A natural preservative synergistic composition, preparation method and application thereof

Abstract

The application belongs to the technical field of preservative technology, and particularly relates to a natural preservative synergistic composition, a preparation method and application. The natural preservative synergistic composition provided by the application is obtained by screening rosemary preservative extract, clove preservative extract, prunella vulgaris preservative extract, perilla leaf preservative extract, perilla seed preservative extract and the like as natural preservative ingredients, introducing the natural preservative ingredients into chemical synthetic preservative ingredients as preservative synergistic ingredients, so that the chemical synthetic preservative ingredients can inhibit the growth and reproduction of microorganisms such as bacteria and fungi in the case of small amount of addition. By adjusting the proportion of the natural plant preservative ingredients such as rosemary preservative extract, clove preservative extract, prunella vulgaris preservative extract, perilla leaf preservative extract and perilla seed preservative extract, and combining the natural plant preservative ingredients with the chemical synthetic preservative ingredients, the preservative effect is further improved, so that the technical problem of lack of natural preservative synergistic agents in the prior art is solved.

Description

Technical Field

[0001] This application belongs to the field of corrosion protection technology, and in particular relates to a natural anti-corrosion synergistic composition, its preparation method and application. Background Technology

[0002] In addition to adding active ingredients to exert their effects, skin care products such as cosmetics, personal care products, and topical preparations use preservatives to inhibit microbial growth and ensure product shelf life and safety.

[0003] Phenoxyethanol, propylparaben, and chlorphenesin are mainstream preservatives in skin care products, boasting advantages such as high efficacy and broad spectrum. However, as chemically synthesized ingredients, their potential risks are constantly being exposed. For example, phenoxyethanol may irritate the skin, and long-term use of parabens such as propylparaben may cause skin allergies or even more serious health problems. Therefore, currently, chemically synthesized preservatives pose safety risks such as skin irritation and sensitization.

[0004] In recent years, with the rise of green consumption trends, consumers' pursuit of "all-natural" products and the advantages of the safety and gentleness of natural plant active ingredients have made natural plant active ingredients an important development trend in the field of preservatives. It is necessary to develop preservative-enhancing active ingredients from existing natural plant active ingredients to improve preservative performance while reducing the amount of chemically synthesized preservatives used in skin care products and reducing safety risks. Summary of the Invention

[0005] In view of this, this application provides a natural preservative-enhancing composition, its preparation method, and its application, to solve the technical problem of the lack of natural preservative-enhancing agents in the prior art.

[0006] The first aspect of this application provides a natural preservative-enhancing composition, comprising at least two of the following: rosemary preservative extract, clove preservative extract, prunella preservative extract, perilla leaf preservative extract, and perilla seed preservative extract; wherein the preservative extract is a preservative extract containing phenolic acid components.

[0007] Preferably, in the natural preservative-enhancing composition, the mass ratio of any two of the preservative extracts among rosemary preservative extract, clove preservative extract, prunella preservative extract, perilla leaf preservative extract, and perilla seed preservative extract is 1~6:1~6; it can be 1~5:1~5, 1~4:1~4, 1~3:1~3, or 1~2:1~2.

[0008] Preferably, in the natural preservative-enhancing composition, the mass ratio of rosemary preservative extract, clove preservative extract, prunella preservative extract, perilla leaf preservative extract, and perilla seed preservative extract is 1~6:1~6:1~6:1~6:1~6:1~6.

[0009] Preferably, in the natural preservative-enhancing composition, the mass ratio of clove preservative extract, prunella vulgaris preservative extract, perilla leaf preservative extract, and perilla seed preservative extract is 1~5:1~5:1~5:1~5.

[0010] Preferably, in the natural preservative-enhancing composition, the mass ratio of rosemary preservative extract, clove preservative extract, and perilla leaf preservative extract is 1~4:1~4:1~4.

[0011] The second aspect of this application provides a method for preparing a natural preservative-enhancing composition, which can prepare the natural preservative-enhancing composition described in the first aspect, comprising the following steps:

[0012] The fatty acids, ethanol and water are mixed evenly to obtain a supramolecular extraction solvent.

[0013] At least two natural plants selected from rosemary, clove, selfheal, perilla leaves, and perilla seeds are added to a supramolecular extraction solvent and vortexed until homogeneous. The mixture is then subjected to ultrasonic extraction, centrifugation, concentration, and drying to obtain a natural preservative and synergistic composition.

[0014] Preferably, the fatty acid in the supramolecular extraction solvent is selected from at least one of short-chain fatty acids (less than 6 carbon atoms), medium-chain fatty acids (6 to 12 carbon atoms), and long-chain fatty acids (more than 12 carbon atoms); preferably, medium-chain fatty acids such as caprylic acid and capric acid are preferred.

[0015] Preferably, the volume ratio of fatty acids, ethanol and water in the supramolecular extraction solvent is 5:10~45:50~85.

[0016] Preferably, during the ultrasonic extraction process, the ratio of natural plant material to supramolecular extraction solvent is 1g:30~50mL, the vortexing time is 1~5min, the ultrasonic time is no more than 30min, and the temperature is 30~80℃.

[0017] Preferably, the centrifugation step after ultrasound includes: adding ethanol to an ultrasound extraction solution containing a natural preservative and synergistic composition, centrifuging to obtain the supernatant, adding water to the supernatant, centrifuging to obtain the lower solution.

[0018] Preferably, the concentration is rotary evaporation concentration and the drying is freeze drying.

[0019] The third aspect of this application provides the application of the natural preservative-enhancing composition described in the first aspect in the preparation of an enhanced composite preservative; the application process includes: using the natural preservative-enhancing composition in combination with a chemical preservative.

[0020] The fourth aspect of this application provides a synergistic composite preservative, comprising: a chemically synthesized preservative component and the natural preservative-synergistic composition described in the first aspect.

[0021] Preferably, in the synergistic composite preservative, the mass ratio of the chemically synthesized preservative component to the natural preservative-synergistic composition is 1~3:1.

[0022] Preferably, the chemically synthesized preservative is selected from at least one of phenoxyethanol, propylparaben, and chlorphenesin.

[0023] The fifth aspect of this application provides the application of the synergistic compound preservative described in the fourth aspect in the preparation of skin care products; the skin care products include at least one of cosmetics, personal care products, and topical preparations.

[0024] The sixth aspect of this application provides an oil-in-water emulsion, including the synergistic composite preservative described in the fourth aspect.

[0025] Compared with the prior art, the natural preservative-enhancing composition and preparation method provided in this application have at least the following beneficial effects:

[0026] 1. The natural preservative-enhancing composition provided in this application, when used in combination with chemically synthesized preservatives, results in a lower minimum inhibitory concentration (MIC), which can effectively inhibit the growth and reproduction of microorganisms such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans even with a small amount of addition.

[0027] 2. The natural preservative synergistic composition provided in this application, after adjusting the different dosage ratios of three, four or five of the five natural preservative synergistic compositions, is used in combination with chemically synthesized preservative components, and the minimum inhibitory concentration (MIC) and FIC value are used as quantitative indicators, or the European Pharmacopoeia EP 5.1.3 external preparations are used as the standard, further discovering a synergistic compound preservative formulation with good antibacterial effect against bacteria and fungi such as Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger.

[0028] 3. The method for preparing a natural preservative-enhancing composition provided in this application can efficiently obtain the natural preservative-enhancing composition by improving the extraction solvent, extraction solvent ratio, extraction process, and mechanical energy of the recovery and enrichment steps. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 The extraction process of natural plant preservative components provided in Example 1 of this application, and the standard curve of gallic acid when detected using the Folin-phenol method;

[0031] Figure 2 The extraction process of natural plant preservative components provided in Example 1 of this application, and the detection results of phenolic acid content of the thirteen preservative extracts obtained;

[0032] Figure 3 The extraction process of natural plant preservative components provided in Example 2 of this application yielded phenolic acid content detection results under different material-liquid ratios, vortex times, ultrasonic extraction times, and temperatures.

[0033] Figure 4 The images show the dilution of the natural plant preservatives provided in Example 2 of this application, with different supernatant-to-water volume ratios. From left to right, the volume ratios of supernatant to water are 1:9, 2:8, 3:7, 4:6, 5:5, 6:4:7:3, 8:2, and 9:1. Image a shows the diluted extract of clove, image b shows the diluted extract of rosemary, image c shows the diluted extract of prunella vulgaris, image d shows the diluted extract of perilla leaves, and image e shows the diluted extract of perilla seeds. Detailed Implementation

[0034] This application provides a natural preservative-enhancing composition, its preparation method, and its application, which addresses the technical problem of the lack of natural preservative-enhancing agents in the prior art.

[0035] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0036] Example 1

[0037] This embodiment provides a method for screening natural plant preservative components. The screening method involves: extracting preservative extracts from natural plants using ultrasound, and using the total phenolic acid content and antibacterial ability against different bacterial strains in the preservative extracts as evaluation criteria to screen for highly effective preservative natural plants; the steps include:

[0038] Steps for selecting plants:

[0039] Referring to the pharmacopoeia, natural plants such as Phyllanthus emblica, Lonicera japonica, Kaempferia galanga, Syzygium aromaticum, Perilla frutescens seeds, Salvia miltiorrhiza, Perilla frutescens stems, Prunella vulgaris, Perilla frutescens leaves, Artemisia capillaris, Rosemary, Taraxacum mongolicum, and Punica granatum peel were selected;

[0040] The steps of ultrasonic extraction:

[0041] Take 6g of powder from each of the following herbs: Phyllanthus emblica, Lonicera japonica, Kaempferia galanga, Syzygium aromaticum, Perilla frutescens seeds, Salvia miltiorrhiza, Perilla frutescens stems, Prunella vulgaris, Perilla frutescens leaves, Artemisia capillaris, Rosemary, Taraxacum mongolicum, and Punica granatum peel. Add 300mL of 60% ethanol solution at a liquid-to-solid ratio of 1:50, sonicate for 30min, centrifuge, filter, and collect the supernatant to obtain thirteen kinds of preservative extract solutions.

[0042] Take 6g of powder from each of the following ingredients: Phyllanthus emblica, Lonicera japonica, Kaempferia galanga, Clove, Perilla frutescens seed, Salvia miltiorrhiza, Perilla frutescens stem, Prunella vulgaris, Perilla frutescens leaf, Artemisia capillaris, Rosemary, Taraxacum mongolicum, and Punica granatum peel. Add 300mL of 60% ethanol solution at a liquid-to-solid ratio of 1:50, sonicate for 30min, centrifuge and filter to obtain the supernatant, concentrate by rotary evaporation, and freeze-dry to obtain freeze-dried powder of thirteen kinds of preservative extracts.

[0043] Determination steps for total phenolic acid content in preservative extract solutions:

[0044] The total phenolic acid content in the preservative extract solution was determined by the Folin-phenol method, and gallic acid was used as the standard sample to obtain the number of milligrams of gallic acid (mg GA / g) equivalent to the phenolic acid content per gram of plant sample.

[0045] Add 0.04 mL of gallic acid aqueous solution with concentrations of 0.05, 0.1, 0.2, 0.3, and 0.4 mg / mL to a test tube, along with 2.46 mL of deionized water and 0.2 mL of Folin-Ciocalteu reagent. Then add 2 mL of 20% sodium carbonate aqueous solution and mix. After 2 hours, measure the absorbance at 750 nm and plot a standard curve. The results are shown below. Figure 1 As shown;

[0046] Thirteen different preservative extract solutions (0.04 mL, 2.46 mL, and 0.2 mL of Folin-Ciocalteu reagent) were added to thirteen test tubes respectively. Then, 2 mL of a 20% sodium carbonate aqueous solution was added and mixed. After 2 hours, the absorbance at 750 nm was measured to obtain the equivalent milligrams of gallic acid (mg GA / g) per gram of plant sample. The results are as follows:Figure 2 As shown.

[0047] Testing steps for the antibacterial activity of freeze-dried preservative extract powder:

[0048] Thirteen different lyophilized preservative extracts were added to thirteen test tubes, each containing 0.25g of lyophilized powder and 20mL of 60% ethanol solution, to prepare thirteen different plant preservative extract stock solutions at a concentration of 12.5mg / mL for later use.

[0049] Using a sterilized inoculation loop, collect *Escherichia coli* CICC10302, *Staphylococcus aureus* CICC20235, or *Pseudomonas aeruginosa* CICC1035 and add them to 5 mL of sterile NB medium. Incubate at 37°C and 160 rpm / min on a shaker for 16–24 h to allow them to reach the logarithmic growth phase. Take the tested bacterial strains that have reached the logarithmic growth phase and dilute the bacterial concentration to 1 × 10⁻⁶ using NB medium. 5 CFU / mL, for later use;

[0050] Using a sterilized inoculation loop, take a small amount of Candida albicans CICC1943 and add it to 5 mL of sterile SDB medium. Incubate at 30°C and 160 rpm / min for 24 h in a constant temperature shaker to allow it to reach the logarithmic growth phase. Take the test strain that has reached the logarithmic growth phase and dilute the bacterial concentration to 1×10⁻⁶ with SDB medium. 5 CFU / mL, for later use;

[0051] Thirteen plant preservative extracts, propylparaben, chlorphenesin, and phenoxyethanol were diluted to concentrations of 6.25 mg / mL, 3.125 mg / mL, 2.5 mg / mL, 1.875 mg / mL, 1.25 mg / mL, 0.625 mg / mL, and 0.3125 mg / mL, respectively, using liquid culture medium. These concentrations were then mixed with bacterial suspensions of the test strains at a volume ratio of 1:1. After 24 h of incubation on a shaker at a constant temperature, 10 μL of the culture medium was added to a petri dish and incubated at 37 °C for 12 h. The presence or absence of bacterial colony growth was observed, and the minimum inhibitory concentration (MIC) at which colony growth occurred was recorded. The results are shown in Table 1.

[0052] from Figure 2It can be seen that the phenolic acid content varies in the solutions of different plant preservative extracts. Extracts from plants such as Phyllanthus emblica, Lonicera japonica, Clove, Perilla frutescens seeds, Salvia miltiorrhiza, Prunella vulgaris, Perilla frutescens leaves, Artemisia capillaris, Rosemary, Taraxacum mongolicum, and Punica granatum peel have relatively high phenolic acid content. Table 1 shows that extracts from Prunella vulgaris, Perilla frutescens leaves, and Rosemary have low effective inhibitory concentrations against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans, while Clove and Perilla frutescens seeds exhibit high inhibitory activity against these microorganisms. Therefore, Rosemary, Clove, Prunella vulgaris, Perilla frutescens leaves, and Perilla frutescens seeds are selected for further research.

[0053] Table 1: Results of Antibacterial Activity Tests of Plant-Based Preservative Extract Mother Liquor and Chemically Synthetic Preservative Components

[0054]

[0055] Example 2

[0056] Since the extraction process can affect the extraction of preservatives from natural plants such as cloves, this embodiment provides an improvement to the extraction process of natural plant preservatives, including an improvement step on the extraction solvent used in the extraction process, an improvement step on the extraction solvent ratio, an improvement step on the extraction process parameters, an extraction step on the natural plant preservatives, and an enrichment step on the natural plant preservatives.

[0057] The improved extraction solvent steps include:

[0058] Dissolve 5 mL of octanoic acid in 10 mL of ethanol, then add 85 mL of water to induce the formation of amphiphilic aggregates. Vortex and centrifuge at 8000 rpm for 15 min to prepare a supramolecular extraction solvent. At the same time, prepare a 35% ethanol-water extraction solvent by mixing 35 mL of ethanol and 65 mL of water, and a 30% ethanol-water extraction solvent by mixing 30 mL of ethanol and 70 mL of water. Weigh 100 mL of ethanol as the ethanol extraction solvent and weigh 100 mL of deionized water as the water extraction solvent.

[0059] Five plant powders, namely clove, rosemary, selfheal, perilla leaf, and perilla seed, were weighed out at 0.2g each. Supramolecular extraction solvent, 35% ethanol-water extraction solvent, 30% ethanol-water extraction solvent, ethanol extraction solvent, and water extraction solvent were added respectively. After vortexing for 3 min, the mixture was sonicated for 20 min. The supernatant was obtained by centrifugation and filtration. The content of phenolic acid active ingredients in the supernatant was determined. The results are shown in Table 2.

[0060] The improved steps for the extraction solvent ratio include:

[0061] Weigh octanoic acid, ethanol, and deionized water in volume ratios of 5:10:85, 5:15:80, 5:20:75, 5:25:70, 5:30:65, 5:35:60, 5:40:55, and 5:45:50 respectively; weigh decanoic acid, ethanol, and deionized water in volume ratios of 5:20:75, 5:25:70, 5:30:65, 5:35:60, 5:40:55, and 5:45:50 respectively.

[0062] The weighed octanoic acid or decanoic acid was dissolved in ethanol, and then water was added to induce the formation of amphiphilic aggregates. The mixture was vortexed and centrifuged at 8000 rpm for 15 min to finally obtain a supramolecular extraction solvent that is divided into two phases, with the upper layer being the supramolecular solvent phase.

[0063] Weigh 0.2g of clove powder, add 10mL of supramolecular extraction solvent with different ratios as described above, vortex for 3min, then sonicate for 20min, add 4mL of ethanol, centrifuge and filter to obtain supernatant, and determine the content of phenolic acid active ingredients in the supernatant. The results are shown in Table 3.

[0064] The steps to improve the extraction process parameters include:

[0065] According to the volume ratio of 5:30:65, octanoic acid, ethanol and deionized water were weighed separately. The weighed octanoic acid was added to ethanol to dissolve, and then water was added and vortexed. The mixture was centrifuged at 8000 rpm for 15 min to obtain the supramolecular extraction solvent.

[0066] Weigh 0.2g of clove powder and add supramolecular extraction solvent at material-to-liquid ratios of 1g:20mL, 1g:30mL, 1g:40mL, 1g:50mL, and 1g:60mL, respectively. Vortex for 3 min, then sonicate for 15 min. Add 4mL of ethanol, centrifuge, and filter to obtain the supernatant. Determine the content of phenolic acid active ingredients in the supernatant. Weigh 0.2g of clove and add 10mL of supramolecular extraction solvent. Vortex for 1 min, 2 min, 3 min, 4 min, and 5 min, respectively, then sonicate for 15 min. Add 4mL of ethanol, centrifuge, and filter to obtain the supernatant. Determine the content of phenolic acid active ingredients in the supernatant. Weigh 0.2g of cloves, add 10mL of supramolecular extraction solvent, vortex for 3min, and then ultrasonically extract for 0min, 5min, 10min, 15min, 20min, 25min, and 30min respectively. Add 4mL of ethanol, centrifuge and filter to obtain the supernatant, and determine the content of phenolic acid active ingredients in the supernatant. Weigh 0.2g of cloves, add 10mL of supramolecular extraction solvent, vortex for 3min, and then ultrasonically extract for 15min at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ respectively. Add 4mL of ethanol, centrifuge and filter to obtain the supernatant, and determine the content of phenolic acid active ingredients in the supernatant. The results are as follows.Figure 3 As shown; from Figure 3 It can be seen that extraction parameters around a material-to-liquid ratio of 1g:40mL, ultrasonic extraction for 25min, and ultrasonic extraction at 60℃ can extract more phenolic acid active ingredients. Further, an orthogonal experiment was conducted around these extraction parameters, using a three-factor, three-level setup as shown in Table 4. The results are shown in Table 5. From the R values ​​in Table 5, it can be seen that the effects of ultrasonic time, ultrasonic temperature, and material-to-liquid ratio on the extraction of phenolic acid active ingredients gradually increase. Therefore, a material-to-liquid ratio of 1g:50mL, an ultrasonic temperature of 60℃, and an ultrasonic time of 25min were selected as the optimal extraction parameters for extracting preservative components from cloves.

[0067] The extraction steps for natural plant preservatives include:

[0068] After improving the extraction solvent, the extraction solvent ratio, and the extraction process parameters, 0.2g of clove powder, rosemary powder, selfheal powder, perilla leaf powder, and perilla seed powder were weighed out, and five portions of each plant powder were weighed out.

[0069] According to the volume ratio of 5:30:65, octanoic acid, ethanol and deionized water were weighed separately. The weighed octanoic acid was added to ethanol to dissolve, and then water was added and vortexed. The mixture was centrifuged at 8000 rpm for 15 min to obtain the supramolecular extraction solvent.

[0070] The weighed plant powder was added to supramolecular extraction solvent at a material-to-liquid ratio of 1g:50mL. After vortexing for 3min, it was ultrasonically extracted at 60℃ for 25min. 4mL of ethanol was added, and the mixture was centrifuged and filtered to obtain the supernatant. The content of phenolic acid active ingredients in the supernatant was determined, and the RSD was calculated. The results are shown in Table 6. As can be seen from Table 6, the improved extraction conditions using the above-mentioned extraction solvent, solvent ratio, and extraction process parameters can efficiently extract phenolic acid active ingredients from clove, rosemary, prunella vulgaris, perilla leaves, and perilla seeds. The extracted phenolic acid active ingredients are higher than those obtained under conventional extraction conditions.

[0071] The enrichment process for natural plant preservatives includes:

[0072] The supramolecular extraction solvent system composed of octanoic acid, ethanol, and water can be affected by adding excess deionized water to change the ratio of ethanol to water. When the ratio of ethanol to water exceeds the critical value, the stable state of the supramolecular extraction solvent is changed, octanoic acid separates from the ethanol and water and floats on the top of the solution, while the phenolic acid active ingredients are mainly present in the ethanol-water solution at the bottom, thus achieving the purpose of enriching the phenolic acid active ingredients.

[0073] Water was added to the supernatant to dilute it according to the volume ratio of supernatant to water of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. The dilution results are as follows.Figure 4 As shown, when the volume ratio of supernatant to water is 1:9, 2:8, 3:7, 4:6, 5:5, and 6:4, octanoic acid can be separated well, yielding an ethanol-water solution containing phenolic acid active ingredients. Subsequently, the supernatant is diluted with water at a volume ratio of 4:6, centrifuged, and the lower solution is collected. Then, it is concentrated by rotary evaporation and freeze-dried to obtain a natural plant preservative extract. The enrichment recovery rate results are shown in Table 7. It can be seen that the phenolic acid compounds in the preservative extract are almost completely recovered, with a recovery rate of over 99%.

[0074] Table 2: Content of phenolic acid active ingredients obtained by extraction with different extraction solvents

[0075]

[0076] Table 3: Content of phenolic acid active ingredients obtained by extraction with different ratios of extraction solvents

[0077]

[0078] Table 4: Parameter settings for orthogonal experiments

[0079]

[0080] Table 5: Content of phenolic acid active ingredients obtained by different extraction process parameters

[0081]

[0082] Table 6: Results of phenolic acid active ingredients extracted from different plants using improved extraction conditions

[0083]

[0084] Table 7: Recovery rate results of enriched natural plant preservative components

[0085]

[0086] Example 3

[0087] This embodiment provides a method for preparing natural plant preservative components, including an extraction solvent preparation step, an extraction step of natural plant preservative components, and an enrichment step of natural plant preservative components.

[0088] The steps for preparing the extraction solvent include:

[0089] Weigh out 500 mL of octanoic acid, ethanol, and deionized water in a volume ratio of 5:30:65. Dissolve the weighed octanoic acid in ethanol, then add water and vortex for 3 min. Centrifuge at 8000 rpm for 15 min to obtain the supramolecular extraction solvent. Prepare five portions in total.

[0090] The extraction steps for natural plant preservatives include:

[0091] Weigh out 10g each of rosemary powder, clove powder, prunella vulgaris powder, perilla leaf powder, and perilla seed powder.

[0092] The weighed rosemary powder, clove powder, prunella vulgaris powder, perilla leaf powder, and perilla seed powder were added to 500 mL of supramolecular extraction solvent at a material-to-liquid ratio of 1 g: 50 mL. After vortexing for 3 min, the mixture was ultrasonically extracted at 60 °C for 25 min. Then, 200 mL of ethanol was added at a volume ratio of 5:2. The mixture was centrifuged and filtered to obtain the supernatant of rosemary preservative extract, clove preservative extract, prunella vulgaris preservative extract, perilla leaf preservative extract, and perilla seed preservative extract.

[0093] The enrichment process for natural plant preservatives includes:

[0094] Water was added to the supernatant of rosemary preservative extract, clove preservative extract, selfheal preservative extract, perilla leaf preservative extract, and perilla seed preservative extract supernatant for dilution at a volume ratio of 4:6. The supernatant was centrifuged and the lower solution was collected. The solution was then concentrated by rotary evaporation and freeze-dried to obtain freeze-dried powders of natural plant preservative extracts such as rosemary preservative extract, clove preservative extract, selfheal preservative extract, perilla leaf preservative extract, and perilla seed preservative extract for later use.

[0095] Experimental Example 1

[0096] This experimental example tested the preservative performance of the rosemary preservative extract, clove preservative extract, prunella preservative extract, perilla leaf preservative extract, and perilla seed preservative extract provided in Example 3, after compounding them with chemically synthesized preservatives. The preservative performance test was a preliminary screening test for the synergistic preservative effect of natural plant preservatives and chemically synthesized preservatives, in order to explore whether natural plant preservatives have the ability to enhance preservative effect and can be used as natural preservative-enhancing functional ingredients.

[0097] In this experimental example, during the preservative performance test, the rosemary preservative extract was named preservative component A, the clove preservative extract was named preservative component B, the selfheal preservative extract was named preservative component C, the perilla leaf preservative extract was named preservative component D, the perilla seed preservative extract was named preservative component E, chlorphenesin was named preservative component P, phenoxyethanol was named preservative component G, and propylparaben was named preservative component F.

[0098] The preliminary screening test for preservative performance is based on the mechanism that natural plant preservatives or chemically synthesized preservatives at 1 / 2 of the minimum inhibitory concentration (MIC) are difficult to effectively inhibit bacteria, namely the principle of sub-inhibition concentration.

[0099] The corrosion resistance test process includes: preparing 1×10 5 CFU of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans bacterial suspensions were prepared. Based on the minimum inhibitory concentration (MIC) in the antibacterial ability results shown in Table 1, 1 / 4 of the MIC of rosemary preservative extract, clove preservative extract, Prunella vulgaris preservative extract, Perilla leaf preservative extract, or Perilla seed preservative extract, and 1 / 4 of the MIC of phenoxyethanol, propylparaben, or chlorphenesin were added to the Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans bacterial suspensions, respectively. After incubation at 37°C for 24 hours, bacterial growth was recorded. Growth was recorded as 1, and no growth was recorded as 0. The results are shown in Table 8.

[0100] As shown in Table 8, the preservative performance test results reveal that the composite preservatives obtained by combining different types of natural plant preservatives and chemically synthesized preservatives exhibit varying antibacterial effects against different microorganisms. For Staphylococcus aureus, both the natural plant preservatives and chemically synthesized preservatives showed a synergistic antibacterial effect, effectively inhibiting its growth. However, for Escherichia coli, neither the natural plant preservatives nor the chemically synthesized preservatives showed any synergistic antibacterial effect and could not effectively inhibit its growth. Conversely, for Pseudomonas aeruginosa and Candida albicans, some natural plant preservatives and chemically synthesized preservatives showed a synergistic antibacterial effect, effectively inhibiting their growth. This indicates that the combination of natural plant preservatives and chemically synthesized preservatives can exhibit good synergistic preservative effects, superior to either a single natural plant preservative or a single chemically synthesized preservative.

[0101] Table 8: Results of the study on the synergistic effect of combined use of natural preservative plant components and chemically synthesized preservative components.

[0102]

[0103] Experimental Example 2

[0104] This experimental example tested the preservative performance of the rosemary preservative extract, clove preservative extract, prunella preservative extract, perilla leaf preservative extract, and perilla seed preservative extract provided in Example 3, after combining them with chemically synthesized preservatives. The preservative performance test was conducted to test the synergistic effect of the combination of natural plant preservatives and chemically synthesized preservatives, in order to explore whether the combination of natural plant preservatives has a superior preservative synergistic effect.

[0105] In this experimental example, during the preservative performance test, the rosemary preservative extract was named preservative component A, the clove preservative extract was named preservative component B, the selfheal preservative extract was named preservative component C, the perilla leaf preservative extract was named preservative component D, the perilla seed preservative extract was named preservative component E, chlorphenesin was named preservative component P, phenoxyethanol was named preservative component G, and propylparaben was named preservative component F.

[0106] The test of the preservative synergy between the combination of natural plant preservative ingredients and chemically synthesized preservative ingredients is to avoid the antagonistic effect of different types of natural plant preservative extracts after combination, and to explore whether there is synergy among different types of natural plant preservative extracts to enhance the antibacterial effect. The sub-inhibition concentration principle is used to explore the synergistic effect of the combination of natural plant preservative ingredients on chemical preservatives.

[0107] The corrosion resistance test process includes: preparing 1×10 5 CFU of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans were prepared. Then, based on the minimum inhibitory concentration (MIC) in Table 1, phenoxyethanol, propylparaben, or chlorphenesin were added at 1 / 4 of the MIC. Additionally, two, three, four, or five preservative extracts were selected from the following: rosemary preservative extract, clove preservative extract, prunella vulgaris preservative extract, perilla leaf preservative extract, and perilla seed preservative extract. When two, three, four, or five preservative extracts were added, the concentrations were 1 / 8, 1 / 12, 1 / 16, or 1 / 20 of the respective MIC. After incubation at 37℃ for 24 hours, bacterial growth was recorded. Growth was recorded as "1", no growth as "0", and "-" indicated that the combination was not tested. The results are shown in Table 9.

[0108] As shown in Table 9, the anti-corrosion performance test results indicate that the combination of natural plant preservative ingredients can effectively inhibit the growth of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. This suggests that the combination of natural plant preservative ingredients, when combined with chemically synthesized preservative ingredients, can exhibit a better synergistic anti-corrosion and enhancement effect.

[0109] Table 9: Results of the study on the synergistic effect of natural plant preservatives combined with chemically synthesized preservatives.

[0110]

[0111] Experimental Example 3

[0112] This experimental example tested the preservative performance of the rosemary preservative extract, clove preservative extract, prunella preservative extract, perilla leaf preservative extract, and perilla seed preservative extract provided in Example 3, combined with chemically synthesized preservatives. The preservative performance test was conducted by combining different ratios of natural plant preservatives with chemically synthesized preservatives to explore whether adjusting the ratio of natural plant preservatives resulted in a superior preservative synergistic effect.

[0113] After screening for combinations of natural plant preservatives that may have synergistic antiseptic effects against three chemically synthesized preservatives, the antibacterial effects of three different bacterial species—Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans—were investigated using a mixture design to establish the synergistic antiseptic effects of five natural plant preservatives (rosemary, clove, selfheal, perilla leaf, and perilla seed) with three chemical preservatives (chlorphenesin, phenoxyethanol, and propylparaben). The minimum inhibitory concentration (MIC) and factor inhibitory concentration (FIC) were used as indicators to quantify the specific antiseptic performance of different preservative formulations.

[0114] The preservative performance testing process included: preparing preservative extract solutions of rosemary, clove, prunella vulgaris, perilla leaf, and perilla seed at a mass concentration of 12.5 mg / mL, respectively; then, according to the volume ratios and the mixing design tables in Tables 10-12, compounding was performed; subsequently, each compound was mixed with three chemically synthesized preservatives at a mass concentration of 12.5 mg / mL in a 1:1 ratio to obtain a stock solution; and determining the MIC of the stock solution: the stock solution was diluted by 1 / 2 times and mixed with a concentration of 1×10⁻⁶. 5 Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans were cultured in a 1:1 volume ratio using CFU. Wells without growth were recorded to determine the MIC of the stock solution, as well as the concentrations of each plant extract and chemically synthesized preservative in the stock solution. The FIC was calculated according to Equation 1. The results are shown in Tables 13-16.

[0115] Formula 1;

[0116] In Equation 1, MIC a MIC is the minimum inhibitory concentration (MIC) when preservative A acts alone. b C is the minimum inhibitory concentration (MIC) of preservative B when used alone. aTo achieve the same antibacterial effect in the compound, the concentration of preservative A (this concentration is usually ≤MIC) should be used. a ), C b To achieve the same antibacterial effect in the compound, the concentration of preservative B (this concentration is usually ≤MIC) should be used. b Furthermore, when FIC ≤ 0.5, a synergistic effect is determined; when 0.5 ≤ FIC ≤ 1, an additive effect is determined; when 1 ≤ FIC ≤ 4, an unrelated effect is determined; and when FIC > 4, an antagonistic effect is determined.

[0117] The preservative performance test results shown in Tables 13-16 indicate that different combinations of natural plant preservatives and chemically synthesized preservatives exhibited good antibacterial effects against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, with minimum inhibitory concentrations (MICs) ranging from 0.10 to 0.39 mg / mL or 0.78 mg / mL, and MICs below 0.05%. The FICI value of ~0.46 indicates that different ratios of natural plant preservatives combined with chemically synthesized preservatives exhibit synergistic antibacterial effects against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. Further, Table 13 shows that to achieve better antibacterial effects against Staphylococcus aureus, a compound of rosemary, clove, prunella vulgaris, perilla leaf, perilla seed preservative extracts, and phenoxyethanol in a mass ratio of 1:1:1:6:1:10 should be used, with a FICI value of 0.05. Table 14 shows that to achieve better antibacterial effects against Pseudomonas aeruginosa, a compound of rosemary, clove, perilla leaf preservative extracts, and phenoxyethanol in a mass ratio of approximately 1:1:4:6 should be used. The FICI of the compound was 0.10. Tables 15 and 16 show that to achieve better antibacterial effects against Candida albicans, a compound of clove, prunella vulgaris, perilla leaf, perilla seed preservative extract, and propylparaben in a mass ratio of approximately 1:5:1:1:8 should be used, with a FICI of 0.23. Overall, the FICI of the five natural plant preservatives (rosemary, clove, prunella vulgaris, perilla leaf, and perilla seed preservative extract) combined with the chemically synthesized preservatives can reach 0.05, demonstrating significant synergistic preservative effects. Therefore, subsequent tests will be conducted on the synergistic preservative effects of different ratios of the five natural plant preservatives combined with the chemically synthesized preservatives.

[0118] Table 10: Formulas of preservative extracts from rosemary, cloves, and perilla leaves

[0119]

[0120] Table 11: Formulas of preservative extracts from cloves, prunella vulgaris, perilla leaves, and perilla seeds

[0121]

[0122] Table 12: Formulas of preservative extracts from rosemary, cloves, prunella vulgaris, perilla leaves, and perilla seeds.

[0123]

[0124] Table 13: Results of the test on the antibacterial and synergistic effects of five natural preservative plant components and three chemically synthesized preservatives against Staphylococcus aureus

[0125]

[0126] Table 14: Results of the test on the antibacterial and synergistic effects of three natural preservative plant components and phenoxyethanol against Pseudomonas aeruginosa.

[0127]

[0128] Table 15: Results of the test on the antibacterial and synergistic effects of four natural preservative plant ingredients and propylparaben on Candida albicans.

[0129]

[0130] Table 16: Results of the test on the antibacterial and synergistic effects of five natural preservative plant components and chlorphenesin on Candida albicans

[0131]

[0132] Experiment Example 4

[0133] This experimental example tested the preservative performance of the rosemary preservative extract, clove preservative extract, prunella preservative extract, perilla leaf preservative extract, and perilla seed preservative extract provided in Example 3, combined with chemically synthesized preservatives. The preservative performance test was conducted by combining the five natural plant preservatives in different proportions with chemically synthesized preservatives to explore whether adjusting the proportions of the five natural plant preservatives resulted in a superior preservative synergistic effect.

[0134] To achieve synergistic antibacterial effects against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, it is necessary to comprehensively consider and balance the ratios of five plant extracts with three chemical preservatives. Based on the preservative synergistic effects test results for the three different bacterial species, and considering the MIC and FIC values ​​of different combinations of the five natural plant preservatives with synthetic chemical preservatives, it is clear that there is no single optimal ratio that simultaneously satisfies all individual optimal points. Therefore, several different combinations were designed, and the final ratio was selected to achieve a synergistic antibacterial effect against all three bacterial species, resulting in a balanced and effective overall effect.

[0135] The preservative performance testing process included: preparing preservative extract solutions of rosemary, clove, prunella vulgaris, perilla leaf, and perilla seed at a mass concentration of 12.5 mg / mL, and then compounding them according to the mixing design table in Table 17 based on the volume ratio. Subsequently, these solutions were compounded with three chemically synthesized preservatives at a mass concentration of 12.5 mg / mL in a 1:1 volume ratio to obtain composite preservative components. The minimum inhibitory concentration (MIC) was tested according to Experiment Example 3, and the FIC value was calculated. The results are shown in Tables 18 to 20.

[0136] As can be seen from the preservative performance test results shown in Tables 18-20, when the mass ratio of the preservative extracts of rosemary, clove, selfheal, perilla leaf, and perilla seed is 1:1:4:3:1, it exhibits better antibacterial ability against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans.

[0137] Table 17: Formulas of five natural plant preservative ingredients in different proportions

[0138]

[0139] Table 18: Results of antibacterial synergistic effect test after compounding five natural plant preservatives with chlorphenesin in different proportions.

[0140]

[0141] Table 19: Results of antibacterial synergistic effect test after compounding five natural plant preservatives with propylparaben in different proportions.

[0142]

[0143] Table 20: Results of antibacterial synergistic effects of five natural plant preservatives in different proportions combined with phenoxyethanol

[0144]

[0145] Experimental Example 5

[0146] This experimental example tested the preservative performance of the rosemary preservative extract, clove preservative extract, prunella preservative extract, perilla leaf preservative extract, and perilla seed preservative extract provided in Example 3, combined with chemically synthesized preservative components, to explore the actual preservative synergistic ability of natural plant preservative components and further clarify whether natural plant preservative components can be used as natural preservative synergistic compositions.

[0147] The corrosion resistance testing process includes:

[0148] Prepare an oil-in-water emulsion as the base, and according to the formulation shown in Table 21, add the natural preservative synergistic composition and chemically synthesized preservative ingredients to the oil-in-water emulsion base in different amounts as test samples. Based on the European Pharmacopoeia (EP5.1.3 standard for external preparations) preservative test, in Table 21, the natural preservative synergistic composition is obtained by compounding rosemary, clove, prunella vulgaris, perilla leaf and perilla seed preservative extracts in a mass ratio of 1:1:1:4:3.

[0149] Two 20g samples were prepared for each test group. Bacterial suspensions were inoculated, and information on the bacterial suspensions is shown in Table 22. The bacterial strains used and the inoculation method were as follows: First, 0.2mL of bacterial suspension was added to the first sample bottle. At 2, 7, 14, and 28 days of storage, TLSH (containing Tween, lecithin, saponins, and histidine) was added and streaked onto TSA medium (tryptone soy agar). For aseptic testing, both TSA and TSA medium containing the neutralizing agent were used to detect potential contamination. The streaked medium was then placed at 30±2℃. Bacterial growth was assessed after incubation at ℃ for 2 days. A second sample vial was inoculated with 0.2 mL of fungal suspension, and streaked onto Sabouraud dextrose agar containing neutralizing agents TLSH (Tween, lecithin, saponins, histidine) at 14 and 28 days. Streaking for sterility testing required the use of both culture media with and without neutralizing agents to identify potential contamination. The streaked media were incubated at 25±2℃ for 2 days, and fungal growth was assessed. The results are shown in Table 23. In Table 23, "-" indicates sterile growth (ca. <10). 2 Cfu / ml); "+" indicates slight growth (ca.10). 2 Cfu / ml); "++" indicates moderate growth (ca.10). 3 Cfu / ml); "++" indicates severe growth (ca.10). 4 Cfu / ml); "++++" indicates severe growth (ca.10). 5 (Cfu / ml), “NG” indicates non-compliance, “A” indicates EP 5.1.3 topical preparation A standard, and “B” indicates EP 5.1.3 topical preparation B standard.

[0150] As shown in Table 23, the performance test results indicate that when the full amount of chemically synthesized preservatives in test sample ① is used alone, its bacterial control meets the high standard of EP 5.1.3 external use Grade A, but its fungal control only meets the basic Grade B standard, making it difficult to effectively inhibit microorganisms. In test sample ②, where the amount of chemically synthesized preservatives is halved, neither bacterial nor fungal control meets the requirements. This demonstrates that reducing the amount of chemically synthesized preservatives directly leads to the complete loss of the preservative system's protective ability against bacteria, fungi, and other microorganisms. Test sample ③, using a combination of plant-based preservatives derived from rosemary, cloves, prunella vulgaris, perilla leaves, and perilla seeds, cannot meet the high standard of EP 5.1.3 external use Grade A for bacterial and fungal control. However, when this plant-based preservative combination is combined with half the amount of chemically synthesized preservatives, bacterial and fungal control meets EP 5.1.3. The high standard of Grade A for external use indicates that when the plant-based preservative composition is combined with half of the chemically synthesized preservatives, the synergistic effect of the plant composition and half of the chemically synthesized preservatives compensates for the protective gap caused by the halved dosage of chemical preservatives. This not only restores the protective ability of the originally completely ineffective preservative system, but also exceeds that of the full-dosage chemical control group, demonstrating a positive synergistic effect of preservative enhancement, rather than a simple additive effect.

[0151] Table 21: Five sets of test samples

[0152]

[0153] Table 22: Inoculated bacterial suspension

[0154]

[0155] Table 23: Corrosion Resistance Results

[0156]

[0157] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A natural preservative-enhancing composition, characterized in that, It includes at least two of the following: rosemary preservative extract, clove preservative extract, prunella preservative extract, perilla leaf preservative extract, and perilla seed preservative extract.

2. The natural preservative-enhancing composition according to claim 1, characterized in that... The mass ratio of any two of the following preservative extracts is 1~6:1~6:1~6.

3. A method for preparing a natural preservative-enhancing composition, characterized in that, A natural preservative-enhancing composition according to any one of claims 1-2 can be prepared by the following steps: The fatty acids, ethanol and water are mixed evenly to obtain a supramolecular extraction solvent. At least two natural plants selected from rosemary, clove, selfheal, perilla leaves, and perilla seeds are added to a supramolecular extraction solvent and vortexed until homogeneous. The mixture is then subjected to ultrasonic extraction, centrifugation, concentration, and drying to obtain a natural preservative and synergistic composition.

4. The method for preparing a natural preservative-enhancing composition according to claim 3, characterized in that, In the supramolecular extraction solvent, the fatty acids are selected from medium-chain fatty acids, and the volume ratio of fatty acids, ethanol and water is 5:10~45:50~85.

5. The method for preparing a natural preservative-enhancing composition according to claim 3, characterized in that, The ratio of natural plant material to supramolecular extraction solvent used is 1g:30~50mL, the vortexing time is 1~5min, the ultrasonic time is no more than 30min, and the temperature is 30~80℃.

6. The method for preparing a natural preservative-enhancing composition according to claim 5, characterized in that, The centrifugation step following ultrasound includes: adding ethanol to an ultrasound extraction solution containing a natural preservative-enhancing composition, centrifuging to obtain the supernatant, adding water to the supernatant, centrifuging to obtain the lower solution.

7. The application of the natural preservative-enhancing composition according to any one of claims 1-4 in the preparation of an enhanced composite preservative, characterized in that, The application process includes: using a combination of natural preservative-enhancing composition and chemical preservative.

8. A synergistic composite preservative, characterized in that, It includes chemically synthesized preservative components and a natural preservative-enhancing composition as described in any one of claims 1-2.

9. The application of the synergistic composite preservative as described in claim 8 in the preparation of skin care products.

10. An oil-in-water emulsion, characterized in that, Includes the synergistic composite preservative as described in claim 8.

Images