Antibacterial Composition Containing Multiple Plant Extracts and Its Application

Abstract

The present invention discloses an antibacterial composition containing multiple plant extracts and its application. The antibacterial composition containing multiple plant extracts comprises raw materials in the following mass percentages: 10-40% of cinnamon bark extract and 60-90% of a composite extract, and the composite extract comprises a mixture of at least three of rosemary extract, myrrh extract, melaleuca alternifolia extract, and ginkgo biloba extract; the antibacterial composition containing multiple plant extracts of the present invention has little irritation, and has the effects of synergistic anti-corrosion, acaricidal, and anti-inflammatory. The antibacterial composition containing multiple plant extracts can be used in cosmetics. The raw materials are all derived from plants, and have the advantages of low dosage, mild raw materials, and natural low irritation.

Description

Technical Field

[0001] The present invention belongs to the field of plant extracts, and particularly relates to an antibacterial composition containing multiple plant extracts and its application. Background Art

[0002] At present, the main methods for removing mites are physical mite removal and chemical mite removal.

[0003] Physical mite removal mainly uses a physical mite removal instrument to remove mites through ultraviolet rays, ultrasonic waves or adsorption. However, the physical mite removal instrument is time-consuming and laborious to use, consumes a large amount of energy, and cannot guarantee the effect.

[0004] Chemical mite removal mainly kills mites through chemical substances such as bromopropylate, fenbutatin oxide, amitraz, etc. Most of the existing chemical mite removal agents are artificially synthesized, have certain potential safety hazards, may cause harm to the human body, and have a single efficacy. While having the efficacy of removing mites, it is difficult to achieve effects such as anti-corrosion or anti-inflammatory. Summary of the Invention

[0005] In view of the above deficiencies in the prior art, the present invention provides an antibacterial composition containing multiple plant extracts. The antibacterial composition containing multiple plant extracts has low irritation and has the synergistic effects of anti-corrosion, mite removal and anti-inflammatory. The antibacterial composition containing multiple plant extracts can be used in cosmetics. The raw materials are all derived from plants and have the advantages of low dosage, mild raw materials, and natural low irritation.

[0006] The purpose of the present invention is to provide an antibacterial composition containing multiple plant extracts, which comprises the following raw materials in mass percentage: 10 - 40% of cinnamon bark extract and 60 - 90% of a composite extract, and the composite extract comprises a mixture of at least three of rosemary extract, myrrh extract, melaleuca alternifolia extract, and ginkgo biloba extract.

[0007] In some embodiments of the present invention, in terms of mass percentage, the dosage of the rosemary extract is 5 - 20% of the antibacterial composition containing multiple plant extracts, the dosage of the myrrh extract is 20 - 40% of the antibacterial composition containing multiple plant extracts, the dosage of the melaleuca alternifolia extract is 5 - 25% of the antibacterial composition containing multiple plant extracts, and the dosage of the ginkgo biloba extract is 15 - 45% of the antibacterial composition containing multiple plant extracts.

[0008] In some embodiments of the present invention, in terms of mass percentage, the dosage of the myrrh extract is 20 - 40% of the antibacterial composition containing multiple plant extracts, the dosage of the melaleuca alternifolia extract is 5 - 25% of the antibacterial composition containing multiple plant extracts, and the dosage of the ginkgo biloba extract is 15 - 45% of the antibacterial composition containing multiple plant extracts.

[0009] In some embodiments of the present invention, by mass percentage, the dosage of the rosemary extract is 5-20% of the antibacterial composition containing various plant extracts, the dosage of the Melaleuca alternifolia extract is 5-25% of the antibacterial composition containing various plant extracts, and the dosage of the ginkgo biloba extract is 15-45% of the antibacterial composition containing various plant extracts.

[0010] In some embodiments of the present invention, by mass percentage, the dosage of the rosemary extract is 5-20% of the antibacterial composition containing various plant extracts, the dosage of the myrrh extract is 20-40% of the antibacterial composition containing various plant extracts, and the dosage of the ginkgo biloba extract is 15-45% of the antibacterial composition containing various plant extracts.

[0011] In some embodiments of the present invention, by mass percentage, the dosage of the rosemary extract is 5-20% of the antibacterial composition containing various plant extracts, the dosage of the myrrh extract is 20-40% of the antibacterial composition containing various plant extracts, and the dosage of the Melaleuca alternifolia extract is 5-25% of the antibacterial composition containing various plant extracts.

[0012] In some embodiments of the present invention, the extraction process of the cinnamon bark extract includes the following steps:

[0013] Take the dry cinnamon bark, crush it, sieve it, and subject the medicinal powder to the first supercritical carbon dioxide extraction, collect the extract, and filter it to obtain the cinnamon bark extract.

[0014] In some embodiments of the present invention, for the first supercritical carbon dioxide extraction, the extraction temperature is 10-20°C, the extraction pressure is 10-30 MPa, the CO2 flow rate is 20-40 L / h, and the extraction time is 2-4 h.

[0015] In some embodiments of the present invention, the extraction process of the rosemary extract includes the following steps:

[0016] Take the dried rosemary, crush it, sieve it, and subject the medicinal powder to the second supercritical carbon dioxide extraction, collect the extract, and filter it to obtain the rosemary extract.

[0017] In some embodiments of the present invention, for the second supercritical carbon dioxide extraction, the extraction temperature is 5-15°C, the extraction pressure is 15-25 MPa, the CO2 flow rate is 10-30 L / h, and the extraction time is 2-4 h.

[0018] In some embodiments of the present invention, the extraction process of the myrrh extract includes the following steps:

[0019] Take myrrh, crush it, sieve it, place the medicinal powder in a steam distillation device, add water, perform steam distillation extraction, let it stand, take the oil layer, remove water, and filter to obtain myrrh extract.

[0020] In some embodiments of the present invention, the mass ratio of myrrh to water is 1:18 - 23.

[0021] In some embodiments of the present invention, the temperature of the steam distillation extraction is 90 - 100 °C, and the time is 2 - 8 hours.

[0022] In some embodiments of the present invention, the extraction process of the Melaleuca viridiflora extract includes the following steps:

[0023] Take Melaleuca viridiflora leaves and place them in a steam distillation device, add water, perform steam distillation extraction, let it stand, take the oil layer, remove water, and filter to obtain Melaleuca viridiflora extract.

[0024] In some embodiments of the present invention, the mass ratio of Melaleuca viridiflora leaves to water is 1:18 - 23.

[0025] In some embodiments of the present invention, the temperature of the steam distillation extraction is 90 - 100 °C, and the time is 2 - 8 hours.

[0026] In some embodiments of the present invention, the extraction process of the Ginkgo biloba extract includes the following steps:

[0027] Take Ginkgo biloba leaves and place them in a steam distillation device, add water, perform steam distillation extraction, let it stand, take the oil layer, remove water, and filter to obtain Ginkgo biloba extract.

[0028] In some embodiments of the present invention, the mass ratio of Ginkgo biloba leaves to water is 1:18 - 23.

[0029] In some embodiments of the present invention, the temperature of the steam distillation extraction is 90 - 100 °C, and the time is 2 - 8 hours.

[0030] Another object of the present invention is to provide the application of the antibacterial composition containing multiple plant extracts in the preparation of daily chemical products, and the daily chemical products contain the antibacterial composition containing multiple plant extracts.

[0031] In some embodiments of the present invention, the application of the antibacterial composition containing multiple plant extracts in the preparation of daily chemical products, the daily chemical products include at least one of body wash, facial mask liquid, lotion, cream, toner, essence, stock solution, facial cleanser, lotion, perfume, makeup remover, liquid foundation, cream foundation, concealer, rouge, lipstick, eyeshadow, blush.

[0032] In some embodiments of the present invention, the application of the antibacterial composition containing multiple plant extracts in the preparation of daily chemical products, calculated by mass percentage, the dosage of the antibacterial composition containing multiple plant extracts is 0.2-1% of the daily chemical products.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) Each component of the antibacterial composition containing multiple plant extracts provided by the present invention is a common plant extract, which has the advantages of safety, mildness, and low irritation, and at the same time has multiple effects.

[0035] (2) There is a synergistic effect among the components of the antibacterial composition containing multiple plant extracts provided by the present invention, which can not only improve the mite removal effect, but also have anti-corrosion and anti-inflammatory properties at the same time. Specific embodiments

[0036] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0037] Example 1

[0038] This example provides an extraction process for cinnamon bark extract. The specific extraction process is as follows:

[0039] Take 1 kg of dry cinnamon bark, crush it with a pulverizer and pass through a 20-mesh sieve. Place the medicinal powder in a supercritical carbon dioxide extraction kettle, turn on the extraction equipment, and set the extraction conditions as follows: extraction temperature 15°C, extraction pressure 20 MPa, CO2 flow rate 30 L / h, extraction time 3 h. After the extraction is completed, collect the extract and filter to obtain cinnamon bark extract.

[0040] Example 2

[0041] This example provides an extraction process for rosemary extract. The specific extraction process is as follows:

[0042] Take 5 kg of fresh whole rosemary plants, wash them and dry them in an oven at 45°C to obtain dry medicinal materials. Crush the dry medicinal materials and pass through a 20-mesh sieve. Place the medicinal powder in a supercritical carbon dioxide extraction kettle, turn on the extraction equipment, and set the extraction conditions as follows: extraction temperature 10°C, extraction pressure 18 MPa, CO2 flow rate 20 L / h, extraction time 3 h. After the extraction is completed, collect the extract and filter to obtain rosemary extract.

[0043] Example 3

[0044] This embodiment provides an extraction process for myrrh extract, and the specific extraction process is as follows:

[0045] Take 1 kg of myrrh, crush it with a pulverizer and sieve it through a 20-mesh sieve. Place it in a steam distillation device at a temperature of 95°C. Based on the weight content, add 18 - 23 times the amount of water. Perform steam distillation extraction for 5 hours. Let the collected oil-water mixture stand for 1 day. After oil-water separation, take the oil layer, add 10 g of anhydrous sodium sulfate, filter and take the filtrate after 12 hours to obtain myrrh extract.

[0046] Example 4

[0047] This embodiment provides an extraction process for Melaleuca viridiflora extract, and the specific extraction process is as follows:

[0048] Take 5 kg of fresh leaves of Melaleuca viridiflora, wash, dry in the shade and place them in a steam distillation device at a temperature of 95°C. Based on the weight content, add 18 - 23 times the amount of water. Perform steam distillation extraction for 5 hours. Let the collected oil-water mixture stand for 1 day. After oil-water separation, take the oil layer, add 10 g of anhydrous sodium sulfate, filter and take the filtrate after 12 hours to obtain Melaleuca viridiflora extract.

[0049] Example 5

[0050] This embodiment provides an extraction process for ginkgo leaf extract, and the specific extraction process is as follows:

[0051] Take 5 kg of fresh ginkgo leaves, wash, dry in the shade and place them in a steam distillation device at a temperature of 95°C. Based on the weight content, add 18 - 23 times the amount of water. Perform steam distillation extraction for 5 hours. Let the collected oil-water mixture stand for 1 day. After oil-water separation, take the oil layer, add 10 g of anhydrous sodium sulfate, filter and take the filtrate after 12 hours to obtain ginkgo leaf extract.

[0052] Preparation of an antibacterial composition containing multiple plant extracts:

[0053] Table 1. Raw material composition of the antibacterial composition containing multiple plant extracts.

[0054]

[0055] Preparation of AES shower gel:

[0056] Table 2. Composition table and production process of AES shower gel.

[0057]

[0058] Antiseptic efficacy test:

[0059] Antiseptic efficacy detection method: Refer to "USP43 (51) Microbiological Antiseptic Efficacy Testing" in the United States Pharmacopeia.

[0060] Experimental instruments, equipment and reagents: Incubator, refrigerator, constant temperature water bath, electronic balance, chemical reagent bottles with screw threads, glass beads, petri dishes (90 mm in diameter), measuring cylinder, pipettes (1 mL, 5 mL), homogenizer, electrothermal constant temperature forced air drying oven, vertical automatic pressure steam sterilizer, wide-mouth transparent glass sample bottles, narrow-mouth transparent glass sample bottles, pH meter or precision pH test paper, magnifying glass, culture media (lecithin polysorbate 80 nutrient agar, rose bengal agar) and reagents, normal saline, liquid paraffin, polysorbate 80, 0.5% triphenyltetrazolium chloride.

[0061] Test strains: Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231, Aspergillus niger ATCC 16404, Burkholderia cepacia ATCC25416, Pseudomonas putida ATCC 17485.

[0062] Experimental steps: (1) Sample treatment: Weigh appropriate amounts of the AES shower gels in each of Examples 11 to 15 and Comparative Examples 6 to 10 into two sterilized narrow-mouth glass sample bottles for standby.

[0063] (2) Preparation of mixed bacterial suspension: Add an appropriate amount of sterile 0.9% sodium chloride aqueous solution to the slants of the activated bacterial and Candida albicans standard strains for elution of the bacterial colonies. Pour the higher-concentration bacterial suspensions into sterile containers of the same size as the McFarland turbidity standards respectively; elute the Aspergillus niger spores with sterile 0.9% sodium chloride aqueous solution containing 0.05% polysorbate 80 to prepare an Aspergillus niger spore suspension, and filter the Aspergillus niger spore suspension to remove mycelia for standby. Dilute the high-concentration single bacterial suspension with an appropriate amount of sterile 0.9% sodium chloride aqueous solution and compare it with the corresponding-concentration McFarland turbidity standard. When the turbidity is the same, it is okay. The standard McFarland turbidity standards need to be shaken well before use. Mix the single bacterial suspensions 1:1 respectively, mix the single fungal suspensions 1:1 respectively, and then mix the bacterial and fungal suspensions 1:1 to obtain the mixed bacterial suspension for standby.

[0064] (3) Counting of the mixed bacterial suspension: Transfer 1 mL of the prepared mixed bacterial suspension to a test tube containing 9 mL of sterile normal saline, mix well to prepare a 1:10 dilution, and so on, successively dilute 1:10 5 、1:10 6 、1:10 7 ; Select 1:10 5 ~1:10 7Pour the diluted gradient solution onto a plate for counting, and calculate the total number of bacterial colonies and the total number of fungal colonies in the mixed bacterial solution. If the concentration of the mixed bacterial suspension is not within the required range for adding bacteria, the sample needs to be re-inoculated and tested.

[0065] (4)Sample inoculation and cultivation: Add 1% of the mixed bacterial suspension to the AES body washes in Examples 11 - 15 and Comparative Examples 6 - 10 on the 0th day, 14th day, and 28th day respectively. The concentration of the bacteria added each time in Examples 11 - 15, Comparative Examples 6 - 10, and the blank control is the same. The initial concentration for the first inoculation: bacterial concentration (5.5×10 5 ), fungal concentration (1.8×10 5 ); The initial concentration for the second inoculation: bacterial concentration (6.7×10 5 ), fungi (2.3×10 5 ); The concentration for the third inoculation: bacteria (7.3×10 5 ), fungi (2.6×10 5 ). Thoroughly mix the test sample with the bacterial suspension. Incubate in an incubator at 22.5°C ± 2.5°C.

[0066] (5)Sample detection and analysis: Take samples for detection on the 14th day after each inoculation. Sample treatment: Inject an appropriate amount of the test sample into sterile physiological saline and dilute it to 1:10, and mix thoroughly. Multiple gradient dilutions can be made as needed, such as 1:100, 1:1000, 1:10000, etc. A new pipette tip should be used for sampling at each dilution; Plate pouring: Use a pipette to aspirate the diluted test sample and inject it into a sterile petri dish, 1 mL per dish. A total of 4 petri dishes are used for each dilution (2 for bacteria and 2 for fungi). Pour the melted lecithin - Tween 80 nutrient agar cooled to about 45°C - 48°C into the bacterial petri dishes, about 15 mL per dish. Immediately rotate the petri dish to thoroughly mix the sample with the lecithin - Tween 80 nutrient agar. After the lecithin - Tween 80 nutrient agar solidifies, invert the petri dish and incubate it in an incubator at 36°C ± 1°C for 72 h ± 2 h. Pour the melted Rose Bengal agar cooled to about 45°C - 48°C into the fungal petri dishes, about 15 mL per dish. Immediately rotate the petri dish to thoroughly mix the sample with the Rose Bengal agar. After the Rose Bengal agar solidifies, invert the petri dish and incubate it at 28°C ± 2°C for 72 h ± 2 h.

[0067] (6) Colony counting: First observe with the naked eye and count the number of colonies. If necessary, use a 5x-10x magnifying glass to check again to prevent omissions. After recording the number of colonies on each plate, calculate the average number of colonies grown on each plate with the same dilution. If there are colonies in the plate that are connected into sheets or flower-like colonies that spread and grow, the plate should not be counted. If the sheet-like colonies account for less than half of the plate, and the number of colonies in the remaining half is evenly distributed, count the colonies in this half of the plate and multiply by 2 to represent the number of colonies in the entire plate.

[0068] Result judgment criteria: (1) Excellent preservative effect: that is, after three additions of bacteria, on the 14th day after each addition, the amount of surviving bacteria is reduced to no more than 0.01% of the initial concentration, and the test is passed; (2) Fair preservative effect: that is, after three additions of bacteria, on the 14th day after each addition, the amount of surviving bacteria is reduced to no more than 0.1% of the initial concentration, and the test is passed; (3) that is, after three additions of bacteria, on the 14th day after each addition, the amount of surviving bacteria is higher than 0.1% of the initial concentration, and the test is not passed.

[0069] The sample is AES shower gel, and the formula and production process are shown in Table 2. The blank control is AES shower gel without adding the antibacterial composition in Table 1.

[0070] Table 3. AES shower gel antiseptic efficacy test data.

[0071]

[0072] It can be seen from Table 3 that the antibacterial composition containing multiple plant extracts of the present invention has a synergistic antiseptic effect.

[0073] Mite killing effect test:

[0074] Mite test (refer to the standard method of mite test of the Ministry of Agriculture and Rural Affairs: NY / T 1151.2-2006).

[0075] (1) Experimental equipment and materials: constant temperature and humidity incubator (temperature (25±1)℃, humidity 70%-80%), covered container (about 30cm×25cm×5cm), beaker (150mL), stereo microscope, disposable plate, white oil and vaseline mixture (1:1), saturated salt water, adult or nymph dust mites, mite feed, and mite counting tools (counter, dissecting needle, brush).

[0076] (2) Preparation of experimental samples: Prepare the mite control samples to be tested.

[0077] (3) Experimental operation: Take disposable petri dishes, add 5 g of the AES body washes of Examples 11 to 15 and Comparative Examples 6 to 10 into each petri dish respectively, and evenly apply a white petrolatum mixture on the upper edge of the inner wall of each petri dish. Add 200 - 300 mites to the center of each petri dish, and then put 0.05 g of mite feed in the center of the petri dish after 30 min. Place them in a constant temperature and humidity incubator. After 48 hours, take out the petri dishes and check and record the number of dead mites under a stereomicroscope, and calculate the acaricidal rate.

[0078] (4) Calculation of acaricidal rate:

[0079] (5) The test system is the AES body wash, and the formula and production process are shown in Table 2. Among them, the blank control is the AES body wash without adding the antibacterial composition in Table 1.

[0080] Table 4. Test results of acaricidal rate.

[0081]

[0082] As can be seen from Table 4, the antibacterial composition containing multiple plant extracts of the present invention has a synergistic acaricidal effect.

[0083] Preparation of skin toner:

[0084] Table 5. Composition table and production process of skin toner.

[0085]

[0086] Anti - inflammatory efficacy test:

[0087] (1) Test name: Test for the soothing efficacy of daily chemical products - Determination of the content of IL - 1β inflammatory factor in vitro and test method for the inflammatory cell model of lipopolysaccharide - induced macrophage cell line.

[0088] (2)Test principle: Bacterial lipopolysaccharide (LPS) binds to the antigen recognition receptor on the surface of macrophages, inducing macrophages to secrete various cytokines such as IL-1β. IL-1β can activate three signaling pathways, namely Caspase protease, JNK, and transcription factor NF-κB, to achieve its biological functions such as cytotoxicity, antiviral activity, immune regulation, and apoptosis. It is one of the important inflammatory factors. LPS-induced RAW264.7 is a classic cell model for studying inflammatory factors. By comparing the differences in the content of IL-1β secreted by RAW264.7 after administering the negative control and the test substance, the effect of the test substance on inhibiting the secretion of IL-1β is evaluated. The content of IL-1β is determined by the enzyme-linked immunosorbent assay (ELISA). The specific principle is as follows: After IL-1β specifically binds to the IL-1β antibody coated on the enzyme-linked immunosorbent assay plate, it binds to the anti-IL-1β antibody labeled with a substrate. After the substrate is catalyzed by the enzyme, a colored product is generated. The content of IL-1β is positively correlated with the depth of the color of the colored product. The optical density value (OD) is measured at a wavelength of 450 nm with an enzyme-linked immunosorbent assay reader, and the content of IL-1β is calculated.

[0089] (3)Instruments and equipment:

[0090] Adjustable pipettes: 1000 μL, 200 μL, 50 μL, 10 μL; CO2 incubator: 37 °C, humidified, 5% CO2 / air; Enzyme-linked immunosorbent assay reader: equipped with a 450 nm filter; Laminar flow hood.

[0091] Micro oscillator; Analytical balance with an accuracy of 0.1 mg; Constant temperature incubator; Vortex shaker; Cell counter or hemocytometer; Inverted microscope; Ultra-low temperature freezer (-80 °C); Low-speed centrifuge.

[0092] (4)Reagents and consumables:

[0093] Cells: The mouse monocyte macrophage leukemia cell line RAW264.7 is selected. The cell strain is derived from the American Type Culture Collection (ATCC), and the selected model is ATCC TIB-71. The cells should be regularly tested to ensure that they are free from mycoplasma contamination and can only be used if they are pollution-free.

[0094] Cell culture reagents and consumables: High-glucose DMEM medium, fetal bovine serum or newborn bovine serum, trypsin / EDTA solution (a 1:1 mixture of 0.25% trypsin solution and 0.02 mol / L EDTA solution), phosphate buffer solution (PBS), cell culture flasks.

[0095] (5) Detection: Bacterial lipopolysaccharide (LPS, from Escherichia coli); 96-well cell culture plate; 1L-1β ELISA detection kit.

[0096] (6) Test method:

[0097] Cell preparation: Cells from cryopreserved cell cultures were inoculated at an appropriate density and passaged at least once before detection. Cells should be inoculated at an appropriate density for the test, and the cell seeding density should ensure that the confluence reaches 45% - 60% 24 hours after inoculation.

[0098] Test article preparation: Unless stability data prove that the stock solution is acceptable, the test article should be freshly prepared and used directly before use. Test articles with limited solubility in water should be dissolved in an appropriate solvent, and the volume of the solvent should be consistent in all cultures, i.e., in the blank control, negative control, and test article, and should be non-cytotoxic.

[0099] (7) Experimental conditions:

[0100] The concentration range of the test article should be determined by cytotoxicity testing, for example, selecting a concentration with a cell viability ≥ 90%. The concentration of LPS should preferably be 1 μg / mL, and the specific stimulation concentration can be adjusted according to the characteristics of the cells cultured in the laboratory. The quality standard that the test should meet is: The upregulation fold of the 1L-1β content in the negative control under the action of LPS compared to the blank control is ≥ 5 times.

[0101] Positive control: A positive control should be used in every test. Recommended positive control substances, such as 100 μg / mL dexamethasone, and the specific dosing concentration can be adjusted according to the characteristics of the cells cultured in each laboratory.

[0102] (8) Experimental procedure:

[0103] Cell digestion and inoculation:

[0104] Trypsin / EDTA was used, and the specific digestion concentration and dosage need to be determined according to the characteristics of the cells in the laboratory: The trypsin concentration should preferably be 0.25%, the trypsin dosage for a T25 culture flask is 1 mL, and the trypsin dosage for a T75 culture flask is 3 mL.

[0105] Observed under a microscope. When most cells became round and were in a suspended state, DMEM medium containing serum about 2 - 3 times the volume of trypsin was added to terminate digestion, and the cells were collected into a centrifuge tube and centrifuged at 800 r / min for 6 min. The rotation speed and centrifugation time can be determined according to the characteristics of the cells in the laboratory.

[0106] After centrifugation, discard the supernatant. Add a certain volume of cell culture medium to the centrifuge tube, and pipette and mix the cells evenly with a bent pipette. Count the cells using a cell counter or a hemocytometer.

[0107] Dilute the cells with cell culture medium to the seeding density (the confluence reaches 45% - 60% 24 h after seeding), and seed them into a 96-well plate with a liquid volume of 200 μL per well.

[0108] After seeding, place it in a CO2 incubator and culture for 24 h ± 2 h.

[0109] Induction and drug administration: Discard the medium in the 96-well plate and carry out the induction and drug administration operations. Add the medium containing a certain concentration of the test substance and LPS to the test substance wells, add the cell culture medium containing LPS to the negative control wells, add the medium containing the positive control and LPS to the positive control wells, and add the cell culture medium to the blank / solvent control wells, 200 μL per well. After drug administration, place the 96-well plate in a CO2 incubator and culture for 24 h ± 2 h.

[0110] Cell supernatant collection: After the incubation culture is completed, collect 200 μL of cell culture supernatant into a 1.5 mL sterile centrifuge tube and store it frozen at -80°C in an ultra-low temperature freezer.

[0111] ELISA detection: ELISA detection should be carried out according to the operation manual of the 1L-1β ELISA detection kit.

[0112] Since the expression level of 1L-1β is relatively high after LPS induction, before the formal detection, a dilution ratio should be set and a preliminary experiment should be carried out to explore and determine the dilution ratio of the ELISA detection experimental group to ensure that the ELISA detection values fall within the standard curve range.

[0113] (9)Calculation of results:

[0114] Calculation of 1L-1β content: It is recommended to use professional curve-making software. Take the concentration of the standard product as the ordinate and the OD450 value as the abscissa to make a standard curve. According to the OD450 value of the sample, find out the corresponding concentration. Or calculate the regression equation of the standard curve with the concentration of the standard product and the OD450 value, substitute the OD450 value of the sample into the equation, and calculate the 1L-1β content of the sample. Finally, take the average value of 3 replicates of each group as the final 1L-1β content result.

[0115] Calculation formula for 1L-1β inhibition rate:

[0116]

[0117] (10)Result reporting:

[0118] The content of 1L-1β should be expressed as: the average value of the content ± standard deviation (SD).

[0119] For the description of the inhibition of the 1L-1β content by the test substance, the test concentration should be included.

[0120] (11) The test substance is a toner, and the formula and production process are shown in Table 5.

[0121] (12) Interpretation of result correlation: On the basis that the test meets the validation of effectiveness, compared with the negative control, the content of 1L-1β decreases for the test substance, indicating that the test substance has the effect of inhibiting the 1L-1β content at the test concentration, which can be used as one of the evidences for the appellation of the raw material of soothing daily chemical products.

[0122] Table 6. Test results of 1L-1β inhibition rate.

[0123]

[0124] It can be seen from the results in Table 6 that the antibacterial composition containing multiple plant extracts of the present invention has the effect of synergistically inhibiting IL-1β.

[0125] Irritation test:

[0126] Experiment name: Chicken embryo chorioallantoic membrane experiment (endpoint evaluation method).

[0127] Experiment principle: The chicken embryo chorioallantoic membrane test is an in vitro evaluation method for eye irritation that has been used earlier. The chorioallantoic membrane (CAM) is a respiratory membrane that surrounds the chicken embryo. This test utilizes the characteristics of the intact, clear, and transparent vascular system of the chorioallantoic membrane in the middle stage of the hatched chicken embryo. A certain amount of the test substance is directly contacted with the chicken embryo chorioallantoic membrane, and after acting for a period of time, the toxic effect indicators of the chorioallantoic membrane are observed, such as changes in bleeding, coagulation, and vascular lysis. These indicators reflect the changes in the morphological structure, color, and permeability of blood vessels and vascular networks, as well as the phenomena of protein denaturation of the chorioallantoic membrane and its degree of damage. Then, a score is combined to evaluate the eye irritation of the test substance. The purpose of this test is to test the ability of the test substance to cause toxic changes in the chicken embryo chorioallantoic membrane, and the standard describes the elements and processes for evaluating the potential eye irritation of the substance to be evaluated.

[0128] Experiment preparation: (1) Source of chicken embryo strain: Fertilized chicken embryos of White Leghorn chicken. TQG chicken embryos should be selected, and the quality of the chicken embryos should meet the requirements of relevant standards. The supplier should have the qualification of "Designated Production Enterprise of TQG Chicken (Egg) for Veterinary Drug Production and Inspection" recognized by the agricultural and rural departments.

[0129] (2) Transportation and storage: Purchase chicken embryos within 7 days old and store them on the egg rack with the air chamber facing up for transportation. The chicken embryos should be transferred or transported without affecting their viability or development, and avoid shaking, unnecessary tilting, knocking, and other mechanical stimuli to the chicken embryos.

[0130] (3) The chicken embryos should be fresh, clean, and intact, with a mass of 50 g to 60 g. When incubated to 9 days old, candling inspection should be carried out, and the unfertilized, non-viable, or defective chicken embryos should be discarded. Severely malformed, cracked, or thin-shelled chicken embryos cannot be used either.

[0131] (4) Incubation conditions: Room temperature is 20°C to 25°C, relative humidity is 45% to 70%. Incubation temperature is 37.5°C ± 0.5°C, relative humidity is 55% to 70%, and the turntable frequency is 3 times / h to 6 times / h. Chicken embryos at 9 days old do not need to be rotated during incubation.

[0132] Test instruments and reagent materials: Automatic incubator, thermostatic and humidity-controlled incubator, double-person ultra-clean workbench, high-pressure steam sterilizer, single-lens reflex camera, LED high-brightness cold light source candler, hand drill for manual punching, medical ophthalmic curved pointed forceps: 10 CM, polytetrafluoroethylene resin ring: 12*9*1.5 mm, pipette: 10 - 100 μl, pipette tips: 10 - 100 μl, electronic timer, wide-mouth conical flask: 100 ml, conical flask: 500 ml, sodium chloride: analytical grade, sterile water.

[0133] Preparation of test substances and control substances: (1) Test substance: Toner, the formula and production process are shown in Table 5. (2) Negative control: Select an aqueous sodium chloride solution with a mass concentration of 0.9% for rinsing after the action of the test substance and negative control. A negative control of 0.9% sodium chloride should be set for each test of the test substance to ensure that the test conditions do not cause irritant reactions. (3) Blank control: Toner without adding the antibacterial composition. (4) Positive control: 10% aqueous acrylamide solution.

[0134] Test procedure: (1) Each test sample should be tested using 10 chicken embryos. For each batch of chicken embryos, negative control, positive control, and solvent control should be set up, with 3 embryos in each group. (2) Endpoint evaluation method: Take 0.3 mL of the test substance and directly apply it to the CAM, ensuring that at least 50% of the CAM surface is covered by the test substance. After 3 minutes of application. (3) CAM preparation: Take out a 10-day-old chicken embryo, use an egg candler to check the air chamber to determine its position, use a manual hole-punching tool with a hand drill to make a small window on the air chamber, use a medical ophthalmic curved pointed forceps to peel off the upper part of the eggshell of the air chamber to expose the white eggshell membrane, use a pipette to drip an appropriate amount of 0.9% sodium chloride solution to moisten the eggshell membrane. After it is completely moist, pour out the 0.9% sodium chloride solution. Carefully use forceps to remove the eggshell membrane. During the removal process, be cautious to ensure that the vascular membrane is not damaged. What shows rich capillaries after removing the eggshell membrane is the CAM. (4) Photograph and record: Use a photographing device to record the situation of the chorioallantoic membrane. If necessary, photograph and record the situations of the three groups of chorioallantoic membranes, namely the CAM that has not contacted the test ring and the sample, the CAM area after placing the test ring, and the test area after adding the sample and culturing for 30 minutes. Through these three groups of pictures, compare and analyze the damage situation and degree of the CAM. (5) Add the test sample: Place a polytetrafluoroethylene resin ring on the chorioallantoic membrane of the chicken embryo as the test area. Use a pipette to transfer 40 μl of the sample to be tested into the polytetrafluoroethylene resin ring, record the time of adding the sample, cover the air chamber with a moistened plastic wrap, and transfer the chicken embryo to a constant temperature and humidity incubator for 30 minutes. (6) Result observation: After culturing for 30 minutes after adding the sample, take out the test chicken embryo, directly take a photograph and observe the degree of vascular damage, and score according to the degree of vascular damage.

[0135] Scoring reference criteria:

[0136] Table 7. Scoring reference criteria for irritation test experiment.

[0137]

[0138] Result analysis: The results of each egg should be evaluated and scored. Finally, remove the highest score and the lowest score from the ten scores obtained. For the remaining 8 valid scores, calculate the irritation score (NC) according to the following formula.

[0139] NC = (X1 + X2 + …… + Xn) / n

[0140] Where: X —— Irritation score

[0141] n —— Number of valid chicken embryos after removing the highest score and the lowest score

[0142] Classify the irritation of the test substance according to the calculated NC value in Table 8.

[0143] Table 8. Classification of irritation of test substance.

[0144]

[0145] Table 9. Results of irritation test.

[0146]

[0147] As can be seen from Table 9, the bacteriostatic composition containing multiple plant extracts of the present invention has low irritation and has the advantages of being safe, mild and having low irritation.

[0148] In the antibacterial composition containing multiple plant extracts of the present invention, the mass percentage content of cinnamon bark extract is in the range of 10 - 40%, and the mass percentage content of the composite extract is in the range of 60 - 90% (for example: the mass percentage content of cinnamon bark extract is 10%, 15%, 20%, 25%, 30%, 35%, 40%, and the mass percentage content of the composite extract is 60%, 65%, 70%, 75%, 80%, 85%, 90%, etc.); and / or other solvents are used to extract rosemary, myrrh, tea tree, and ginkgo leaves (for example: solvents such as ethanol, methanol, petroleum ether, ethyl acetate, etc. are used to extract rosemary, myrrh, tea tree, and ginkgo leaves); and / or by mass percentage, the dosage of the rosemary extract is 5 - 20% of the antibacterial composition containing multiple plant extracts, the dosage of the myrrh extract is 20 - 40% of the antibacterial composition containing multiple plant extracts, the dosage of the tea tree extract is 5 - 25% of the antibacterial composition containing multiple plant extracts, and the dosage of the ginkgo leaf extract is 15 - 45% of the antibacterial composition containing multiple plant extracts; and / or by mass percentage, the dosage of the myrrh extract is 20 - 40% of the antibacterial composition containing multiple plant extracts, the dosage of the tea tree extract is 5 - 25% of the antibacterial composition containing multiple plant extracts, and the dosage of the ginkgo leaf extract is 15 - 45% of the antibacterial composition containing multiple plant extracts; and / or by mass percentage, the dosage of the rosemary extract is 5 - 20% of the antibacterial composition containing multiple plant extracts, the dosage of the tea tree extract is 5 - 25% of the antibacterial composition containing multiple plant extracts, and the dosage of the ginkgo leaf extract is 15 - 45% of the antibacterial composition containing multiple plant extracts; and / or by mass percentage, the dosage of the rosemary extract is 5 - 20% of the antibacterial composition containing multiple plant extracts, the dosage of the myrrh extract is 20 - 40% of the antibacterial composition containing multiple plant extracts, and the dosage of the ginkgo leaf extract is 15 - 45% of the antibacterial composition containing multiple plant extracts; and / or by mass percentage, the dosage of the rosemary extract is 5 - 20% of the antibacterial composition containing multiple plant extracts, the dosage of the myrrh extract is 20 - 40% of the antibacterial composition containing multiple plant extracts, and the dosage of the tea tree extract is 5 - 25% of the antibacterial composition containing multiple plant extracts; the obtained antibacterial composition containing multiple plant extracts all has the effects of synergistic anti-corrosion, anti-mite, and anti-inflammatory.

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that after reading the specification of this application, they can still modify the specific implementation manners of the present invention or make equivalent substitutions, but these modifications or changes do not depart from the protection scope of the pending claims of this invention application.

Claims

1. An antibacterial composition containing multiple plant extracts, characterized in that, It consists of raw materials with the following mass percentages: 10 - 40% of cinnamon bark extract and 60 - 90% of compound extract, and the compound extract is selected from myrrh extract, melaleuca alternifolia extract, and ginkgo leaf extract; By mass percentage, the dosage of the myrrh extract is 20 - 40% of the antibacterial composition containing various plant extracts, the dosage of the melaleuca alternifolia extract is 5 - 25% of the antibacterial composition containing various plant extracts, and the dosage of the ginkgo leaf extract is 15 - 45% of the antibacterial composition containing various plant extracts; The extraction process of the cinnamon bark extract includes the following steps: Take dry cinnamon bark, crush it, sieve it, subject the medicinal powder to first supercritical carbon dioxide extraction, collect the extract, filter it to obtain the cinnamon bark extract; The extraction process of the myrrh extract includes the following steps: Take myrrh, crush it, sieve it, place the medicinal powder in a steam distillation device, add water, carry out steam distillation extraction, let it stand, take the oil layer, remove water, filter it to obtain the myrrh extract; The extraction process of the melaleuca alternifolia extract includes the following steps: Take melaleuca alternifolia leaves, place them in a steam distillation device, add water, carry out steam distillation extraction, let it stand, take the oil layer, remove water, filter it to obtain the melaleuca alternifolia extract; The extraction process of the ginkgo leaf extract includes the following steps: Take ginkgo leaves, place them in a steam distillation device, add water, carry out steam distillation extraction, let it stand, take the oil layer, remove water, filter it to obtain the ginkgo leaf extract.

2. The antibacterial composition containing multiple plant extracts according to claim 1, characterized in that, For the first supercritical carbon dioxide extraction, the extraction temperature is 10 - 20°C, the extraction pressure is 10 - 30 MPa, the CO2 flow rate is 20 - 40 L / h, and the extraction time is 2 - 4 h.

3. The antibacterial composition containing multiple plant extracts according to claim 1, characterized in that, The mass ratio of the myrrh to water is 1:18 - 23.

4. The antibacterial composition containing multiple plant extracts according to claim 1, characterized in that, The mass ratio of the melaleuca alternifolia leaves to water is 1:18 - 23.

5. The antibacterial composition containing multiple plant extracts according to claim 1, characterized in that, The mass ratio of the ginkgo leaves to water is 1:18 - 23.

6. The application of the antibacterial composition containing multiple plant extracts according to any one of claims 1 to 5 in the preparation of daily chemical products, characterized in that, The daily chemical product contains the antibacterial composition containing various plant extracts.

7. Use of the antibacterial composition containing multiple plant extracts according to claim 6 in the preparation of daily chemical products, characterized in that, The daily chemical product includes at least one of body wash, lotion, cream, essence, facial cleanser, lotion, perfume, rouge, lipstick, eyeshadow, and blush.

8. Use of the antibacterial composition containing multiple plant extracts according to claim 6 in the preparation of daily chemical products, characterized in that, The daily chemical product includes at least one of facial mask liquid, essence, makeup remover, toner, liquid foundation, cream foundation, and concealer.

9. Use of the antibacterial composition containing multiple plant extracts according to any one of claims 7 to 8 in the preparation of daily chemical products, characterized in that, By mass percentage, the dosage of the antibacterial composition containing various plant extracts is 0.2 - 1% of the daily chemical product.

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