Compound essential oil, anti-corrosion system containing same and application of anti-corrosion system

Abstract

The invention provides compound essential oil, an anti-corrosion system containing the compound essential oil and application of the anti-corrosion system. The compound essential oil consists of the following components: wild chrysanthemum essential oil, jasmine essential oil, pogostemon cablin essential oil and citral cinnamomum camphora leaf essential oil. The compound essential oil has the effect of remarkably inhibiting the growth of propionibacterium acnes, staphylococcus aureus and malassezia within a relatively long time. The invention also provides a compound preservative system, and the preservative system contains the compound essential oil, 1, 2-hexanediol and caprylyl glycol.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a compound essential oil with antibacterial effect, a preservative system containing the compound essential oil, and uses thereof. Background Art

[0002] Essential oils are extracted from leaves, flowers, seeds, fruits, roots, bark, resin, wood core and other parts of plants by steam distillation, cold pressing, liposorption, supercritical carbon dioxide extraction and other methods. Since essential oils are of natural origin, they are widely considered to be closer to nature than chemical synthetic products and are a healthier and more sustainable choice. Essential oils are also complex mixtures of various chemical components. The volatile oils, phenols, alcohols, esters and other components contained in them give essential oils multiple effects such as antibacterial, anti-inflammatory, antioxidant and analgesic. The combination of essential oils and non-additive preservatives belongs to a non-additive preservative system, which can synergistically enhance the antibacterial effect while reducing the amount of non-additive preservatives used to reduce skin irritation.

[0003] Three methods are commonly used internationally to evaluate the antiseptic and antibacterial properties: determination of the minimum inhibitory concentration (MIC), antiseptic challenge test, and linear regression method. Among them, the antiseptic challenge test is to add a certain amount of microorganisms to the preservative sample. On the following 0 / 7 / 14 / 21 / 28 days, the antiseptic performance is evaluated by measuring the content of microorganisms in the sample, and the experimental cycle is as long as 28 days. The antiseptic challenge test is a regulatory requirement of many industries (such as cosmetics, medicine, and food) and meets international standards. It is not only scientific and reliable in evaluating antiseptic and antibacterial properties, but also provides important support for product development, regulatory compliance, and market competition. Summary of the invention

[0004] The present invention aims to provide a compound essential oil with antibacterial effect, which comprises the following ingredients: wild chrysanthemum essential oil, jasmine essential oil, patchouli essential oil and citral camphor leaf essential oil.

[0005] Preferably, the wild chrysanthemum essential oil, jasmine essential oil, patchouli essential oil and citral camphor leaf essential oil of the present invention are prepared by the following steps: the wild chrysanthemum essential oil is prepared from the wild chrysanthemum of the Asteraceae plant ( Chrysanthemum indicum L. ) flowers as raw materials, extracted by steam distillation; Jasmine essential oil is made from Chinese jasmine ( Jasminum officinalis sambac ) flowers as raw materials, extracted by supercritical carbon dioxide; Patchouli essential oil is made from Patchouli ( Patchouli ) above ground parts as raw materials, extracted by steam distillation; Citral camphor leaf essential oil is made from citral camphor tree ( Cinnamomum camphora ) Fresh leaves are used as raw materials and extracted by water distillation.

[0006] Preferably, the volume ratio of the wild chrysanthemum essential oil, jasmine essential oil, patchouli essential oil and citral-type camphor leaf essential oil contained in the compound essential oil of the present invention is 4:1:2:3.

[0007] The present invention studies the antibacterial effect of the compound essential oil of the present invention, and the result shows that the antibacterial effect of the compound essential oil of the present invention is higher than that of the single essential oil, and the single essential oils produce a synergistic effect. In addition, the compound essential oil overcomes the defect of the narrow antibacterial spectrum of the single essential oil. For example, the antibacterial rate of the wild chrysanthemum essential oil on Staphylococcus aureus at a concentration of 50 mg / ml is 68%, and the antibacterial rate of the same concentration on Propionibacterium acnes is close to 95%. The antibacterial effect of the compound essential oil on three bacteria, Staphylococcus aureus, Propionibacterium acnes and Malassezia, within a specific concentration range is not much different, all of which are higher than 90%, and has a broad-spectrum antibacterial effect.

[0008] On the other hand, the present invention uses essential oil as a natural preservative substance from plant sources and adds polyols to form a compound preservative composition, namely, a "preservative system". The present invention provides a preservative system, which contains the above-mentioned compound essential oil.

[0009] Preferably, the preservative system of the present invention is composed of the above-mentioned compound essential oil, 1,2-hexanediol and caprylyl glycol, wherein the volume ratio of the compound essential oil, 1,2-hexanediol and caprylyl glycol is (1~10):2:1.

[0010] Preferably, in the preservative system of the present invention, the volume ratio of the compound essential oil, 1,2-hexanediol and caprylyl glycol is 10:2:1.

[0011] The antiseptic system of the present invention can reduce the colony by nearly 100% (sterile growth) on the 7th day of co-incubation with bacteria, can effectively kill Escherichia coli and Staphylococcus aureus, and no colony formation is observed during the 28-day experiment; the antiseptic system of the present invention can reduce the colony by nearly 90% on the 7th day of co-incubation with fungi, can effectively prevent the growth of Candida albicans, and no colony formation is observed during the 28-day experiment. The above results indicate that the antiseptic system of the present invention has a strong antibacterial effect.

[0012] In another aspect, the present invention provides use of the above-mentioned compound essential oil and preservative system in cosmetics.

[0013] Preferably, the content of the preservative system of the present invention in the cosmetics is 0.05%-2%.

[0014] More preferably, the content of the preservative system of the present invention in the cosmetic is 0.5%.

[0015] Advantages of the preservative system of the present invention: The preservative system of the present invention combines a compound essential oil with antibacterial activity from a plant source and a chemical preservative component, polyol, to obtain a preservative system with synergistic effects of plant essential oil preservatives and chemical preservatives. Selecting compound essential oils and polyols to be compounded in a certain ratio can reduce the amount of polyol used, thereby reducing the risk of irritation to the skin caused by a large amount of polyol; at the same time, the active ingredients of plant essential oils have whitening, anti-aging, repair and other effects, making the preservative system multifunctional. In addition, plant essential oils have a special fragrance and a fragrance-enhancing effect, all of which highlight the advantages of essential oils in the application of additive-free preservative systems in cosmetics. Compounding compound essential oils with polyols, the additive-free preservative system after compounding can achieve the effects of improving safety of use, increasing antibacterial strength, and broadening the antibacterial spectrum. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : The antibacterial rates of different concentrations of compound essential oils and their single essential oil components against Staphylococcus aureus.

[0017] Figure 2 : The antibacterial rates of different concentrations of compound essential oils and their single essential oil components against Propionibacterium acnes.

[0018] Figure 3 : The inhibition rates of different concentrations of compound essential oils and their single essential oil components against Malassezia.

[0019] Figure 4 : The antibacterial rate of each compound essential oil of different concentrations against Staphylococcus aureus; p<0.05, p<0.001.

[0020] Figure 5 : The antibacterial rate of different concentrations of compound essential oils on Propionibacterium acnes.

[0021] Figure 6 : The antibacterial rate of different concentrations of compound essential oils against Malassezia.

[0022] Figure 7 : The inhibitory effect of each preservative system on Staphylococcus aureus and Escherichia coli.

[0023] Figure 8 : The inhibitory effect of each preservative system on Candida albicans.

[0024] Fig. 9 : Detection results of wild chrysanthemum essential oil by gas chromatography-mass spectrometry.

[0025] Fig.10 : Detection results of jasmine essential oil by gas chromatography-mass spectrometry.

[0026] Fig.11: GC-MS detection results of patchouli essential oil.

[0027] Fig.12 : Detection results of citral camphor leaf essential oil by gas chromatography-mass spectrometry. DETAILED DESCRIPTION

[0028] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0029] The essential oils used in the following Examples 2-4 are all commercially available or extracted by the method of Example 1 or 2.

[0030] Wild chrysanthemum essential oil: purchased from Mujiuye (Shanghai) Technology Co., Ltd., batch number: TCM02611012310, enterprise standard: relative density (20℃): 0.960~1.015; refractive index (20℃): 1.475~1.525. The wild chrysanthemum essential oil cosmetic raw material safety information reporting code is 007637-02571-1356.

[0031] Jasmine essential oil: purchased from Mujiuye (Shanghai) Technology Co., Ltd., batch number: BEO08612012310, enterprise standard: relative density (20℃): 0.925~0.998; refractive index (20℃): 1.481~1.525. The safety information reporting code for jasmine essential oil cosmetic raw materials is 004723-02571-4822.

[0032] Patchouli essential oil: purchased from Mujiuye (Shanghai) Technology Co., Ltd., batch number: BEO02561012310, enterprise standard: relative density (20℃): 0.949~0.983; refractive index (20℃): 1.505~1.512. The patchouli essential oil cosmetic raw material safety information reporting code is 002634-02571-5566.

[0033] Citral camphor leaf essential oil: purchased from Mujiuye (Shanghai) Technology Co., Ltd., batch number: BEO02241012408, enterprise standard: relative density (20℃): 0.875~0.890; refractive index (20℃): 1.468~1.474. The safety information reporting code for camphor leaf essential oil cosmetic raw materials is: 008504-02571-9021.

[0034] [Example 1] Preparation of unilateral essential oil The equipment used in the steam distillation method is a water-sealed still, which consists of: Distillation kettle: The overall appearance is cylindrical, with a spherical bottom and the upper opening is the feed port; Gooseneck: Conical cover with the top of the still, connected to the condenser; Condenser: It is an aluminum tube condenser, which condenses the steam and cools the distillate; Oil-water separator: Made of aluminum, it is both a container for receiving distillate and a separator for essential oil and water.

[0035] The process flow of extracting essential oils by steam distillation: feeding, adding water, distillation, cooling, oil-water separation, essential oil, and packaging. 1.1 Preparation of wild chrysanthemum essential oil

[0036] Wild chrysanthemum essential oil is made from wild chrysanthemum ( Chrysanthemum indicum L. ) flowers as raw materials, and the volatile oil is extracted by steam distillation.

[0037] During the flowering period of wild chrysanthemum from October to December every year, pick wild chrysanthemums when the buds begin to open but not fully bloom from the morning dew to about 10 am. After picking, spread the wild chrysanthemums to dry in the sun or dry at low temperature. Place the treated wild chrysanthemums evenly in the inner tank of the distillation equipment, and control the filling amount to 70%-80% of the tank volume to ensure that the steam can fully penetrate the flower material layer. Add pure water equal to the volume of wild chrysanthemums to the outer distillation tank to ensure that steam can continue to be generated and cover the raw materials at the bottom of the inner tank. Turn on the heating device, the extraction temperature is 90~96°C, the condenser temperature is 15~25°C, the distillation time is 60~90min, and the heating operation is terminated when the essential oil content in the condensate after distillation is significantly reduced. The condensed mixed liquid flows into the oil-water separator through the condenser. The mixed liquid needs to stand for 24 hours and naturally stratify. Use a dropper or a separation funnel to separate the wild chrysanthemum essential oil on the upper layer from the pure dew on the lower layer. The separated essential oil is filtered through filter paper or filter cloth to remove particles or other impurities suspended in the essential oil to obtain wild chrysanthemum essential oil. 1.2 Preparation of jasmine essential oil

[0038] Jasmine essential oil is made from Chinese jasmine ( Jasminum officinalis sambac ) flowers as raw materials, and the volatile oil is obtained by supercritical carbon dioxide extraction.

[0039] At dusk, choose jasmine flowers that are moderately open, and avoid flowers that are too young or over-mature. After picking, place the flowers in a well-ventilated, sun-free drying room for 1-2 days, and then use a dryer to heat and dry at 45°C. After the flowers are dried, the petals will be brittle and hard, without a sense of moisture. Put the dried jasmine flowers into a grinder for processing, and crush them into coarse powder that passes through a 10-20 mesh sieve. Use a sandbag to pack the weighed jasmine coarse powder, put it into the extractor, and seal the top cover. Turn on the thermostatic bath power. Open the valve of the carbon dioxide cylinder, so that the gas passes through the dryer along the pipeline, cools the condenser (immersed in an ice water bath), and the plunger pump, and enters the booster buffer bottle. After the pressure of the booster buffer bottle greatly exceeds the experimental pressure, open valve V1 to boost the extractor to the experimental pressure, close valve V1, and let the jasmine flowers pre-soak for 10 minutes. Supercritical CO 2 Fluid Extraction. CO 2 The fluid flow rate is 25L / H-40L / H, the extraction temperature is 40°C, the extraction pressure is 20MPa-25MPa, the separation temperature is 35°C, the separation pressure is 4.5MPa-5.5MPa, and the extraction time is 1.5h-2h to obtain jasmine essential oil. 1.3 Preparation of Patchouli Essential Oil

[0040] Patchouli essential oil is made from Patchouli ( Patchouli ) The above-ground part is used as raw material to obtain volatile oil through steam distillation.

[0041] During the patchouli flowering period from July to September, select fresh, dry, uncontaminated patchouli plants and wash them. Disassemble the distiller and clean all parts to ensure that there is no oil or impurities. Add 2 / 3 to 3 / 4 of distilled water to the distiller and place it on the heating plate. Heat the heating plate until the water begins to boil. When the water in the distiller begins to boil, place the patchouli plants in the distiller and make sure they are all submerged in the water. After waiting for the water to begin to boil, connect the distiller with a cooling tube and place the tube at the other end into the collection bottle. The steam will condense through the cooling tube and flow into the collection bottle. Wait for 24 hours, the essential oil will float on the upper layer of the water, and use a dropper or separation funnel to separate it from the water. After collecting the essential oil, use filter paper or filter cloth to filter to remove particles or other impurities suspended in the essential oil to obtain patchouli essential oil. 1.4 Preparation of citral camphor leaf essential oil

[0042] Citral camphor leaf essential oil is made from citral camphor tree ( Cinnamomum camphora ) Fresh leaves are used as raw materials, and the volatile oil is extracted by water distillation.

[0043] Fresh camphor leaves are picked from citral camphor trees planted in Yongxiu County, Jiujiang City, Jiangxi Province from June to September, the fresh camphor leaves are washed and placed on the upper sieve plate of an essential oil extractor; a proper amount of water is added to the bottom of the distiller and heated to boiling, the generated steam passes through the sieve plate from bottom to top and reaches the position of the fresh camphor leaves, so that the components in the fresh camphor leaves flow out to form oil-water mixed steam; the oil-water mixed steam enters the oil-water separator through a condenser, and after distillation for 2 hours, the liquid mixture in the oil-water separator is allowed to stand and separate, and the upper oil phase is taken to obtain the citral camphor leaf essential oil.

[0044] [Example 2] Preparation of compound essential oil According to the following formula, the single essential oils are mixed evenly in proportion to obtain compound essential oil 1 to compound essential oil 4.

[0045] Compound essential oil 1: wild chrysanthemum essential oil + jasmine essential oil + patchouli essential oil + citral camphor leaf essential oil, volume ratio 1:4:3:2; that is, the content of wild chrysanthemum essential oil in compound essential oil 1 is 10%, the content of jasmine essential oil is 40%, the content of patchouli essential oil is 30%, and the content of citral camphor leaf essential oil is 20%.

[0046] Compound essential oil 2: wild chrysanthemum essential oil + jasmine essential oil + patchouli essential oil + citral camphor leaf essential oil, the ratio is 2:3:4:1; that is, the content of wild chrysanthemum essential oil in compound essential oil 2 is 20%, the content of jasmine essential oil is 30%, the content of patchouli essential oil is 40%, and the content of citral camphor leaf essential oil is 10%.

[0047] Compound essential oil 3: wild chrysanthemum essential oil + jasmine essential oil + patchouli essential oil + citral camphor leaf essential oil, the ratio is 3:2:1:4; that is, the content of wild chrysanthemum essential oil in compound essential oil 3 is 30%, the content of jasmine essential oil is 20%, the content of patchouli essential oil is 10%, and the content of citral camphor leaf essential oil is 40%.

[0048] Compound essential oil 4: wild chrysanthemum essential oil + jasmine essential oil + patchouli essential oil + citral camphor leaf essential oil, the ratio is 4:1:2:3; that is, the content of wild chrysanthemum essential oil in compound essential oil 4 is 40%, the content of jasmine essential oil is 10%, the content of patchouli essential oil is 20%, and the content of citral camphor leaf essential oil is 30%.

[0049] [Example 3] Inhibitory effect of compound essential oil on bacteria This example studies the inhibitory effects of the compound essential oils 1 to 4 (Example 2) of the present invention on three bacteria, Staphylococcus aureus, Propionibacterium acnes, and Malassezia. 3.1 Test materials

[0050] 3.1.1 Test strains: Staphylococcus aureus (ATCC6538), Propionibacterium acnes (SC5314), and Malassezia (ATCC14521) were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0051] 3.1.2 Test drugs and reagents: wild chrysanthemum essential oil, jasmine essential oil, patchouli essential oil, citral camphor leaf essential oil, normal saline, PBS, surfactant HS15, BHI medium, tryptone soy agar medium (TSA), MH broth medium.

[0052] 3.1.3 Consumables and instruments: 10 cm culture dish, sterile inoculation loop, 96-well plate, multifunctional microplate reader, anaerobic generation bag, shaker, biochemical incubator, clean bench. 3.2 Test methods

[0053] 3.2.1 Preparation of single essential oil mixed solution and compound essential oil mixed solution Dissolve the single essential oil or compound essential oil 1 to compound essential oil 4 in a solution containing 2% surfactant HS15 to form a mixed solution with a mass concentration of 800 mg / ml: add 800 mg of the single essential oil or compound essential oil 1 to compound essential oil 4 to 1 ml of a solution containing 2% surfactant HS15, and mix well.

[0054] 3.2.2 Preparation of strains 3.2.2.1 Cultivation of Staphylococcus aureus: Before culturing, take out the frozen strains, and after reviving, use an inoculation loop to inoculate the bacteria onto the TSA agar plate by streaking, and culture at 37°C for 24-48 h until obvious colonies appear, which will be used as the working plate. The working plate should be replaced every 20 days to ensure the activity of the colonies and stored at 4°C. In order to obtain the Staphylococcus aureus liquid after the logarithmic growth period, pick a small round colony on the working plate each time, place it in 1ml TSA liquid culture medium, shake the bacteria overnight at a speed of 250rpm and a temperature of 37°C, and harvest the strains for experiments the next day.

[0055] 3.2.2.2 Cultivation of Propionibacterium acnes: Propionibacterium acnes is an anaerobic bacterium and needs to be cultured in an anaerobic environment. An opened anaerobic bag is placed in the anaerobic culture bag to create an anaerobic environment, and the oxygen content in the culture bag is observed using an anaerobic indicator. Before culture, take out the frozen strain, and after recovery, use an inoculation loop to inoculate the bacteria onto the BHI agar plate by streaking, and place it in an anaerobic environment at 37°C for 48-72h until there are obvious colonies as working plates. The working plates are replaced every 20 days to ensure the activity of the colonies and stored at 4°C. In order to obtain the Propionibacterium acnes bacterial liquid after the logarithmic growth period, a small round colony on the working plate is picked each time, placed in 1ml BHI liquid culture medium, and shaken overnight in an anaerobic environment at a speed of 250 rpm and a temperature of 37°C. The strains are harvested the next day for experiments.

[0056] 3.2.2.3 Cultivation of Malassezia: Before culturing, take out the frozen strains, and after reviving, use an inoculation loop to inoculate the bacteria onto the MH agar plate by streaking, and culture at 30°C for 24-48 h until obvious colonies appear, which is used as the working plate. The working plate is replaced every 20 days to ensure the activity of the colonies and stored at 4°C. To obtain the Malassezia culture after the logarithmic growth period, pick a small round colony on the working plate each time, place it in 1ml MH liquid culture medium, shake the bacteria overnight at a speed of 250rpm and a temperature of 30°C, and harvest the strains for experiments the next day.

[0057] 3.2.3 Determination of the antibacterial rate of each single essential oil and compound essential oil against Staphylococcus aureus, Propionibacterium acnes and Malassezia: The micro-dilution method was used to test the antibacterial rate of plant essential oil samples. The specific steps are as follows: (1) Add 100 µl of culture medium to each well of a 96-well plate; (2) Add 100µl of the prepared single essential oil mixed solution or compound essential oil mixed solution to the first row, and then dilute the essential oil by two times. That is, after adding the single essential oil mixed solution or compound essential oil mixed solution to the first well, use a pipette to blow and mix thoroughly, then draw 100µl and add it to the second well and blow and mix thoroughly, repeat this to the last well, and draw 100µl from the last well and throw it away, and repeat 3 times for each essential oil concentration. At this time, each test well should contain 100µl of solution; (3) Add 100 µl of diluted Staphylococcus aureus, Propionibacterium acnes or Malassezia to each well (the concentration of the diluted bacterial solution is 2×10 6 cfu / ml), the final concentration of the bacterial solution added to each well was 1×10 6 cfu / ml, the final dilution multiple of the single essential oil mixed solution or compound essential oil mixed solution in the first well is 4 times; (4) Set up negative control (containing essential oil and culture medium at different dilutions to remove drug background OD600) and positive control (100µl culture medium + 100µl bacterial solution, with a final bacterial solution concentration of 1×10 6 cfu / ml); (5) After incubation at 37°C for 24 hours, measure the OD600 of each well using a microplate reader and calculate the bacterial viability.

[0058] Inhibition rate (%) = 100%-[OD600(test)-OD600(negative)] / [OD600(positive)-OD600(negative)] 3.3 Test results

[0059] 3.3.1 Antibacterial effect of compound essential oil on Staphylococcus aureus The antibacterial effects of different concentrations of compound essential oils and their single components on Staphylococcus aureus are shown in Tables 1 and Figure 1 The results showed that the compound essential oil and its single components had different degrees of inhibitory effects on Staphylococcus aureus. The antibacterial rate of compound essential oil 4 at each concentration was higher than that of single essential oil. The antibacterial rate of compound essential oil 4 against Staphylococcus aureus in the concentration range of 25mg / ml and 50mg / ml could reach 90%; the antibacterial rate of compound essential oil 1 against Staphylococcus aureus in the concentration range of 25mg / ml-200mg / ml was higher than that of single essential oil; the antibacterial rates of compound essential oils 2 and 3 against Staphylococcus aureus in the concentration range of 6.25mg / ml-12.5mg / ml were both lower than 50%, and the antibacterial rates against Staphylococcus aureus in the concentration range of 25mg / ml-100mg / ml were lower than 90%, but slightly higher than that of single essential oil.

[0060] Table 1 The antibacterial rate of compound essential oils and their single components at different concentrations against Staphylococcus aureus (mean, unit: %)

[0061] 3.3.2 Antibacterial effect of compound essential oil on Propionibacterium acnes The antibacterial effects of different concentrations of compound essential oils and their single components on Propionibacterium acnes are shown in Tables 2 and Figure 2 The results showed that the compound essential oil and its single components had different degrees of inhibition of Propionibacterium acnes. The antibacterial rate of compound essential oil 1 against Propionibacterium acnes at a concentration of 25mg / ml-100mg / ml could reach 90%; the antibacterial rates of compound essential oils 2 and 4 against Propionibacterium acnes in the concentration range of 50mg / ml-200 mg / ml were both higher than 90%; the antibacterial rate of compound essential oil 3 against Propionibacterium acnes in the concentration range of 100mg / ml-200mg / ml was higher than 90%.

[0062] Table 2 The antibacterial rate of compound essential oils and their single components at different concentrations against Propionibacterium acnes (mean, unit: %)

[0063] 3.3.3 Antibacterial effect of compound essential oil on Malassezia The antibacterial effects of different concentrations of compound essential oils and their single components on Malassezia are shown in Tables 3 and Figure 3The results showed that the compound essential oil and its single components had different degrees of inhibition of Malassezia. The antibacterial rate of compound essential oil 4 at each concentration was higher than that of single essential oil. The antibacterial rates of compound essential oils 1 and 2 against Malassezia in the concentration range of 12.5 mg / ml-200 mg / ml were higher than 50% and lower than 90%; the antibacterial rate of compound essential oil 3 against Malassezia in the concentration range of 6.25 mg / ml-200 mg / ml was close to 90%; the antibacterial rate of compound essential oil 4 against Malassezia in the concentration range of 6.25 mg / ml-25 mg / ml was higher than 50%, and the antibacterial rate of compound essential oil 4 against Malassezia in the concentration range of 50-200 mg / ml was higher than 90%.

[0064] Table 3 Inhibition rate of different concentrations of compound essential oils and their single components against Malassezia (mean, unit: %)

[0065] 3.3.4 Comparison of the antibacterial effects of four compound essential oils on Staphylococcus aureus, Propionibacterium acnes and Malassezia Results Figure 4-Figure 6 The comparison of the two groups of data used independent samples. t test( t- test). The antibacterial effects of different concentrations of the compound essential oils on Staphylococcus aureus, Propionibacterium acnes and Malassezia were different to varying degrees. In the concentration range of 6.25-50 mg / ml, the antibacterial effect of compound essential oil 4 on Staphylococcus aureus was significantly enhanced compared with the other groups ( p <0.05; p <0.001); there was no significant difference between the antibacterial effects of the four compound essential oils of the present invention on Propionibacterium acnes and on Malassezia.

[0066] 3.4 Experimental Conclusion Comprehensive comparison shows that the antibacterial effect of compound essential oil 4 shows obvious advantages over other formulas. Therefore, we selected compound essential oil 4 with a concentration of 50 mg / ml for subsequent experiments.

[0067] [Example 4] Antibacterial test of a preservative system containing compound essential oil 4 and polyols 4.1 Test materials

[0068] 4.1.1 Test strains: Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 25922), and Candida albicans (ATCC 10231) were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0069] 4.1.2 Test drugs and reagents: wild chrysanthemum essential oil, jasmine essential oil, patchouli essential oil, citral camphor leaf essential oil, 1,2-hexanediol, caprylyl glycol, normal saline, PBS, surfactant HS15, tryptone soy agar medium (TSA), Sabouraud dextrose agar medium (SDA).

[0070] 4.1.3 Consumables and instruments: inoculation loop, culture dish, biochemical incubator, inverted microscope, clean bench. 4.2 Test methods

[0071] 4.2.1 Preparation of bacterial solution for experiment Preparation of mixed bacterial solution of Staphylococcus aureus and Escherichia coli: Take an appropriate amount of Staphylococcus aureus and Escherichia coli from the preserved test tube slant (or the strain preservation tube) and inoculate it on the tryptone soy agar (TSA) slant, and culture it at 37°C for 18h-24h. Use an inoculation loop to take the first-generation culture, streak it on the TSA plate, and culture it at 37°C for 18h-24h. Pick out the typical colonies from the above-mentioned second-generation culture, streak it on the TSA agar slant, and culture it at 37°C for 18h-24h, which is the third-generation culture. Pipette an appropriate amount of sterile saline into the slant test tube, blow and wash repeatedly to wash off the bacterial moss. Transfer the wash solution to another sterile test tube and vortex to mix for 20 seconds. Dilute it to about 1.0×10 8 The prepared bacterial suspension should be used within 2 hours or stored at 2℃~8℃ for no more than 24 hours.

[0072] Preparation of Candida albicans bacterial solution: Take an appropriate amount of Candida albicans cells from the preserved test tube slant (or strain storage tube) and inoculate it on the Sabouraud dextrose agar (SDA) slant, and culture it at 28 ℃ for 18h-24h. Use an inoculation loop to take the first-generation culture, streak it on the SDA plate, and culture it at 28 ℃ for 18h-24h. Pick out the typical colonies from the above-mentioned second-generation culture, streak it on the SDA agar slant, and culture it at 28 ℃ for 18h-24h, which is the third-generation culture. Pipette an appropriate amount of sterile PBS buffer and add it to the slant test tube, blow and wash repeatedly to wash off the bacterial moss. Transfer the wash solution to another sterile test tube and vortex to mix for 20 seconds. Dilute it to about 1.0×10 7 The prepared bacterial suspension should be used within 2 hours or stored at 2℃~8℃ for no more than 24 hours.

[0073] 4.2.2 Preparation of the mixed solution of compound essential oil 4 contained in the compound preservative system A 50 mg / ml mixed solution of compound essential oil 4 was used as the compound essential oil mother solution added to the compound preservative system. Preparation of a 50 mg / ml mixed solution of compound essential oil 4: 50 mg of compound essential oil 4 was added to 1 ml of a solution containing 2% surfactant HS15, and mixed to obtain a 50 mg / ml mixed solution of compound essential oil 4.

[0074] 4.2.3 Compound anticorrosion system Compounding scheme 1: The preservative system is a mixed solution of compound essential oil 4 (1%) + 1,2-hexanediol (0.2%) + caprylyl glycol (0.1%). Specific operation: Take 20 ml of tryptone soy agar medium (for mixed bacterial solution of Staphylococcus aureus and Escherichia coli) / Sabouraud glucose agar medium (for Candida albicans), add 0.2 ml of the mixed solution of compound essential oil 4 prepared in 4.2.2, 0.04 ml of 1,2-hexanediol and 0.02 ml of caprylyl glycol, and mix well.

[0075] Compounding solution 2: The preservative system is a mixed solution of compound essential oil 4 (0.3%) + 1,2-hexanediol (0.2%) + caprylyl glycol (0.1%). Specific operation: Take 20 ml of tryptone soy agar medium (for mixed bacterial solution of Staphylococcus aureus and Escherichia coli) / Sabouraud glucose agar medium (for Candida albicans), add 0.06 ml of the mixed solution of compound essential oil 4 prepared in 4.2.2, 0.04 ml of 1,2-hexanediol and 0.02 ml of caprylyl glycol, and mix well.

[0076] Compounding solution 3: The preservative system is compound essential oil 4 (0.1%) + 1,2-hexanediol (0.2%) + caprylyl glycol (0.1%). Specific operation: Take 20 ml of tryptone soy agar medium (for mixed bacterial solution of Staphylococcus aureus and Escherichia coli) / Sabouraud glucose agar medium (for Candida albicans), add 0.02 ml of the mixed solution of compound essential oil 4 prepared in 4.2.2, 0.04 ml of 1,2-hexanediol and 0.02 ml of caprylyl glycol, and mix well.

[0077] 4.2.4 Anticorrosion efficiency test of compound anticorrosion system - anticorrosion challenge test The antiseptic challenge test simulates the risk of microbial contamination in actual use of the product by adding specific microorganisms to the product, and scientifically evaluates its antiseptic and antibacterial capabilities. The test provides quantitative data showing the growth or reduction trend of microorganisms in the product, thereby accurately evaluating the antiseptic effect.

[0078] Antiseptic performance test of compound solution 1: add 0.1ml of mixed bacterial solution of Staphylococcus aureus and Escherichia coli to 10ml of the antiseptic system of compound solution 1, and the final bacterial suspension concentration is 10 6 cfu / mL; add 0.1ml of Candida albicans bacterial solution to 10ml of the preservative system of compound solution 1, and the final bacterial suspension concentration is 10 5 cfu / mL. After culturing at 37°C (Staphylococcus aureus and Escherichia coli) and 28°C (Candida albicans) for a specific period of time (7, 14, 21, 28 days), 0.1 ml of the sample was taken and placed in 2 sterile plates for plate counting.

[0079] The testing steps for the anti-corrosion efficacy of compounding scheme 2 and compounding scheme 3 are the same as compounding scheme 1. 4.3 Test results

[0080] 4.3.1 Antibacterial activity of the compound preservative system containing compound essential oils and polyols against Staphylococcus aureus and Escherichia coli The results are shown in Table 4 and Figure 7 . The results show that the colony count of compound scheme 1 decreased by nearly 100% (sterile growth) on the 7th day, and it can effectively kill Escherichia coli and Staphylococcus aureus. No colony formation was observed during the 28-day experiment, indicating that it has a strong antibacterial and preservative effect; while for compound schemes 2 and 3, the colonies in the culture dish gradually increased with the extension of the culture time during the 28-day experiment. The above results indicate that compound scheme 1 has obvious advantages over compound schemes 2 and 3 in inhibiting bacterial activity.

[0081] Table 4 Colony concentrations of Staphylococcus aureus and Escherichia coli

[0082] 4.3.2 Antibacterial effect of the compound preservative system containing compound essential oils and polyols on Candida albicans Results Figure 8 .Depend on Figure 8 It can be seen that the fungal colonies of compound schemes 1, 2 and 3 were reduced by nearly 90% on the 7th day, which can effectively prevent the growth of Candida albicans in the culture medium, and no colony formation was observed during the 28-day experiment, indicating that they have a strong inhibitory effect on fungi. There is no significant difference in the antifungal activity of compound schemes 1, 2 and 3. 4.4 Experimental Conclusion

[0083] In summary, this experiment found that the preservative system of compound scheme 1 had a strong antibacterial effect on Escherichia coli, Staphylococcus aureus, and Candida albicans, and the preservative systems of compound schemes 2 and 3 had a strong antibacterial effect on Candida albicans.

[0084] [Example 5] Detection of the four kinds of single essential oils of the present invention by gas chromatography-mass spectrometry In this embodiment, the quality of each single essential oil of the present invention is tested by gas chromatography-mass spectrometry (GC-MS) so as to control the quality of the raw materials of the compound essential oil of the present invention.

[0085] 5.1 Preparation of test samples Pipette 0.5 mL of the essential oil to be tested into a 1.5 mL centrifuge tube and centrifuge for 1 min; take 50 μL of the supernatant, weigh it, add ethyl acetate to make an essential oil solution with a concentration of 100 mg / mL; add anhydrous sodium sulfate powder at a mass ratio of 1:10, mix well, let stand, and centrifuge; take 10 μL of the supernatant and dilute it 100 times with ethyl acetate to obtain a solution with a concentration of 1 mg / mL; centrifuge the solution to be tested for 1 min, take the supernatant, and detect it by gas chromatography-mass spectrometry.

[0086] 5.2 Detection methods Chromatographic column: Agilent HP-5 MS UI (30 m×0.25 mm×0.25 μm) Carrier gas: high purity helium Flow rate: 1.0 mL / min Inlet temperature: 250℃ Split ratio: 10:1 Injection volume: 1 μL Heating program: 50 ℃—180 ℃ (3 ℃ / min)—300 ℃ (20 ℃ / min, hold for 5 min) Ion source: EI+ Ion source temperature: 230°C Scan mode: Full ion scan Scan range: m / z 40 -600 Solvent delay: 4 min 5.3 Test results Test results such as Figure 9-12 The raw data of GC / MS were analyzed by Agilent MassHunter Qualitative Analysis (VB.07.00) software to obtain the peak information of each component (retention time, integrated peak area, total peak area percentage, and peak height).

Claims

1. A compound essential oil having an antibacterial effect, characterized in that: The compound essential oil consists of the following ingredients: wild chrysanthemum essential oil, jasmine essential oil, patchouli essential oil and citral camphor leaf essential oil; the volume ratio of the wild chrysanthemum essential oil, jasmine essential oil, patchouli essential oil and citral camphor leaf essential oil is selected from any one of the following groups: 1:4:3:2, 2:3:4:1, 3:2:1:4, 4:1:2:3 and 4:1:2:

3.

2. The compound essential oil according to claim 1, characterized in that The wild chrysanthemum essential oil is a volatile oil extracted from the flowers of wild chrysanthemum by steam distillation; the jasmine essential oil is a volatile oil extracted from the flowers of Chinese jasmine by supercritical carbon dioxide extraction; the patchouli essential oil is a volatile oil extracted from the aerial parts of patchouli by steam distillation; the citral camphor leaf essential oil is a volatile oil extracted from the fresh leaves of citral camphor trees by water distillation.

3. The compound essential oil according to claim 1, characterized in that The volume ratio of the wild chrysanthemum essential oil, the jasmine essential oil, the patchouli essential oil and the citral type camphor leaf essential oil is 4:1:2:

3.

4. An anti-corrosion system, characterized in that: Contains the compound essential oil as claimed in claim 1.

5. The anticorrosion system according to claim 4, characterized in that: The preservative system also includes 1,2-hexanediol and caprylyl glycol.

6. The anticorrosion system according to claim 5, characterized in that: The volume ratio of the compound essential oil, 1,2-hexanediol and caprylyl glycol is (1-10):2:

1.

7. The anticorrosion system according to claim 6, characterized in that: The volume ratio of the compound essential oil, 1,2-hexanediol and caprylyl glycol is 10:2:

1.

8. Use of the compound essential oil according to any one of claims 1 to 3 or the preservative system according to any one of claims 4 to 7 in cosmetics.

9. The use according to claim 8, characterized in that The content of the preservative system in the cosmetics is 0.05%-2%.

10. The use according to claim 9, characterized in that The content of the preservative system in the cosmetics is 0.5%.

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