A composite essential oil and a preparation method and application thereof

Abstract

The application belongs to the field of essential oils, and particularly relates to a compound essential oil and a preparation method and application thereof, wherein the compound essential oil is prepared by compounding several single essential oils with anti-inflammatory effects, and the compound essential oil with synergistic antibacterial effect is screened from the compounded essential oils through a checkerboard dilution method. Compared with the prior art, the application solves the problem that there is no research on the compound essential oil compounded by bitter orange essential oil and thyme essential oil in the prior art, realizes the compounding of single essential oils, produces synergistic effect, and has better antibacterial and anti-inflammatory performance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of essential oils, and particularly relates to a composite essential oil and a preparation method and application thereof. BACKGROUND

[0002] Plant essential oil is a kind of aromatic substance extracted from the plant body, which widely exists in the roots, stems, leaves, flowers and fruits of plants, and is a colorless or light yellow fat-soluble liquid with volatility. The composition of plant essential oil is complex and diverse, mainly including terpene derivatives, ketone derivatives, alcohol derivatives and aldehyde derivatives, etc. Due to the different compositions, the plant essential oil has different biological activities, such as anti-inflammatory performance, antibacterial performance, antioxidant performance, antitumor performance, etc.

[0003] Bitter orange belongs to Rutaceae and Citrus. The essential oil can be obtained by distillation from the flowers, leaves, fruits and peels of bitter orange, and the bitter orange essential oil has anti-inflammatory, antibacterial, antioxidant and anti-anxiety performances, and is widely used in aromatherapy and cosmetic products.

[0004] Thyme is a semi-shrub of Labiatae, commonly known as ground pepper and mosquito grass, and mainly distributed in the northwest region of China. The thyme essential oil is a light yellow oil-soluble liquid obtained by steam distillation, with sweet smell and good anti-inflammatory, bacteriostatic, antioxidant and antiviral effects.

[0005] It has been proved by current research that plant essential oil has good anti-inflammatory performance and high-efficiency broad-spectrum bacteriostatic activity, and the composite essential oil can play a combined bacteriostatic effect through the synergistic effect between the components, not only the addition of the bacteriostatic effect of single component, but also the synergistic effect between the bacteriostatic substances can increase the selectivity of bacteriostasis to improve the bacteriostatic effect, so as to reduce the skin inflammation caused by microbial infection and skin microecological disorder, and play a better anti-inflammatory activity. At present, the single-component research of bitter orange essential oil and thyme essential oil has made progress, but the effect of the composite essential oil compounded by the two has not been studied. SUMMARY

[0006] The present application aims to solve at least one of the above problems, and provides a composite essential oil and a preparation method and application thereof, so as to solve the lack of research on the composite essential oil compounded by bitter orange essential oil and thyme essential oil in the prior art, realize the compounding of single-component essential oil, produce synergistic effect, and have better bacteriostatic and anti-inflammatory performance.

[0007] The object of the present application is achieved by the following technical scheme.

[0008] The first aspect of the present application discloses a preparation method of a composite essential oil, which is prepared by compounding several single essential oils with anti-inflammatory effects, and the composite essential oil with synergistic antibacterial effect is screened from the compounded essential oil by the checkerboard dilution method.

[0009] Preferably, the composite essential oil is compounded by at least two single essential oils selected from chamomile essential oil, angelica essential oil, citronella essential oil, vetiver essential oil, thyme essential oil, clary sage essential oil and bitter orange essential oil.

[0010] Preferably, the composite essential oil is compounded by bitter orange essential oil and thyme essential oil.

[0011] Preferably, the volume ratio of bitter orange essential oil to thyme essential oil is 1:2 to 1:8.

[0012] Preferably, the volume ratio of bitter orange essential oil to thyme essential oil is 1:8.

[0013] The second aspect of the present application discloses a composite essential oil prepared by any of the above-mentioned methods.

[0014] Preferably, the single essential oils used include chamomile essential oil, angelica essential oil, citronella essential oil, vetiver essential oil, thyme essential oil, clary sage essential oil and bitter orange essential oil.

[0015] Preferably, the single essential oils used are bitter orange essential oil and thyme essential oil, and the volume ratio of bitter orange essential oil to thyme essential oil is 1:2 to 1:8.

[0016] The third aspect of the present application discloses an application of the composite essential oil in the field of antibacterial, and the acting bacteria are Staphylococcus aureus, Escherichia coli and Candida albicans.

[0017] The fourth aspect of the present application discloses an application of the composite essential oil in the field of anti-inflammatory.

[0018] The fifth aspect of the present application discloses an application of the composite essential oil in the field of cosmetics. The composite essential oil is used in the base ingredients of cosmetics, and its synergistic antibacterial effect can be used as a natural preservative in cosmetics to assist in improving the preservative effect of cosmetics.

[0019] Preferably, the application of the complex essential oil in the efficacy skincare. The complex essential oil is further compounded with other components as one of the formula compositions of the efficacy skincare, to obtain the efficacy cosmetics with anti-inflammatory and antibacterial effects. The application of the complex essential oil in the efficacy cosmetics: anti-inflammatory performance, removal of post-acne erythema, broad-spectrum antibacterial performance, and acne removal effect.

[0020] In addition, the complex essential oil also has the possibility of application in the food field: as a natural preservative to assist in improving the preservative effect and providing a certain fragrance; due to the use of plant essential oils for compounding, the safety is higher.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] At present, the determination of the complex essential oil system is mostly simple miscible blending of single essential oil, and the final complex system is determined according to the odor and efficacy. The checkerboard dilution method is to carry out gradient dilution and compounding by using the minimum inhibitory concentration (MIC) of two drugs, and to judge the synergistic, additive, irrelevant and antagonistic effects between drugs by calculating the fractional inhibitory concentration (FIC) index. It is one of the most commonly used joint drug sensitivity methods, but it is less used in essential oil compounding system. The checkerboard dilution method is used to scientifically determine the type and amount of complex essential oil, which can effectively reduce the use amount while ensuring the synergistic antibacterial effect between essential oils, and ensure the safety, effectiveness and comfort of the application.

[0023] 1. The complex essential oil with synergistic effect provided by the application is selected from several essential oils with the best performance in seven essential oils with known good anti-inflammatory performance, and the following experiments are carried out, which has excellent antibacterial and anti-inflammatory performance and can be used in light industry field, such as cosmetic anti-allergic and antibacterial products and food field.

[0024] 2. The complex essential oil with synergistic effect provided by the application has good anti-inflammatory performance, and through the study of the combined antibacterial effect of single essential oil, the complex essential oil with more significant antibacterial effect is finally prepared, which can make up for the low antibacterial performance of single essential oil.

[0025] 3. The complex essential oil with synergistic effect provided by the application has simple preparation method and is suitable for large-scale production. DETAILED DESCRIPTION

[0026] Figure 1 is the hyaluronidase inhibition rate chart of single essential oil and positive control (disodium glycyrrhizinate);

[0027] Figure 2 is the antibacterial circle chart of bitter orange essential oil, thyme essential oil and complex essential oil with different proportions on escherichia coli, staphylococcus aureus and candida albicans;

[0028] Figure 3 Figure 1 shows the inhibition rate of hyaluronidase by bitter orange essential oil, thyme essential oil and compound essential oil.

[0029] Figure 4 This is a schematic diagram of the chessboard dilution method. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0031] Unless otherwise specified in the following description, the reagents used are commercially available products and the methods used are conventional.

[0032] Hyaluronidase inhibition rate test:

[0033] Hyaluronidase is a specific hydrolase of hyaluronic acid and is closely related to the occurrence of skin inflammation and allergies. Inhibiting hyaluronidase activity can ensure the normal content and function of hyaluronic acid. High molecular weight hyaluronic acid plays an important role in regulating scarless wound healing and can significantly reduce inflammatory responses. However, the degradation products of hyaluronic acid can increase inflammatory responses during wound healing. The degradation of hyaluronic acid depends on the activity of hyaluronidase, and the inhibition of hyaluronidase activity can serve as an indicator of a substance's anti-inflammatory response. Therefore, the hyaluronidase inhibition rate can be used as an indicator to evaluate the anti-inflammatory activity of a substance; the higher the hyaluronidase inhibition rate, the stronger the anti-inflammatory activity.

[0034] The hyaluronidase inhibition rates of chamomile essential oil, angelica essential oil, lemongrass essential oil, vetiver essential oil, thyme essential oil, clary sage essential oil, and bitter orange essential oil were determined using a dipotassium glycyrrhizate solution (20 μL / mL) of the same concentration. The specific steps are as follows:

[0035] The essential oil sample was dissolved in a small amount of DMSO and then diluted with water to a final concentration of 20 μL / mL. The experiment was divided into a sample group (T), a sample control group (t), a negative control group (C), and a blank control group (c). Three replicates were used, and the mean value was taken. The specific operating steps are as follows:

[0036] 1. Add 100 μL of calcium chloride solution to groups T, t, C, and c respectively; add 50 μL of hyaluronidase to groups T and C respectively, and add 50 μL of acetate buffer to groups t and c respectively; finally, add 100 μL of sample to groups T and t respectively, and add 100 μL of sample solvent to groups C and c respectively, shake well, and incubate at 37℃ for 40 min.

[0037] 2. Add 200 μL of sodium hyaluronate to groups T and C respectively, and add 200 μL of distilled water to groups t and c respectively, and keep warm at 37℃ for 1 h;

[0038] 3. Add 100 μL of sodium hydroxide solution and 100 μL of acetylacetone solution to groups T, t, C, and c respectively, mix well, and then boil in a water bath at 100°C for 15 min, and immediately cool to room temperature.

[0039] 4. Add 500 μL of p-dimethylaminobenzaldehyde to groups T, t, C, and c respectively, mix thoroughly, and incubate at 37℃ for 10 min; take 200 μL of each group T, t, C, and c onto the enzyme label strip and measure its absorbance at 585 nm.

[0040] The formula for calculating the hyaluronidase inhibition rate is as follows:

[0041]

[0042] In the formula, T is the absorbance of the sample tube; t is the absorbance of the sample control tube; C is the absorbance of the negative control tube; and c is the absorbance of the blank control tube.

[0043] The experimental results are shown in Table 1.

[0044] Table 1. Hyaluronidase inhibition rate of different essential oils at the same concentration.

[0045]

[0046]

[0047] Arrange the essential oils in descending order of hyaluronidase inhibition rate, such as... Figure 1 As shown in the figure. Among the essential oils selected in the experiment, angelica essential oil had the best anti-inflammatory properties, followed by bitter orange essential oil and thyme essential oil, while clary sage essential oil had the lowest average inhibition rate and the worst anti-inflammatory properties.

[0048] Minimum inhibitory concentration (MIC) determination:

[0049] The minimum inhibitory concentration (MIC) was determined as the lowest concentration that inhibited the growth of the test microorganisms, and was measured using a two-fold dilution method. The microorganisms selected for the experiment were *Escherichia coli*, *Staphylococcus aureus*, *Pseudomonas aeruginosa*, and *Candida albicans*. Based on the results, the MICs of *Angelica sinensis* essential oil, bitter orange essential oil, and thyme essential oil were further determined. The specific implementation steps are as follows:

[0050] Take a sterile 96-well plate and first dilute the essential oils with culture medium to prepare sample solutions with an initial concentration of 320 μL / mL. Add 100 μL of liquid culture medium sequentially to columns 2-11 of the 96-well plate. Add 200 μL of the essential oil sample solution to column 1. Take 100 μL / mL of the solution and add it to column 2, mixing thoroughly. Repeat the above steps to serially dilute up to column 10. After mixing thoroughly, discard 100 μL of the mixture from column 10. Remove *Escherichia coli*, *Staphylococcus aureus*, *Pseudomonas aeruginosa*, and *Candida albicans* from the refrigerator and perform serial dilutions to ensure that the bacterial suspension contains bacteria on the order of 10T. 10 Add 100 μL of diluted bacterial suspension to columns 1-11, and 200 μL of liquid culture medium to column 12. Column 11 serves as a positive control, and column 12 as a blank control. Perform three parallel experiments, placing the 96-well plates with added samples in a 37°C incubator (bacteria) or a 28°C shaker (fungus) for 24 h or 48 h, and measuring the absorbance at OD = 600 nm before and after incubation.

[0051] Calculate: OD 培养后 -OD 培养前 If the difference is less than 0.05, the concentration of the essential oil sample in that well is considered to have an antibacterial effect. The maximum concentration with an antibacterial effect is the minimum antibacterial concentration of the essential oil.

[0052] The results are shown in Table 2.

[0053] Table 2. Minimum inhibitory concentrations of three essential oils against bacteria.

[0054]

[0055]

[0056] Note: If microorganisms do not grow at all and the liquid in the well is clear and transparent, it is marked as "-"; if microorganisms grow slowly and the liquid in the well is slightly turbid, it is marked as "+"; if microorganisms grow rapidly and the liquid in the well is very turbid, it is marked as "++".

[0057] Table 2 shows that Angelica sinensis essential oil has the best antibacterial effect among the three essential oils. Its MIC values ​​against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans are 1.25 μL / mL, 1.25 μL / mL, 2.5 μL / mL, and 2.5 μL / mL, respectively, demonstrating good inhibitory effects on both bacteria and fungi. Thyme essential oil and bitter orange essential oil have MIC values ​​of 40 μL / mL and 160 μL / mL against Staphylococcus aureus, respectively; MIC values ​​against Escherichia coli and Pseudomonas aeruginosa are 2.5 μL / mL and 10 μL / mL, and 20 μL / mL and 20 μL / mL, respectively; and MIC values ​​against Candida albicans are 20 μL / mL and 80 μL / mL, respectively. Their inhibitory effects on Gram-negative bacteria are better than those on Gram-positive bacteria.

[0058] Determination of the combined antibacterial index (FIC):

[0059] According to the MIC values of the obtained angelica essential oil, thyme essential oil, and bitter orange essential oil against different bacterial strains, pairwise combination antibacterial experiments were conducted on the above three essential oils. According to the determination results of the minimum inhibitory concentration of single essential oil, the checkerboard dilution method was used to test the combined antibacterial activity of the compound essential oil. The experimental data results are shown in Tables 3 to 6, and the specific implementation steps are as follows:

[0060] Taking thyme essential oil and bitter orange essential oil as an example, take a sterile 96-well plate. A 3×3 grid is the compounding of the two essential oils. Add 100 μL of culture medium in sequence. According to Figure 4 as shown, add 10 μL of thyme essential oil and bitter orange essential oil with a specified concentration to each well. Finally, add 80 μL of bacterial suspension in sequence, which is the checkerboard dilution of thyme essential oil and bitter orange essential oil. Complete the compounding of thyme essential oil and angelica essential oil, and angelica essential oil and bitter orange essential oil in the same way. Add 100 μL of bacterial suspension and 100 μL of culture medium to the 10th column as a positive control, and add 200 μL of culture medium to the 11th column as a blank control. Set 2 groups of parallel experiments.

[0061] The grading antibacterial concentration FIC index was used to evaluate the combined antibacterial effect of the compound essential oil. The formula is as follows:

[0062]

[0063] Note: In this formula, A and B refer to any pairwise combination of thyme essential oil, bitter orange essential oil, and angelica essential oil.

[0064] In this experiment, the following criteria were used to judge the experimental results: when FIC ≤ 0.5, it shows a synergistic effect; when 0.5 < FIC ≤ 1, it is an additive effect; when 1 < FIC ≤ 2, it shows an irrelevant effect; when FIC > 2, it is an antagonistic effect.

[0065] The results are shown in Tables 3 to 6.

[0066] Table 3 Compound results of the three essential oils against Escherichia coli

[0067]

[0068] Table 4 Compound results of the three essential oils against Staphylococcus aureus

[0069]

[0070]

[0071] Table 5 Compound results of the three essential oils against Pseudomonas aeruginosa

[0072]

[0073] Table 6. Results of the combination of three essential oils against Candida albicans

[0074]

[0075] As shown in Table 3, the antibacterial experiment of compound essential oils revealed that the FIC values ​​of the compound essential oils of thyme and bitter orange and the compound essential oils of angelica and bitter orange were 0.375 for Escherichia coli, while the FIC value of the compound essential oil of thyme and angelica was 0.5. This indicates that the compound essential oils of thyme, bitter orange and angelica can achieve synergistic effects when compounded in pairs.

[0076] As shown in Table 4, when thyme, bitter orange, and angelica essential oils are combined in pairs for Staphylococcus aureus, the FIC value is 0.25, indicating a good synergistic effect.

[0077] As shown in Table 5, the thyme and angelica blend essential oils showed an additive effect against Pseudomonas aeruginosa. The thyme and bitter orange blend essential oils and the angelica and bitter orange blend essential oils did not show any combined antibacterial effect in the experiment. The reason is that no minimum inhibitory concentration was found under any blend ratio.

[0078] As shown in Table 6, all three blends exhibited synergistic effects. The blends of thyme and bitter orange essential oils and thyme and angelica showed better synergistic effects, with an FIC index of 0.25. The FIC index of the blend of angelica and bitter orange essential oils was 0.375.

[0079] The results showed that the concentration of essential oils required to exert their antibacterial effect when used in combination was significantly lower than that when used alone, and no antagonistic effect was observed in the experiment. All three blends exhibited synergistic effects against *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans*, with the synergistic effect of the thyme and bitter orange blend being particularly superior. Therefore, thyme and bitter orange essential oils were selected as the preferred blend, and the size of the inhibition zone was subsequently tested to determine the antibacterial effect of the blended essential oils.

[0080] Example 1

[0081] Based on the results of the aforementioned analysis, a compound essential oil with a volume ratio of 1:8 was prepared using bitter orange and thyme essential oils, and the inhibition zone of the three bacterial strains was determined using the filter paper disc method.

[0082] Example 2

[0083] The difference between this embodiment and Embodiment 1 is that the volume ratio of bitter orange and thyme essential oils is 1:4.

[0084] Example 3

[0085] The difference between this embodiment and Embodiment 1 is that the volume ratio of bitter orange and thyme essential oils is 1:2.

[0086] Comparative Example 1

[0087] The difference between this comparative example and Example 1 is that the volume ratio of bitter orange and thyme essential oils is 1:1.

[0088] Comparative Example 2

[0089] The difference between this comparative example and Example 1 is that the volume ratio of bitter orange and thyme essential oils is 2:1.

[0090] Comparative Example 3

[0091] The difference between this comparative example and Example 1 is that the volume ratio of bitter orange and thyme essential oils is 4:1.

[0092] Comparative Example 4

[0093] The difference between this comparative example and Example 1 is that the volume ratio of bitter orange and thyme essential oils is 8:1.

[0094] Comparative Example 5

[0095] The difference between this comparative example and Example 1 is that the antibacterial zone of the single bitter orange essential oil was measured.

[0096] Comparative Example 6

[0097] The difference between this comparative example and Example 1 is that the inhibition zone of the single thyme essential oil was measured.

[0098] inhibition zone determination (filter paper method):

[0099] The specific implementation steps are as follows:

[0100] Prepare a solid culture medium, sterilize it, pour it out, and cool it to solidify to obtain a sterile solid culture medium. Prepare compound essential oils with different volume ratios or use single essential oils. Soak sterilized circular filter paper discs in the single and compound essential oils for 30 minutes.

[0101] Remove the test bacteria and dilute the bacterial suspension so that the number of bacteria in the diluted bacterial suspension is on the order of 10. 10 To determine the antibacterial effect, add 100 μL of diluted bacterial suspension to the surface of the culture medium and spread it evenly with a spreader. Place a soaked filter paper disc in the center of the culture medium. Incubate the petri dish in a 37°C incubator (for bacteria) or a 28°C shaker for 24 or 48 hours. Measure the diameter of the inhibition zone after incubation to determine the antibacterial effect.

[0102] An inhibition zone diameter ≤ 7mm is considered to have no antibacterial effect; 7mm < inhibition zone diameter < 10mm is considered to be minimally sensitized; 10mm < inhibition zone diameter < 20mm is considered to be moderately sensitized; and an inhibition zone diameter ≥ 20mm is considered to be highly sensitized.

[0103] The results of Examples 1-3 and Comparative Examples 1-6 are shown in Table 7 and Figure 2 As shown, the compound essential oils exhibited different antibacterial properties at different blending ratios. For *E. coli*, bitter orange essential oil showed low sensitivity, while thyme essential oil showed high sensitivity. When the volume ratio of bitter orange essential oil to thyme essential oil was 8:1 and 4:1, it showed moderate sensitivity; at all other ratios, it showed high sensitivity. Furthermore, at a V(bitter orange essential oil):V(thyme essential oil) ratio of 1:8, the inhibition zone diameter reached (39.25±0.25) mm, slightly higher than the (36.75±0.15) mm diameter of thyme essential oil, and significantly higher than the (7.75±0.25) mm diameter of bitter orange essential oil.

[0104] For Staphylococcus aureus and Candida albicans, a ratio of 1:8 (V:V:Thyme essential oil) showed high sensitivity, with inhibition zone diameters of (43.60±0.80) mm and (43.60±0.40) mm, respectively. This significantly enhanced the antibacterial ability of bitter orange essential oil, and was slightly higher than the inhibition zone diameters of (40.40±1.10) mm and (39.60±1.30) mm of thyme essential oil. Therefore, the optimal ratio of the compound essential oils was determined to be 1:8 (V:V:Thyme essential oil).

[0105] Table 7. Inhibition zone diameters of three tested bacteria at different compound volume ratios.

[0106]

[0107]

[0108] Note: "-" indicates the absence of an inhibition zone.

[0109] Experimental Example 1

[0110] Hyaluronidase inhibition rate test:

[0111] Based on the results obtained from Examples 1-3 and Comparative Examples 1-6, the final volume ratio of bitter orange essential oil and thyme essential oil was determined to be 1:8. The hyaluronidase inhibition rate of the compound essential oil was then determined.

[0112] Experimental results are as follows Figure 3 As shown, at a concentration of 20 μL / mL, the hyaluronidase inhibition rate of the compound essential oil was 54.49%. Compared with the single essential oil, the compound essential oil had a lower inhibition rate than bitter orange essential oil and a slightly higher inhibition rate than thyme. The results indicate that the compound essential oil can improve the anti-inflammatory properties of the single essential oil to a certain extent.

[0113] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing a compound essential oil, characterized in that, The compound essential oil described above was prepared by blending bitter orange essential oil and thyme essential oil at a volume ratio of 1:

8. The diameter of the inhibition zone of the compound essential oil against Escherichia coli was (39.25±0.25) mm, and the diameters of the inhibition zones against Staphylococcus aureus and Candida albicans were (43.60±0.80) mm and (43.60±0.40) mm, respectively.

2. A compound essential oil, characterized in that, Prepared by the method as described in claim 1.

3. The application of the compound essential oil as described in claim 2 in the preparation of antibacterial products, characterized in that, The target bacteria are Staphylococcus aureus, Escherichia coli, and Candida albicans.

4. The application of the compound essential oil as described in claim 2 in the preparation of cosmetics.

5. The application according to claim 4, characterized in that, Application of this compound essential oil in the preparation of functional skin care products.

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