Application of clove light oil component and cinnamon heavy oil component in combination in preparing anti-Aspergillus niger medicine

Abstract

The invention discloses a plant essential oil composition with anti-Aspergillus 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 12.5:1 to 0.39:1. The invention finds through a large number of experimental studies that the clove light oil component and the cinnamon heavy oil component obtained by re-distillation and refining, especially in a volume ratio of 3.1:1, 12.5:1 and 0.78:1, respectively, have a good synergistic effect on Aspergillus flavus, Aspergillus terreus and Aspergillus fumigatus, and can be used to prepare an anti-Aspergillus drug.

Description

Technical Field

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

[0002] Aspergillus is widely distributed in nature and is a conditional pathogen. It can invade the human body when the human immune function declines, causing invasive aspergillosis, which has become the second deep fungal infection after candidiasis. Invasive aspergillosis has a high incidence, low early diagnosis rate, and a mortality rate of up to 30%-95%, which seriously endangers human health and imposes a huge economic burden on both the country and individuals. Aspergillus fumigatus is the main pathogenic fungus of aspergillosis, accounting for 46%-60% of Aspergillus infections, followed by Aspergillus flavus, Aspergillus niger and Aspergillus terreus. The commonly used drugs for the treatment of Aspergillus infections are polyenes, azoles and echinocandins. Polyenes are highly toxic and echinocandins are expensive, making azoles the first-line drugs for the treatment of Aspergillus infections. However, studies have shown that Aspergillus' resistance to azoles has gradually increased, which has increased the difficulty of clinical treatment and seriously threatened the life safety of patients. Therefore, the development of new anti-Aspergillus agents has become a clinical problem that needs to be solved urgently.

[0003] Clove is the dried flower bud of Eugenia caryophyllata Thunb., a plant of the Myrtaceae family. It has the effects of warming the middle and lowering adverse reactions, tonifying the kidney and boosting yang. Clove contains a variety of chemical components, of which volatile oil is the main active ingredient, with a content of about 15%-20%. It has a variety of biological activities such as antibacterial, insect repellent, and antioxidant. Due to its antibacterial and antioxidant effects, clove volatile oil is directly used in the food industry as a natural preservative, reducing the use of synthetic preservatives, antioxidants, etc.

[0004] Cinnamon is the dried bark of Cinnamomum cassia Presl, a plant of the Lauraceae family, and is widely used in the pharmaceutical and food industries. Modern pharmacological studies have shown that cinnamon has antibacterial, antioxidant, and antiseptic biological activities. Cinnamon volatile oil is the main active ingredient in cinnamon, which has antibacterial and antipyretic pharmacological effects.

[0005] The clove-cinnamon drug pair originated from Dingguisan. Under the guidance of traditional Chinese medicine theory, long-term clinical practice has proved that the combination of the two is a classic cold-dispelling and analgesic drug pair. However, there is no report on the application of clove light oil component and cinnamon heavy oil component and their combined use in anti-Aspergillus. 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 use in anti-Aspergillus 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 anti-aspergillus 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 having anti-Aspergillus activity, the volume ratio of the clove light oil component to the cinnamon heavy oil component is 12.5:1 to 0.39:1.

[0010] As a preferred embodiment, in the above-mentioned plant essential oil composition with anti-Aspergillus activity, the volume ratio of the clove light oil component to the cinnamon heavy oil component is 12.5:1, 3.1:1, 0.78:1 or 0.39:1.

[0011] As a particularly preferred embodiment, in the above-mentioned plant essential oil composition having anti-Aspergillus activity, the volume ratios of the clove light oil component and the cinnamon heavy oil component are 12.5:1, 3.1:1, and 0.78:1.

[0012] The plant essential oil composition with anti-Aspergillus 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 anti-Aspergillus activity, the clove light oil component is prepared by the following method:

[0019] Clove essential oil extraction: add 10-12 times the amount of water to a certain amount of clove powder, soak for 1-1.5 hours, steam distill for 1-5 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: add 10-12 times the amount of water to a certain amount of cinnamon powder, soak for 1-1.5 hours, extract by steam distillation for 1-2 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 invention finds through a large number of experiments that the clove light oil component and the cinnamon heavy oil component combined in a specific volume ratio have significant activities against Aspergillus flavus, Aspergillus terreus, Aspergillus fumigatus or Aspergillus niger.

[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 anti-Aspergillus 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 12.5:1, 3:1 and 0.78:1 has a synergistic anti-Aspergillus effect. In particular, it has a good synergistic effect on Aspergillus flavus, Aspergillus terreus and Aspergillus fumigatus, and can be used to prepare an anti-Aspergillus drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the MIC determination chart of clove light oil component against Aspergillus flavus and Aspergillus terreus.

[0029] Figure 2 This is the MIC determination chart of clove light oil component against Aspergillus fumigatus and Aspergillus niger.

[0030] Figure 3 This is a graph showing the MIC determination of cinnamon heavy oil components against Aspergillus flavus and Aspergillus terreus.

[0031] Figure 4It is a graph showing the MIC determination of cinnamon heavy oil components against Aspergillus fumigatus and Aspergillus niger.

[0032] Figure 5 This is the MIC determination chart of the combination of clove light oil component and cinnamon heavy oil component against Aspergillus flavus.

[0033] Figure 6 This is a graph showing the MIC determination of a combination of a light clove oil component and a heavy cinnamon oil component against Aspergillus terreus.

[0034] Figure 7 This is a graph showing the MIC determination of a combination of a light clove oil component and a heavy cinnamon oil component against Aspergillus fumigatus.

[0035] Figure 8 This is a graph showing the MIC determination of a combination of a light clove oil component and a heavy cinnamon oil component against Aspergillus niger. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific implementations, but these embodiments should not be construed as limiting the present invention.

[0037] Example 1

[0038] A plant essential oil composition with anti-Aspergillus activity, the plant essential oil composition with anti-Aspergillus activity consists of a clove light oil component and a cinnamon heavy oil component.

[0039] (1) The clove light oil component is prepared by the following method:

[0040] (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;

[0041] (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;

[0042] (2) The cinnamon heavy oil component is prepared by the following method:

[0043] (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;

[0044] (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.

[0045] Example 2 Determination of Minimum Inhibitory Concentration (MIC) of Clove and Cinnamon Essential Oils

[0046] 1. Activation of bacterial strains: Use the streak method to inoculate the bacterial strains onto the sterilized culture medium, and culture the mold at 28°C for 48 hours to obtain the activated test strains for use.

[0047] 2. Pretreatment of test solution:

[0048] Take the clove light oil component prepared in step (1.2) of Example 1 above and the cinnamon heavy oil component prepared in step (2.2), filter and sterilize them through a 0.22 μM filter membrane, dilute the clove essential oil to 64 μL / mL and the cinnamon essential oil to 32 μL / mL with a 1% Tween-80 distilled water solution, and set aside.

[0049] 3. Preparation of test bacterial suspension: Pipette 5 mL of 0.85% saline and transfer it to Sabouraud agar (SDA) medium where Aspergillus colonies (A. flavus, A. terreus, A. fumigatus or A. niger) are grown. Gently wipe the colonies with a sterile cotton swab to form a suspension. Use a sterile pipette to transfer the Aspergillus suspension to a test tube, filter through 8 layers of sterile gauze to remove hyphae and culture medium residues, vortex oscillate for 15 seconds, dilute appropriately, and adjust the bacterial suspension to 1.8-2.2 McFarland units using a turbidimeter.

[0050] 4. MIC determination: The MIC was determined by the microbroth dilution method (96-well plate method). 50 μL of sterilized RPMI 1640 medium (fungus) was added to each well of the 96-well plate; the first column was the positive control, with 50 μL of RPMI 1640 medium and test bacterial solution added; the second column was the negative control, with 50 μL of RPMI 1640 medium and test bacterial solution added; 1640 medium and Tween 80, 50 μL each (fungus); 50 μL of the test bacterial solution was added to each well of the 3rd to 12th columns, and then 50 μL of the essential oil diluted in turn was added, wherein the concentrations of the clove light oil component were 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03 μL / mL, and the concentrations of the cinnamon heavy oil component were 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03, 0.15 μL / mL; after the drug and bacterial solution were loaded, the plate was covered and the Aspergillus was cultured at 28°C for 3 to 4 days; the lowest concentration at which the culture medium was clear and no bacterial growth was observed by naked eye was determined as the minimum inhibitory concentration of the drug, and at the same time, the OD was measured every 6 hours. 560nmMeasure the absorbance of the mold for determination.

[0051] 5. Evaluation of the antibacterial activities of the light clove oil component and the heavy cinnamon oil component

[0052] Based on the MIC values of the above two essential oils, a series of concentrations of essential oils were prepared and 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.

[0053] FICI = MIC A (Combined) / MIC A (Used alone)+MIC B (Combined) / MIC B (Used alone)

[0054] 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.

[0055] 6. Experimental results

[0056] 6.1. The antibacterial MIC results of the light clove oil component and the heavy cinnamon oil component are shown in Table 1 and Figures 1 to 4 as follows.

[0057] Table 1 MIC (μL / mL) of the light clove oil component and the heavy cinnamon oil component

[0058]

[0059] 6.2. The combined FIC indices and the compounding effects of various volume ratios of the light clove oil component and the heavy cinnamon oil component in inhibiting mold are shown in Table 2 and Table 3 and Figures 5 to 8 as follows.

[0060] Table 2 FIC indices of the optimal compounding of the light clove oil component and the heavy cinnamon oil component

[0061]

[0062] Table 3 Optimal compounding effects of various volume ratios of the light clove oil component and the heavy cinnamon oil component

[0063] strain Clove Enhancement Multiple Cinnamon booster Compounding effect Optimum volume ratio Aspergillus flavus 16 4 Collaboration 3.1∶1 Aspergillus terreus 4 4 Collaboration 12.5∶1 Aspergillus fumigatus 64 4 Collaboration 0.78∶1 Aspergillus niger 64 2 Add 0.39∶1 .

[0064] 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 when combined in volume ratios of 3.1:1, 12.5:1 and 0.78:1, respectively, have a good synergistic effect on Aspergillus flavus, Aspergillus terreus and Aspergillus fumigatus, and can be used to prepare anti-Aspergillus drugs.

Claims

1. Application of a combination of a clove light oil component and a cinnamon heavy oil component in a volume ratio of 3.1:1 in the preparation of an anti-aflatoxin drug; The clove light oil component is prepared by the following method: Clove essential oil extraction: add 10-12 times the amount of water to a certain amount of clove powder, soak for 1-1.5 hours, steam distill for 1-5 hours, centrifuge, collect the essential oil, and store at low temperature 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 10-12 times the amount of water, soak for 1-2 hours, steam distill for 1-2 hours, centrifuge, collect the essential oil, and store at low temperature 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. Use of a combination of a clove light oil component and a cinnamon heavy oil component in a volume ratio of 12.5:1 in the preparation of a drug for resisting Aspergillus terreus; The clove light oil component is prepared by the following method: Clove essential oil extraction: add 10-12 times the amount of water to a certain amount of clove powder, soak for 1-1.5 hours, steam distill for 1-5 hours, centrifuge, collect the essential oil, and store at low temperature 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 10-12 times the amount of water, soak for 1-2 hours, steam distill for 1-2 hours, centrifuge, collect the essential oil, and store at low temperature 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.

3. Use of a combination of a clove light oil component and a cinnamon heavy oil component in a volume ratio of 0.78:1 in the preparation of a drug against Aspergillus fumigatus; The clove light oil component is prepared by the following method: Clove essential oil extraction: add 10-12 times the amount of water to a certain amount of clove powder, soak for 1-1.5 hours, steam distill for 1-5 hours, centrifuge, collect the essential oil, and store at low temperature 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 10-12 times the amount of water, soak for 1-2 hours, steam distill for 1-2 hours, centrifuge, collect the essential oil, and store at low temperature 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.

4. The use according to any one of claims 1 to 3, characterized in that: The clove light oil component and the cinnamon heavy oil component are prepared into tablets, granules, capsules, pills, powders, ointments or oral liquids with pharmaceutically acceptable carriers.

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