A plant essential oil composition with anti-Candida activity and its preparation method and application

Abstract

The present invention discloses a plant essential oil composition having anti-Candida activity and a preparation method thereof. The composition comprises a clove light oil component and a cinnamon heavy oil component in a volume ratio of 1:1 to 128:1. Through extensive experimental studies, the present invention has found that the combination of undistilled and refined clove essential oil and cinnamon essential oil does not have a good anti-Candida effect and lacks a significant synergistic effect. However, the combination of a clove light oil component obtained by secondary distillation and refined clove and a cinnamon heavy oil component in a volume ratio of 1:1, 8:1, or 16:1 has a synergistic anti-Candida effect, particularly against Candida tropicalis, Candida krusei, and Candida albicans. The composition can be used to prepare an anti-Candida drug.

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 anti-Candida activity, a preparation method and an application thereof. Background Art

[0002] Candida spp. are the most common pathogens of invasive fungal infections, primarily infecting the frail, immunocompromised, or immunocompromised, causing candidemia. Candidemia in adults and neonates carries a high mortality rate, posing a serious threat to human health. Over 90% of invasive Candida infections are caused by species such as Candida albicans, Candida glabrata, Candida tropicalis, and Candida krusei. Among them, Candida albicans is the most common pathogen causing bloodstream infections, and the proportion of infections with species such as Candida tropicalis, Candida glabrata, and Candida krusei is also increasing. The limited availability of anti-Candida drugs, the increasing prevalence of drug resistance, and significant adverse reactions have made the development of new anti-Candida agents and the reduction of drug resistance and adverse reactions urgent clinical challenges.

[0003] Natural products possess multiple biological activities, including antibacterial, anti-inflammatory, and antioxidant activities. They are also known for their resistance to drug resistance, low toxicity, lack of residual activity, and minimal side effects, making them an important source of new antimicrobial agents. Combination therapy can improve drug efficacy while reducing the dosage of individual drugs and minimizing adverse drug reactions. Therefore, exploring new anti-Candida agents using natural products as raw materials and combining them with other drugs could provide an effective strategy for ameliorating Candida infections.

[0004] Cloves are the dried flower buds of Eugenia caryophyllata Thunb., a plant of the Myrtaceae family. They are used in medicine, fragrances, and food preservatives. Their chemical components include phenols, flavonoids, and volatile oils. The volatile oil content is approximately 15-20%, one of its main active ingredients, exhibiting antiviral, antibacterial, anticancer, antioxidant, and anti-inflammatory activities.

[0005] Cinnamon, the dried bark of the Lauraceae plant Cinnamomum cassia, has the effects of tonifying heat and yang, guiding heat back to its source, dispelling cold and relieving pain, and warming and unblocking the meridians. It contains a large amount of volatile oil and has a fragrant aroma, making it a popular spice. Furthermore, due to its antioxidant and antibacterial properties, it is used as a natural preservative. Its volatile oil, one of its main active ingredients, exhibits antibacterial, insecticidal, and anticancer activities.

[0006] The clove-cinnamon drug pair can enhance the warming-yang and expelling-cold effects of both, but there are currently no reports on the application of the light clove oil component and the heavy cinnamon oil component in anti-Candida. Summary of the Invention

[0007] Purpose of the invention: The purpose of the present invention is to screen out the optimal volume ratio of clove light oil component and cinnamon heavy oil component through a large number of experiments for use in anti-Candida application.

[0008] Technical solution: In order to achieve the above objectives, the technical solution adopted by the present invention is:

[0009] The invention discloses a plant essential oil composition with anti-candida activity, which consists of a clove light oil component and a cinnamon heavy oil component.

[0010] As a preferred embodiment, in the above-mentioned plant essential oil composition having anti-Candida activity, the volume ratio of the clove light oil component to the cinnamon heavy oil component is 1:1 to 128:1.

[0011] As a more preferred embodiment, in the above-mentioned plant essential oil composition having anti-Candida activity, the volume ratio of the clove light oil component to the cinnamon heavy oil component is 1:1, 8:1, 16:1, or 128:1.

[0012] As a particularly preferred embodiment, in the above-mentioned plant essential oil composition having anti-Candida activity, the volume ratio of the clove light oil component to the cinnamon heavy oil component is 1:1, 8:1 or 16:1.

[0013] The plant essential oil composition with anti-Candida activity of the present invention, wherein the clove light oil component is prepared by the following method:

[0014] Clove essential oil extraction: Clove powder is soaked in water, extracted by steam distillation, centrifuged, and the essential oil is collected and stored away from light;

[0015] Clove essential oil refining: Clove 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 clove light oil component is collected;

[0016] The cinnamon heavy oil component is prepared by the following method:

[0017] Extraction of cinnamon essential oil: Cinnamon powder is added with water, soaked, extracted by steam distillation, centrifuged, and the essential oil is collected and stored in the dark.

[0018] 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.

[0019] As a preferred embodiment, the above-mentioned plant essential oil composition with anti-Candida activity, wherein the clove light oil component is prepared by the following method:

[0020] 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 in the dark at 4°C.

[0021] Clove essential oil refining: Clove essential oil was charged into a molecular distillation feed bottle, liquid nitrogen was charged into the cold trap, the refrigeration device was turned on, the reflux water device temperature was set to 5°C, the scraping speed was 240 r / min, the material flow rate was 1 mL / min, the distillation temperature was 110°C, and the distillation pressure was 10 Pa, and the clove light oil component was collected;

[0022] The cinnamon heavy oil component is prepared by the following method:

[0023] Extraction of cinnamon essential oil: a certain amount of cinnamon powder was added with 10 times the amount of water, soaked for 1 hour, steam distilled for 2 hours, centrifuged, and the essential oil was collected and stored at 4°C in the dark.

[0024] Cinnamon essential oil refining: Cinnamon essential oil was charged into a molecular distillation feed bottle, liquid nitrogen was charged into the cold trap, the refrigeration device was turned on, the reflux water device temperature was set to 5°C, the scraping speed was 240 r / min, the material flow rate was 1 mL / min, the distillation temperature was 70°C, and the distillation pressure was 90 Pa. The cinnamon heavy oil component was collected.

[0025] The present invention has found 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 Candida tropicalis, Candida albicans, Candida krusei or Candida glabrata.

[0026] 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.

[0027] Beneficial effects:

[0028] The present invention has found that the combination of undistilled and refined clove essential oil and cinnamon essential oil has a poor anti-Candida effect, lacking a significant synergistic effect. However, the combination of a light clove oil component obtained through secondary distillation and a heavy cinnamon oil component in a volume ratio of 1:1, 8:1, or 16:1 exhibits a synergistic anti-Candida effect. This combination exhibits particularly strong synergistic effects against Candida tropicalis, Candida krusei, and Candida albicans, and can be used to prepare an anti-Candida drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a graph showing the MIC determination of clove light oil components against Candida tropicalis and Candida krusei.

[0030] Figure 2 This is a graph showing the MIC determination of clove light oil components against Candida albicans and Candida glabrata.

[0031] Figure 3 This is a graph showing the MIC determination of cinnamon heavy oil components against Candida tropicalis and Candida krusei.

[0032] Figure 4 This is a graph showing the MIC determination of cinnamon heavy oil components against Candida albicans and Candida glabrata.

[0033] Figure 5 This is a graph showing the MIC determination of a combination of a light clove oil component and a heavy cinnamon oil component against Candida tropicalis.

[0034] 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 Candida krusei.

[0035] 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 Candida glabrata.

[0036] 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 Candida albicans. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with specific embodiments, but these embodiments should not be construed as limiting the present invention.

[0038] Example 1

[0039] A plant essential oil composition with anti-Candida activity, comprising a clove light oil component and a cinnamon heavy oil component.

[0040] (1) The clove light oil component is prepared by the following method:

[0041] (1.1) Clove essential oil extraction: 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 the clove essential oil, and store in the dark at 4°C;

[0042] (1.2) Clove essential oil refining: Clove essential oil was charged into a molecular distillation feed bottle, liquid nitrogen was charged into the cold trap, the refrigeration device was turned on, the reflux water device temperature was set to 5°C, the scraping speed was 240 r / min, the material flow rate was 1 mL / min, the distillation temperature was 110°C, and the distillation pressure was 10 Pa, and the clove light oil component was collected;

[0043] (2) The cinnamon heavy oil component is prepared by the following method:

[0044] (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 in the dark at 4°C;

[0045] (2.2) Cinnamon essential oil refining: Cinnamon essential oil was placed in a molecular distillation feed bottle, liquid nitrogen was placed in a cold trap, the refrigeration device was turned on, the reflux water device temperature was set to 5°C, the scraping speed was 240 r / min, the material flow rate was 1 mL / min, the distillation temperature was 70°C, and the distillation pressure was 90 Pa, and the cinnamon heavy oil component was collected.

[0046] Example 2 Minimum inhibitory concentration (MIC) determination of clove and cinnamon essential oils

[0047] 1. Activation of strains: Use the streak method to inoculate the strain onto the sterilized culture medium. Culture the Candida at 28°C for 48 hours to obtain the activated test strain for later use.

[0048] 2. Pretreatment of test solution:

[0049] 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) were sterilized by filtration through a 0.22 μM filter membrane. The clove essential oil was diluted to 64 μL / mL and the cinnamon essential oil was diluted to 32 μL / mL with 1% Tween-80 distilled water solution and set aside.

[0050] 3. Preparation of test bacterial suspension: Candida: Take single colonies of Candida tropicalis, Candida albicans, Candida krusei and Candida glabrata and dilute them with sterile 0.85% saline to a suspension of 1.8 to 2.2 McFarland units; dilute the above bacterial suspension 1000 times with RPMI 1640 medium to obtain a standby bacterial suspension with a concentration of 1×10 3 ~5×10 3 cfu / mL.

[0051] 4. MIC determination: MIC was determined by 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 bacteria solution added; the second column was the negative control, with 50 μL of RPMI 1640 medium and test bacteria solution added. 50 μL each of 1640 medium and Tween 80 (fungus); 50 μL of the test bacterial solution was added to each well of columns 3-12, and then 50 μL of the diluted essential oil was added in sequence, wherein the concentrations of the light clove 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 heavy cinnamon oil component 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 Candida was cultured at 28°C for 48 hours; 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. At the same time, the OD values ​​were measured every 6 hours. 560nmMeasure the absorbance of Candida and make a judgment.

[0052] 5. Evaluation of the antibacterial activities of the light clove oil component and the heavy cinnamon oil component

[0053] 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 in pairs. The fractional inhibitory concentration index FICI was used as the judgment basis.

[0054] FICI = MIC A (Combined) / MIC A (Used alone)+MIC B (Combined) / MIC B (Used alone)

[0055] 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.

[0056] 6. Experimental results

[0057] 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 shown.

[0058] Table 1 MIC (μL / mL) of the two essential oils

[0059]

[0060] 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 on inhibiting Candida are shown in Table 2 and Table 3 and [[ID=Z38]] Figures 5 to 8 as shown:

[0061] Table 2 FIC index of the optimal compounding of the light clove oil component and the heavy cinnamon component

[0062]

[0063] Table 3 Optimal compounding effects of various volume ratios of the light clove oil component and the heavy cinnamon component

[0064] strain Clove synergistic multiple Cinnamon Enhancement Multiples Compounding effect Optimal volume ratio Candida tropicalis 4 4 Collaboration 8∶1 Candida krusei 64 4 Collaboration 1∶1 Candida glabrata 2 32 Add 128∶1 Candida albicans 4 4 Collaboration 16∶1 .

[0065] The above experimental results demonstrate that the present invention has discovered that the clove light oil component and the cinnamon heavy oil component, obtained by redistillation and refining, particularly when combined in a volume ratio of 1:1, 8:1, or 16:1, exhibit synergistic anti-Candida effects. The synergistic effects are particularly pronounced against Candida tropicalis, Candida krusei, and Candida albicans, and can be used to prepare anti-Candida drugs.

Claims

1. Use of a plant essential oil composition composed of a light clove oil component and a heavy cinnamon oil component in the preparation of a drug against Candida tropicalis or Candida krusei; the volume ratio of the light clove oil component to the heavy cinnamon oil component is 8:1 or 1:1; The clove essential oil extraction method comprises the following steps: adding a certain amount of clove medicinal material powder to 12 times the amount of water, soaking for 1.5 hours, steam distilling for 5 hours, centrifuging, collecting the essential oil, and storing at 4°C in the dark; Clove essential oil refining: Clove essential oil was charged into a molecular distillation feed bottle, liquid nitrogen was charged into the cold trap, the refrigeration device was turned on, the reflux water device temperature was set to 5°C, the scraping speed was 240 r / min, the material flow rate was 1 mL / min, the distillation temperature was 110°C, and the distillation pressure was 10 Pa, and the clove light oil component was collected; The cinnamon heavy oil component is prepared by the following method: Extraction of cinnamon essential oil: a certain amount of cinnamon powder was added with 10 times the amount of water, soaked for 1 hour, steam distilled for 2 hours, centrifuged, and the essential oil was collected and stored at 4°C in the dark. Cinnamon essential oil refining: Cinnamon essential oil was charged into a molecular distillation feed bottle, liquid nitrogen was charged into the cold trap, the refrigeration device was turned on, the reflux water device temperature was set to 5°C, the scraping speed was 240 r / min, the material flow rate was 1 mL / min, the distillation temperature was 70°C, and the distillation pressure was 90 Pa. The cinnamon heavy oil component was collected.

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