CN118697680A - Antibacterial composition containing multiple plant extracts and application thereof - Google Patents

Abstract

The invention discloses a bacteriostatic composition containing multiple plant extracts and application thereof, wherein the bacteriostatic composition containing multiple plant extracts comprises the following raw materials in percentage by mass: 10-40% of cinnamon bark extract and 60-90% of compound extract, wherein the compound extract comprises at least three mixtures of rosemary extract, myrrh extract, melaleuca alternifolia extract and ginkgo leaf extract; the antibacterial composition containing multiple plant extracts has small irritation, has the effects of synergistic corrosion prevention, mite removal and anti-inflammatory, can be used in cosmetics, is prepared from plants, and has the advantages of low consumption, mild raw materials, natural low irritation and the like.

Description

Antibacterial composition containing multiple plant extracts and application thereof

Technical Field

The invention belongs to the field of plant extracts, and in particular relates to an antibacterial composition containing multiple plant extracts and application thereof.

Background Art

At present, there are two main methods of mite removal: physical mite removal and chemical mite removal.

Physical mite removal mainly uses physical mite removal devices to remove mites through ultraviolet rays, ultrasonic waves or adsorption. However, physical mite removal devices are time-consuming and labor-intensive to use, consume a lot of energy, and the effect cannot be guaranteed.

Chemical mite removal mainly kills mites through chemical substances such as bromocriptine, fenbutatin, and amitraz. Most of the existing chemical mite removal agents are artificially synthesized, which poses certain safety hazards and may cause harm to the human body. They also have a single effect. While having the effect of removing mites, it is difficult for them to have antiseptic or anti-inflammatory effects.

Summary of the invention

In view of the deficiencies in the above-mentioned 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 synergistic antiseptic, mite removal and anti-inflammatory effects. The antibacterial composition containing multiple plant extracts can be used in cosmetics. The raw materials are all derived from plants, and it has the advantages of low dosage, mild raw materials, and natural low irritation.

The object of the present invention is to provide an antibacterial composition containing multiple plant extracts, comprising the following raw materials in percentage by weight: 10-40% of cinnamon bark extract and 60-90% of a composite extract, wherein the composite extract comprises a mixture of at least three of rosemary extract, myrrh extract, niaouli extract and ginkgo leaf extract.

In some embodiments of the present invention, the amount of the rosemary extract is 5-20% of the antibacterial composition containing multiple plant extracts, the amount of the myrrh extract is 20-40% of the antibacterial composition containing multiple plant extracts, the amount of the Niaouli extract is 5-25% of the antibacterial composition containing multiple plant extracts, and the amount of the Ginkgo biloba extract is 15-45% of the antibacterial composition containing multiple plant extracts.

In some embodiments of the present invention, the amount of the myrrh extract is 20-40% of the antibacterial composition containing multiple plant extracts, the amount of the niaouli extract is 5-25% of the antibacterial composition containing multiple plant extracts, and the amount of the ginkgo leaf extract is 15-45% of the antibacterial composition containing multiple plant extracts.

In some embodiments of the present invention, the amount of the rosemary extract is 5-20% of the antibacterial composition containing multiple plant extracts, the amount of the Niaouli extract is 5-25% of the antibacterial composition containing multiple plant extracts, and the amount of the Ginkgo biloba extract is 15-45% of the antibacterial composition containing multiple plant extracts.

In some embodiments of the present invention, the amount of the rosemary extract is 5-20% of the antibacterial composition containing multiple plant extracts, the amount of the myrrh extract is 20-40% of the antibacterial composition containing multiple plant extracts, and the amount of the ginkgo leaf extract is 15-45% of the antibacterial composition containing multiple plant extracts.

In some embodiments of the present invention, the amount of the rosemary extract is 5-20% of the antibacterial composition containing multiple plant extracts, the amount of the myrrh extract is 20-40% of the antibacterial composition containing multiple plant extracts, and the amount of the Niaouli extract is 5-25% of the antibacterial composition containing multiple plant extracts.

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

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.

In some embodiments of the present invention, the first supercritical carbon dioxide extraction has an extraction temperature of 10-20°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.

In some embodiments of the present invention, the extraction process of the rosemary extract comprises the following steps:

The dried rosemary is crushed and sieved, the medicinal material powder is subjected to a second supercritical carbon dioxide extraction, the extract is collected and filtered to obtain the rosemary extract.

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.

In some embodiments of the present invention, the extraction process of the myrrh extract comprises the following steps:

The myrrh powder is crushed and sieved, and the powder is placed in a steam distillation device, water is added, and the powder is extracted by steam distillation. The powder is allowed to stand, and the oil layer is taken, and the water is removed and filtered to obtain the myrrh extract.

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

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.

In some embodiments of the present invention, the extraction process of the Niaouli extract comprises the following steps:

The Niaouliu leaves are placed in a steam distillation device, water is added, the leaves are extracted by steam distillation, the leaves are allowed to stand, the oil layer is taken, water is removed, and the leaves are filtered to obtain the Niaouliu extract.

In some embodiments of the present invention, the mass ratio of the Niaouliu leaves to water is 1:18-23.

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.

In some embodiments of the present invention, the extraction process of the ginkgo leaf extract comprises the following steps:

The ginkgo leaf blades are placed in a steam distillation device, water is added, the extraction is performed by steam distillation, the extraction is allowed to stand, the oil layer is taken, the water is removed, and the extraction is filtered to obtain the ginkgo leaf extract.

In some embodiments of the present invention, the mass ratio of the ginkgo leaf leaf to water is 1:18-23.

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.

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

In some embodiments of the present invention, the antibacterial composition containing multiple plant extracts is used in the preparation of daily chemical products, and the daily chemical products include at least one of shower gel, facial mask liquid, lotion, cream, toner, essence, stock solution, facial cleanser, lotion, perfume, makeup remover, liquid foundation, foundation cream, concealer cream, rouge, lipstick, eye shadow, and blush.

In some embodiments of the present invention, the antibacterial composition containing multiple plant extracts is used in the preparation of daily chemical products, and the amount of the antibacterial composition containing multiple plant extracts is 0.2-1% of the daily chemical products in terms of mass percentage.

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

(1) The components of the antibacterial composition containing multiple plant extracts 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.

(2) The components of the antibacterial composition containing multiple plant extracts 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

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.

Example 1

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

Take 1 kg 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.

Example 2

This embodiment provides an extraction process for rosemary extract, and the specific extraction process is as follows:

Take 5 kg of fresh rosemary whole plant, wash it and dry it in a 45°C oven to obtain dry medicinal materials, grind the dry medicinal materials through a 20-mesh sieve, place the medicinal material powder in a supercritical carbon dioxide extraction kettle, start the extraction equipment, and set the extraction conditions as: extraction temperature 10°C, extraction pressure 18 MPa, _CO2 flow rate 20 L/h, extraction time 3h, collect the extract after the extraction, and filter to obtain rosemary extract.

Example 3

This embodiment provides an extraction process of myrrh extract, and the specific extraction process is as follows:

Take 1 kg of myrrh, grind it with a grinder, pass it through a 20-mesh sieve, place it in a steam distillation device, set the temperature to 95°C, add 18 to 23 times the weight of water, and extract it with steam distillation for 5 hours. Let the collected oil-water mixture stand for 1 day, take the oil layer after oil-water separation, add 10 g of anhydrous sodium sulfate, and filter the filtrate after 12 hours to obtain the myrrh extract.

Example 4

This embodiment provides an extraction process of Niaouliu Thunbergia extract, and the specific extraction process is as follows:

Take 5 kg of fresh leaves of Niaouli, wash and dry them in the shade, then place them 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, take the oil layer after oil-water separation, add 10 g of anhydrous sodium sulfate, and filter the filtrate after 12 hours to obtain the Niaouli extract.

Example 5

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

Take 5kg of fresh ginkgo leaves, wash and dry them in the shade, and place them 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, and filter the filtrate after 12 hours to obtain the ginkgo leaf extract.

Preparation of antibacterial composition containing various plant extracts:

Table 1. Raw material composition of antibacterial compositions containing various plant extracts.

Preparation of AES Shower Gel:

Table 2. AES shower gel ingredients and production process.

Antiseptic efficacy test:

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

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 blower 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.

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.

Experimental steps: (1) Sample treatment: Weigh an appropriate amount of the AES shower gel of each of Examples 11 to 15 and Comparative Examples 6 to 10 into two sterilized narrow-mouthed glass sample bottles for later use.

(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, and set aside.

(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 1:10 ⁵ , 1:10 ⁶ , and 1:10 ⁷ ; select dilutions of 1:10 ⁵ ~1:10 ⁷ gradient and pour plates for counting to 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 bacterial addition, the sample needs to be re-tested.

(4) Sample inoculation and culture: 1% mixed bacterial suspension was added to the AES shower gels of Examples 11-15 and Comparative Examples 6-10 on the 0th day, the 14th day and the 28th day respectively. The concentration of bacteria added each time in Examples 11-15, Comparative Examples 6-10 and the blank control was the same. The initial concentration of the first bacterial addition was: bacterial concentration (5.5×10 ⁵ ), fungal concentration (1.8×10 ⁵ ); the initial concentration of the second bacterial addition was: bacterial concentration (6.7×10 ⁵ ), fungal concentration (2.3×10 ⁵ ); the third bacterial addition was: bacterial concentration (7.3×10 ⁵ ), fungal concentration (2.6×10 ⁵ ). The sample was fully mixed with the bacterial suspension. The sample was placed in an incubator at 22.5°C±2.5°C for culture.

(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. It can be diluted in multiple gradients 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.

(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 are 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.

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.

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.

Table 3. AES shower gel antiseptic efficacy test data.

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

Mite killing effect test:

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

(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).

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

(3) Experimental operation: Take a disposable plate, add 5g of AES shower gel of Examples 11 to 15 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.

(4) Calculation of mite killing rate:

(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.

Table 4. Mite killing rate test results.

It can be seen from Table 4 that the antibacterial composition containing multiple plant extracts of the present invention has a synergistic mite removal effect.

Preparation of toner:

Table 5. Toner ingredients and production process.

Anti-inflammatory efficacy test:

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

(2) Test principle: Bacterial lipopolysaccharide (LPS) binds to the antigen recognition receptors on the surface of macrophages, inducing macrophages to secrete 1L-1β and other cytokines. 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, and 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 on 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.

(3) Instruments and equipment:

Adjust the pipette: 1000 μL, 200 μL, 50 μL, 10 μL; CO ₂ incubator: 37 ℃, humidification, 5% CO ₂ /air; ELISA reader: equipped with 450 nm filter; ultra-clean workbench.

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.

(4) Reagents and consumables:

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.

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.

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

(6) Test methods:

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.

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.

(7) Experimental conditions:

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.

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.

(8) Experimental steps:

Cell digestion and inoculation:

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.

Observe under a microscope. When most of the cells become round and suspended, add about 2 to 3 times the volume of trypsin-containing DMEM medium 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 laboratory cells.

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.

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.

After inoculation, place the culture in a CO ₂ incubator for 24 h±2 h.

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 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 CO ₂ incubator for 24 h±2 h.

Collection of cell supernatant: 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.

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

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.

(9) Calculation results:

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.

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

(10) Result report:

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

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

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

(12) Interpretation 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.

Table 6.1L-1β inhibition rate test results.

From the results in Table 6, it can be seen that the antibacterial composition containing multiple plant extracts of the present invention has the effect of synergistically inhibiting IL-1β.

Irritation test:

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

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.

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 be qualified as a "Designated Production Enterprise of TQG Chickens (Eggs) for Veterinary Drug Production and Inspection" approved by the Ministry of Agriculture and Rural Affairs.

(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.

(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.

(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.

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 illuminator, 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.

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.

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. After 3 minutes of action. (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. 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.

Scoring reference standards:

Table 7. Reference standards for scoring of irritation test experiments.

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.

NC=（X1+X2+……+Xn）/n

Where: X——stimulation score

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

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

Table 8. Irritation classification of the test substances.

Table 9. Irritation test results.

It can be seen from Table 9 that the antibacterial composition containing multiple plant extracts of the present invention has low irritation and has the advantages of being safe, mild and low in irritation.

The mass percentage of cinnamon bark extract in the antibacterial composition containing multiple plant extracts of the present invention is within the range of 10-40%, and the mass percentage of the composite extract is within the range of 60-90% (e.g., the mass percentage of cinnamon bark extract is 10%, 15%, 20%, 25%, 30%, 35%, 40%, and the mass percentage of the composite extract is 60%, 65%, 70%, 75%, 80%, 85%, 90%, etc.); and/or other solvents are used to extract rosemary, myrrh, niaouli, and ginkgo leaves (e.g., using ethanol, methanol, petroleum ether, ethyl acetate, etc. solvents to extract rosemary, myrrh, niaouli, and ginkgo leaves); Melaleuca alternifolia and Ginkgo biloba leaves); and/or in terms of mass percentage, the amount of the rosemary extract is 5-20% of the antibacterial composition containing multiple plant extracts, the amount of the myrrh extract is 20-40% of the antibacterial composition containing multiple plant extracts, the amount of the Niaouliu truncatula extract is 5-25% of the antibacterial composition containing multiple plant extracts, and the amount of the Ginkgo biloba extract is 15-45% of the antibacterial composition containing multiple plant extracts; and/or in terms of mass percentage, the amount of the myrrh extract is 20-40% of the antibacterial composition containing multiple plant extracts, the amount of the Niaouliu truncatula extract is The dosage 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 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 niaouli 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 in terms of mass percentage, the dosage of the rosemary extract is 5-20% of the antibacterial composition containing multiple plant extracts , the amount of the myrrh extract is 20-40% of the antibacterial composition containing multiple plant extracts, and the amount of the ginkgo leaf extract is 15-45% of the antibacterial composition containing multiple plant extracts; and/or in terms of mass percentage, the amount of the rosemary extract is 5-20% of the antibacterial composition containing multiple plant extracts, the amount of the myrrh extract is 20-40% of the antibacterial composition containing multiple plant extracts, and the amount of the niaouli extract is 5-25% of the antibacterial composition containing multiple plant extracts; the obtained antibacterial composition containing multiple plant extracts has synergistic antiseptic, mite removal and anti-inflammatory effects.

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 (10)

1. An antibacterial composition containing multiple plant extracts, characterized in that it comprises the following raw materials in percentage by weight: 10-40% of cinnamon bark extract and 60-90% of a composite extract, wherein the composite extract comprises a mixture of at least three of rosemary extract, myrrh extract, niaouli extract, and ginkgo leaf extract. 2. The antibacterial composition containing multiple plant extracts as claimed in claim 1, characterized in that, in terms of mass percentage, the amount of the rosemary extract is 5-20% of the antibacterial composition containing multiple plant extracts, the amount of the myrrh extract is 20-40% of the antibacterial composition containing multiple plant extracts, the amount of the niaouli extract is 5-25% of the antibacterial composition containing multiple plant extracts, and the amount of the ginkgo leaf extract is 15-45% of the antibacterial composition containing multiple plant extracts. 3. The antibacterial composition containing multiple plant extracts as claimed in claim 1, characterized in that, in terms of mass percentage, the amount of the myrrh extract is 20-40% of the antibacterial composition containing multiple plant extracts, the amount of the niaouli extract is 5-25% of the antibacterial composition containing multiple plant extracts, and the amount of the ginkgo leaf extract is 15-45% of the antibacterial composition containing multiple plant extracts. 4. The antibacterial composition containing multiple plant extracts as claimed in claim 1, characterized in that, in terms of mass percentage, the amount of the rosemary extract is 5-20% of the antibacterial composition containing multiple plant extracts, the amount of the Niaouli extract is 5-25% of the antibacterial composition containing multiple plant extracts, and the amount of the Ginkgo biloba extract is 15-45% of the antibacterial composition containing multiple plant extracts. 5. The antibacterial composition containing multiple plant extracts as claimed in claim 1, characterized in that, in terms of mass percentage, the amount of the rosemary extract is 5-20% of the antibacterial composition containing multiple plant extracts, the amount of the myrrh extract is 20-40% of the antibacterial composition containing multiple plant extracts, and the amount of the ginkgo leaf extract is 15-45% of the antibacterial composition containing multiple plant extracts. 6. The antibacterial composition containing multiple plant extracts according to claim 1, characterized in that, in terms of mass percentage, the amount of the rosemary extract is 5-20% of the antibacterial composition containing multiple plant extracts, the amount of the myrrh extract is 20-40% of the antibacterial composition containing multiple plant extracts, and the amount of the Niaouli extract is 5-25% of the antibacterial composition containing multiple plant extracts. 7. The antibacterial composition containing multiple plant extracts according to claim 1, wherein 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 rosemary extract comprises the following steps: The dried rosemary is crushed and sieved, the medicinal powder is subjected to a second supercritical carbon dioxide extraction, the extract is collected and filtered to obtain a rosemary extract; The extraction process of the myrrh extract comprises the following steps: Take myrrh, grind it, sieve it, put the powder of the medicinal material into a steam distillation device, add water, extract it by steam distillation, let it stand, take the oil layer, remove water, filter it, and obtain the myrrh extract; The extraction process of the Niaouli extract comprises the following steps: Place Niaouliu leaves in a steam distillation device, add water, perform steam distillation extraction, let stand, take the oil layer, remove water, filter, and obtain Niaouliu extract; The extraction process of the ginkgo leaf extract comprises the following steps: The ginkgo leaf blades are placed in a steam distillation device, water is added, the extraction is performed by steam distillation, the extraction is allowed to stand, the oil layer is taken, the water is removed, and the extraction is filtered to obtain the ginkgo leaf extract. 8. The antibacterial composition containing multiple plant extracts according to claim 7, characterized in that the first supercritical carbon dioxide extraction has an extraction temperature of 10-20°C, an extraction pressure of 10-30 MPa, a CO ₂ flow rate of 20-40 L/h, and an extraction time of 2-4 h; The second supercritical carbon dioxide extraction has an extraction temperature of 5-15°C, an extraction pressure of 15-25MPa, a CO ₂ flow rate of 10-30L/h, and an extraction time of 2-4h; The mass ratio of myrrh to water is 1:18-23; The mass ratio of the Niaouli leaf to water is 1:18-23; The mass ratio of the ginkgo leaf leaf to water is 1:18-23. 9. The use of the antibacterial composition containing multiple plant extracts as described in any one of claims 1 to 8 in the preparation of daily chemical products, characterized in that the daily chemical products contain the antibacterial composition containing multiple plant extracts; the daily chemical products include at least one of shower gel, facial mask liquid, emulsion, cream, toner, essence, stock solution, facial cleanser, lotion, perfume, makeup remover, liquid foundation, foundation cream, concealer cream, rouge, lipstick, eye shadow, and blush. 10. Use of the antibacterial composition containing multiple plant extracts as claimed in claim 9 in the preparation of daily chemical products, characterized in that, in terms of mass percentage, the amount of the antibacterial composition containing multiple plant extracts is 0.2-1% of the daily chemical products.