molecular distillation feed bottle, liquid nitrogen is put into the cold trap, the refrigeration device is turned on, distillation is carried out, and the cinnamon heavy oil component is collected.

[0018] As a preferred embodiment, the above-mentioned plant essential oil composition with antibacterial activity, wherein the clove light oil component is prepared by the following method:

[0019] Clove essential oil extraction: add 12 times the amount of water to a certain amount of clove powder, soak for 1.5 hours, steam distill for 5 hours, centrifuge, collect the essential oil, and store at 4°C away from light;

[0020] Clove essential oil refining: Clove essential oil is loaded into a molecular distillation feed bottle, liquid nitrogen is loaded into a cold trap, the refrigeration device is turned on, the temperature of the reflux water device is set to 5°C, the scraping speed is 240r / min, the material flow rate is 1mL / min, the distillation temperature is 110°C, the distillation pressure is 10Pa, and the clove light oil component is collected;

[0021] The cinnamon heavy oil component is prepared by the following method:

[0022] Extraction of cinnamon essential oil: a certain amount of cinnamon powder, add 10 times the amount of water, soak for 1 hour, steam distill for 2 hours, centrifuge, collect the essential oil, and store at 4°C away from light;

[0023] Cinnamon essential oil refining: cinnamon essential oil is loaded into a molecular distillation feed bottle, liquid nitrogen is loaded into the cold trap, the refrigeration device is turned on, the temperature of the reflux water device is set to 5°C, the scraping speed is 240r / min, the material flow rate is 1mL / min, the distillation temperature is 70°C, the distillation pressure is 90Pa, and the cinnamon heavy oil component is collected.

[0024] The present invention screens antibacterial experiments of volatile oils of different proportions and finds that the clove light oil component and the cinnamon heavy oil component combined in a specific volume ratio have significant antibacterial activity against Enterococcus faecium, Corynebacterium fasciatum, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and the like. In particular, it has a good synergistic effect on Enterococcus faecium, Corynebacterium fasciatum and Escherichia coli, and achieves a very good and unexpected technical effect.

[0025] The invention can prepare the clove light oil component and the cinnamon heavy oil component with a pharmaceutically acceptable carrier into tablets, granules, capsules, pills, powders, ointments, oral liquids or injections.

[0026] Beneficial effects:

[0027] The present invention has found that the combination of undistilled and refined clove essential oil and cinnamon essential oil has poor antibacterial effect and no obvious synergistic effect, while the combination of clove light oil component and cinnamon heavy oil component obtained by distillation and refining at a volume ratio of 3.3:1, 8:1 or 9.5:1 has a synergistic antibacterial effect. In particular, it has a good synergistic effect on Enterococcus faecium, Corynebacterium fasciatum and Escherichia coli, and can be used to prepare antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the MIC determination chart of clove light oil component against Enterococcus faecalis.

[0029] Figure 2 This is the MIC determination chart of clove light oil component against Staphylococcus aureus and Corynebacterium striatum.

[0030] Figure 3 This is the MIC determination chart of clove light oil component against Escherichia coli.

[0031] Figure 4 This is a graph showing the MIC determination of clove light oil components against Klebsiella pneumoniae.

[0032] Figure 5 This is a graph showing the MIC determination of cinnamon heavy oil components against Enterococcus faecium.

[0033] Figure 6 This is a graph showing the MIC determination of cinnamon heavy oil components against Staphylococcus aureus and Corynebacterium striatum.

[0034] Figure 7 This is a graph showing the MIC determination of cinnamon heavy oil components against Escherichia coli.

[0035] Figure 8 This is a graph showing the MIC determination of cinnamon heavy oil components against Klebsiella pneumoniae.

[0036] Fig. 9This is a graph showing the MIC determination of a combination of a light clove oil component and a heavy cinnamon oil component against Enterococcus faecium.

[0037] Fig.10 This is a graph showing the MIC determination of a combination of a light clove oil component and a heavy cinnamon oil component against Staphylococcus aureus.

[0038] Fig.11 This is a graph showing the MIC determination of a combination of a light clove oil component and a heavy cinnamon oil component against Corynebacterium vesiculosus.

[0039] Fig.12 This is a graph showing the MIC determination of a combination of a light clove oil component and a heavy cinnamon oil component against Escherichia coli.

[0040] Fig.13 This is a graph showing the MIC determination of a combination of a light clove oil component and a heavy cinnamon oil component against Klebsiella pneumoniae. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with specific implementations, but these embodiments should not be construed as limiting the present invention.

[0042] Example 1

[0043] The invention discloses a plant essential oil composition with antibacterial activity, which consists of a clove light oil component and a cinnamon heavy oil component.

[0044] (1) The clove light oil component is prepared by the following method:

[0045] (1.1) Extraction of clove essential oil: Take a certain amount of clove powder, add 12 times the amount of water, soak for 1.5 hours, extract by steam distillation for 5 hours, centrifuge, collect clove essential oil, and store at 4°C away from light;

[0046] (1.2) Clove essential oil refining: Clove essential oil was loaded into a molecular distillation feed bottle, liquid nitrogen was loaded into a cold trap, the refrigeration device was turned on, the temperature of the reflux water device was set to 5°C, the scraping speed was 240r / min, the material flow rate was 1mL / min, the distillation temperature was 110°C, the distillation pressure was 10Pa, and the clove light oil component was collected;

[0047] (2) The cinnamon heavy oil component is prepared by the following method:

[0048] (2.1) Extraction of cinnamon essential oil: Take a certain amount of cinnamon powder, add 10 times the amount of water, soak for 1 hour, extract by steam distillation for 2 hours, centrifuge, collect cinnamon essential oil, and store at 4°C away from light;

[0049] (2.2) Refining of cinnamon essential oil: Cinnamon essential oil was loaded into a molecular distillation feed bottle, liquid nitrogen was loaded into the cold trap, the refrigeration device was turned on, the temperature of the reflux water device was set to 5°C, the scraping speed was 240r / min, the material flow rate was 1mL / min, the distillation temperature was 70°C, the distillation pressure was 90Pa, and the cinnamon heavy oil component was collected.

[0050] Example 2 Determination of Minimum Inhibitory Concentration (MIC) of Clove and Cinnamon Essential Oils

[0051] 1. Activation of bacterial strains: Use the streak method to inoculate the bacterial strains onto the sterilized culture medium, and culture the bacteria at 35°C for 24 hours to obtain the activated test strains for use.

[0052] 2. Pretreatment of the test solution: The clove light oil component and the cinnamon heavy oil component were sterilized by filtration through a 0.22 μM filter membrane, and the clove light oil component was diluted to 64 μL / mL and the cinnamon heavy oil component was diluted to 32 μL / mL with a 1% Tween-80 distilled water solution for later use.

[0053] 3. Preparation of test bacterial suspension: Bacteria: Take a single colony and dilute it with sterile 0.45% saline to a 0.5 McFarland unit bacterial suspension, that is, the bacterial suspension concentration is about 10 8 cfu / mL. The above bacterial solution was diluted 1000 times with sterile saline to obtain a standby bacterial suspension with a concentration of about 1×10 5 cfu / mL.

[0054] 4. MIC determination: MIC was determined by microbroth dilution method (96-well plate method). 50 μL of sterilized MH broth medium (bacteria) was added to each well of the 96-well plate; the first column was the positive control, and 50 μL of MH broth medium and test bacterial solution (bacteria) were added; the second column was the negative control, and 50 μL of MH broth medium and Tween 80 (bacteria) were added; 50 μL of the test bacterial solution was added to each well of columns 3-12, and then 50 μL of the essential oil diluted in succession was added in sequence, where the concentrations of the clove light oil component were 16, 8, 4, 2, 1, The concentrations of the cinnamon heavy oil components were 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03 and 0.15 μL / mL respectively. After the drug and bacterial solution were loaded, the plate was covered and the bacteria were placed in a 35°C incubator for 24 hours. The minimum inhibitory concentration of the drug was determined as the lowest concentration at which the culture medium was clear and no bacterial growth was observed by naked eye.

[0055] 5. Evaluation of antibacterial activity of clove light oil component and cinnamon heavy oil component

[0056] Based on the MIC values of the two essential oils, a series of concentrations of essential oils were prepared. The checkerboard method was used, namely 2MIC, 1 / MIC, 1 / 2MIC, 1 / 4MIC, 1 / 8MIC, 1 / 16MIC, 1 / 32MIC, 1 / 64MIC, and they were combined pairwise. The fractional inhibitory concentration index FICI was used as the judgment basis.

[0057] FICI = MIC A (Combined) / MIC A (Used alone)+MIC B (Combined) / MIC B (Used alone)

[0058] The judgment criteria for FICI are as follows: when FICI ≤ 0.5, there is a synergistic effect between the two drugs; when 0.5 < FICI ≤ 1.0, there is an additive effect between the two drugs; when 1.0 < FICI < 4.0, there is no relevant effect between the two drugs; when FICI ≥ 4.0, the two drugs have an antagonistic effect. Based on the FICI judgment criteria, the optimal compound ratio of the drugs was calculated using the test drug combination with the smallest FICI value.

[0059] 6. Experimental results

[0060] 6.1 The MIC determination values of the light clove oil component and the heavy cinnamon oil component are shown in Table 1 and Figures 1 to 8 as follows.

[0061] Table 1 MIC (μL / mL) of the light clove oil component and the heavy cinnamon oil component

[0062] bacteria Clove light oil Cinnamon Heavy Oil Enterococcus faecium 2.00±0.00 0.25±0.00 Corynebacterium striatum 1.67±0.58 0.50±0.00 Staphylococcus aureus 2.00±0.00 0.50±0.00 Escherichia coli 2.00±0.00 0.21±0.06 Klebsiella pneumoniae 2.00±0.00 0.125±0.000 .

[0063] 6.2 Optimal compound FIC index of the light clove oil component and the heavy cinnamon oil component

[0064] 6.2.1 The determination values of the FIC index for the optimal compound to inhibit bacteria are shown in Table 2, Table 3 and Figures 9 to 13 as follows.

[0065] Table 2 FIC index of the optimal compound of the light clove oil component and the heavy cinnamon component

[0066]

[0067] Table 3 Optimal compound effect of each volume ratio of the light clove oil component and the heavy cinnamon component

[0068] strain Clove Enhancement Multiple Cinnamon booster Compounding effect Optimum volume ratio Enterococcus faecium 4 4 Collaboration 8∶1 Staphylococcus aureus 2 2 Add 4∶1 Corynebacterium striatum 4 4 Collaboration 3.3∶1 Escherichia coli 4 4 Collaboration 9.5∶1 Klebsiella pneumoniae 2 64 Add 512∶1 .

[0069] The above experimental results show that the present invention has found that the clove light oil component and the cinnamon heavy oil component obtained by re-distillation and refining, especially the combination of the two in a volume ratio of 3.3:1, 8:1 or 9.5:1, have a good synergistic antibacterial effect on Corynebacterium striatum, Enterococcus faecalis and Escherichia coli, and can be used to prepare antibacterial drugs.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a clove light oil component and a cinnamon heavy oil component in a volume ratio of 3.3:1, 8:1 or 9.5:1 in the preparation of drugs against Corynebacterium fasciatum, Enterococcus faecium or Escherichia coli; The clove light oil component is prepared by the following method: Clove essential oil extraction: add 12 times the amount of water to a certain amount of clove powder, soak for 1.5 hours, steam distill for 5 hours, centrifuge, collect the essential oil, and store at 4°C away from light; Clove essential oil refining: Clove essential oil is loaded into a molecular distillation feed bottle, liquid nitrogen is loaded into a cold trap, the refrigeration device is turned on, the temperature of the reflux water device is set to 5°C, the scraping speed is 240r / min, the material flow rate is 1mL / min, the distillation temperature is 110°C, the distillation pressure is 10Pa, and the clove light oil component is collected; The cinnamon heavy oil component is prepared by the following method: Extraction of cinnamon essential oil: a certain amount of cinnamon powder, add 10 times the amount of water, soak for 1 hour, steam distill for 2 hours, centrifuge, collect the essential oil, and store at 4°C away from light; Cinnamon essential oil refining: cinnamon essential oil is loaded into a molecular distillation feed bottle, liquid nitrogen is loaded into the cold trap, the refrigeration device is turned on, the temperature of the reflux water device is set to 5°C, the scraping speed is 240r / min, the material flow rate is 1mL / min, the distillation temperature is 70°C, the distillation pressure is 90Pa, and the cinnamon heavy oil component is collected.

2. The use according to claim 1, characterized in that: The clove light oil component and the cinnamon heavy oil component are prepared with a pharmaceutically acceptable carrier into a spray, tablet, granule, capsule, pill, powder, ointment, mixture or injection.

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