Compound bacteriostatic agent as well as preparation method and application thereof

Abstract

The invention discloses a compound bacteriostatic agent as well as a preparation method and application thereof. The compound bacteriostatic agent is prepared from the following components: an aloe arborescens extract and eugenol. The concentration of the aloe arborescens extract is greater than or equal to 1mg / mL; the concentration of the eugenol is greater than or equal to 0.125 mg / mL; the solvent of the compound bacteriostatic agent is dipropylene glycol. The compound bacteriostatic agent provided by the invention can well inhibit the growth of escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, candida albicans and aspergillus niger. More importantly, the aloe arborescens extract and eugenol have a synergistic effect on escherichia coli, staphylococcus aureus, pseudomonas aeruginosa and candida albicans, and have an additive effect on aspergillus niger. Therefore, when the aloe arborescens extract and the eugenol are used in a combined manner, the inhibition effect on the five bacteria can be achieved by using lower concentrations of the aloe arborescens extract and the eugenol.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a compound antibacterial agent and a preparation method and application thereof. Background Art

[0002] Cosmetics contain a variety of ingredients that are conducive to the growth of microorganisms. During the production, filling and consumer use, microorganisms can easily multiply in large numbers, causing the cosmetics to spoil. In order to control the amount of microorganisms in cosmetics, companies generally add one or more preservatives to solve the problem of microbial contamination. Therefore, preservatives are an indispensable component of cosmetics, which can ensure that cosmetics are not contaminated by microorganisms and deteriorate during production, storage and use. Currently, chemical synthetic preservatives mainly based on phenoxyethanol are commonly used in cosmetics. Although they inhibit the growth of microorganisms, chemical preservatives are one of the main causes of facial allergies when consumers use cosmetics. In addition, excessive accumulation of preservatives can easily cause thinning of the stratum corneum.

[0003] Therefore, with the development of the cosmetics industry, attention has now turned to the research of natural preservatives. At present, a variety of plant extracts are used in chemical, pharmaceutical and food fields. The active ingredients of these extracts are highly safe and also include other functions, such as antioxidant, anti-inflammatory, whitening, etc.; and more and more consumers tend to choose products containing natural ingredients. Therefore, preservatives based on plant extracts are becoming a new prospect and new market choice in the current field of preservation.

[0004] There are more than 500 varieties of aloe vera, and representative varieties reported for medicinal use include Aloe vera, Aloe arborescens, Aloe arborescens, etc. Among them, Aloe vera is one of the most intensively studied and widely used varieties, and has been listed as a commonly used Chinese medicine in the Chinese Pharmacopoeia, while research on Aloe arborescens is relatively rare.

[0005] For example, patent CN105963477A provides a pure plant-based wash-free disinfectant gel and a preparation method thereof: the pure plant-based wash-free disinfectant gel, according to the mass percentage, comprises 2-3% anthraquinone compounds in aloe vera leaf extract, 8-9% polysaccharides in aloe vera leaf extract, 15-20% phenolic compounds, phytohormones, organic acids, vitamins, and active enzymes in aloe vera leaf extract, 2.5-3% amino acids in aloe vera leaf extract, 0.8-1.2% minerals in aloe vera leaf extract, 5-8% glycerol, 0.7-0.8% limonene, 0.7-0.9% terpinene, 0.4-0.5% p-cymene, 0.3-0.4% sesquiterpenes, 1-1.5% terpinene tetraol, 1-2% eucalyptol, 1-1.5% sesquiterpenoids, and the rest are water and unavoidable impurities. The disinfectant gel can be used to clean and disinfect hands without water and is safe for use by pregnant women and children.

[0006] However, in recent years, research has focused on aloin in aloe vera, and research on the extraction and efficacy of flavonoids in aloe arborescens is relatively lacking. At present, the development of aloe vera deep processing industry in my country is relatively weak, and the lack of relevant research makes it difficult to fully explore the huge medicinal value of aloe arborescens. Aloe arborescens is planted in large quantities in northern my country, but its huge medicinal value has not yet been effectively developed. Summary of the invention

[0007] In order to solve the problems existing in the above-mentioned prior art, the purpose of the present application is to provide a compound antibacterial agent and its preparation method and application.

[0008] Eugenol, also known as 4-allylguaiacol, is a colorless to pale yellow liquid with a molecular formula of C10H12O2 and a relative molecular mass of 164.210. It is slightly soluble in water and miscible with organic solvents such as ethanol, acetone, ethyl acetate, ether, and chloroform. Its main function is to serve as the main flavoring agent of some flavors. It can also be used as a edible flavoring. At the same time, due to its strong bactericidal power, it can be used as an antibacterial agent.

[0009] Aloe arborescens Miller, also known as wood sword aloe, antler aloe, upright aloe, woody aloe, and large aloe, is native to South Africa and has a considerable output in Japan. The raw leaves of Aloe arborescens Miller taste bitter and can relieve stomach pain, nausea, and constipation. Although it is native to South Africa, it is used worldwide to treat and relieve minor wounds, irritations, and burns because of its wound healing and anti-inflammatory properties in the gel in its leaves. The leaves of Aloe arborescens Miller are roughly composed of three parts: leaf epidermis, mesophyll, and mucus. The mucus part contains the innermost transparent gel, the outermost green leaf skin, and the yellow viscous juice secreted by the vascular cells when the leaves are cut. By ultrasonic extraction of Aloe arborescens Miller, a mixture of active ingredients such as flavonoids, glycosides, and anthraquinones can be obtained. These active ingredients have antioxidant, anti-inflammatory, whitening, antibacterial and other effects in cosmetics, so Aloe arborescens extract can be used as an effective ingredient and antibacterial ingredient in cosmetics.

[0010] The extraction process of Aloe arborescens is the basis for exploring its functions. The research on the performance of Aloe arborescens has important practical significance in practical application development. Therefore, this study has important practical significance for developing the medicinal value of Aloe arborescens. Based on this, a compound antibacterial agent containing Aloe arborescens will be designed.

[0011] Based on the above objectives, this application provides the following technical solutions:

[0012] One of the technical solutions of the present application provides a compound antibacterial agent, which comprises the following components:

[0013] Aloe arborescens extract, eugenol.

[0014] Furthermore, the concentration of the Aloe arborescens extract is ≥1 mg / mL.

[0015] Furthermore, the concentration of the eugenol is ≥0.125 mg / mL.

[0016] More preferably, the concentration of the Aloe arborescens extract is 1 mg / mL to 32 mg / mL; and the concentration of the eugenol is 0.125 mg / mL to 16 mg / mL.

[0017] Furthermore, the compound antibacterial agent also includes a solvent, and the solvent is dipropylene glycol.

[0018] The second technical solution of the present application provides a method for preparing the composite antibacterial agent, comprising the following steps:

[0019] (1) Preparing Aloe arborescens extract powder: taking Aloe arborescens powder as a raw material, performing alcohol extraction, and concentrating the filtrate by rotary evaporation and then freeze-drying to obtain Aloe arborescens extract powder;

[0020] (2) Preparing a compound antibacterial agent: dissolving the Aloe arborescens extract powder and eugenol obtained in step (1) with dipropylene glycol to prepare a compound antibacterial agent.

[0021] Furthermore, the alcohol extraction in step (1) is carried out under the assistance of ultrasound; the volume fraction of ethanol used is 40% to 100%; the time of the ultrasound assistance is 20 to 60 minutes; and the temperature of the ultrasound assistance is 20 to 40°C.

[0022] Furthermore, the temperature of the ultrasonic assistance is preferably 40° C.; the time of the ultrasonic assistance is preferably 40 min; and the volume fraction of ethanol is preferably 70%.

[0023] The third technical solution of the present application provides the use of the composite antibacterial agent in the preparation of a preservative.

[0024] Furthermore, the compound antibacterial agent inhibits the growth and reproduction of bacteria and / or fungi; the bacteria is at least one of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa; the fungi is at least one of Candida albicans and Aspergillus niger.

[0025] Furthermore, the Aloe arborescens extract and eugenol have a synergistic effect on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, and an additive effect on Aspergillus niger.

[0026] Preferably, when the bacterium inhibited by the antibacterial agent is Escherichia coli, the concentration of the Aloe arborescens extract is ≥1 mg / mL, and the concentration of the eugenol is ≥0.125 mg / mL.

[0027] Preferably, when the bacterium inhibited by the antibacterial agent is Staphylococcus aureus, the concentration of the Aloe arborescens extract is ≥1 mg / mL, and the concentration of the eugenol is ≥0.125 mg / mL.

[0028] Preferably, when the bacterium inhibited by the antibacterial agent is Pseudomonas aeruginosa, the concentration of the Aloe arborescens extract is ≥1 mg / mL, and the concentration of the eugenol is ≥0.125 mg / mL.

[0029] Preferably, when the fungus inhibited by the antibacterial agent is Candida albicans, the concentration of the Aloe arborescens extract is ≥4 mg / mL, and the concentration of the eugenol is ≥0.25 mg / mL.

[0030] Preferably, when the fungus inhibited by the antibacterial agent is Aspergillus niger, the concentration of the Aloe arborescens extract is ≥4 mg / mL, and the concentration of the eugenol is ≥0.25 mg / mL.

[0031] Furthermore, the preservative is added to cosmetics to control the amount of microorganisms in the cosmetics and prevent the cosmetics from deteriorating.

[0032] Compared with the prior art, the compound antibacterial agent provided by the present invention has at least the following beneficial effects:

[0033] The present invention provides that aloe arborescens extract and eugenol have antibacterial effects through experiments. The present invention finds through combined antibacterial experiments that the compound of aloe arborescens extract and eugenol has a synergistic antibacterial effect on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans; and has an additive antibacterial effect on Aspergillus niger. While reducing the concentration of aloe arborescens extract and eugenol to reduce irritation, a more outstanding antibacterial effect is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the standard curve of rutin;

[0035] Figure 2 The effect of ultrasonic temperature on the extraction rate of Aloe arborescens;

[0036] Figure 3 The effect of ultrasound time on the extraction rate of Aloe arborescens;

[0037] Figure 4 The effect of ethanol volume fraction on the extraction rate of Aloe arborescens;

[0038] Figure 5 This is a schematic diagram of the checkerboard dilution method;

[0039] In the figure:

[0040] A1=1 / 2MIC A, A2=1 / 4MIC A , A3=1 / 8MIC A ;

[0041] B1=1 / 2MIC B , B2=1 / 4MIC B , B3=1 / 8MIC B . DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0043] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0044] Materials used in this application:

[0045] (1) Eugenol: purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; product number: 48317454.

[0046] (2) Arborescent Aloe: This is commercially available Aloe vera powder produced in Shijiazhuang, Hebei Province, and grown for more than ten years.

[0047] Example 1 Preparation of Aloe arborescens Extract and Calculation of Flavonoid Content

[0048] This example provides a method for preparing an Aloe arborescens extract using ultrasound-assisted alcohol extraction, and provides a method for determining the flavonoid content in the Aloe arborescens extract.

[0049] The preparation method of the Aloe arborescens extract is as follows:

[0050] (1) Accurately weigh 2 g of Aloe arborescens powder and place it in a conical flask.

[0051] (2) 70% ethanol (V / V, solvent is deionized water) was added at a solid-liquid ratio of 1:15, and ultrasonication was performed for 40 minutes at a temperature of 30° C. and a power of 225 W.

[0052] (3) The liquid after ultrasonication is centrifuged and filtered, and a portion of the filtered liquid is retained to obtain the Aloe arborescens extract, and the extraction rate of flavonoids in the Aloe arborescens is calculated according to the rutin standard curve. The other portion of the filtered liquid (Aloe arborescens extract) is concentrated by rotary evaporation and freeze-dried to obtain the Aloe arborescens extract powder for later use.

[0053] In this embodiment, rutin is used as a standard substance, and the method for determining the flavonoid content in the Aloe arborescens extract comprises the following steps:

[0054] (1) Drawing of rutin standard curve:

[0055] Accurately weigh 5 mg of rutin standard in a 25 mL volumetric flask, add 60% volume fraction of ethanol, put it in a water bath and heat until dissolved, then cool to room temperature, then dilute with 60% ethanol, shake well, and obtain a 0.2 mg / mL rutin standard solution. According to the aluminum nitrate colorimetric method, 0, 1, 2, 3, 4, 5, and 6 mL of rutin standard solution are transferred to a volumetric flask, and 10, 9, 8, 7, 6, 5, and 4 mL of 60% ethanol are added to dilute. Then add 0.5 mL of 5% sodium nitrite solution, shake well, and let it stand for 6 minutes, add 0.5 mL of 10% aluminum nitrate solution, shake well, let it stand for 6 minutes, add 2 mL of 4% sodium hydroxide solution, shake well, let it stand for 15 minutes, use 60% ethanol as a blank control, and measure its absorbance at 510 nm after the reaction is completed. Wherein, the absorbance is set as the ordinate y, the concentration is set as the abscissa x, and the rutin standard curve is drawn with x and y, the regression equation is obtained by linear fitting, and the correlation coefficient R is calculated. 2 .

[0056] Rutin standard curve Figure 1 As shown: The equation y = 0.403x + 0.0067, R 2 =0.999.

[0057] (2) Calculation of flavonoids in Aloe arborescens extract: Use a pipette to draw 0.5 mL of crude Aloe arborescens extract, add 70% ethanol to dilute 10 times, mix well, and use a pipette to draw 2.5 mL of the dilution, add 0.125 mL of 5% sodium nitrite solution, shake well, and let stand for 6 minutes, add 0.125 mL of 10% aluminum nitrate solution, shake well, let stand for 6 minutes, add 0.5 mL of 4% sodium hydroxide solution and react for 15 minutes, use the corresponding reagent as a blank control, and measure its absorbance at 510 nm after the reaction is completed. Then calculate the extraction rate of Aloe arborescens flavonoids according to the following formula.

[0058]

[0059] Where:

[0060] c is the mass concentration of Aloe arborescens flavonoids (mg / mL);

[0061] V is the volume of the extract solution (mL);

[0062] n is the dilution factor;

[0063] m is the mass of Aloe arborescens powder (g).

[0064] Subsequent experiments were performed based on the measurement method of this example.

[0065] Example 2 Optimization of Process Conditions for Aloe Arborescens Flavonoids

[0066] This example selects three factors that affect the flavonoid extraction rate, including ultrasonic temperature, ultrasonic time and ethanol volume fraction. The extraction conditions of the above-mentioned Example 1 are used as fixed levels, and the flavonoid content in Aloe arborescens is used as a reference index to conduct a single factor experiment, as shown in Table 1. The specific method is as follows:

[0067] (1) Investigate the effect of ultrasonic temperature on the flavonoid content of the extract:

[0068] Accurately weigh 2 g of Aloe arborescens powder, the volume fraction of ethanol is 70%, the solid-liquid ratio is 1:15, the ultrasonic time is 40 min, and the ultrasonic temperatures are set to 20°C, 25°C, 30°C, 35°C, and 40°C respectively. Extraction is carried out under this condition, and each experiment is repeated three times in parallel.

[0069] The experimental results are as follows Figure 2 As shown in the figure, when the ultrasonic temperature is gradually increased from 20℃ to 40℃, the flavonoid extraction rate shows a gradual upward trend, that is, the flavonoid extraction rate reaches the maximum value when the ultrasonic temperature is 40℃, so 40℃ is selected as the optimal ultrasonic temperature.

[0070] In addition, the figure shows that when the ultrasonic temperature is 35°C, the flavonoid extraction rate decreases, but when it reaches 40°C, the extraction rate suddenly increases. The possible reason is that the temperature increase may accelerate the speed of the chemical reaction, thereby increasing the extraction rate. If the reaction is an exothermic reaction, the temperature increase will move the chemical equilibrium in the reverse direction, causing the reactant conversion rate to decrease, and the extraction rate will also decrease. When the temperature reaches a certain level, it may trigger other chemical reactions, which may promote the extraction of solutes, so the extraction rate will increase again.

[0071] (2) Investigate the effect of ultrasound time on the flavonoid content of the extract:

[0072] Accurately weigh 2 g of Aloe arborescens powder, the volume fraction of ethanol is 70%, the solid-liquid ratio is 1:15, the ultrasonic temperature is 30°C, and the ultrasonic time is 20 min, 30 min, 40 min, 50 min, and 60 min respectively. Extraction is carried out under this condition, and each experiment is repeated three times in parallel.

[0073] The experimental results are as follows Figure 3 As shown in the figure, as the ultrasonic time gradually increased from 20 min to 60 min, the flavonoid extraction rate showed a trend of first increasing and then decreasing. When the ultrasonic time was 40 min, the flavonoid extraction rate reached the highest value, so 40 min was selected as the optimal ultrasonic time.

[0074] (3) Investigate the effect of ethanol volume fraction on the flavonoid content of the extract: Accurately weigh 2 g of Aloe arborescens powder, select 40%, 55%, 70%, 85%, and 100% ethanol volume fractions, respectively, with a solid-liquid ratio of 1:15, ultrasonic temperature of 30°C, and ultrasonic time of 40 min. Extraction was carried out under this condition, and each experiment was repeated three times in parallel.

[0075] The experimental results are as follows Figure 4 As shown in the figure, with the continuous increase of ethanol volume fraction, the flavonoid extraction rate shows a trend of first increasing and then decreasing. When the ethanol volume fraction reaches 70%, the flavonoid extraction rate reaches the highest value, so 70% is selected as the optimal ethanol volume fraction.

[0076] (4) According to the above single factor experimental results, ultrasonic temperature (A), ultrasonic time (B), and ultrasonic time (C) were selected as the three factors of the orthogonal experiment to investigate the effect of the three-factor three-level interaction on the flavonoid extraction rate of Aloe arborescens. The orthogonal factor table is shown in Table 1, and the orthogonal experiment result analysis table is shown in Table 2.

[0077] Table 1 Orthogonal factor level table

[0078]

[0079] Table 2 Orthogonal test results analysis table

[0080]

[0081]

[0082] From the R value in the orthogonal test analysis results table in Table 2, it can be seen that the influence of each orthogonal test factor on the flavonoid yield of Aloe arborescens is ranked from large to small as follows: ultrasonic temperature > ultrasonic time > ethanol volume ratio. Through range analysis, it can be determined that the best extraction process optimized by orthogonal test is A3B2C2, that is, the ultrasonic temperature is 40℃, the ultrasonic time is 40min, and the ethanol volume fraction is 70%. Under this condition, the optimal extraction rate of flavonoids in Aloe arborescens can be obtained.

[0083] According to the above orthogonal test results, the optimal extraction conditions were obtained, namely, extraction at an ultrasonic temperature of 40°C, an ultrasonic time of 40 min, and an ethanol volume fraction of 70%. After repeating the experiment three times, the extraction rates of flavonoids were 12.64%, 12.75%, and 13.31%, respectively, with an average of 12.9%.

[0084] Example 3 Minimum Inhibitory Concentration (MIC) Determination

[0085] Minimum inhibitory concentration (MIC) refers to the lowest drug concentration that can inhibit the growth and reproduction of microorganisms (such as bacteria, fungi, etc.). MIC is an important indicator for measuring the inhibitory effect of antimicrobial drugs, antibiotics or antimicrobial agents on specific microorganisms. This example uses the microbroth dilution method to determine the minimum inhibitory concentration, and the specific method is as follows:

[0086] (1) Preparation of nutrient broth liquid culture medium: Nutrient broth is used for general bacterial culture, transfer, rejuvenation and bacterial enrichment. Its ingredients include 10.0 g / L peptone, 3.0 g / L beef extract powder, and 5.0 g / L sodium chloride. Accurately weigh 0.9 g of powder and place it in a conical flask. Then add 50 mL of distilled water according to the product instructions, sterilize at 121°C for 15 minutes, and set aside.

[0087] (2) Prepare potato liquid culture medium (containing chloramphenicol): Potato liquid culture medium (containing chloramphenicol) is a culture medium used for the enrichment of molds and yeasts. Its main components include 6.0 g / L potato extract powder, 20.0 g / L glucose, and 0.1 g / L chloramphenicol. Accurately weigh 1.305 g of powder and place it in a conical flask. Then add 50 mL of distilled water according to the product instructions, sterilize at 121°C for 15 minutes, and set aside.

[0088] (3) Dilute the sample with liquid culture medium in a 96-well plate by the two-fold dilution method so that wells 1 to 8 contain 100 μL of drug-containing culture medium (either Aloe arborescens extract or eugenol solution) at concentrations of 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 mg / mL, respectively.

[0089] Then, a concentration of 107 CFU / mL of bacterial suspension (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa) or 10 6 100 μL of fungal suspension (Candida albicans, Aspergillus niger) with CFU / mL; 100 μL of sample solution, including Aloe arborescens extract and eugenol solution, was added to well No. 9, and then 100 μL of liquid culture medium was added as a negative control of the experiment.

[0090] Then measure OD 600 The 96-well plate containing the bacterial suspension was incubated at 37°C for 24 h, and the 96-well plate containing the fungal suspension was incubated at 28°C for 48 h, and the OD was measured again. 600 The lowest concentration with ΔOD ≤ 0.05 was taken as the MIC result.

[0091] The experiment was repeated three times for each sample.

[0092] The experimental results are shown in Tables 3 and 4:

[0093] Table 3 MIC results of Aloe arborescens extract against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger (OD 600 )

[0094]

[0095] Note: “+” means only a small amount of colonies grew, and “-” means no colonies grew.

[0096] As shown in Table 3, the extract of Aloe arborescens inhibits the growth of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger. The minimum inhibitory concentration (MIC) for Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Aspergillus niger is 8 mg / mL; the minimum inhibitory concentration (MIC) for Candida albicans is 16 mg / mL.

[0097] Table 4 MIC results of eugenol solution against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger (OD 600 )

[0098]

[0099]

[0100] Note: “+” means only a small amount of colonies grew, and “-” means no colonies grew.

[0101] As shown in Table 4, the eugenol solution inhibited the growth of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger, and the minimum inhibitory concentration MIC for Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger was 1 mg / mL.

[0102] Example 4 Determination of combined antibacterial index

[0103] Based on the MIC results, the checkerboard dilution method was used to detect the inhibition of the compound antibacterial agent made of Aloe arborescens extract and eugenol on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger. The specific operation is as follows:

[0104] (1) A 96-well plate was divided into 3×3 grids. Aloe arborescens extract and eugenol were diluted to a certain concentration according to the minimum inhibitory concentration (MIC) results. 1 / 2, 1 / 4, and 1 / 8 MIC of Aloe arborescens extract were added to the three horizontal columns, and 1 / 8, 1 / 4, and 1 / 2 MIC of eugenol solution were added to the three vertical columns, respectively. Figure 5 (In the figure, A represents Aloe arborescens extract; B represents eugenol solution).

[0105] (2) Add 10% ethanol to each well of the 9-grid. 7 CFU / mL of bacterial suspension (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa) or 10 6 100 μL of fungal suspension (Candida albicans, Aspergillus niger) with CFU / mL.

[0106] (3) Determination of OD 600 After the OD value was obtained, the 96-well plate containing the bacterial suspension was incubated at 37°C for 24 h, and the 96-well plate containing the fungal suspension was incubated at 28°C for 48 h, and the OD value was measured again. 600 The combined inhibition index (FIC) of each well was determined by measuring the OD value and calculating the difference method. The experiment was repeated three times and the average value was taken.

[0107] The FIC value is used as the main means to evaluate antimicrobial drug interactions because it can decipher all possible combined effects, including antagonism, addition, indifference, and synergy. Calculation of the FIC index: FIC = MIC of drug A when used in combination for bacteriostasis / MIC of drug A when used alone + MIC of drug B when used in combination / MIC of drug B when used alone. When FIC ≤ 0.5, it indicates a synergistic effect; when 0.5 < FIC ≤ 1, it is an additive effect; when 1 < FIC ≤ 2, it is an indifferent effect; when FIC > 2, it is an antagonistic effect. A synergistic effect means that the bacteriostatic effect after combination is more outstanding than when used alone; addition means that the antibacterial ability after combination is determined by the sum of the effects when used alone; an indifferent effect means that whether used in combination or alone, the inhibitory effect on the bacterial strain remains unchanged; an antagonistic effect means that the antibacterial ability after combination is lower than the effect when using one or several of the components alone.

[0108] The results of this example are shown in Table 5:

[0109] Table 5 FIC results of the combined use of Aloe arborescens Mill. extract and eugenol extract against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger (OD 600 )

[0112]

[0113]

[0114] The FIC was calculated by combining the mean data of single drug use in Tables 3 and 4 and the mean data of combined drug use in Table 5. As shown in Table 5, the FIC index of the combined antibacterial effect of 1 mg / mL (1 / 8×MIC) Aloe arborescens extract and 0.125 mg / mL (1 / 8MIC) eugenol solution on Escherichia coli was 0.25, indicating that the combined use of Aloe arborescens extract and eugenol had a synergistic inhibitory effect on the growth of Escherichia coli; the FIC index of the combined antibacterial effect of 1 mg / mL (1 / 8×MIC) Aloe arborescens extract and 0.125 mg / mL (1 / 8MIC) eugenol solution on Staphylococcus aureus was 0.25, indicating that the combined use of Aloe arborescens extract and eugenol had a synergistic inhibitory effect on the growth of Staphylococcus aureus; The FIC index of the combined inhibition against Pseudomonas aeruginosa was 0.25, indicating that the combined use of Aloe arborescens extract and eugenol had a synergistic inhibitory effect on the growth of Pseudomonas aeruginosa; the FIC index of the combined inhibition against Candida albicans by 4 mg / mL (1 / 4×MIC) Aloe arborescens extract and 0.25 mg / mL (1 / 4MIC) eugenol solution was 0.5, indicating that the combined use of Aloe arborescens extract and eugenol had a synergistic inhibitory effect on the growth of Candida albicans; the FIC index of the combined inhibition against Candida albicans by 4 mg / mL (1 / 2×MIC) Aloe arborescens extract and 0.25 mg / mL (1 / 4MIC) eugenol solution was 0.75, indicating that the combined use of Aloe arborescens extract and eugenol had an additive inhibitory effect on the growth of Candida albicans.

[0115] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A compound antibacterial agent, characterized in that: The compound antibacterial agent comprises the following ingredients: Aloe arborescens extract, eugenol; The concentration of the Aloe arborescens extract is ≥1 mg / mL; the concentration of the eugenol is ≥0.125 mg / mL.

2. A compound antibacterial agent according to claim 1, characterized in that: The concentration of the Aloe arborescens extract is 1 mg / mL to 32 mg / mL; the concentration of the eugenol is 0.125 mg / mL to 16 mg / mL.

3. A compound antibacterial agent according to claim 1, characterized in that: The compound antibacterial agent also includes a solvent, and the solvent is dipropylene glycol.

4. The method for preparing a composite antibacterial agent according to claim 1, characterized in that: The following steps are involved: (1) Preparing Aloe arborescens extract powder: taking Aloe arborescens powder as a raw material, performing alcohol extraction, and concentrating the filtrate by rotary evaporation and then freeze-drying to obtain Aloe arborescens extract powder; (2) Preparing a compound antibacterial agent: dissolving the Aloe arborescens extract powder and eugenol obtained in step (1) with dipropylene glycol to prepare a compound antibacterial agent.

5. The method for preparing a composite antibacterial agent according to claim 4, characterized in that: The alcohol extraction in step (1) is carried out under the assistance of ultrasound; the volume fraction of ethanol used is 40% to 100%; the time of the ultrasound assistance is 20 to 60 minutes; and the temperature of the ultrasound assistance is 20 to 40°C.

6. Use of the compound antibacterial agent as claimed in claim 1 in the preparation of preservatives.

7. The use of a compound antibacterial agent according to claim 6 in the preparation of a preservative, characterized in that: The compound antibacterial agent inhibits the growth and reproduction of bacteria and / or fungi; The bacteria is at least one of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa; The fungus is at least one of Candida albicans and Aspergillus niger.

8. The use of a compound antibacterial agent according to claim 7 in the preparation of a preservative, characterized in that: When the bacterium inhibited by the antibacterial agent is Escherichia coli, the concentration of the Aloe arborescens extract is ≥1 mg / mL, and the concentration of the eugenol is ≥0.125 mg / mL; When the bacterium inhibited by the antibacterial agent is Staphylococcus aureus, the concentration of the Aloe arborescens extract is ≥1 mg / mL, and the concentration of the eugenol is ≥0.125 mg / mL; When the bacterium inhibited by the antibacterial agent is Pseudomonas aeruginosa, the concentration of the Aloe arborescens extract is ≥1 mg / mL, and the concentration of the eugenol is ≥0.125 mg / mL.

9. The use of a compound antibacterial agent according to claim 7 in the preparation of a preservative, characterized in that: When the fungus inhibited by the antibacterial agent is Candida albicans, the concentration of the Aloe arborescens extract is ≥4 mg / mL, and the concentration of the eugenol is ≥0.25 mg / mL; When the fungus inhibited by the antibacterial agent is Aspergillus niger, the concentration of the Aloe arborescens extract is ≥4 mg / mL, and the concentration of the eugenol is ≥0.25 mg / mL.

10. Use of a compound antibacterial agent according to claim 7 in the preparation of a preservative, characterized in that: The preservatives are added to cosmetics.

Images