Composition as well as preparation method and application thereof in preparation of product with acne removing effect

Abstract

The invention relates to the technical field of acne removal, in particular to a composition, a preparation method of the composition and application of the composition in preparation of a product with an acne removal effect. According to the composition composed of the hinokii extract and the sophorolipid, the two components in the composition have a synergistic effect in the aspects of resisting bacteria and removing acnes, and the raw materials in the composition are low in cost and easy to obtain; the formed composition is an active matter which can regulate skin microbial flora to inhibit excessive breeding of propionibacterium acnes as a main target, and has the characteristics of remarkable acne removal effect, good recurrence resistance effect, stability and durability.

Description

Technical Field

[0001] This invention relates to the field of acne treatment technology, and more particularly to compositions thereof, methods of preparation thereof, and their application in the preparation of products with acne-removing effects. Background Technology

[0002] Acne is a common skin condition primarily caused by excessive sebum secretion, overgrowth of Propionibacterium acnes, hyperkeratosis of the hair follicle opening, and the release of inflammatory factors leading to localized redness and swelling. The excessive proliferation of Propionibacterium acnes causes hyperkeratosis of the hair follicle, resulting in a closed follicle opening and the creation of an anaerobic environment. This allows the bacteria to further proliferate, releasing irritants such as lipase, lysozyme, and protease, which damage the inner wall cells of the hair follicle. This triggers an immune response, further amplifying the inflammatory cascade, leading to vasodilation and symptoms such as redness and swelling.

[0003] Current acne treatments primarily focus on acid peels or anti-inflammatory methods. Acid peels utilize acidic substances (such as salicylic acid and glycolic acid) to promote the exfoliation of the skin's stratum corneum, helping to unclog pores and reduce blackhead formation, thereby treating acne. However, acid peels can cause breakouts during treatment, leading to short-term discomfort. Furthermore, repeated acid peels may damage the skin's barrier function, making the skin more sensitive and potentially developing into sensitive skin. Anti-inflammatory treatments use medications or ingredients with anti-inflammatory properties (such as antibiotics and anti-inflammatory drugs) to reduce redness and inflammation caused by acne, making pimples appear smaller and creating a false impression of quick results. However, this treatment method does not fundamentally solve the acne problem, and consumers often experience recurring breakouts, causing long-term skin health problems.

[0004] Excessive sebum secretion is caused by individual constitution and hormonal imbalances, making it difficult for topical cosmetics to address. Therefore, inhibiting Propionibacterium acnes is crucial. However, currently available antibacterial ingredients have several shortcomings. Firstly, some effective antibacterial ingredients are expensive, increasing treatment costs and limiting their widespread application. Secondly, some antibacterial ingredients lack stability and are easily affected by environmental factors during storage and use, affecting the sustainability and reliability of treatment effects. Furthermore, the antibacterial effects of existing ingredients are limited, often only inhibiting the growth of Propionibacterium acnes to a certain extent, failing to completely eradicate it, leading to recurrent acne and hindering long-term stable treatment results.

[0005] For example, piocin, a quasi-drug approved ingredient by the Japanese Ministry of Pharmaceuticals and Healthcare products, is highly recognized and widely used in cosmetics. It has in vitro data showing that even very low dosages can inhibit bacterial growth. However, this ingredient still has the following problems: ① High cost, with a purchase price of 500,000-1,000,000 RMB / kg, and a cost of 25-50 RMB / kg for formulas using it alone. ② Poor stability, easily yellowing under light and heat. ③ Requires pre-processing with polyols at elevated temperatures, adding extra time and labor. ④ Difficult to preserve after pre-processing, requiring refrigeration. ⑤ The widespread use of piocin has not solved the problem of recurrent acne breakouts in acne patients, as Propionibacterium acnes continues to dominate the bacterial flora.

[0006] Given the drawbacks of existing antibacterial ingredients, such as high price, poor stability, and limited efficacy, there is still a need to find suitable active ingredients for acne treatment. Combining existing substances is a common approach, but indiscriminate combination cannot produce synergistic effects and is unlikely to improve the overall acne treatment effect. Ideally, synergistic effects should enhance the inhibition of Propionibacterium acnes, improve the skin microenvironment, promote skin barrier repair, reduce inflammatory responses, and lower skin sensitivity. Therefore, screening for combinations of active ingredients that can produce synergistic effects is a pressing technical challenge that needs to be addressed. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a composition and a method for preparing the same, and its application in the preparation of products with acne-removing effects.

[0008] The composition provided by this invention consists of cypress extract and sophorolipid.

[0009] This invention screens antibacterial components and found in the experimental group that when cypress extract is used as an antibacterial agent and combined with sophorolipids that can improve the skin barrier, it can produce a synergistic effect in multiple aspects such as antibacterial and anti-inflammatory effects, and the effect is better than the combination of other components.

[0010] In some embodiments, the mass ratio of the cypress extract to sophorolipid is (0.5-1.5):(1-5).

[0011] As a feasible example, the mass ratio of the cypress extract to sophorolipid is (0.5-1):(1-3), or (0.5-1):(1-5), or (0.5-1):(3-5), or (0.5-1.5):(1-3), or (0.5-1.5):(1-5), or (0.5-1.5):(3-5), or (1-1.5):(1-3), or (1-1.5):(1-5), or (1-1.5):(3-5).

[0012] Alternatively, as a feasible example, the mass ratio of the cypress extract to sophorolipid is 0.5:(1-3), or 0.5:(1-5), or 0.5:(3-5), or 1:(1-3), or 1:(1-5), or 1:(3-5), or 1.5:(1-3), or 1.5:(1-5), or 1.5:(3-5).

[0013] Alternatively, as a feasible example, the mass ratio of the cypress extract to sophorolipid is (0.5–1):1, or (0.5–1):3, or (0.5–1):5, or (0.5–1.5):1, or (0.5–1.5):3, or (0.5–1.5):5, or (1–1.5):1, or (1–1.5):3, or (1–1.5):5.

[0014] In a specific embodiment, the mass ratio of the cypress extract to sophorolipid is 1.5:(1-5). More specifically, the mass ratio of the cypress extract to sophorolipid is 0.5:1, or 1:1, or 1:3, or 1.5:5, or 0.5:5, or 1.5:1.

[0015] Taking the mass ratio of cypress extract and sophorolipid as 1:1 as an example, the two have a synergistic effect in antibacterial properties, as verified by the FIC index method and the Bliss independent model.

[0016] Furthermore, the present invention also provides a nano-suspension prepared from excipients and the composition as described above.

[0017] This invention screened excipients and preparation methods for nanosuspensions. Results showed that different preparation methods affected the appearance and stability of the formulation. Nanosuspensions prepared under suitable conditions had an average particle size not exceeding 25 nm, exhibited good resistance to high and low temperatures and light exposure, and demonstrated good stability. Preferably, the excipients in the nanosuspension were 2-methyl-2,4-pentanediol and / or PEG-40 hydrogenated castor oil.

[0018] In some embodiments, the nanosuspension is prepared from cypress extract, sophorolipid, PEG-40 hydrogenated castor oil and 2-methyl-2,4-pentanediol, wherein the mass ratio of cypress extract, sophorolipid, PEG-40 hydrogenated castor oil and 2-methyl-2,4-pentanediol is (0.5-1.5):(1-5):(1-5):(5-10).

[0019] In a specific embodiment, the mass ratio of the cypress extract, sophorolipid, PEG-40 hydrogenated castor oil, and 2-methyl-2,4-pentanediol is 0.5:1:1:5, or 1:1:5:5, or 1:3:3:5, or 1.5:5:2:8, or 1.5:1:2:10, or 1:1:5:5.

[0020] In this invention, the method for preparing the nano-suspension includes:

[0021] After mixing cypress extract and 2-methyl-2,4-pentanediol, sophorolipid was added, and PEG-40 hydrogenated castor oil was added while stirring. The mixture was then homogenized by ultrasound and high pressure to obtain the nano-suspension.

[0022] In some embodiments, the mixing conditions for the cypress extract and 2-methyl-2,4-pentanediol include heating to 60°C-70°C with stirring at 600-800 rpm. Preferably, the mixing conditions include heating to 60°C with stirring at 700 rpm.

[0023] In some embodiments, the temperature is lowered to 30–50°C before adding sophorolipids; preferably, the temperature at which sophorolipids are added is 40°C. Preferably, after adding the sophorolipids, stirring is maintained at 600–800 rpm for 5 minutes; more preferably, stirring is maintained at 700 rpm for 5 minutes. Preferably, the sophorolipids are added completely within 5 minutes, and the addition rate is 5 drops / second.

[0024] In some embodiments, after adding PEG-40 hydrogenated castor oil, the continued stirring includes stirring at 1000 rpm for 1-2 minutes, the ultrasonication conditions include ultrasonication at 600 W for 5-10 minutes, and the high-pressure homogenization conditions include homogenization at 1000-1200 Pa for 3-5 times. Preferably, the continued stirring includes stirring at 1000 rpm for 1 minute, the ultrasonication conditions include ultrasonication at 600 W for 7 minutes, and the high-pressure homogenization conditions include homogenization at 1000 Pa for 5 times.

[0025] The preparation method provided by this invention is simple and easy to implement. The nano-suspension prepared by this method has uniform particle size, transparent color, stable properties, and good preservation of the physiological activities of cypress extract and sophorolipid.

[0026] The present invention also provides the application of the composition, the nanosuspension, or the nanosuspension prepared by the preparation method in the preparation of anti-acne products.

[0027] In this invention, the anti-acne treatment includes antibacterial, anti-inflammatory, oil-controlling, and / or acne-improving effects.

[0028] In some embodiments, the antibacterial activity includes activity against *Propionibacterium acnes*. In specific embodiments, the antibacterial activity includes inhibiting the growth of *Propionibacterium acnes* and / or inhibiting the formation of *Propionibacterium acnes* biofilms.

[0029] In some embodiments, the anti-inflammatory effect includes inhibiting the levels of inflammatory factors. These inflammatory factors include TNF-α and / or IL-6.

[0030] In some embodiments, oil control refers to inhibiting the secretion of oil by the sebaceous glands.

[0031] In some embodiments, the improvement of acne symptoms includes improving redness and swelling, improving pain, and / or reducing the number of acne lesions.

[0032] Furthermore, the present invention also provides an anti-acne product comprising the composition as described above, the nano-suspension as described above, or the nano-suspension prepared by the preparation method as described above.

[0033] In this invention, the anti-acne product includes, but is not limited to, at least one of ointments, creams, gels, lotions, tinctures, solutions, masks, patches, sprays, or foams. In some embodiments, the anti-acne product comprises a nano-suspension as described above. Preferably, the mass fraction of the nano-suspension in the anti-acne product is 0.5 wt% to 1.5 wt%. For example, it is 0.5 wt%, 0.8 wt%, 1.0 wt%, 1.2 wt%, or 1.5 wt%.

[0034] In a specific embodiment, the anti-acne product comprises 0.5wt% to 1.5wt% of the aforementioned nano-suspension and blank base material.

[0035] In this invention, the excipients in the blank base material include, but are not limited to, at least one of the following: humectant, chelating agent, thickener, solvent, surfactant, neutralizer, preservative or fragrance.

[0036] As a feasible example, the moisturizer includes, but is not limited to, at least one of butylene glycol, 1,3-propanediol, 2-methyl-2,4-pentanediol, hexanediol, glycerin, hyaluronic acid, sorbitol, or panthenol.

[0037] As a feasible example, the chelating agent includes, but is not limited to, at least one of EDTA, disodium EDTA, tetrasodium EDTA, citric acid, tartaric acid, and sodium phytate.

[0038] As a feasible example, the thickener includes, but is not limited to, at least one of xanthan gum, carbomer 981, sodium carboxymethyl cellulose (CMC-Na), hydroxyethyl cellulose (HEC), guar gum, sodium polyacrylate, and carrageenan.

[0039] As a feasible example, the solvent includes, but is not limited to, at least one of water, ethanol, isopropanol, propylene glycol, and polysorbate-80.

[0040] As a feasible example, the surfactant includes, but is not limited to, at least one of PEG-40 hydrogenated castor oil, sodium lauryl ether sulfate (SLES), and Tween.

[0041] As a feasible example, the neutralizing agent includes, but is not limited to, at least one of triethanolamine, aminomethylpropanol (AMP), and sodium hydroxide.

[0042] As a feasible example, the preservatives include, but are not limited to, at least one of phenoxyethanol, methylparaben, chlorphenesin, and sodium dehydroacetate.

[0043] In addition, the product may also include other skin-care active substances, including but not limited to at least one of dipotassium glycyrrhizate, salicylic acid, retinol, niacinamide, tea tree oil, and allantoin.

[0044] Preferably, the blank base material comprises the following components by mass fraction: 4% butanediol, 2% 1,3-propanediol, 0.03% EDTA, 1% preservative, 0.5% 2-methyl-2,4-pentanediol, 0.18% xanthan gum, 14.85% purified water, 0.16% carbomer 981, 0.1% purified water, 0.1% neutralizer, 0.1% 75% dipotassium glycyrrhizate, 0.04% PEG-40 hydrogenated castor oil, 0.004% fragrance, with the balance being water.

[0045] For example, the fragrance is selected from at least one of menthol, lavender essential oil, rose essence, and lemon essence.

[0046] Furthermore, the present invention also provides a method for preparing the product as described above, comprising: mixing the blank base material and the nano suspension, stirring at 300-700 rpm for 1-10 min, to obtain the product. Preferably, stirring at 500 rpm for 5 min.

[0047] Furthermore, the present invention also provides a method for treating acne, which includes applying the product as described above to the skin surface.

[0048] As a feasible example, the application method includes, but is not limited to, at least one of the following: spot application, wet compress, massage, full-face application, and localized thick application.

[0049] This invention provides a composition consisting of cypress extract and sophorolipid. The two components in this composition have a synergistic effect in antibacterial and acne-removing properties. Furthermore, the raw materials in this composition are inexpensive and readily available. The resulting composition is an active ingredient that primarily targets the regulation of the skin's microbiome to inhibit the excessive growth of Propionibacterium acnes. It exhibits significant acne-removing efficacy, good anti-recurrence effect, and stable and long-lasting properties. Detailed Implementation

[0050] This invention provides a composition, its preparation method, and its application in the preparation of products with acne-removing effects. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0051] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0052] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0053] The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, or B exists alone. A and B can be singular or plural.

[0054] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0055] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0056] The test materials used in this invention are all common commercially available products, and can be purchased on the market. Among them:

[0057] The Chamaecyparis obtusa var. formosana (Hayata) Rehd. extract is obtained by steam distillation from the trunk of the Chamaecyparis obtusa var. formosana (Hayata) Rehd., a plant belonging to the Cupressaceae family. The main component is physalisol. Physalisol can also be obtained through chemical synthesis. Sophorolipids were purchased from Shenzhen Viki Technology, PEG-40 hydrogenated castor oil from BASF, and 2-methyl-2,4-pentanediol from Solvay; alternatives can be found at suppliers of similar chemicals.

[0058] Compared with the prior art, the composition and nanosuspension provided by the present invention have at least one of the following beneficial effects:

[0059] The ingredients are mild and safe, using pure natural cypress as the key active ingredient. It is green, natural, safe and mild. Sophorolipid is prepared by bio-fermentation (prepared by the supplier, Shenzhen Weiqi), which is low cost, green and pollution-free. In the daily chemical industry, cypress extract is mostly used for its preservative effect, but compared with chemical antibacterial agents, it has problems such as high dosage, high cost and insufficient preservative effect, which greatly limits its application. This invention is the first attempt to use the combination of cypress extract and sophorolipid in acne care and found that it has a synergistic antibacterial effect.

[0060] With a unique efficacy design, this product addresses the pain point of competing products on the market that only solve current acne problems with acid peeling and strong anti-inflammatory effects, without providing long-term and sustained results. Its efficacy mechanism focuses on inhibiting and balancing bacteria at the source, while anti-inflammatory conditioning and skin care, oil control and softening of the stratum corneum are used as auxiliary measures. It has the effect of continuous and gentle regulation, and prevents recurrence.

[0061] The innovative features of the antibacterial synergistic effect are prominent. It uses the less common plant cypress as the key raw material source, enriches and purifies the target components, and uses bio-fermented sophorolipids in synergy. It is the first time that the two can synergistically inhibit the biofilm formation of Propionibacterium acnes, and have a synergistic effect on the inhibition of bacteria.

[0062] This multi-dimensional acne-fighting and antibacterial combination demonstrates efficacy in three dimensions: oil control, anti-inflammation, and antibacterial properties. Specifically, in terms of anti-inflammation, both ingredients synergistically inhibit different inflammatory factors, targeting both early-stage and late-stage inflammatory activation. Sophorolipids inhibit the early-stage inflammatory activator TNF-α, while cypress extract specifically inhibits IL-6, the skin redness and swelling factor caused by late-stage inflammatory amplification, effectively blocking the amplification of inflammation. Combined with oil control and antibacterial properties, it addresses acne from multiple angles. Furthermore, due to its synergistic effect, it also provides anti-inflammatory, repairing, and redness-reducing benefits, showing good results in post-acne repair.

[0063] The innovative formulation process allows for the preparation of nano-suspensions with an average particle size as low as 14nm, which facilitates rapid penetration into the skin follicles, effectively targeting the target microbial flora and further enhancing the acne treatment effect.

[0064] It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments:

[0065] Example 1: Screening of Antibacterial Agents

[0066] The antibacterial solution was prepared into a 10% dispersion and then serially diluted with phosphate-buffered saline (PBS) to create test solutions of different concentrations. These test solutions were then mixed with double-concentration MH agar medium and poured into Petri dishes to prepare agar plates containing different concentrations of antibacterial (bacteriostatic) agents. Simultaneously, a pipette was used to pipette plates containing approximately 10% bacterial count. 7 A bacterial suspension of CFU / ml was inoculated onto these plates, with approximately 10 CFU / ml per plate. 4 CFU / ml. Incubate the plates upside down in a 35°C incubator for 18-24 hours, then observe the growth of *Propionibacterium acnes*. The minimum concentration of antibacterial agent required to completely inhibit colonies is the MIC.

[0067] Table 1 MIC values ​​of each component

[0068]

[0069] Note: "+" indicates the presence of Propionibacterium acnes.

[0070] "-" indicates that Propionibacterium acnes is not growing.

[0071] As can be seen from Table 1, the antibacterial effect of sophorolipids alone is limited, while the cypress extract has the lowest MIC and the best inhibitory ability against Propionibacterium acnes. When cypress extract and sophorolipids are used in combination at a ratio of 1:1, the antibacterial ability can be improved.

[0072] The FIC index method is a method that calculates the FIC index by measuring the minimum inhibitory concentration (MIC) of two or more antimicrobial drugs used in combination and comparing it with the MIC when used alone. This allows the determination of whether there is a synergistic, additive, irrelevant, or antagonistic effect between the drugs.

[0073]

[0074] Synergy: FIC index ≤ 0.5 indicates that the antibacterial activity when the two drugs are used in combination is significantly greater than the sum of the activities when used alone. Additive effect: 0.5 < FIC index ≤ 1 indicates that the antibacterial activity when the two drugs are used in combination is equal to the sum of the activities when used alone. Irrelevant effect: 1 < FIC index ≤ 2 indicates that the antibacterial activity when the two drugs are used in combination is equivalent to the activity when used alone, without obvious interaction. Antagonistic effect: FIC index > 2 indicates that the antibacterial activity when the two drugs are used in combination is significantly lower than the activity when used alone.

[0075] The MIC of the cypress extract is 0.005%, and the MIC of sophorolipid is 0.1%

[0076] Cypress extract + sophorolipid = 0.00375%, cypress extract:sophorolipid = 1:1. Therefore, when used in combination, the MIC of the cypress extract is 0.001875%, and the MIC of sophorolipid is 0.001875%;

[0077] FIC = 0.001875% / 0.005% + 0.001875% / 0.1% = 0.375 + 0.01875 = 0.39375 < 0.5

[0078] It can be seen that, compared with the groups using the cypress extract and sophorolipid alone, a synergistic effect is produced when the two are used in combination.

[0079] Example 2

[0080] Table 2 Components and their contents in the examples and the control group

[0081] sample Cypress extract Sophorolipids PEG-40 hydrogenated castor oil 2-Methyl-2,4-pentanediol Experimental group 1 0.5 1 1 5 Experimental group 2 1 1 5 5 Experimental group 3 1 3 3 5 Experimental group 4 1.5 5 5 10 Experimental group 5 0.5 5 2 8 Experimental group 6 1.5 1 2 10 Experimental group 7 1 1 5 5 Control group 1 / / 5 / Control group 2 / / / 5 Control group 3 / / 5 5 Control group 4 / 1 5 5 Control group 5 1 / 5 5 Control group 6 1 1 0 5 Control group 7 1 1 5 0 control group 8 0.5 6 5 5 Control group 9 1.5 0.5 5 5 control group 10 1 1 5 5 Control group 11 1 1 5 5 control group 12 1 1 5 5

[0082] 2.1 The preparation method of the dosage forms (including Experimental Groups 1-7 and Control Groups 1-9) includes:

[0083] S1. Accurately weigh the cypress extract and 2-methyl-2,4-pentanediol components, mix them, heat to 60°C under stirring at 700 rpm, and continuously stir until at least a small amount of white particles are suspended;

[0084] S2. Lower the temperature to 40°C, slowly add the sophorolipid component at 5 drops / s, continue stirring at the same rotation speed for 5 min (added within 5 min), disperse evenly, and keep the white particles suspended;

[0085] S3. Quickly add PEG-40 hydrogenated castor oil, stir and disperse at 1000 rpm for 1 min, ultrasonic at 600 W for 7 min, add it into a high-pressure homogenizer and homogenize 5 times at a pressure of 1000 pa, and discharge to obtain a clear and transparent 10-50 nm nano-suspension sample.

[0086] 2.2 The steps for dissolving by ordinary stirring and heating (control group 10) include:

[0087] Each component was accurately weighed and mixed. The mixture was heated to 80°C with stirring at 800 rpm and kept at that temperature for 30 minutes. At least a small amount of white particles were suspended in the mixture. The mixture was then cooled to obtain control group 10.

[0088] 2.3 The steps of ultrasonic dissolution + centrifugal dispersion and homogenization (control group 11) include:

[0089] S1. Accurately weigh each component, mix them, heat to 80°C while stirring at 800 rpm, and keep stirring and warm for 10 minutes until at least a small amount of white particles are suspended.

[0090] S2, stir and disperse at 1000 rpm for 1 min, sonicate at 600W for 7 min to obtain a small amount of white particles in suspension, and cool to obtain control group 11.

[0091] 2.4 The steps of ordinary dissolution + centrifugal dispersion and homogenization (control group 12) include:

[0092] S1. Accurately weigh each component, mix them, heat to 80°C while stirring at 800 rpm, and keep stirring and warm for 10 minutes until at least a small amount of white particles are suspended.

[0093] S2. Immediately homogenize at high speed (7000 rpm for 6 min), homogenize twice to obtain a small amount of white particles in suspension, and cool to obtain control group 12.

[0094] Performance verification

[0095] I. Physicochemical Test Results

[0096] Each experimental group and control group listed in Table 2 was prepared into a 1% solution by adding pure water. The appearance, odor, pH, conductivity, particle size, and stability were then tested. In addition, a control group (designated as control group 13) was set up, in which piocin was dissolved in 1,3-propanediol (0.001% by mass) for the experiment. The results are shown in the table below:

[0097] Table 3

[0098]

[0099]

[0100] Physicochemical stability analysis: The results show that the experimental group was transparent in appearance, light yellow in color, and the particle size was less than 25 nm. All stability tests were passed.

[0101] Control groups 1-4 were transparent and had good stability, but no particle size D50 was detected. Control group 5 differed from control group 4 in that it contained cypress extract, which had poor solubility and produced a white emulsion that could not be effectively dissolved. Stability tests showed varying degrees of cypress extract precipitation, which was more pronounced at low temperatures. This indicates that the synergistic solubilization of the components in the composition is crucial and can effectively ensure the solubility of the active ingredients.

[0102] The control groups 8 and 9 were light yellow, transparent liquids. Their stability and particle size results were similar to those of the experimental groups, indicating that this method can effectively solubilize the liquids.

[0103] The control group (10-12), prepared using a different process, appeared as a milky white, turbid liquid with large particle sizes (D50 of 3-5 micrometers), and stability tests revealed the precipitation of pale yellow particles. This indicates that the formulation process and parameters are crucial; without a suitable formulation process, stable samples cannot be prepared.

[0104] The control group 13 was a sample of piocin, which was found to be completely soluble in polyols. It showed poor stability and discoloration due to heat and light exposure.

[0105] II. Comparison of MIC for inhibiting Propionibacterium acnes and inhibition rate of Propionibacterium acnes biofilm formation

[0106] 2.1 Bacterial Information: Propionibacterium acnes ATCC 11827, Generation 3

[0107] 2.2 MIC Experiment: Samples from the experimental and control groups in Table 1, along with piocin, were prepared into 10% dispersions. These dispersions were then serially diluted with phosphate-buffered saline (PBS) to create test solutions of varying concentrations. These test solutions were then mixed with double-concentration MH agar medium and poured into Petri dishes to prepare agar plates containing different concentrations of antibacterial (bacteriostatic) agents. Simultaneously, a pipette was used to pipette samples containing approximately 10 μL of bacteria. 7 A bacterial suspension of CFU / ml was inoculated onto these plates, with approximately 10 CFU / ml per plate. 4 CFU. Incubate the plates upside down in a 35°C incubator for 18-24 hours, then observe the growth of Propionibacterium acnes. The minimum concentration of antibacterial agent required to completely inhibit colonies is the MIC.

[0108] 2.3 Biomembrane formation inhibition experiment:

[0109] In a 96-well plate, add the test solution and culture medium (20 μL), then add 180 μL of Propionibacterium acnes bacterial suspension (1 × 10⁻⁶). 8After shaking for 1 minute, the plate was incubated under culture conditions. After 24 hours, the supernatant was removed, and PBS (200 μL) was added to wash the biofilm formed at the bottom of the 96-well plate to remove airborne bacteria. The plate was then dried at room temperature. 0.1% crystal violet solution (200 μL) was added to each well, and the plate was incubated with shaking for 20 minutes. The plate was then washed three times with PBS (200 μL) and dried at room temperature. 99% ethanol (200 μL) was added to each well, and the plate was incubated with shaking for 1 hour. The supernatant (100 μL) was transferred to a new 96-well plate, and the absorbance (OD) was measured at 595 nm. 595 ).

[0110] Average inhibition rate = (1 - sample group OD) 595 / Control group OD 595 )*100%

[0111] Table 4

[0112]

[0113]

[0114] 2.3.1 Analysis of MIC Experimental Test Results

[0115] As shown in Table 4, the MIC values ​​of experimental groups 1-6 were all below 25 μg / mL, indicating significant antibacterial effects. Control groups 1-3 showed no antibacterial effect from solubilizers and solvents. Control groups 4-5 confirmed that the key antibacterial component was cypress extract, with a MIC of 37.5, and sophorolipids, with a MIC of 500. In control group 6, the MIC of cypress extract and sophorolipids was significantly reduced to 50, indicating a synergistic antibacterial effect, possibly related to the surfactant properties of sophorolipids. In control group 7, the addition of PEG-40 hydrogenated castor oil to the combination of cypress extract and sophorolipids reduced the MIC to 25, further confirming that PEG-40 hydrogenated castor oil had a synergistic effect on the antibacterial effects of cypress extract and sophorolipids.

[0116] According to the FIC index method, the MIC of cypress extract was 50 μg / ml, and the MIC of sophorolipid was 1000 μg / ml. The FIC values ​​of each experimental group and control group were calculated, and the results are as follows:

[0117] Table 5

[0118]

[0119] The results showed that the test samples in experimental groups 1 to 6 of this invention produced a synergistic effect, indicating that the ratio of the two in the test samples of these experimental groups was appropriate.

[0120] 2.3.2 Analysis of Experimental Results on Biomembrane Inhibition Rate

[0121] Table 4 shows that the experimental group achieved a biofilm inhibition rate of over 30%, indicating a significant effect. Control groups 1-3 confirmed that neither the solubilizer nor the solvent had any effect on inhibiting biofilm synthesis. Control groups 4-5 confirmed that the key component inhibiting biofilm formation was sophorolipid, with an inhibition rate of 14.21%, which may be related to the surfactant effect of sophorolipid. In control group 6, the inhibition rate significantly increased to 27.25% when combined with cypress extract, indicating that the two have a synergistic effect on inhibiting biofilm formation. The mechanism may be that sophorolipid inhibits biofilm formation, which helps cypress extract enter the bacterial cells, affecting bacterial metabolism and further influencing biofilm formation.

[0122] Using PEG-40 hydrogenated castor oil and 2-methyl-2,4-pentanediol as the matrix, and based on the Bliss independent model, the biofilm formation inhibition rate in experimental group 2 was considered the effect of the two-drug combination, while the biofilm formation inhibition rates in control groups 4 and 5 were considered the effects of the two single drugs, respectively. The CI value was calculated using the following formula:

[0123]

[0124] CI=33.57%÷(14.21%+0.96%-14.21%×0.96%)=2.23

[0125] CI>1 indicates synergy, CI=1 indicates addition, and CI<1 indicates antagonism. It can be seen that the combined use of cypress extract and sophorolipid can produce a synergistic effect.

[0126] Taking 2-methyl-2,4-pentanediol as the matrix, and based on the Bliss independent model, the biofilm formation inhibition rate in experimental group 2 was considered the effect of the two-drug combination, while the biofilm formation inhibition rates in control groups 3 and 6 were considered the effects of the two single drugs, respectively. The CI value was calculated using the following formula:

[0127]

[0128] CI=33.57%÷(27.25%+0.13%-27.25%×0.13%)=1.23

[0129] CI>1 indicates synergy, CI=1 indicates additive effect, and CI<1 indicates antagonism. This shows that adding PEG-40 hydrogenated castor oil to the extracts of *Platycladus orientalis* and sophorolipids further enhances the antibacterial effect, producing a synergistic effect. This indicates that PEG-40 hydrogenated castor oil has a further synergistic effect on the antibacterial activity of *Platycladus orientalis* and sophorolipids.

[0130] III. Results of Oil Control Experiment: SZ95 Cell Inhibition of Oil Secretion

[0131] Excessive sebum secretion, sebum composition imbalance, and lipid peroxidation are considered the main factors contributing to skin problems in oily skin. Sebum secretion is related to the activity of 5α-reductase in sebaceous glands. 5α-reductase can convert testosterone into dihydrotestosterone (DHT). DHT binds to the receptor AR, increasing the activity of sebaceous glands and stimulating them to secrete oil.

[0132] This experiment used human sebaceous gland cells SZ95 to construct an in vitro acne model. The sebum secretion of SZ95 cells was used to simulate the sebum secretion process of human skin. Nile red dye was used to fluoresce neutral lipids. The sebum content of sebaceous gland cells was qualitatively and quantitatively detected by fluorescence microscopy and fluorescence quantitative methods. The effects of the test substances (each experimental group, control group and Pioneer) on inhibiting sebum secretion were observed, thereby evaluating the oil-controlling efficacy of the test substances.

[0133] The experimental steps include:

[0134] Take SZ95 cells in the logarithmic growth phase and add 2×10⁻⁶ cells to each well of a 24-well culture plate. 4 Cells were cultured in H-DMEM medium containing 5% FBS at 37℃, 5% CO2, and saturated humidity for 24 h. The culture medium was then aspirated. The negative control group received 0.5 mL of culture medium, while the model control group, test sample group, and positive control group received 0.5 mL of testosterone culture medium, 0.5 mL of testosterone + 0.5 mL of sample solution, and 0.5 mL of testosterone culture medium + 0.5 mL of LPA-Zn solution, respectively, to induce cell modeling for 24 h. The culture medium was then aspirated, and each well was washed with 0.5 mL of PBS solution. The cells were then stained with Nile Red solution diluted in PBS and incubated at 37℃ in the dark for 10 min. After washing with PBS, the Nile Red fluorescence intensity was detected and photographed using a multi-mode microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 565 nm. The results are shown in Table 6.

[0135] Table 6

[0136]

[0137] Ps* showed a significant difference relative to the model group (p < 0.05).

[0138] Results analysis, based on data from the negative control group, positive control group, and model group, showed that the addition of testosterone significantly increased sebum secretion, indicating successful model establishment; the addition of PCA-Zn significantly decreased sebum secretion, but the model showed no abnormalities.

[0139] The test data of experimental group 1-6 show that it has a significant reduction compared with the model group, and has a good oil control effect;

[0140] The test data of the control group 1-4 show no difference from the data of the model group, indicating no oil control effect;

[0141] According to the test data of the control group 4-5, the cypress extract is the key oil-controlling ingredient, while sophorolipid has basically no oil-controlling effect.

[0142] The test data of control group 5-7 were further reduced compared with control group 3-4, indicating that PEG-40 hydrogenated castor oil and polyol synergistically have a further reducing effect on oil control;

[0143] The control group of 8-9 further confirmed that the cypress extract had a significant inhibitory effect, but it had an inhibition peak. At an inappropriate ratio, it could not further improve the oil control effect. On the contrary, the control group of 5-7 showed better oil control feedback.

[0144] Data from the control group (13 groups) showed that Pioneer had a certain oil-controlling effect, but it did not reach a significant difference compared to the model group. In fact, it was weaker than the experimental groups.

[0145] In summary, piracetam had a weaker oil-controlling effect compared to the experimental group, cypress was the key oil-controlling component, sophorolipid had no oil-controlling effect, and the oil-controlling effect was further enhanced when the two were combined with PEG-40 hydrogenated castor oil, and the synergistic effect of 2-methyl-2,4-pentanediol was also achieved.

[0146] IV. Anti-inflammatory Experiment Results: Inhibition Rate of Macrophage Secretion of Inflammatory Factors

[0147] Inflammation is one of the most common clinical conditions, a defensive response of the body to remove harmful stimuli and repair damaged tissues. When human immune cells are exposed to inflammatory factors, some small-molecule soluble proteins or peptides with specific immunomodulatory functions, capable of transmitting information between cells, are secreted by the body and participate in or cause inflammatory responses. These substances are called inflammatory factors, including NO, TNF-α, IL-6, PGE-2, IL-1, and TGF-β1. NO, IL-6, TNF-α, and TGF-β1 are all important cellular activity factors in inflammatory responses, playing direct or indirect roles in cellular inflammation and influencing each other. TNF-α is the first key inflammatory factor after Propionibacterium acnes stimulates the hair follicle wall and initiates TL-2 expression, serving as a characteristic indicator of further stimulating the expression of other inflammatory factors and amplifying the inflammatory response. IL-6 is a key factor leading to vasodilation and skin redness and swelling, and is an important factor in the later stages of acne inflammation.

[0148] This experiment used LPS-induced mouse macrophages RAW264.7 as an in vitro inflammatory cell model to evaluate the in vitro anti-inflammatory efficacy of the test substance by measuring the relative expression levels of inflammatory factors (TNF-α and IL-6) mRNA.

[0149] First, the safe concentration of the test sample was confirmed by CKK cytotoxicity assay. Based on the safe concentration, the test concentration was uniformly set at 0.25%. A negative control group, a positive control group (50 nm dexamethasone), a model control group (LPS), and a test sample group (0.025%) were set up. Cells were cultured in a conventional manner, and the cell suspension was seeded into a 6-well cell culture plate and returned to the incubator for 18-24 h of culture.

[0150] Remove the culture plate and discard the original culture medium in the wells. Add culture medium with LPS as a stimulus to the test sample group and the model group, and add culture medium to the negative control group. Incubate for 18 h. Then remove the solution from the wells. Add the test sample to the test sample group, and add culture medium to the model group and the negative control group. Incubate for 24 h. Disrupt the cells in each group to collect RNA, reverse transcribe it into cDNA, store at -80℃, and finally perform real-time PCR to determine the expression levels of TNF-α and IL-6.

[0151] Table 7

[0152]

[0153] Note: *** indicates a highly significant p-value compared to the model group, ** indicates a significant p-value compared to the model group, and * indicates a significant p-value compared to the model group.

[0154] The results show that after LPS induction, the expression of inflammatory factors in the model group increased significantly, indicating successful modeling. After treatment with the positive control group, the expression of inflammatory factors decreased significantly, indicating that the model data are effective.

[0155] TNF-α represents the initial inflammatory factor in acne inflammation and plays a crucial role in the early initiation of inflammation. In the experimental groups, all showed good inhibitory effects, similar to the positive control. Control groups 4 and 5 were single-component controls of sophorolipid and cypress, respectively. The results showed that sophorolipid had a better inhibitory effect on TNF-α than cypress, indicating that sophorolipid can have a good inhibitory effect in the initial stage of inflammation. Control groups 6-7 showed that the solvent had a certain influence after the two were combined, resulting in a further synergistic effect. In control groups 8-9, adjusting the ratio of the two components did not yield better inhibitory effects and did not reach the level of the experimental group, indicating that the component ratio in the experimental group had priority value. Control group 13 also achieved levels similar to the experimental group and the positive control, indicating that piracetam also has a good inhibitory effect on TNF-α.

[0156] IL-6 represents the redness and swelling amplified by the inflammatory cascade in acne and is a key inflammatory factor in the later stages of acne. Data shows that the experimental and positive control groups had similar inhibitory effects on IL-6. Control groups 4-5 consisted of a single component of sophorolipid and cypress extract. Results showed that cypress extract significantly inhibited IL-6 better than sophorolipid. Control groups 6-7 showed that the combination of both with PEG-40 hydrogenated castor oil and 2-methyl-2,4-pentanediol further reduced IL-6 expression. Control groups 8-9, even with adjustments to the ratio of the two components, did not achieve better inhibitory effects and did not reach the level of the experimental group, indicating that the component ratio in the experimental group has priority value.

[0157] The control group 13 showed a significant difference compared to the experimental group and the positive control group, indicating that the effect of Pioneer on inhibiting IL-6 was weak and could not effectively suppress red and swollen pimples.

[0158] V. Results of acne treatment test and microbial abundance test in the population: left and right face comparison test.

[0159] Ninety volunteers aged 20-30 years with obvious facial acne symptoms such as redness, swelling, and raised bumps, as well as closed comedones (based on the Pillsbury four-level modified acne scale, level 1 symptoms were selected) were recruited and signed informed consent forms. The volunteers were randomly divided into three groups of 30 each. One group used the blank base material, another group used the product containing the experimental group's two samples prepared as described below, and the positive control group contained the Pioneerol sample. After 7 and 14 days of product use, a consumer questionnaire was used to assess the volunteers' responses on the following dimensions: reduction in acne redness and swelling, resolution of acne redness and swelling, reduction in acne pain, disappearance of acne pain, partial disappearance of acne, and substantial disappearance of acne.

[0160] Volunteers were required to use two sets of samples for up to 8 weeks to track acne and pimple recurrence and verify the effectiveness of the acne-fighting formulation in the product.

[0161] Table 8 Blank base material formulation and process

[0162]

[0163]

[0164] Experimental Group 7: Take 99g of blank base material from Table 5, add 1g of sample from Experimental Group 2, stir at 500rpm for 5min, dispense into 25g portions as test material, and distribute to volunteers.

[0165] Positive control group: 0.003% piocin (polyol preheated and dispersed) was added to the blank base, stirred at 500 rpm for 5 min, and dispensed into 25g portions for volunteers.

[0166] Table 9

[0167]

[0168] Table 10

[0169]

[0170] The results after 2 weeks of use on individuals with existing acne and pimples showed that the control group had virtually no effect on improving acne and pimples. The experimental group and the positive control group showed significant improvement compared to the control group. The experimental group 7 showed good feedback on the improvement of acne and inflammation, while the positive control group showed better feedback on the improvement of acne, but was relatively weaker in treating inflammatory acne.

[0171] Further follow-up on the recurrence of acne and pimples for 2 months revealed that nearly 60% of the 30 participants in the control group experienced recurrence of acne and inflammatory pimples. In the experimental group 7, with continued use, the number of new acne and pimples continued to decrease, and the recurrence rate after 2 months was approximately 16%, indicating that the use of this product composition can continuously improve the effect of the raw material on the inhibition of recurrence. In the positive control group, after 2 months of long-term use, the proportion of inflammatory recurrence was 50%, and the proportion of acne recurrence was 30%, indicating that Pioneer has a good inhibitory effect on acne recurrence, but a weaker inhibitory effect on inflammatory pimple recurrence, and its overall inhibitory effect on acne recurrence is weaker than that of the experimental group 7.

[0172] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition comprising cypress extract and sophorolipid.

2. The composition according to claim 1, characterized in that, The mass ratio of the cypress extract to sophorolipid is (0.5-1.5):(1-5).

3. A nano suspension prepared from excipients and the composition according to claim 1 or 2.

4. The nano-suspension according to claim 3, characterized in that, It was prepared from cypress extract, sophorolipid, PEG-40 hydrogenated castor oil and 2-methyl-2,4-pentanediol, wherein the mass ratio of cypress extract, sophorolipid, PEG-40 hydrogenated castor oil and 2-methyl-2,4-pentanediol was (0.5-1.5):(1-5):(1-5):(5-10).

5. A method for preparing the nano-suspension according to claim 3 or 4, comprising: After mixing cypress extract and 2-methyl-2,4-pentanediol, sophorolipid was added, and PEG-40 hydrogenated castor oil was added while stirring. The mixture was then homogenized by ultrasound and high pressure to obtain the nano-suspension.

6. The preparation method according to claim 5, characterized in that, The mixing conditions for the cypress extract and 2-methyl-2,4-pentanediol include heating to 60°C-70°C while stirring at 600-800 rpm. After adding the sophorolipid, stir at 600-800 rpm for 5 minutes; After adding PEG-40 hydrogenated castor oil, the continued stirring includes stirring at 1000 rpm for 1 to 2 minutes, the ultrasonic conditions include ultrasonication at 600W for 5 to 10 minutes, and the high-pressure homogenization conditions include homogenization at 1000 to 1200 Pa for 3 to 5 times.

7. The use of the composition of claim 1 or 2, the nanosuspension of claim 3 or 4, or the nanosuspension prepared by the preparation method of claim 5 or 6 in the preparation of anti-acne products.

8. The application according to claim 7, characterized in that, The anti-acne treatment includes antibacterial, anti-inflammatory, oil-controlling, and / or acne-improving effects; The antibacterial properties include inhibiting the growth of Propionibacterium acnes and / or inhibiting the formation of Propionibacterium acnes biofilm; The improvements in acne symptoms include reducing redness and swelling, reducing pain, and / or reducing the number of acne lesions.

9. An anti-acne product comprising the composition of claim 1 or 2, the nanosuspension of claim 3 or 4, or the nanosuspension prepared by the method of claim 5 or 6.

10. A method for preparing the product of claim 9, comprising: The blank base material and the nano suspension are mixed and stirred at 300-700 rpm for 1-10 minutes to obtain the product.

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