Centella asiatica leaf extract with soothing effect and application of centella asiatica leaf extract

Abstract

A preparation method of the centella asiatica leaf extract comprises the following steps: adding crushed and sieved centella asiatica leaf powder, an auxiliary carbon source and an auxiliary nitrogen source into water, adjusting the pH value to 5.0-7.0, and sterilizing to obtain a fermentation culture medium; inoculating the activated compound microorganism strain into the fermentation culture medium for fermentation culture; and then sterilizing, centrifuging and filtering to obtain the product. The compound microbial strains comprise lactobacillus rhamnosus, lactobacillus reuteri and bacillus subtilis. According to the centella asiatica leaf extract disclosed by the invention, the centella asiatica leaves are subjected to fermentation culture by adopting a compound microorganism strain of the rhamnosus lactobacillus, the lactobacillus reuteri and the bacillus subtilis, and the obtained centella asiatica leaf extract has remarkable anti-inflammatory activity and soothing effect. Through verification, the content of TNF-alpha and IL-6 can be remarkably reduced, and the hyaluronidase inhibition rate can be remarkably increased.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a Centella asiatica leaf extract with soothing effects and its application. Background Technology

[0002] Centella asiatica (L.) Urban, a perennial herb belonging to the genus Centella of the family Apiaceae, is a traditional medicinal plant. It is widely distributed, primarily in Jiangsu, Zhejiang, Anhui, Guangdong, Guangxi, and Hunan provinces. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it is described as cold in nature, bitter and pungent in taste, and affecting the liver, spleen, and kidney essence. It is used to clear heat and dampness, detoxify, and reduce swelling. It is commonly used to treat damp-heat jaundice, carbuncles, boils, and injuries from falls. The main chemical components of Centella asiatica leaves are triterpenoids, volatile oils, and polyacetylenes, containing various active pharmaceutical ingredients such as asiaticoside, hydroxyasiaticoside, and asiatic acid, which have anti-inflammatory, wound-healing, antioxidant, and skin-soothing effects.

[0003] In recent years, with the development of more and more medicinal plants, Centella asiatica has been widely used in cosmetics and pharmaceuticals. The raw materials added are mostly Centella asiatica extract, Centella asiatica extract powder, or high content of asiaticoside. These extracts are generally obtained through complex extraction processes. However, traditional extraction methods (such as water extraction and alcohol extraction) may have the following shortcomings: (1) The extraction rate of some active ingredients is not high or the molecular weight is large, making it difficult to be absorbed through the skin; (2) It may not be able to completely release the potential active substances in plant cells; (3) The stability of the extracted substances needs to be improved.

[0004] Microbial fermentation technology offers a new avenue for the in-depth development of plant raw materials. Microorganisms typically contain several active enzymes, including amylase, β-glucosidase, cellulase, protease, and lipase. Under the action of these enzymes, microorganisms decompose or convert underutilized substrates into compatible components. They can also increase antioxidant content by promoting the production or conversion of active ingredients into their metabolites, or break down large molecules into smaller molecules, allowing for better absorption and efficacy by the human body. Natural product extracts prepared through microbial fermentation can effectively reduce the inherent toxicity of some natural products. Studies have shown that fermentation can break down large molecules into more easily absorbed small molecules, generating new active metabolites, thereby significantly enhancing various functional activities. Microbial fermentation broth also features low cost and low allergenicity, making it a current research and development hotspot.

[0005] However, there are currently few reports on Centella asiatica leaves, especially on fermented products made from specific microbial strains to obtain fermented liquid products with synergistic effects of soothing and repair.

[0006] In recent years, influenced by factors such as environmental pollution, increased life stress, and unhealthy lifestyles, the number of people with sensitive skin problems has increased dramatically, and the age of onset is getting younger. Skin sensitivity and susceptibility to inflammation have received widespread public attention. Based on the International Forum for the Study of Pruritus (IFSI) and the "Chinese Expert Consensus on the Diagnosis and Treatment of Sensitive Skin" (2017 edition), sensitive skin is defined as a syndrome in which the skin, after being exposed to minor external stimuli, experiences paroxysmal or periodic burning, flushing, stinging, itching, and tightness, with or without persistent erythema. Sensitive skin most commonly occurs on the face and is a highly intolerant skin condition. Typically, the skin experiences varying degrees of burning, paroxysmal redness, stinging, and itching after exposure to minor stimuli such as temperature changes, seasonal changes, chemicals, or psychological stress, with or without erythema. It often cannot tolerate ordinary skincare products, causing significant distress to those with this condition. Exposure to seasonal ultraviolet radiation, photoaging, and post-sun inflammation are also issues that require attention. The treatment principles are to control symptoms, promote skin barrier repair, reduce inflammation, and decrease neurovascular hyperreactivity, aiming to improve skin tolerance. Repairing the damaged skin barrier is the fundamental measure for treating various skin inflammation problems; therefore, proper skincare is crucial. The principles should be followed: moderate cleansing, soothing moisturizing, and strict sun protection. The selection of soothing skincare products is also particularly important.

[0007] Currently, there are relatively few fermented Centella asiatica leaf active ingredient products on the market, and they have varying degrees of problems. The application of fermented Centella asiatica leaf active ingredients still needs further research. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the purpose of this invention is to provide a Centella asiatica leaf extract with a soothing effect and its application.

[0009] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for preparing a Centella asiatica leaf extract with a soothing effect, comprising the following steps: S1. Crush the Centella asiatica leaves and sieve them; S2. Add the sieved Centella asiatica leaf powder, auxiliary carbon source, and auxiliary nitrogen source to water, adjust the pH to 5.0-7.0, and then sterilize to obtain the sterilized fermentation medium. S3. Inoculate the activated compound microbial strains into the fermentation medium for fermentation culture; S4. After fermentation and culture, sterilize, then centrifuge and filter to obtain Centella asiatica leaf extract with soothing effects. In step S3, the composite microbial strains include Lactobacillus rhamnosus, Lactobacillus reuteri, and Bacillus subtilis.

[0010] As a preferred embodiment, the ratio of viable bacteria of Lactobacillus rhamnosus, Lactobacillus reuteri, and Bacillus subtilis is 1:1:1 to 4:1:4.

[0011] As a further preferred embodiment, the ratio of viable bacteria of Lactobacillus rhamnosus, Lactobacillus reuteri and Bacillus subtilis is 1:1:1 to 2:1:2, more preferably 1:1:1 to 1:1:2, and most preferably 1:1:1.

[0012] As a preferred embodiment, the Lactobacillus rhamnosus is Lactobacillus rhamnosus LR663, the Lactobacillus reuteri is Lactobacillus reuteri CCFM1154, and the Bacillus subtilis is BNCC188080.

[0013] As a preferred embodiment, the *Lactobacillus rhamnosus*, *Lactobacillus reuteri*, and *Bacillus subtilis* are activated before inoculation, and the activation process includes the following steps: Lactobacillus rhamnosus and Lactobacillus reuteri were inoculated into MRS liquid medium and cultured at 37℃ and 100 rpm for 24–36 h to obtain activated Lactobacillus rhamnosus and activated Lactobacillus reuteri. Bacillus subtilis was inoculated into LB liquid medium and cultured at 37°C and 100 rpm for 24–36 h to obtain activated Bacillus subtilis.

[0014] As a further preferred embodiment, the activated *Lactobacillus rhamnosus*, *Lactobacillus reuteri*, or *Bacillus subtilis* each have a cell concentration of 10-1. 6 ~10 7 CFU / mL.

[0015] As a preferred embodiment, in step S1, the Centella asiatica leaves are crushed and then passed through a 60-80 mesh sieve.

[0016] As a preferred embodiment, in step S2, the auxiliary carbon source is glucose and the auxiliary nitrogen source is peptone.

[0017] As a preferred embodiment, in step S2, the amount of Centella asiatica leaf powder added is 50-70 g / L; The amount of auxiliary carbon source and auxiliary nitrogen source added is 1-5% of the dry weight of Centella asiatica leaf powder.

[0018] As a preferred option, in step S2, the pH is adjusted by adding phosphate.

[0019] As a further preferred embodiment, the phosphate includes at least one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, and disodium hydrogen phosphate.

[0020] As a preferred embodiment, in step S3, the inoculation amount of the composite microbial strain is 5-10%.

[0021] As a preferred embodiment, in step S3, the fermentation conditions are: fermentation at 25–37°C under aerobic conditions for 48–72 hours.

[0022] As a preferred embodiment, in steps S2 and S4, the sterilization conditions are: sterilization at 121°C and 0.1 MPa for 20 min.

[0023] As a preferred embodiment, in step S4, the centrifugation conditions are: centrifugation at 5000-12000 rpm / min for 5-15 min; The supernatant obtained after centrifugation was filtered through a 0.45–3 µm polypropylene filter membrane.

[0024] In a first aspect, the present invention provides the application of Centella asiatica leaf extract prepared according to the aforementioned method in the preparation of cosmetics with soothing effects.

[0025] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention utilizes a composite microbial strain of *Lactobacillus rhamnosus*, *Lactobacillus reuteri*, and *Bacillus subtilis* to ferment and culture *Centella asiatica* leaves. The resulting *Centella asiatica* leaf extract (the fermented product) exhibits significant anti-inflammatory activity and soothing effects. Verification has shown that it can significantly reduce the levels of TNF-α and IL-6 (TNF-α levels as low as 21864.34 pg / mL and IL-6 levels as low as 922.92 pg / mL), and significantly increase the hyaluronidase inhibition rate (hyaluronidase inhibition rate as high as 88.849%).

[0026] 2) By further optimizing the ratio of viable bacteria of Lactobacillus rhamnosus, Lactobacillus reuteri and Bacillus subtilis, this invention can further improve the anti-inflammatory activity and soothing effect of Centella asiatica leaf extract. Detailed Implementation

[0027] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0028] The terms "preferred," "more preferably," and "more suitable" used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0029] It should be understood that, except in any operational instance or otherwise indicated, all figures representing the amounts of ingredients used, for example, in the specification and claims, should be understood to be modified in all cases by the term "about". Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximations varying with the desired performance to be obtained according to the invention. It is not at all an attempt to limit the application of the doctrine of equivalents to the scope of the claims; each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques.

[0030] Although the numerical ranges and parameters illustrating the broad scope of the invention are approximate, the values ​​listed in the specific examples are reported as precisely as possible. However, any numerical value inherently contains some error that is necessarily caused by the standard deviation found in their respective test measurements.

[0031] This application provides a general and / or specific description of the materials and experimental methods used in the experiments.

[0032] In the following examples, all raw materials and reagents used were commercially available products or prepared by existing methods, and the present invention does not impose any particular limitations. For example, *Lactobacillus rhamnosus* LR663 was purchased from Suzhou Beini Flower International Trade Co., Ltd., *Lactobacillus reuteri* CCFM1154 was purchased from Suzhou Beini Flower International Trade Co., Ltd., *Bacillus subtilis* BNCC188080 was purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd., and *Lactobacillus plantarum* HCS03-001 was purchased from Jiangxi Renren Health Microecological Technology Co., Ltd.

[0033] Example 1: Preparation of Centella Asiatica Leaf Extract A 1) Take 50 g of dried Centella asiatica leaves (originating from Yulin, Guangxi), crush them, pass them through a 60-mesh sieve, add 1 L of deionized water, then add 2.5 g of glucose and 1.0 g of peptone, and adjust the pH to 6.0 with potassium dihydrogen phosphate. The resulting fermentation medium is sterilized at 121℃ and 0.1 MPa for 20 min.

[0034] 2) *Lactobacillus rhamnosus* LR663 and *Lactobacillus reuteri* CCFM1154 were inoculated into MRS liquid medium, while *Bacillus subtilis* BNCC188080 was inoculated into LB liquid medium. The cultures were incubated at 37°C and 100 rpm for 24–36 h to obtain activated *Lactobacillus rhamnosus* LR663, *Lactobacillus reuteri* CCFM1154, and *Bacillus subtilis* BNCC188080 bacterial suspensions. The bacterial concentration in each suspension was increased to 10⁻⁶. 6 ~10 7 Follow-up steps are performed when CFU / mL is reached; 3) After cooling the sterilized fermentation medium, inoculate it at a 5% inoculum with the activated *Lactobacillus rhamnosus* LR663, *Lactobacillus reuteri* CCFM1154, and *Bacillus subtilis* BNCC188080 bacterial suspensions obtained in step 2) (viable cell ratio 1:1:1). Then, allow it to ferment statically at 36℃ for 48 h. After fermentation, sterilize at 121℃ and 0.1 MPa for 20 min. Centrifuge at 8000 rpm / min for 10 min, collect the supernatant, and filter it through a 0.45 μm polypropylene membrane to obtain *Centella asiatica* leaf extract A.

[0035] Example 2: Preparation of Centella Asiatica Leaf Extract B The preparation method is the same as in Example 1, except that the ratio of viable bacteria of Lactobacillus rhamnosus LR663, Lactobacillus reuteri CCFM1154 and Bacillus subtilis BNCC188080 is changed to 2:1:1 to obtain Centella asiatica leaf extract B.

[0036] Example 3: Preparation of Centella Asiatica Leaf Extract C The preparation method is the same as in Example 1, except that the ratio of viable bacteria of Lactobacillus rhamnosus LR663, Lactobacillus reuteri CCFM1154 and Bacillus subtilis BNCC188080 is changed to 1:1:2 to obtain Centella asiatica leaf extract C.

[0037] Example 4: Preparation of Centella Asiatica Leaf Extract D The preparation method is the same as in Example 1, except that the ratio of viable bacteria of Lactobacillus rhamnosus LR663, Lactobacillus reuteri CCFM1154 and Bacillus subtilis BNCC188080 is changed to 3:1:2 to obtain Centella asiatica leaf extract D.

[0038] Example 5: Preparation of Centella Asiatica Leaf Extract E The preparation method is the same as in Example 1, except that the ratio of viable bacteria of Lactobacillus rhamnosus LR663, Lactobacillus reuteri CCFM1154 and Bacillus subtilis BNCC188080 is changed to 1:1:3 to obtain Centella asiatica leaf extract E.

[0039] Example 6: Preparation of Centella Asiatica Leaf Extract F The preparation method is the same as in Example 1, except that the ratio of viable bacteria of Lactobacillus rhamnosus LR663, Lactobacillus reuteri CCFM1154 and Bacillus subtilis BNCC188080 is changed to 4:1:4 to obtain Centella asiatica leaf extract F.

[0040] Example 7: Preparation of Centella Asiatica Leaf Extract G 1) Take 70 g of dried Centella asiatica leaves (originating from Yulin, Guangxi), crush them, pass them through an 80-mesh sieve, add 1 L of deionized water, then add 2.1 g of glucose and 3.5 g of peptone, and adjust the pH to 7.0 with potassium dihydrogen phosphate. The resulting fermentation medium is sterilized at 121℃ and 0.1 MPa for 20 min.

[0041] 2) *Lactobacillus rhamnosus* LR663 and *Lactobacillus reuteri* CCFM1154 were inoculated into MRS liquid medium, while *Bacillus subtilis* BNCC188080 was inoculated into LB liquid medium. The cultures were incubated at 37°C and 100 rpm for 24–36 h to obtain activated *Lactobacillus rhamnosus* LR663, *Lactobacillus reuteri* CCFM1154, and *Bacillus subtilis* BNCC188080 bacterial suspensions. The bacterial concentration in each suspension was increased to 10⁻⁶. 6 ~10 7 Follow-up steps are performed when CFU / mL is reached; 3) After cooling the sterilized fermentation medium, inoculate it with the activated *Lactobacillus rhamnosus* LR663, *Lactobacillus reuteri* CCFM1154, and *Bacillus subtilis* BNCC188080 bacterial suspensions obtained in step 2) at an 8% inoculation rate (viable cell ratio 1:1:1). Then, allow it to ferment statically at 25°C for 72 h. After fermentation, sterilize at 121°C and 0.1 MPa for 20 min. Centrifuge at 10000 rpm / min for 5 min, collect the supernatant, and filter it through a 0.45 μm polypropylene membrane to obtain *Centella asiatica* leaf extract G.

[0042] Example 8: Preparation of Centella Asiatica Leaf Extract H 1) Take 60 g of dried Centella asiatica leaves (originating from Yulin, Guangxi), crush them, pass them through a 60-mesh sieve, add 1 L of deionized water, then add 0.6 g of glucose and 0.6 g of peptone, and adjust the pH to 5.0 with potassium dihydrogen phosphate. The resulting fermentation medium is sterilized at 121℃ and 0.1 MPa for 20 min.

[0043] 2) *Lactobacillus rhamnosus* LR663 and *Lactobacillus reuteri* CCFM1154 were inoculated into MRS liquid medium, while *Bacillus subtilis* was inoculated into LB liquid medium. The cultures were incubated at 37°C and 100 rpm for 24–36 h to obtain activated *Lactobacillus rhamnosus* LR663, *Lactobacillus reuteri* CCFM1154, and *Bacillus subtilis* BNCC188080 bacterial suspensions. The bacterial concentration in each suspension was increased to 10⁻⁶. 6 ~10 7 Follow-up steps are performed when CFU / mL is reached; 3) After cooling the sterilized fermentation medium, inoculate it at a 10% inoculum with the activated *Lactobacillus rhamnosus* LR663, *Lactobacillus reuteri* CCFM1154, and *Bacillus subtilis* BNCC188080 bacterial suspensions obtained in step 2) (viable cell ratio 1:1:1). Then, allow it to ferment statically at 30℃ for 60 h. After fermentation, sterilize at 121℃ and 0.1 MPa for 20 min. Centrifuge at 5000 rpm / min for 15 min, collect the supernatant, and filter it through a 0.45 μm polypropylene membrane to obtain *Centella asiatica* leaf extract H.

[0044] Comparative Example 1: Centella Asiatica Fermentation Blank Extract Except for omitting step 2) and not inoculating Lactobacillus rhamnosus LR663, Lactobacillus reuteri CCFM1154 and Bacillus subtilis BNCC188080 in step 3), the other steps were carried out according to the preparation method in Example 1 to obtain Centella asiatica fermentation blank extract.

[0045] Comparative Example 2: Centella Asiatica Leaf Aqueous Extract Take 50 g of dried Centella asiatica leaves from the same batch, add 1 L of deionized water, heat and reflux at 90°C for 2 hours, filter, and concentrate the filtrate to the same volume as the fermentation liquid in Example 1 to obtain Centella asiatica leaf water extract.

[0046] Comparative Example 3: Fermentation extract of a single strain (Lactobacillus rhamnosus) The preparation method is the same as in Example 1, except that only activated Lactobacillus rhamnosus LR663 was inoculated at a 5% inoculum to obtain Centella asiatica leaf extract I.

[0047] Comparative Example 4: Fermentation extract of a single strain (Lactobacillus reuteri CCFM1154) The preparation method is the same as in Example 1, except that only activated Lactobacillus reuteri CCFM1154 was inoculated at a 5% inoculum to obtain Centella asiatica leaf extract J.

[0048] Comparative Example 5: Fermentation extract of a single strain (Bacillus subtilis BNCC188080) The preparation method is the same as in Example 1, except that: only activated Bacillus subtilis BNCC188080 is inoculated at a 5% inoculum amount to obtain Centella asiatica leaf extract K.

[0049] Comparative Example 6: Fermentation extract of a compound strain (Lactobacillus rhamnosus LR663 and Lactobacillus reuteri CCFM1154) The preparation method is the same as in Example 1, except that only activated Lactobacillus rhamnosus LR663 and Lactobacillus reuteri CCFM1154 (live count ratio 1:1) are inoculated at a 5% inoculum amount to obtain Centella asiatica leaf extract L.

[0050] Comparative Example 7: Fermentation extract of a compound strain (Lactobacillus rhamnosus LR663, Lactobacillus plantarum HCS03-001, and Bacillus subtilis BNCC188080) The preparation method is the same as in Example 1, except that: activated Lactobacillus rhamnosus LR663 bacterial suspension, Lactobacillus plantarum HCS03-001 bacterial suspension (the activation step is: Lactobacillus plantarum HCS03-001 is inoculated into MRS liquid medium and cultured at 37℃ and 100 rpm for 24-36 h to obtain activated Lactobacillus plantarum HCS03-001 bacterial suspension) and Bacillus subtilis BNCC188080 bacterial suspension (live count ratio 1:1:1) are inoculated at a 5% inoculation amount to obtain Centella asiatica leaf extract M.

[0051] Comparative Example 8: Fermentation extract of a compound microbial strain (Lactobacillus rhamnosus LR663, Lactobacillus reuteri CCFM1154, and Bacillus subtilis BNCC188080) The preparation method is the same as in Example 1, except that: activated Lactobacillus rhamnosus LR663 bacterial suspension, Lactobacillus reuteri CCFM1154 bacterial suspension and Bacillus subtilis bacterial suspension (live count ratio 0.5:1:0.5) were inoculated at a 5% inoculum amount to obtain Centella asiatica leaf extract N.

[0052] Comparative Example 9: Fermentation extract of a compound microbial strain (Lactobacillus rhamnosus LR663, Lactobacillus reuteri CCFM1154, and Bacillus subtilis BNCC188080) The preparation method is the same as in Example 1, except that: activated Lactobacillus rhamnosus LR663 bacterial suspension, Lactobacillus reuteri CCFM1154 bacterial suspension and Bacillus subtilis BNCC188080 bacterial suspension (live count ratio 5:1:1) were inoculated at a 5% inoculum amount to obtain Centella asiatica leaf extract O.

[0053] Efficacy verification: Validation Example 1: Effect of fermentation product on LPS-induced macrophage cytokines (1) Sample preparation The Centella asiatica leaf extract (AH) prepared in Examples 1-8, the Centella asiatica fermentation blank extract prepared in Comparative Example 1, the Centella asiatica leaf aqueous extract prepared in Comparative Example 2, and the Centella asiatica leaf extract (IO) prepared in Comparative Examples 3-9 were added to PBS solution to prepare various test samples at a final concentration of 0.1%. Dexamethasone was used as a positive control and was added to PBS solution to prepare a positive control sample.

[0054] (2) Cell culture According to the appropriate inoculation density (1×10 5 RAW264.7 macrophages (purchased from Beina Chuanglian) were seeded into 24-well plates and cultured in an incubator (37℃, 5%CO2, 95%RH) for about 24 h.

[0055] (3) Cell-based drug delivery When the cell deposition rate in the 24-well plates reached 40%–50%, the cells were administered to three groups, with each group having three replicates. Cell culture medium containing each test sample (final concentration of each test sample was 0.10%, serving as the experimental group), a positive control sample (100 μg / mL dexamethasone, serving as the positive control group), and an equal volume of PBS solution (serving as the model group) was added. The blank control group also received an equal volume of PBS solution in cell culture medium. Each group had three replicates. After administration, the 24-well plates were incubated in an incubator (37℃, 5% CO2) for 2 h.

[0056] (4) Macrophage stimulation After 2 h of grouped drug administration and culture, LPS working solution prepared with the corresponding test substance working solution was added to the well plates of each administered experimental group, positive control group, and model group according to the experimental design. The well plates were shaken left and right to mix the drug in the well plates. The final concentration of LPS was 1 μg / mL. The plates were then incubated in an incubator (37℃, 5% CO2) for 22 h. No LPS working solution was added to the blank control group.

[0057] (5) Determination of the effect of LPS on the content of inflammatory cytokines in macrophages After incubation, the cell culture supernatant from each group was collected into EP tubes and stored at -80°C. TNF-α and IL-6 were detected and analyzed according to the ELISA kit instructions. Before the formal detection, a dilution ratio needed to be set and a preliminary experiment conducted to determine the optimal dilution ratio for the ELISA detection groups, ensuring that the ELISA values ​​fell within the range of the standard curve. The test results are shown in Table 1.

[0058] Table 1 Results of macrophage inflammatory cytokine assay Note: Compared with the model group, # indicates p < 0.05, ## indicates p < 0.01, and ### indicates p < 0.001; compared with Example 1 group, This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001.

[0059] As shown in Table 1, compared with the blank group, the TNF-α content in the model group was 26525.08±939.84 pg / mL ( P <0.01), and the IL-6 content was 1439.41±16.5 pg / mL ( P <0.001 indicates successful model establishment. Compared with the model group, the levels of TNF-α and IL-6 in the positive control group were significantly decreased ( P <0.001).

[0060] The comparison results between Examples 1-6 and Comparative Examples 1-2 show that the TNF-α and IL-6 contents of Centella asiatica leaf extracts A-F prepared in Examples 1-6 are significantly lower than those of the Centella asiatica fermentation blank extract prepared in Comparative Example 1 and the Centella asiatica leaf water extract prepared in Comparative Example 2. This indicates that by using microbial fermentation, the TNF-α and IL-6 contents can be significantly reduced, thereby achieving a better anti-inflammatory effect.

[0061] As can be seen from the comparison results between Examples 7 and 8 and Example 1, the preparation conditions of Example 1 can significantly reduce the content of TNF-α and IL-6, thereby achieving better anti-inflammatory activity.

[0062] The comparison results between Example 1 and Comparative Examples 3-7 show that the TNF-α and IL-6 contents of Centella asiatica leaf extract A prepared in Example 1 are significantly lower than those of Centella asiatica leaf extracts I-M prepared in Comparative Examples 3-7. This indicates that the fermentation of the compound microbial strain of Lactobacillus rhamnosus LR663, Lactobacillus reuteri CCFM1154 and Bacillus subtilis BNCC188080 in this invention can significantly improve the anti-inflammatory activity.

[0063] The comparison results between Examples 1-6 and Comparative Examples 8-9 show that when the ratio of viable bacteria of *Lactobacillus rhamnosus* LR663, *Lactobacillus reuteri* CCFM1154, and *Bacillus subtilis* BNCC188080 in the composite microbial strains exceeded the range of 1:1:1-4:1:4, the contents of TNF-α and IL-6 were significantly increased. Furthermore, compared with the *Centella asiatica* fermentation blank extract prepared in Comparative Example 1 and the *Centella asiatica* leaf aqueous extract prepared in Comparative Examples 8-9, the N and O contents of the *Centella asiatica* leaf extract prepared in Comparative Examples 8-9 were higher than those of the *Centella asiatica* fermentation blank extract and the *Centella asiatica* leaf aqueous extract, while the TNF-α content was not significantly different. This indicates that the *Centella asiatica* leaf extract prepared in Comparative Examples 8-9 did not improve the anti-inflammatory activity compared with the *Centella asiatica* fermentation blank extract and the *Centella asiatica* leaf aqueous extract.

[0064] Verification of the in vitro soothing efficacy experiment in Example 2 The Centella asiatica leaf extract (AH) prepared in Examples 1-8, the Centella asiatica fermentation blank extract prepared in Comparative Example 1, the Centella asiatica leaf water extract prepared in Comparative Example 2, and the Centella asiatica leaf extract (IO) prepared in Comparative Examples 3-9 were used as test samples. The samples were tested according to laboratory method HMC-WI-029 "Hyaluronidase Inhibition Rate" and T / JSRH 003-2023 "Test of Anti-allergic Activity of Cosmetic Raw Materials: Hyaluronidase Inhibition Method". When the hyaluronidase inhibition rate of the positive control (1.0% dipotassium glycyrrhizate) is >50%, the reaction system is considered effective; when the hyaluronidase inhibition rate of the sample is higher than that of the negative control (distilled water) and there is a significant difference (…), the reaction system is considered effective. P If the value is less than 0.05, the test sample can be considered to have a certain soothing effect.

[0065] Table 2. Hyaluronidase inhibition rate results (unit / %) Note: Hyaluronidase inhibition rate is rounded to three decimal places.

[0066] The hyaluronidase inhibition rate of the sample was significantly higher than that of the negative control (p<0.05), indicating that the test sample has a certain soothing effect. This indicates a significant difference between the sample or positive control and the negative control (p<0.001).

[0067] The test results are shown in Table 2. As can be seen from the results in Table 2, compared with the fermented blank extract of Centella asiatica leaves prepared in Comparative Example 1 and the aqueous extract of Centella asiatica leaves prepared in Comparative Example 2, the hyaluronidase inhibition rates of Centella asiatica leaf extracts A-F prepared in Examples 1-6 were significantly improved. The hyaluronidase inhibition rates of Centella asiatica leaf extracts A-F reached 80.651-88.849%, while the hyaluronidase inhibition rates of the fermented blank extract of Centella asiatica leaves and the aqueous extract of Centella asiatica leaves were only 68.553% and 70.456%, respectively. This indicates that by using microbial fermentation, the hyaluronidase inhibition rate can be significantly improved, thereby achieving a better soothing effect.

[0068] The results of hyaluronidase inhibition rate comparisons between the Centella asiatica fermentation blank extract prepared in Comparative Example 1, Centella asiatica leaf extract A prepared in Example 1, and Centella asiatica leaf extracts I-M prepared in Comparative Examples 3-7 showed that the hyaluronidase inhibition rate of Centella asiatica leaf extract A was as high as 88.849%, significantly higher than that of the Centella asiatica fermentation blank extract. However, the hyaluronidase inhibition rate of Centella asiatica leaf extracts I-M was not significantly improved compared to the Centella asiatica fermentation blank extract, and even the hyaluronidase inhibition rate of Centella asiatica leaf extract J was significantly lower than that of the Centella asiatica fermentation blank extract. This indicates that only by specifically using a compound microbial strain of *Lactobacillus rhamnosus* LR663, *Lactobacillus reuteri* CCFM1154, and *Bacillus subtilis* BNCC188080 for fermentation can the hyaluronidase inhibition rate be significantly improved, resulting in better soothing effects.

[0069] The comparison results between Examples 1-6 and Comparative Examples 8-9 show that when the viable cell ratio of *Lactobacillus rhamnosus* LR663, *Lactobacillus reuteri* CCFM1154, and *Bacillus subtilis* BNCC188080 is 1:1:1-4:1:4, the hyaluronidase inhibition rate of *Centella asiatica* leaf extracts A-F can reach over 79.543%. However, when the viable cell ratio exceeds this range, the hyaluronidase inhibition rates of *Centella asiatica* leaf extracts N-O are only 73.641% and 72.503%, respectively. This indicates that further optimization of the viable cell ratio of the three strains can significantly improve the hyaluronidase inhibition rate and achieve better soothing effects.

[0070] As can be seen from the comparison results between Example 1 and Examples 7-8, the preparation conditions of Example 1 can significantly improve the hyaluronidase inhibition rate, thereby achieving a better soothing effect.

[0071] The above experimental results show that the Centella asiatica leaf extract prepared in this invention has anti-inflammatory and soothing effects, and can be used to prepare cosmetics with soothing effects.

[0072] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. A method for preparing a Centella asiatica leaf extract with a soothing effect, characterized in that, Includes the following steps: S1. Crush the Centella asiatica leaves and sieve them; S2. Add the sieved Centella asiatica leaf powder, auxiliary carbon source, and auxiliary nitrogen source to water, adjust the pH to 5.0-7.0, and then sterilize to obtain the sterilized fermentation medium. S3. Inoculate the activated compound microbial strains into the fermentation medium for fermentation culture; S4. After fermentation and culture, sterilize, then centrifuge and filter to obtain Centella asiatica leaf extract with soothing effects. In step S3, the composite microbial strains include Lactobacillus rhamnosus, Lactobacillus reuteri, and Bacillus subtilis.

2. The method for preparing the Centella asiatica leaf extract with soothing effect according to claim 1, characterized in that, The ratio of viable counts of *Lactobacillus rhamnosus*, *Lactobacillus reuteri*, and *Bacillus subtilis* is 1:1:1 to 4:1:

4.

3. The method for preparing the Centella asiatica leaf extract with soothing effect according to claim 1, characterized in that, The Lactobacillus rhamnosus is Lactobacillus rhamnosus LR663, the Lactobacillus reuteri is Lactobacillus reuteri CCFM1154, and the Bacillus subtilis is BNCC188080.

4. The method for preparing the Centella asiatica leaf extract with soothing effect according to any one of claims 1-3, characterized in that, The *Lactobacillus rhamnosus*, *Lactobacillus reuteri*, and *Bacillus subtilis* were activated before inoculation. The activation process included the following steps: Lactobacillus rhamnosus and Lactobacillus reuteri were inoculated into MRS liquid medium and cultured at 37°C and 100 rpm for 24–36 h to obtain activated Lactobacillus rhamnosus and activated Lactobacillus reuteri. Bacillus subtilis was inoculated into LB liquid medium and cultured at 37°C and 100 rpm for 24–36 h to obtain activated Bacillus subtilis.

5. The method for preparing the Centella asiatica leaf extract with soothing effect according to claim 1, characterized in that, In step S1, the Centella asiatica leaves are crushed and then passed through a 60-80 mesh sieve.

6. The method for preparing the Centella asiatica leaf extract with soothing effect according to claim 1, characterized in that, In step S2, the auxiliary carbon source is glucose, and the auxiliary nitrogen source is peptone; In step S2, the amount of Centella asiatica leaf powder added is 50-70 g / L; The amount of auxiliary carbon source and auxiliary nitrogen source added is 1-5% of the dry weight of Centella asiatica leaf powder.

7. The method for preparing the Centella asiatica leaf extract with soothing effect according to claim 1, characterized in that, In step S2, the pH is adjusted by adding phosphate.

8. The method for preparing the Centella asiatica leaf extract with soothing effect according to claim 1, characterized in that, In step S3, the inoculation amount of the composite microbial strain is 5-10%.

9. The method for preparing the Centella asiatica leaf extract with soothing effect according to claim 1, characterized in that, In step S3, the fermentation conditions are: fermentation at 25-37°C under aerobic conditions for 48-72 hours.

10. The use of a Centella asiatica leaf extract prepared by the method according to any one of claims 1-9 in the preparation of cosmetics with a soothing effect.

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