A multifunctional plant antibacterial agent and its application

Abstract

The invention discloses a multifunctional plant bacteriostatic agent and application thereof. The multifunctional plant bacteriostatic agent comprises the following raw materials in percentage by weight: 10-60% of the bacteriostatic agent, and the bacteriostatic agent comprises a mixture of at least two of cinnamon bark extract, lemon fruit extract, jasmine extract and camellia extract. The multifunctional plant bacteriostatic agent has low irritation, synergistic antiseptic, mite removal and anti-inflammatory effects, is used in daily chemical products, and has the advantages of low dosage, mild raw materials, natural low irritation and the like.

Description

Technical Field

[0001] The invention belongs to the field of plant extracts, and in particular relates to a multifunctional plant antibacterial agent and application thereof. Background Art

[0002] In recent years, with the improvement of living standards, people pay more and more attention to the maintenance of their skin, and the use of cosmetics and skin care products has become more frequent. Preservatives are an indispensable ingredient in cosmetics. Adding preservatives can inhibit the growth of microorganisms in cosmetics. Selecting efficient and safe preservatives is one of the keys to the development of cosmetic formulas. However, traditional chemical preservatives, such as methylparaben and ethylparaben, are prone to cause skin problems. Therefore, the importance of developing natural extract preservatives is becoming increasingly prominent.

[0003] However, the antibacterial performance of a single natural extract is poor. Either the types of antibacterial strains are limited, or it is difficult to have good antibacterial effects on most strains at the same time. In addition, it is impossible to have good antibacterial effects while having good mite removal and anti-inflammatory effects.

[0004] Therefore, in order to solve the problems in the prior art, an antibacterial agent with the functions of antiseptic, antibacterial, mite removal and anti-inflammatory is prepared, which is of great significance for further expanding the application of antibacterial agents in the fields of daily chemical products and the like. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the multifunctional plant antibacterial agent of the present invention has low irritation, synergistic antiseptic, mite removal and anti-inflammatory effects, and is used in daily chemical products, and has the advantages of low dosage, mild raw materials, and natural low irritation.

[0006] The object of the present invention is to provide a multifunctional plant antibacterial agent, comprising the following raw materials in percentage by weight: 10-60% of an antibacterial agent, wherein the antibacterial agent comprises a mixture of at least two of cinnamon bark extract, lemon fruit extract, jasmine extract and camellia extract.

[0007] In some embodiments of the present invention, the multifunctional plant antibacterial agent comprises the following raw materials in percentage by weight: 15-45% of the antibacterial agent, wherein the antibacterial agent comprises a mixture of at least two of cinnamon bark extract, lemon fruit extract, jasmine extract and camellia extract.

[0008] In some embodiments of the present invention, the multifunctional plant antibacterial agent comprises the following raw materials in percentage by weight: 20-35% of the antibacterial agent, wherein the antibacterial agent comprises a mixture of at least two of cinnamon bark extract, lemon fruit extract, jasmine extract and camellia extract.

[0009] In some embodiments of the present invention, the multifunctional plant antibacterial agent further comprises 40-90% of a solvent.

[0010] In some embodiments of the present invention, the multifunctional plant antibacterial agent further comprises 55-85% of a solvent.

[0011] In some embodiments of the present invention, the multifunctional plant antibacterial agent further comprises 65-80% of a solvent.

[0012] In some embodiments of the present invention, the solvent includes at least one of 1,3-butanediol, 1,3-propylene glycol, 1,2-propylene glycol, 1,2-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, isopentyl glycol, isohexanediol, 1,2-hexanediol, dipropylene glycol, water, glycerol, methylpropanediol, and phenylethyl alcohol.

[0013] In some embodiments of the present invention, the solvent comprises phenylethanol.

[0014] In some embodiments of the present invention, the mass ratio of the cinnamon bark extract to the lemon fruit extract is 10-30:1-10.

[0015] In some embodiments of the present invention, the mass ratio of the cinnamon bark extract to the jasmine extract is 10-30:0.5-8.

[0016] In some embodiments of the present invention, the mass ratio of the cinnamon bark extract to the camellia extract is 8-28:2-13.

[0017] In some embodiments of the present invention, the mass ratio of the lemon fruit extract to the jasmine flower extract is 1-10:0.5-8.

[0018] In some embodiments of the present invention, the mass ratio of the lemon fruit extract to the camellia extract is 1-10:2-13.

[0019] In some embodiments of the present invention, the mass ratio of the jasmine extract to the camellia extract is 0.5-8:2-13.

[0020] In some embodiments of the present invention, the mass ratio of the cinnamon bark extract, the jasmine extract and the camellia extract is 10-30:0.5-8:2-13.

[0021] In some embodiments of the present invention, the mass ratio of the cinnamon bark extract, the lemon fruit extract and the camellia extract is 10-30:1-10:2-13.

[0022] In some embodiments of the present invention, the mass ratio of the cinnamon bark extract, the lemon fruit extract and the jasmine extract is 10-30:1-10:0.5-8.

[0023] In some embodiments of the present invention, the mass ratio of the cinnamon bark extract, lemon fruit extract, jasmine extract and camellia extract is 10-30:0.5-8:0.5-8:2-13.

[0024] In some embodiments of the present invention, the extraction process of the cinnamon bark extract comprises the following steps:

[0025] The dried cinnamon bark is crushed and sieved, the medicinal material powder is subjected to a first supercritical carbon dioxide extraction, the extract is collected and filtered, and the cinnamon bark extract is obtained.

[0026] In some embodiments of the present invention, the first supercritical carbon dioxide extraction has an extraction temperature of 5-15° C., an extraction pressure of 10-30 MPa, a CO2 flow rate of 20-40 L / h, and an extraction time of 2-4 h.

[0027] In some embodiments of the present invention, the extraction process of the lemon fruit extract comprises the following steps:

[0028] The lemon fruit is placed in a steam distillation device, water is added, the first steam distillation is performed, the oil layer is taken, the water is removed, and the lemon fruit extract is obtained by filtering.

[0029] In some embodiments of the present invention, the mass ratio of the lemon fruit to water is 1:18-23.

[0030] In some embodiments of the present invention, the temperature of the first steam distillation extraction is 90-100° C., and the time is 2-8 hours.

[0031] In some embodiments of the present invention, the extraction process of the jasmine extract comprises the following steps:

[0032] The dried jasmine flowers are crushed and sieved, the medicinal material powder is subjected to a second supercritical carbon dioxide extraction, the extract is collected and filtered, and the jasmine flower extract is obtained.

[0033] In some embodiments of the present invention, the second supercritical carbon dioxide extraction has an extraction temperature of 5-15° C., an extraction pressure of 15-25 MPa, a CO2 flow rate of 10-30 L / h, and an extraction time of 2-4 h.

[0034] In some embodiments of the present invention, the extraction process of the camellia extract comprises the following steps:

[0035] The camellia is placed in a steam distillation device, water is added, a second steam distillation is performed, the oil layer is taken, water is removed, and filtering is performed to obtain a camellia extract.

[0036] In some embodiments of the present invention, the mass ratio of camellia to water is 1:18-23.

[0037] In some embodiments of the present invention, the temperature of the second steam distillation extraction is 90-100° C., and the time is 2-8 hours.

[0038] Another object of the present invention is to provide an application of the multifunctional plant antibacterial agent in the preparation of daily chemical products, wherein the daily chemical products contain the multifunctional plant antibacterial agent, and the cosmetics include at least one of facial mask liquid, emulsion, cream, toner, shower gel, essence, stock solution, facial cleanser, lotion, perfume, makeup remover, foundation liquid, foundation cream, concealer cream, rouge, lipstick, eye shadow, and blush.

[0039] In some embodiments of the present invention, the amount of the multifunctional plant antibacterial agent is 0.2-1% of the daily chemical product in terms of mass percentage.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) The components of the multifunctional plant antibacterial agent provided by the present invention are all common plant extracts, which have the advantages of being safe, mild, and low in irritation, and at the same time have multiple functions.

[0042] (2) The components of the multifunctional plant antibacterial agent provided by the present invention have a synergistic effect, which can not only improve the mite removal effect, but also have antiseptic and anti-inflammatory effects. DETAILED DESCRIPTION

[0043] In order to enable those skilled in the art to better understand the technical solutions of 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 part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0044] Example 1

[0045] This embodiment provides an extraction process for cinnamon bark extract, and the specific extraction process is as follows:

[0046] Take 1kg of dried cinnamon bark, grind it with a grinder and pass it through a 20-mesh sieve. Place the medicinal powder in a supercritical carbon dioxide extraction kettle, start the extraction equipment, and set the extraction conditions to: extraction temperature 15°C, extraction pressure 20MPa, CO2 flow rate 30L / h, extraction time 3h. After the extraction is completed, collect the extract, filter it, and obtain cinnamon bark extract.

[0047] Example 2

[0048] This embodiment provides an extraction process for lemon fruit extract, and the specific extraction process is as follows:

[0049] Take 5kg of fresh lemon fruit, cut it into 5mm thin slices, dry it in the sun to remove most of the moisture, place it in a steam distillation device, the temperature is 95°C, add 18 to 23 times the weight of water, and steam distill for 5 hours. Let the collected oil-water mixture stand for 1 day, take the oil layer after oil-water separation, add 10g of anhydrous sodium sulfate, filter it after 12 hours, take the filtrate, and obtain lemon fruit extract.

[0050] Example 3

[0051] This embodiment provides an extraction process for jasmine extract, and the specific extraction process is as follows:

[0052] Take 5kg of fresh jasmine flowers, wash them and dry them in a 45℃ oven to obtain dried flowers. Grind the dried flowers and pass them through a 20-mesh sieve. Place the medicinal powder in a supercritical carbon dioxide extraction kettle. Start the extraction equipment. The extraction conditions are set as follows: extraction temperature (10℃), extraction pressure (18MPa), CO2 flow rate (20L / h), extraction time (3h). After the extraction, collect the extract, filter it, and obtain jasmine extract.

[0053] Example 4

[0054] This embodiment provides an extraction process of camellia extract, and the specific extraction process is as follows:

[0055] Take 1kg of dried camellia and place it in a steam distillation device at 95°C. Add 18 to 23 times the weight of water and perform steam distillation extraction for 5 hours. Let the collected oil-water mixture stand for 1 day. After the oil and water are separated, take the oil layer, add 10g of anhydrous sodium sulfate, filter after 12 hours, and take the filtrate to obtain the camellia extract.

[0056] Preparation of multifunctional plant antibacterial agent:

[0057] Table 1. Raw material composition of multifunctional plant antibacterial agent.

[0058]

[0059] According to the raw materials and mass percentages in Table 1, add the above extract and phenylethanol respectively into a 250mL flask with a thermometer and a stirrer, raise the temperature to 40°C, stir for 30-50 minutes until completely mixed, cool to room temperature, filter out insoluble impurities, and obtain a multifunctional plant antibacterial agent.

[0060] Preparation of AES Shower Gel:

[0061] Table 2. AES shower gel ingredients and production process.

[0062]

[0063] Antiseptic efficacy test:

[0064] Preservative efficacy test method: refer to "United States Pharmacopoeia USP43 (51) Microbial Preservative Efficacy Test".

[0065] Experimental instruments and reagents: constant temperature incubator, refrigerator, constant temperature water bath, electronic balance, thread-mouth screw-mouth chemical reagent bottles, glass beads, culture dishes (diameter 90 mm), measuring cylinders, pipettes (1 mL, 5 mL), homogenizers, electric constant temperature blast drying ovens, vertical automatic pressure steam sterilizers, wide-mouth transparent glass sample bottles, narrow-mouth transparent glass sample bottles, pH markers or precision pH test paper, magnifying glasses, culture media (lecithin Tween 80 nutrient agar, Bengal red agar) and reagents, physiological saline, liquid paraffin, Tween 80, 0.5% triphenyltetrazolium chloride.

[0066] Tested bacteria: 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.

[0067] Experimental steps: (1) Sample treatment: Weigh an appropriate amount of the AES shower gel of each of Examples 15 to 24 and Comparative Examples 6 to 10 into two sterilized narrow-mouthed glass sample bottles for later use.

[0068] (2) Preparation of mixed bacterial suspension: Add an appropriate amount of sterile 0.9% sodium chloride aqueous solution to the activated bacteria and the standard strain of Candida albicans to elute the bacterial moss, and pour the higher concentration bacterial suspension into sterile containers of the same size as the McFarland turbidimetric tube; use a sterile 0.9% sodium chloride aqueous solution containing 0.05% Tween-80 to elute the Aspergillus niger spores to prepare an Aspergillus niger spore suspension, filter the Aspergillus niger spore suspension to remove the mycelium, and set aside. Add an appropriate amount of sterile 0.9% sodium chloride aqueous solution to the high-concentration single bacterial solution for dilution, and compare it with the corresponding concentration of the McFarland turbidimetric tube. If the turbidity is consistent, it is ready. Shake well before using the standard McFarland turbidimetric tube. Mix the single bacterial suspension in a ratio of 1:1, and the single fungal suspension in a ratio of 1:1, and then mix the bacterial and fungal suspensions in a ratio of 1:1 to obtain a mixed bacterial suspension, which is set aside.

[0069] (3) Counting of mixed bacterial suspension: Pipette 1 mL of the prepared mixed bacterial suspension into a finger-shaped bottle containing 9 mL of sterile saline, mix thoroughly, and make a 1:10 dilution. Repeat this process to dilute the suspension 1:10. 5 , 1:10 6 , 1:10 7 ; Select 1:10 5 ~1:107 Pour the gradient dilutions onto plates for counting, and calculate the total number of bacterial colonies and fungal colonies in the mixed bacterial suspension. If the concentration of the mixed bacterial suspension is not within the required range for adding bacteria, the sample needs to be re-tested.

[0070] (4) Sample inoculation and culture: 1% of the mixed bacterial suspension was added to the AES shower gels of Examples 15 to 24 and Comparative Examples 6 to 10 on day 0, day 14, and day 28, respectively. The concentration of bacteria added each time in Examples 15 to 24, Comparative Examples 6 to 10, and the blank control was the same. The initial concentration of the first addition of bacteria was: bacterial concentration (5.5×10 5 ), fungal concentration (1.8×10 5 ); Second bacterial addition starting concentration: bacterial concentration (6.7×10 5 ), fungi (2.3×10 5 ), the third bacterial concentration: bacteria (7.3×10 5 ), fungi (2.6×10 5 ), mix the sample and bacterial suspension thoroughly. Place in an incubator at 22.5℃±2.5℃ for cultivation.

[0071] (5) Sample testing and analysis: Samples were collected and tested on the 14th day after each inoculation. Sample processing: Take an appropriate amount of sample and inject it into sterile saline, dilute to 1:10, and mix thoroughly. Multiple gradient dilutions can be made as needed, such as 1:100, 1:1000, 1:10000, etc., and a different pipette tip should be used for sampling at each dilution. Plate pouring: Use a pipette to draw up the sample dilution and inject it into a sterile plate, 1 mL per plate, and a total of 4 plates for each dilution (two bacterial plates and two fungal plates). Pour the melted and cooled lecithin-Tween 80 nutrient agar to about 45℃-48℃ into the bacterial plate, about 15 mL per plate, and then rotate the plate to mix the sample and lecithin-Tween 80 nutrient agar thoroughly. After the lecithin-Tween 80 nutrient agar solidifies, turn the plate over and place it in a 36℃±1℃ incubator for 72h±2h. Pour the melted Red Bengal (Red Tiger) agar cooled to about 45℃-48℃ into the fungal plate, about 15mL per plate, and then rotate the plate to mix the sample and Red Bengal (Red Tiger) agar thoroughly. After the Red Bengal (Red Tiger) agar solidifies, turn the plate over and incubate it at 28℃±2℃ for 72h±2h.

[0072] (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.

[0073] 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.

[0074] The sample is AES shower gel, and the formula and production process are shown in Table 2. Among them, the blank control is AES shower gel without adding the multifunctional plant antibacterial agent in Table 1.

[0075] Table 3. AES shower gel antiseptic efficacy test data.

[0076] It can be seen from Table 3 that the multifunctional plant antibacterial agent of the present invention has a synergistic antiseptic effect.

[0077] Mite killing effect test:

[0078] Mite test (refer to the standard method of mite test: NY / T 1151.2-2006).

[0079] (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).

[0080] (2) Preparation of experimental samples: Prepare the mite control samples to be tested.

[0081] (3) Experimental operation: Take a disposable plate, add 5g of AES shower gel of Examples 15 to 24 and Comparative Examples 6 to 10 respectively, and evenly apply the white vaseline mixture on the upper edge of the inner wall of each plate. Add 200-300 mites to the center of each plate, and after 30 minutes, put 0.05g of mite feed in the center of the plate. Place it in a constant temperature and humidity incubator. After 48 hours, take out the plate and check under a stereo microscope to record the number of dead mites and calculate the mite killing rate.

[0082] (4) Calculation of mite killing rate:

[0083] (5) The test system is AES shower gel. The formula and production process are shown in Table 2. The blank control is AES shower gel without the antibacterial composition in Table 1.

[0084] Table 4. Mite killing rate test results.

[0085]

[0086] It can be seen from Table 4 that the multifunctional plant antibacterial agent of the present invention has a synergistic mite removal effect.

[0087] Preparation of toner:

[0088] Table 5. Toner ingredients and production process.

[0089]

[0090] Anti-inflammatory efficacy test:

[0091] (1) Test name: Daily chemical product soothing efficacy test - in vitro 1L-1β inflammatory factor content determination method of lipopolysaccharide-induced macrophage inflammatory cell model test method.

[0092] (2) Test principle: Bacterial lipopolysaccharide (LPS) binds to the antigen recognition receptors on the surface of macrophages, inducing macrophages to secrete multiple cytokines such as 1L-1β. 1L-1β can activate three signaling pathways: Caspase, JNK, and transcription factor NF-kB, to achieve its biological functions such as cytotoxicity, antiviral, immunomodulation, 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 difference in the secretion of 1L-1β by RAW264.7 after administration of the negative control and the test substance, the effect of the test substance in inhibiting the secretion of 1L-1β was evaluated. The 1L-1β content was determined by enzyme-linked immunosorbent assay (ELISA). The specific principle is: 1L-1β specifically binds to the 1L-1β antibody coated on the ELISA plate, and then binds to the anti-1L-1β antibody labeled with a substrate. After the substrate is catalyzed by the enzyme, a colored product is generated. The 1L-1β content is positively correlated with the color depth of the colored product. The optical density value (OD) is measured at a wavelength of 450 nm using an ELISA instrument to calculate the 1L-1β content.

[0093] (3) Instruments and equipment:

[0094] Adjust the pipette: 1000 μL, 200 μL, 50 μL, 10 μL; CO2 incubator: 37 ℃, humidification, 5% CO2 / air; microplate reader: equipped with 450 nm filter; ultra-clean workbench.

[0095] Micro-oscillator; analytical balance with an accuracy of 0.1 mg; constant temperature incubator; vortex shaker; cell counter or hemacytometer; inverted microscope; ultra-low temperature refrigerator (-80 ℃); low-speed centrifuge.

[0096] (4) Reagents and consumables:

[0097] Cells: Mouse mononuclear macrophage leukemia cell line RAW264.7 was selected. The cell line was from the American Type Culture Collection (ATCC), and the model was ATCC TIB-71. Cells should be tested regularly to ensure that they are free of mycoplasma contamination. Only those without contamination can be used.

[0098] Cell culture reagents and consumables: high-glucose DMEM medium, fetal bovine serum or newborn bovine serum, trypsin / EDTA solution (0.25% trypsin solution and 0.02 mol / L EDTA solution mixed in a ratio of 1:1), phosphate buffered saline (PBS), cell culture flask.

[0099] (5) Detection: bacterial lipopolysaccharide (LPS, from Escherichia coli); 96-well cell culture plate; 1L-1β ELISA detection kit.

[0100] (6) Test methods:

[0101] Cell preparation: Cell cultures from cryopreserved cultures are seeded at an appropriate density and passaged at least once before testing. Cells should be seeded at an appropriate density for the test, and the cell seeding density should ensure that the confluence reaches 45%~60% 24 hours after seeding.

[0102] Preparation of test substances: Unless stability data are available to demonstrate that the stock solution is acceptable, the test substance should be prepared freshly before use. Test substances with limited solubility in water should be dissolved in an appropriate solvent, the volume of which should be consistent in all cultures, i.e., in blank controls, negative controls, and test substances, and should be non-cytotoxic.

[0103] (7) Experimental conditions:

[0104] The concentration of the test substance should be determined by cytotoxicity testing, for example, the concentration of cell viability ≥ 90% should be selected. The concentration of LPS should be 1 μg / mL, and the specific stimulation concentration can be adjusted according to the characteristics of laboratory cultured cells. The quality standard that the test should meet is: the 1L-1β content of the negative control under the action of LPS is increased by ≥ 5 times compared with the blank control.

[0105] Positive control: Each test should be performed with a positive control. Positive controls such as 100 μg / mL dexamethasone are recommended, and the specific dosing concentration can be adjusted according to the characteristics of the cells cultured in each laboratory.

[0106] (8) Experimental steps:

[0107] Cell digestion and inoculation:

[0108] Use trypsin / EDTA. The specific digestion concentration and dosage should be determined according to the characteristics of laboratory cells: the trypsin concentration should be 0.25%, the trypsin dosage for T25 culture flask is 1 mL, and the trypsin dosage for T75 culture flask is 3 mL.

[0109] Under microscope, when most of the cells become round and suspended, add DMEM medium containing serum at a volume of about 2 to 3 times that of trypsin to terminate digestion, collect the cells into a centrifuge tube, and centrifuge at 800 r / min for 6 min. The speed and centrifugation time can be determined according to the characteristics of the cells in the laboratory.

[0110] After centrifugation, discard the supernatant, add a certain volume of cell culture medium to the centrifuge tube, mix the cells by pipetting with an elbow pipette, and count the cells with a cell counter or hemocytometer.

[0111] The cells were diluted with cell culture medium to the seeding density (the degree of confluence reached 45%-60% 24 h after seeding) and seeded into a 96-well plate with a volume of 200 μL per well.

[0112] After inoculation, place the cells in a CO2 incubator and culture for 24 h±2 h.

[0113] Induction and administration: Discard the culture medium in the 96-well plate and carry out the induction and administration operation. Add culture medium containing a certain concentration of the test substance and LPS to the test substance wells, add cell culture medium containing LPS to the negative control wells, add culture medium containing positive control and LPS to the positive control wells, and add cell culture medium to the blank / solvent control wells, 200 μL per well. After the administration, place the 96-well plate in a CO2 incubator for 24 h±2 h.

[0114] Cell supernatant collection: After the incubation, collect 200 μL of cell culture supernatant into a 1.5 mL sterile centrifuge tube and store it in a -80°C ultra-low temperature refrigerator.

[0115] ELISA test: ELISA test should be performed according to the operating instructions of the 1L-1β ELISA test kit.

[0116] Because the expression level of 1L-1β is high after LPS induction, the dilution ratio should be set and a preliminary experiment should be conducted before the formal test to determine the dilution ratio of the ELISA test group to ensure that the ELISA test value falls within the range of the standard curve.

[0117] (9) Calculation results:

[0118] Calculation of 1L-1β content: It is recommended to use professional curve making software, with the concentration of the standard as the ordinate and the OD450 value as the abscissa, to make a standard curve, and find out the corresponding concentration according to the OD450 value of the sample. Or use the concentration of the standard and the OD450 value to calculate the regression equation of the standard curve, 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 the three replicate wells in each group as the final 1L-1β content result.

[0119] The calculation formula of 1L-1β inhibition rate is:

[0120]

[0121] (10) Result report:

[0122] The content of 1L-1β should be expressed as: mean value ± standard deviation (SD) of the content.

[0123] For statements about whether the test substance inhibits 1L-1β content, the test concentration should be included.

[0124] (11) The test substance is toner. The formula and production process are shown in Table 5.

[0125] (12) Interpretation of the relevance of the results: On the basis that the test meets the validity verification requirements, the 1L-1β content of the test substance decreased compared with the negative control, indicating that the test substance has the effect of inhibiting the 1L-1β content at this test concentration, which can be used as one of the evidences for the soothing daily chemical product raw material title.

[0126] Table 6.1L-1β inhibition rate test results.

[0127]

[0128] From the results in Table 6, it can be seen that the multifunctional plant antibacterial agent of the present invention has the effect of synergistically inhibiting IL-1β.

[0129] Experiment name: Chicken embryo allantoic membrane test (end point evaluation method).

[0130] Experimental principle: The chicken embryo chorioallantoic membrane test is an early in vitro eye irritation assessment method. The chorioallantoic membrane (CAM) is a respiratory membrane that surrounds the chicken embryo. This test uses the characteristics of the intact, clear and transparent vascular system of the chorioallantoic membrane of the mid-stage incubated chicken embryo. A certain amount of the test substance is directly exposed to the chicken embryo chorioallantoic membrane. After a period of action, the toxic effect indicators of the chorioallantoic membrane are observed, such as changes in bleeding, coagulation and vascular melting. These indicators reflect the changes in the morphological structure, color and permeability of blood vessels and vascular networks, as well as the phenomena of chorioallantoic membrane protein denaturation and the 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. The standard describes the elements and processes for evaluating the potential eye irritation of the evaluated substance.

[0131] Experimental preparation: (1) Source of chicken embryo strain: Fertilized chicken embryos of White Leghorn chicken should be TQG chicken embryos. The quality of the chicken embryos should meet the requirements of relevant standards. The supplier should have the qualification of "TQG chicken (egg) designated production enterprise for veterinary drug production and inspection" approved by the agricultural department.

[0132] (2) Transportation and storage: Purchase chicken embryos within 7 days of age and store them on egg racks with the air chamber facing upwards. The chicken embryos should be transferred or transported without affecting the activity or development of the embryos, and avoid shaking, unnecessary tilting, knocking, and other mechanical stimulation of the chicken embryos.

[0133] (3) The chicken embryos should be fresh, clean, and intact, weighing 50g to 60g. When the eggs are 9 days old, they should be inspected and unfertilized, inactive, or defective chicken embryos should be discarded. Severely deformed, broken, or thin-shelled chicken embryos should not be used.

[0134] (4) Incubation conditions: room temperature 20℃~25℃, relative humidity 45%~70%. Incubation temperature 37.5℃±0.5℃, relative humidity 55%~70%, turntable frequency 3 times / h~6 times / h. Rotation is not necessary when incubating 9-day-old chicken embryos.

[0135] Experimental instruments and reagent materials: fully automatic incubator, constant temperature and humidity incubator, double clean workbench, high-pressure steam sterilizer, SLR camera, LED high-brightness cold light source egg candling device, hand-twisted drill manual punching tool, medical ophthalmic curved pointed tweezers: 10CM, polytetrafluoroethylene resin ring: 12*9*1.5mm, pipette: 10-100μl, pipette tip: 10-100μl, electronic timer, wide-mouth conical flask: 100ml, conical flask: 500ml, sodium chloride: analytical grade, sterile water.

[0136] Preparation of test substance and control substance: (1) Test substance: Toner, the formula and production process are shown in Table 5. (2) Negative control: A sodium chloride aqueous solution with a mass concentration of 0.9% is selected for rinsing after the test substance is applied and for 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 will not cause irritation. (3) Blank control: Toner without adding antibacterial composition. (4) Positive control: 10% acrylamide aqueous solution.

[0137] Test process: (1) Each test sample should be tested with 10 chicken embryos. Each batch of chicken embryos should have 3 negative controls, 3 positive controls, and 3 solvent controls. (2) Endpoint evaluation method: Take 0.3 mL of the test substance and directly apply it to the CAM to ensure that at least 50% of the CAM surface is covered by the test substance for 3 minutes. (3) CAM preparation: Take out a 10-day-old chicken embryo, use an egg candling device to observe the air chamber, determine the position of the air chamber, use a manual drilling tool to open a small window on the air chamber, use medical ophthalmic curved tip tweezers to peel off the eggshell part of the upper layer of the air chamber to expose the white eggshell membrane, and add an appropriate amount of 0.9% sodium chloride solution with a pipette to moisten the eggshell membrane. After it is completely moistened, pour out the 0.9% sodium chloride solution. Carefully remove the eggshell membrane with tweezers, and be cautious during the removal process to ensure that the vascular membrane is not damaged. The CAM is the one with abundant capillaries after removing the eggshell membrane. (4) Photo recording: Use a camera to record the condition of the chorioallantoic membrane. If necessary, take photos to record the condition of three groups of chorioallantoic membranes, namely the CAM without contact with the test ring and sample, the CAM area after the test ring is placed, and the test area after the sample is added and cultured for 30 minutes. Through these three groups of pictures, the damage and degree of the CAM are compared and analyzed. (5) Adding the sample: Place the polytetrafluoroethylene resin ring on the chorioallantoic membrane of the chicken embryo as the test area, use a pipette to transfer 40μl of the sample to be tested into the polytetrafluoroethylene resin ring, record the time of adding the sample and cover the air chamber with a moistened plastic wrap, and move the chicken embryo to a constant temperature and humidity chamber for culture for 30 minutes. (6) Result observation: After 30 minutes of culture, take out the test chicken embryo, take photos directly and observe the degree of vascular damage, and score according to the degree of vascular damage.

[0138] Scoring reference standards:

[0139] Table 7. Reference standards for scoring of irritation test experiments.

[0140]

[0141] Result analysis: The results of each egg should be evaluated and scored. Finally, the highest and lowest scores will be removed from the ten scores, leaving 8 valid scores. The stimulation score (NC) will be calculated according to the following formula.

[0142] NC=(X1+X2+……+Xn) / n

[0143] Where: X——stimulation score

[0144] n——The number of effective chicken embryos after removing the highest and lowest scores

[0145] The irritation of the test substances was classified according to the calculated NC values ​​in Table 8.

[0146] Table 8. Irritation classification of the test substances.

[0147]

[0148] Table 9. Irritation test results.

[0149]

[0150] It can be seen from Table 9 that the multifunctional plant antibacterial agent of the present invention has low irritation and has the advantages of being safe, mild and low in irritation.

[0151] The mass percentage of the antibacterial agent in the multifunctional plant antibacterial agent of the present invention is within the range of 10-60%, and the mass percentage of the solvent is within the range of 40-90% (e.g., the mass percentage of the antibacterial agent is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, and the mass percentage of the solvent is 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, etc.); and / or other solvents are used to extract cinnamon bark, lemon fruit, jasmine, and camellia (e.g., water, ethyl acetate, ethanol, etc.). cinnamon bark, lemon fruit, jasmine, camellia are extracted with solvents such as alcohol, methanol, acetone, petroleum ether, ether, chloroform, ethyl acetate, and dichloroethane); and / or other solvents are selected for multifunctional plant antibacterial agents (such as 1,3-butanediol, 1,3-propylene glycol, 1,2-propylene glycol, 1,2-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, isopentyl glycol, isohexanediol, 1,2-hexanediol, dipropylene glycol, water, glycerol, methyl propanediol, etc.); and / or the mass ratio of cinnamon bark extract to lemon fruit extract is 10-30:1-10 and / or the mass ratio of cinnamon bark extract to jasmine extract is in the range of 10-30:0.5-8; and / or the mass ratio of cinnamon bark extract to camellia extract is in the range of 8-28:2-13; and / or the mass ratio of lemon fruit extract to jasmine extract is in the range of 1-10:0.5-8; and / or the mass ratio of lemon fruit extract to camellia extract is in the range of 1-10:2-13; and / or the mass ratio of jasmine extract to camellia extract is in the range of 0.5-8:2-13; and / or cinnamon bark extract, The mass ratio of jasmine extract to camellia extract is within the range of 10-30:0.5-8:2-13; and / or the mass ratio of cinnamon bark extract, lemon fruit extract to camellia extract is within the range of 10-30:1-10:2-13; and / or the mass ratio of cinnamon bark extract, lemon fruit extract to jasmine extract is within the range of 10-30:1-10:0.5-8; and / or the mass ratio of cinnamon bark extract, lemon fruit extract, jasmine extract to camellia extract is within the range of 10-30:0.5-8:0.5-8:2-13. The obtained multifunctional plant antibacterial agent has the effects of synergistic antiseptic, mite removal and anti-inflammatory.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that after reading the specification of this application, the technicians can still modify or replace the specific implementation mode of the present invention with equivalents, but these modifications or changes do not deviate from the scope of protection of the pending claims of the present application.

Claims

1. A multifunctional plant antibacterial agent, characterized in that: The raw materials include the following mass percentages: 10-60% of antibacterial agent, wherein the antibacterial agent includes a mixture of at least two of cinnamon bark extract, lemon fruit extract, jasmine extract and camellia extract.

2. The multifunctional plant antibacterial agent according to claim 1, characterized in that Also includes 40-90% solvent.

3. The multifunctional plant antibacterial agent according to claim 2, characterized in that The solvent includes at least one of 1,3-butanediol, 1,3-propylene glycol, 1,2-propylene glycol, 1,2-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,5-pentanediol, isopentyl glycol, isohexanediol, 1,2-hexanediol, dipropylene glycol, water, glycerol, methylpropanediol, and phenylethyl alcohol.

4. The multifunctional plant antibacterial agent according to claim 1, characterized in that The mass ratio of the cinnamon bark extract to the lemon fruit extract is 10-30:1-10; The mass ratio of the cinnamon bark extract to the jasmine extract is 10-30:0.5-8; The mass ratio of the cinnamon bark extract to the camellia extract is 8-28:2-13; The mass ratio of the lemon fruit extract to the jasmine flower extract is 1-10:0.5-8; The mass ratio of the lemon fruit extract to the camellia extract is 1-10:2-13; The mass ratio of the jasmine extract to the camellia extract is 0.5-8:2-13.

5. The multifunctional plant antibacterial agent according to claim 1, characterized in that The mass ratio of the cinnamon bark extract, the jasmine extract and the camellia extract is 10-30:0.5-8:2-13; The mass ratio of the cinnamon bark extract, the lemon fruit extract and the camellia extract is 10-30:1-10:2-13; The mass ratio of the cinnamon bark extract, the lemon fruit extract and the jasmine extract is 10-30:1-10:0.5-8; The mass ratio of the cinnamon bark extract, the lemon fruit extract, the jasmine extract and the camellia extract is 10-30:0.5-8:0.5-8:2-13.

6. The multifunctional plant antibacterial agent according to claim 1, characterized in that The extraction process of the cinnamon bark extract comprises the following steps: The dried cinnamon bark is crushed and sieved, the medicinal powder is subjected to a first supercritical carbon dioxide extraction, the extract is collected and filtered to obtain a cinnamon bark extract; The extraction process of the lemon fruit extract comprises the following steps: Put lemon fruit in a steam distillation device, add water, perform steam distillation extraction, let stand, take the oil layer, remove water, filter, and obtain lemon fruit extract; The extraction process of the jasmine extract comprises the following steps: The dried jasmine flowers are crushed and sieved, the medicinal powder is subjected to a second supercritical carbon dioxide extraction, the extract is collected and filtered to obtain a jasmine flower extract; The extraction process of the camellia extract comprises the following steps: The camellia is placed in a steam distillation device, water is added, a second steam distillation is performed, the oil layer is taken, water is removed, and filtering is performed to obtain a camellia extract.

7. The multifunctional plant antibacterial agent according to claim 6, characterized in that The first supercritical carbon dioxide extraction has an extraction temperature of 5-15°C, an extraction pressure of 10-30MPa, a CO2 flow rate of 20-40L / h, and an extraction time of 2-4h; The second supercritical carbon dioxide extraction has an extraction temperature of 5-15°C, an extraction pressure of 15-25MPa, a CO2 flow rate of 10-30L / h, and an extraction time of 2-4h; The mass ratio of the lemon fruit to water is 1:18-23; The mass ratio of the camellia to water is 1:18-23.

8. The multifunctional plant antibacterial agent according to claim 6, characterized in that: The first steam distillation extraction is performed at a temperature of 90-100° C. for 2-8 hours; The second steam distillation extraction is performed at a temperature of 90-100° C. and for a time of 2-8 hours.

9. The use of the multifunctional plant antibacterial agent according to any one of claims 1 to 8 in the preparation of daily chemical products, characterized in that: The cosmetics contain the multifunctional plant antibacterial agent, and the cosmetics include at least one of facial mask liquid, emulsion, cream, toner, shower gel, essence, stock solution, cleanser, lotion, perfume, makeup remover, liquid foundation, foundation cream, concealer cream, rouge, lipstick, eye shadow, and blush.

10. The use according to claim 9, characterized in that Calculated by mass percentage, the dosage of the multifunctional plant antibacterial agent is 0.2-1% of the daily chemical product.

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