Centella asiatica fermentation product and application thereof

Abstract

The invention discloses a centella asiatica fermentation product and application thereof. According to the method, specific strains are adopted, and the probiotic fermentation process of centella asiatica is optimized, so that the content of bioactive substances in fermented centella asiatica is greatly increased, and the bioavailability and functional characteristics of the centella asiatica are remarkably improved. The centella asiatica fermentation product disclosed by the invention has excellent effects on inhibiting bacteria, resisting aging, relieving, moisturizing, scavenging free radicals, resisting oxidation and resisting inflammation, and is expected to be widely applied to cosmetics, daily necessities, food, health care products and medicines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant ingredients for skin care and health care, and in particular relates to a Centella asiatica fermentation product and application thereof. BACKGROUND

[0002] Centella asiatica is a plant with important medicinal and health care values, and is widely used in the fields of cosmetics, food and medicine. The rich active ingredients contained in Centella asiatica include pentacyclic triterpenes, flavonoids and polyphenols, which have the effects of antioxidant, anti-inflammatory, antibacterial, promoting wound healing and improving skin barrier function. The main active ingredients are pentacyclic triterpenes, including Asiaticoside, Madecassoside, Asiatic acid and Madecassic acid, etc. These pentacyclic triterpenes have the effects of antioxidant, anti-inflammatory, promoting wound healing, anti-aging and improving skin barrier function. However, the active ingredients of Centella asiatica may have problems such as low bioavailability and poor stability under traditional extraction methods, which limits its further development and application.

[0003] At present, the application research on Centella asiatica mainly focuses on direct extraction or chemical synthesis of its active ingredients, and although there are many patents on using probiotic fermentation to improve the bioactivity and efficacy of Centella asiatica, they do not pay attention to the changes of its derivatives. For example, Asiaticoside and Madecassoside are deglycosylated by β-D-glucosidase and α-L-rhamnosidase to generate Asiatic acid and Madecassic acid, which have stronger anti-inflammatory and antioxidant capacity. In addition, the current research on the effects of active ingredients of Centella asiatica is basically limited to its anti-inflammatory and antioxidant functions.

[0004] It can be seen that the development and utilization of active ingredients in Centella asiatica and its fermentation products in terms of derivative activity and other functions need to be further deepened. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to optimize the probiotic fermentation process of Centella asiatica, increase the content of bioactive substances in Centella asiatica after fermentation, and improve its bioavailability and functional characteristics.

[0006] In one aspect, the present application provides a Centella asiatica fermentation product, which is obtained by fermenting a Centella asiatica-containing raw material using Lactiplantibacillus plantarum.

[0007] Preferably, the Lactiplantibacillus plantarum is MY-9, and the preservation number is CCTCC M 20251284.

[0008] In one or more embodiments, the fermentation is with NH4CI as the nitrogen source.

[0009] Preferably, the fermentation is with NH4CI as the nitrogen source at a concentration of 0.5-5.0 g / L. More preferably, the fermentation is with NH4CI as the nitrogen source at a concentration of 0.5-1.5 g / L. Further preferably, the fermentation is with NH4CI as the nitrogen source at a concentration of 1 g / L.

[0010] In one or more embodiments, the fermentation is with peptone as the nitrogen source.

[0011] Preferably, the fermentation is with peptone as the nitrogen source at a concentration of 1.0-10 g / L. More preferably, the fermentation is with peptone as the nitrogen source at a concentration of 5.0 g / L.

[0012] In one or more embodiments, the fermentation is with yeast extract as the nitrogen source.

[0013] Preferably, the fermentation is with yeast extract as the nitrogen source at a concentration of 1.0-10 g / L. More preferably, the fermentation is with yeast extract as the nitrogen source at a concentration of 5.0 g / L.

[0014] In one or more embodiments, the fermentation is with peptone and yeast extract as the nitrogen source.

[0015] Preferably, the fermentation is with peptone and yeast extract as the nitrogen source at a total concentration of 1.0-10 g / L. More preferably, the fermentation is with peptone and yeast extract as the nitrogen source at a total concentration of 5.0 g / L.

[0016] Preferably, the fermentation is with peptone and yeast extract as the nitrogen source at a concentration of 2.0-3.0 g / L of peptone and 2.0-3.0 g / L of yeast extract. More preferably, the fermentation is with peptone and yeast extract as the nitrogen source at a concentration of 2.5 g / L of peptone and 2.5 g / L of yeast extract.

[0017] In another aspect, the present application provides use of the Centella asiatica fermentation product as described in any one of the embodiments herein in a health product, food product, cosmetic product, or daily use product as one or more ingredients selected from the group consisting of a bacteriostatic ingredient, an anti-inflammatory ingredient, an antioxidant ingredient, an anti-wrinkle ingredient, an anti-aging ingredient, a soothing ingredient, and a moisturizing ingredient.

[0018] In another aspect, the present application provides a use of the Centella asiatica fermentation product as described in any one of the embodiments herein in the preparation of a medicament for inhibiting the activity of MMP-1, MMP-9 or KLK7, or for treating abnormal expression of HAS1, HAS2, TNF-a, IL-6, IL-8, IL-1a, IL-1b, PGE2, COX-2, IL-10 or IL-37, or abnormal protein secretion of TNF-a, IL-6, IL-8, IL-1a, IL-1b, PGE2, COX-2, IL-10 or IL-37.

[0019] Preferably, the abnormal expression is abnormal expression at mRNA level or abnormal expression at protein level.

[0020] In another aspect, the present application provides a composition comprising the Centella asiatica fermentation product as described in any one of the embodiments herein and a suitable amount of excipients; the composition is a pharmaceutical composition, a health product composition, a food composition, a cosmetic composition or a daily necessity composition.

[0021] In another aspect, the present application provides a use of the composition as described in any one of the embodiments herein in a health product, a food, a cosmetic or a daily necessity, the composition being used as one or more ingredients selected from the group consisting of bacteriostatic ingredients, anti-inflammatory ingredients, antioxidant ingredients, anti-wrinkle ingredients, anti-aging ingredients, soothing ingredients and moisturizing ingredients.

[0022] In another aspect, the present application provides a use of the composition as described in any one of the embodiments herein in the preparation of a medicament for inhibiting the activity of MMP-1, MMP-9 or KLK7, or for treating abnormal expression of HAS1, HAS2, TNF-a, IL-6, IL-8, IL-1a, IL-1b, PGE2, COX-2, IL-10 or IL-37, or abnormal protein secretion of TNF-a, IL-6, IL-8, IL-1a, IL-1b, PGE2, COX-2, IL-10 or IL-37.

[0023] In another aspect, the present application provides a fermentation method of Centella asiatica, the method comprising fermenting a Centella asiatica-containing raw material using Lactiplantibacillus plantarum

[0024] Preferably, the Lactiplantibacillus plantarum is MY-9, with the accession number CCTCC M 20251284.

[0025] In one or more embodiments, the fermentation uses NH4Cl as the nitrogen source.

[0026] In another aspect, the present application provides a Lactiplantibacillus plantarum MY-9, the preservation number of which is CCTCC M 20251284.

[0027] In another aspect, the present application provides use of the Lactiplantibacillus plantarum MY-9 as described in any of the embodiments herein in fermentation of Centella asiatica.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] 1. When the Lactiplantibacillus plantarum MY-9 of the present application is used for fermentation in a Centella asiatica-containing culture medium, 1 g / L NH4Cl is used as a nitrogen source, which can make the MY-9 logarithmic growth phase bacterial concentration grow fastest, and the contents of asiaticoside, asiatic acid, madecassic acid and total saponins significantly increase.

[0030] 2. The Centella asiatica MY-9 fermentation broth has a significant inhibitory effect on S. aureus and C. acnes and their biofilm formation; when diluted to 3.13% to 50.0% of the original concentration, the inhibitory effect on the biofilms of S. aureus and C. acnes is still very significant.

[0031] 3. When diluted to 25% or 2.5% of the original concentration, the Centella asiatica MY-9 fermentation broth has a significant inhibitory effect on MMP-1 and MMP-9; when diluted to 25% of the original concentration, the inhibitory effect of the Centella asiatica MY-9 fermentation broth on MMP-1 and MMP-9 is significantly better than that of 0.01 nM of Iloperidone.

[0032] 4. When diluted to 25% or 2.5% of the original concentration, the Centella asiatica MY-9 fermentation broth has a significant inhibitory effect on KLK7.

[0033] 5. When diluted to 25% or 2.5% of the original concentration, the Centella asiatica MY-9 fermentation broth can significantly improve the mRNA relative expression levels of HAS1 and HAS2, and the improvement on HAS1 is better than that of ascorbic acid.

[0034] 6. When diluted to 3.13%–50% of the original concentration, or undiluted, Centella asiatica MY-9 fermentation broth can significantly scavenge ABTS and DPPH free radicals in vitro; when diluted to 2.5% of the original concentration, Centella asiatica MY-9 fermentation broth exhibits significant antioxidant effects in HaCat cells, including reversing abnormal changes in SOD activity, GSH-Px activity, GSH content, and MDA content during oxidative stress.

[0035] 7. When diluted to 2.5% of the original concentration, Centella asiatica MY-9 fermentation broth showed significant activity in reversing abnormal cytokine expression in an inflammatory cell model constructed by UVB irradiation, including reducing the expression and secretion of pro-inflammatory factors and promoting the expression and secretion of anti-inflammatory factors. Attached Figure Description

[0036] Figure 1 This is a graph showing the changes in viable bacterial count and pH value of MY-9 under different nitrogen source conditions over time. Figure A corresponds to no nitrogen source; Figure B corresponds to YE nitrogen source; Figure C corresponds to Tryptone nitrogen source; Figure D corresponds to a combination of YE and Tryptone nitrogen sources; Figure E corresponds to 1 g / L NH4Cl nitrogen source; Figure F corresponds to 3 g / L NH4Cl nitrogen source; Figure G corresponds to 5 g / L NH4Cl nitrogen source; Figure H corresponds to 1 g / L (NH4)2SO4 nitrogen source; Figure I corresponds to 3 g / L (NH4)2SO4 nitrogen source; and Figure J corresponds to 5 g / L (NH4)2SO4 nitrogen source.

[0037] Figure 2 These are heatmaps showing the changes in active ingredients and enzyme activity in the fermentation broth of MY-9 under different nitrogen source conditions. Figure A shows the changes in the content of active ingredients and enzyme activity, while Figure B shows the relative changes in the content of active ingredients.

[0038] Figure 3 The figures show the inhibitory effect of Centella Asiatica MY-9 fermentation broth on bacteria. Figure A shows the inhibitory effect of Centella Asiatica MY-9 fermentation broth on Staphylococcus aureus; Figure B shows the inhibitory effect of Centella Asiatica MY-9 fermentation broth on Propionibacterium acnes.

[0039] Figure 4 This study investigated the effects of Centella asiatica MY-9 fermentation broth on bacterial biofilm formation. Figure A shows the effect of Centella asiatica MY-9 fermentation broth on the biofilm formation ability of *S. aureus*, and Figure B shows the effect of Centella asiatica MY-9 fermentation broth on the biofilm formation ability of *C. acnes*.

[0040] Figure 5 The figures show the inhibitory effect of Centella asiatica MY-9 fermentation broth on matrix metalloproteinases. Figure A shows the inhibitory effect of Centella asiatica MY-9 fermentation broth on MMP-9, and Figure B shows the inhibitory effect of Centella asiatica MY-9 fermentation broth on MMP-1.

[0041] Figure 6 This is the inhibitory effect of Centella Asiatica MY-9 fermentation broth on KLK7.

[0042] Figure 7 This study investigated the effects of Centella asiatica MY-9 fermentation broth on the expression levels of HAS1 and HAS2 mRNA. Figure A shows the effect of Centella asiatica MY-9 fermentation broth on HAS1 mRNA expression level; Figure B shows the effect of Centella asiatica MY-9 fermentation broth on HAS2 mRNA expression level.

[0043] Figure 8 These are the results of the free radical scavenging ability determination of Centella asiatica MY-9 fermentation broth. Figure A shows the results of the ABTS free radical scavenging ability determination of Centella asiatica MY-9 fermentation broth; Figure B shows the results of the DPPH free radical scavenging ability determination of Centella asiatica MY-9 fermentation broth.

[0044] Figure 9 This is the result of measuring the intracellular antioxidant capacity (CAA value) of HaCat cells in Centella asiatica MY-9 fermentation broth.

[0045] Figure 10 These are the results of the intracellular antioxidant capacity determination of HaCat cells from Centella Asiatica MY-9 fermentation broth. Figure A shows the effect on SOD, Figure B shows the effect on GSH-Px activity, Figure C shows the effect on GSH content, and Figure D shows the effect on MDA content.

[0046] Figure 11 This study investigated the effects of Centella Asiatica MY-9 fermentation broth on the expression levels of cytokine mRNA. Figure A shows the effect on TNF-α, Figure B on IL-6, Figure C on IL-8, Figure D on IL-1β, Figure E on IL-α, Figure F on COX-2, Figure G on IL-10, and Figure H on IL-37.

[0047] Figure 12 This study investigated the effects of Centella Asiatica MY-9 fermentation broth on cytokine secretion levels. Figure A shows the effect on TNF-α, Figure B on IL-6, Figure C on IL-8, Figure D on IL-1β, Figure E on IL-α, Figure F on PGE2, Figure G on IL-10, and Figure H on IL-37.

[0048] Depositing matter

[0049] The Lactiplantibacillus plantarum MY-9 of this invention was deposited at the China Center for Type Culture Collection (CCTCC) on June 6, 2025, with accession number CCTCC M 20251284. Detailed Implementation

[0050] The rationale behind this invention's use of probiotics to ferment Centella asiatica is as follows: During fermentation, probiotics (such as lactic acid bacteria and bifidobacteria) can degrade macromolecules through enzymatic hydrolysis, releasing or transforming them into more bioactive components. For example, fermentation makes the polysaccharides and flavonoids in Centella asiatica easier to absorb, while also enhancing their antioxidant and anti-inflammatory activities. Furthermore, probiotics can produce various metabolites during fermentation, such as organic acids, polypeptides, and short-chain fatty acids, further enhancing the functional properties of Centella asiatica.

[0051] On the other hand, probiotics (such as lactic acid bacteria and bifidobacteria) can secrete various enzymes during fermentation, such as β-glucosidase, esterase, and oxidoreductase. These enzymes can catalyze the glycoside hydrolysis, carboxyl modification, and redox reactions of pentacyclic triterpenoids, thereby generating secondary metabolites with stronger biological activity and better absorption. For example:

[0052] Glycoside hydrolysis: Through the action of β-D-glucosidase and α-L-rhamnosidase, asiaticoside and hydroxyasiaticoside can be deglycosylated to convert into asiatic acid and hydroxyasiatic acid, respectively, the latter having stronger anti-inflammatory and antioxidant capabilities.

[0053] Structural modification: Esterases and decarboxylases can further modify pentacyclic triterpenoids, enhancing their lipid solubility and skin permeability, thus increasing their absorption efficiency in topical skincare products or functional foods.

[0054] Redox transformation: Some lactic acid bacteria and bifidobacteria can produce NADH / NADPH-dependent redox enzymes during metabolism, which regulate the oxidation state of pentacyclic triterpenoids and enhance their antioxidant and anti-aging effects.

[0055] Example 1: Lactiplantibacillus plantarum MY-9

[0056] Lactiplantibacillus plantarum MY-9 was obtained by the applicant of this invention through screening and isolation, and its 16S rDNA sequence is shown in SEQ ID NO: 1.

[0057] Example 2: Nitrogen source screening of MY-9 fermented Centella asiatica

[0058] In this embodiment, *Lactiplantibacillus plantarum* MY-9 was used to ferment *Centella asiatica*, and fermentation media containing different nitrogen sources were employed. The fermentation media contained *Centella asiatica* powder, with water as the solvent, and the ratio of *Centella asiatica* powder to water was 1:10 (w / w). It also contained 20 g / L glucose, and the nitrogen sources were 1 g / L, 3 g / L and 5 g / L NH4Cl, 1 g / L, 3 g / L and 5 g / L (NH4)2SO4, 5 g / L yeast extract (YE), 5 g / L tryptone, or 2.5 g / L yeast extract + 2.5 g / L tryptone (YE / Tryptone).

[0059] Growth conditions

[0060] Lactobacillus plantarum MY-9 was cultured in MRS liquid medium at 37℃ and 200rpm for 16h, and then... 7 CFU / mL was inoculated into the fermentation medium and then cultured at 37℃ and 200 rpm. The pH value and viable cell count of the fermentation broth were measured at 0, 4, 8, 12, 16, 20 and 24 h during the fermentation process. The viable cell count was determined by plate counting method.

[0061] Growth curves of *Lactobacillus plantarum* MY-9 under different nitrogen source conditions are as follows: Figure 1 As shown. Without a nitrogen source ( Figure 1 (A) During the logarithmic growth phase (0–8 h), the bacterial cells rapidly grew to 9.09 lg CFU / mL, an increase of 1.71 lg CFU / mL from 0 h. At 24 h, the cell density decreased to 8.91 lg CFU / mL, and the pH dropped from 5.10 at 0 h to 3.79 at 24 h. Although the rate of pH decrease slowed after 8 h, it continued to decline, indicating that the cells were still producing acidic metabolites. When a single organic nitrogen source was added ( Figure 1 (B~C), the logarithmic growth phase was prolonged to 12h, and the bacterial concentrations in the YE group and Tryptone group reached 9.30 and 9.35 lg CFU / mL, respectively, representing increases of 1.89 and 1.81 lg CFU / mL compared to 0h. The combined use of YE and Tryptone prolonged the logarithmic growth phase to 16h (B~C). Figure 1 (D), the bacterial concentration was 9.48 lg CFU / mL, an increase of 1.92 lg CFU / mL from 0 h, and remained at a high concentration of 9.24 lg CFU / mL until 24 h. When 1, 3, and 5 g / L of inorganic nitrogen source NH4Cl were added ( Figure 1(E-G) The logarithmic growth phase was extended to 12 hours, and the bacterial concentration increased by 2.22, 2.02, and 2.00 lg CFU / mL compared to 0 hours, respectively. After 24 hours, the bacterial concentration decreased to 8.75, 8.87, and 8.64 lg CFU / mL. After 12 hours, the pH slowly decreased to about 3.70, the same as at 24 hours, indicating that the cells were still in a state of producing acidic metabolites. When 1, 3, and 5 g / L of inorganic nitrogen source (NH4)2SO4 were added ( Figure 1 After the logarithmic growth phase (H-J) was extended to 12 hours, the bacterial concentration increased by 2.00, 2.07, and 1.93 lg CFU / mL compared to 0 hours, respectively. By 24 hours, the bacterial concentration decreased to 8.42, 8.28, and 8.30 lg CFU / mL, respectively. The pH decrease gradually slowed after 12 hours, and by 24 hours, the pH gradually decreased to around 3.60, indicating that the cells were still producing acidic metabolites. Overall, with 1 g / L NH4Cl as the nitrogen source, strain MY-9 showed the fastest rate of bacterial concentration increase during the logarithmic growth phase. All groups exhibited acid production. After 12 hours, the rate of pH decrease slowed, but by 24 hours, the pH continued to drop to around 3.6-3.7, indicating that the cells were still capable of producing acidic metabolites.

[0062] Active substance production

[0063] Lactobacillus plantarum MY-9 was cultured in MRS liquid medium at 37℃ and 200rpm for 16h, and then... 7 CFU / mL was inoculated into the fermentation medium and then fermented at 37℃ and 200rpm. After 24 hours of fermentation, the conversion of active substances and the effect on enzyme activity under different nitrogen source conditions were detected by HPLC. An unfermented group was set up, in which MY-9 or nitrogen source was not added. A nitrogen-free group was also set up, in which MY-9 was added but no nitrogen source was added.

[0064] The liquid phase detection conditions for asiaticoside, asiatic acid, hydroxyasiaticoside and hydroxyasiatic acid were as follows: mobile phase: acetonitrile: aqueous phase (4 mM γ-cyclodextrin + 0.2% phosphoric acid) = 20:80, flow rate: 1 mL / min, temperature: 30℃, and detection wavelength: 205 nm.

[0065] Enzyme activity is determined indirectly by measuring the amount of substrate produced. β-d-glucosidase hydrolyzes p-NPG to produce the substrate p-NP, and α-l-rhamnosidase hydrolyzes p-NPR to produce the substrate p-NP. Enzyme activity is determined by measuring the concentration of p-NP using liquid chromatography. The liquid chromatography conditions are: mobile phase: 320 mL acetonitrile + 675 mL water + 5 mL acetic acid; flow rate: 0.8 mL / min; temperature: [temperature missing].

[0066] Test results as follows Figure 2As shown, total saponins refer to the total content of asiaticoside, asiatic acid, asiaticoside, and asiatic acid. Compared with the unfermented group, the asiaticoside and total saponin contents of each group increased significantly after fermentation with MY-9. Among them, the increase in saponin content was most significant under the condition of 1 g / L NH4Cl, and the enzyme activity was also at a high level. Compared to unfermented Centella asiatica, the Centella asiatica culture medium containing 1 g / L NH4Cl fermented with MY-9 showed a 3.53-fold increase in asiaticoside content, and increases in asiatic acid and asiaticoside content by 74.3% and 56.4%, respectively. Although the asiaticoside content decreased by 26.2%, the total saponin content increased by 88.5%. Compared to the MY-9 fermentation process without added nitrogen source, the asiaticoside content increased by 112%, the asiaticoside content increased by 1.55%, the asiatic acid content increased by 32.5%, the asiaticoside content increased by 24.6%, and the total saponin content increased by 51.9%.

[0067] Based on the growth status and bioactive substance yield of the strains, 1 g / L NH4Cl was determined as the nitrogen source for fermenting Centella asiatica.

[0068] Example 3: Antibacterial effect of Centella asiatica MY-9 fermentation broth

[0069] 1. Inhibitory effect against Staphylococcus aureus and Propionibacterium acnes

[0070] S. aureus and C. acnes were cultured in LB liquid medium at 37°C with shaking for 16 h, and then diluted with fresh LB liquid medium to a final bacterial concentration of 10. 6 CFU / mL. The 24-hour fermentation broth from the unfermented group and the 1 g / L NH4Cl group in Example 2 was serially diluted with LB medium to 50.0%, 25.0%, 12.5%, 6.25%, 3.13%, and 1.56% of the original fermentation broth concentration, respectively. Then, 100 μL of each gradient concentration of fermentation broth was added to 100 μL of diluted *S. aureus* bacterial culture and incubated at 37°C for 24 h. A blank control was also set up, in which 100 μL of diluted bacterial culture was added to 100 μL instead of the fermentation broth. The OD of each well was measured. 600 The antibacterial rate is calculated using the following formula.

[0071]

[0072] In addition, compared with the blank control, the minimum inhibitory concentration (MIC) of the fermentation broth is defined as the concentration of the fermentation broth at which no pathogenic bacteria cells are observed to grow with the naked eye.

[0073] The antibacterial rate results are as follows Figure 3 As shown, under the same dilution concentration, the antibacterial rate of Centella asiatica MY-9 fermented broth was significantly higher than that of unfermented Centella asiatica (p<0.05). Furthermore, the MIC values ​​of unfermented Centella asiatica against both pathogenic bacteria were 50%, while the MIC of Centella asiatica MY-9 fermented broth was 25%, indicating that the MY-9 fermented broth had a more significant antibacterial effect than the unfermented Centella asiatica. These results demonstrate that the ability of MY-9-fermented Centella asiatica to inhibit Staphylococcus aureus and Propionibacterium acnes was significantly enhanced.

[0074] 2. Inhibitory effect on biofilm formation of Staphylococcus aureus and Propionibacterium acnes

[0075] S. aureus and C. acnes were cultured in LB liquid medium at 37°C with shaking for 16 h, and then diluted with fresh LB liquid medium to a final bacterial concentration of 10. 6 CFU / mL. The 24-hour fermentation broth from the unfermented group and the 1 g / L NH4Cl group in Example 2 was serially diluted with LB medium to 50.0%, 25.0%, 12.5%, 6.25%, 3.13%, and 1.56% of the original fermentation broth concentration, respectively. Then, 100 μL of each gradient concentration of fermentation broth was added to 100 μL of diluted *S. aureus* bacterial culture and incubated at 37°C for 24 h. A blank control was also provided, in which 100 μL of LB liquid medium was added to 100 μL of diluted bacterial culture instead of fermentation broth. After incubation, the culture medium was discarded, and each well was washed twice with sterile PBS buffer to remove airborne bacteria. Then, 200 μL of 99% methanol was added to each well for fixation for 20 min, the methanol was discarded, and the wells were air-dried. 200 μL of 1% crystal violet solution was added to each well for staining for 20 min, followed by gentle washing with distilled water several times to remove unbound dye. Then, 200 μL of 33% glacial acetic acid was added, and the stained biofilm was dissolved by shaking. After 30 min, the absorbance was measured at a wavelength of 590 nm to assess the biofilm formation.

[0076] Biomembrane inhibition assay results are as follows Figure 4 As shown, the fermentation broth of Centella asiatica MY-9 diluted to 1.56%–50.0% of the original concentration significantly inhibited the biofilm formation of *S. aureus*, and the fermentation broth diluted to 3.13%–50.0% of the original concentration also significantly inhibited the biofilm formation of *C. acnes*. This inhibitory effect was concentration-dependent. Compared with unfermented Centella asiatica, the fermentation broth diluted to 3.13%–50.0% of the original concentration significantly inhibited biofilm formation (p<0.05).

[0077] Example 4: Inhibitory effect of Centella asiatica MY-9 fermentation broth on MMP-1 and MMP-9

[0078] The 24-hour fermentation broth from the unfermented group and the 1 g / L NH4Cl group in Example 2 were diluted to 25% and 2.5% of their original concentrations, respectively, and used as test samples. The incubated MMP-1 / MMP-9 was diluted 5-fold with buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w / v) Brij-35, pH 7.5) and immediately placed on ice, following the steps in Tables 1 and 2. A positive control group was also set up, except that the test sample was replaced with the same volume of 0.1 nM or 0.01 nM ilomasta solution in the inhibitor group A1. The microplate reader was maintained at a constant temperature of 37°C. After starting the reaction, continuous measurements were performed at an excitation wavelength of 490 nm, an emission wavelength of 525 nm, and a gain of 75, measuring fluorescence intensity every 1 minute for 1 hour. The inhibition rate was calculated using the following formula.

[0079]

[0080] Among them, F TN1 : at time T N1 Fluorescence value at F; TN0 : at time T N0 Fluorescence value under T; N0 : Time 0; T N1 : The Nth moment; S B1 S: The slope calculated from the enzyme control group; A1 S: The slope calculated from the inhibitor group; B0 The slope calculated from the blank sample group.

[0081] Table 1: Inhibition experiments of MMP-1

[0082]

[0083] Table 2: Inhibition experiments of MMP-9

[0084]

[0085]

[0086] like Figure 5As shown, different concentrations of Centella asiatica MY-9 fermentation broth (25% and 2.5%) all exhibited inhibitory effects on MMP-1 and MMP-9. For MMP-9, both the Centella asiatica MY-9 fermentation broth and unfermented Centella asiatica significantly inhibited MMP-9 activity. Although there was no significant difference in inhibition rate between the two at the same concentration (p>0.05), the activity of the Centella asiatica MY-9 fermentation broth was slightly higher than that of the unfermented Centella asiatica. The inhibitory effect at a 25% concentration was significantly better than that of 0.01 nM ilomastartrazine (p<0.05). For MMP-1, the Centella asiatica MY-9 fermentation broth showed a significant inhibitory effect, while the unfermented Centella asiatica showed almost no inhibitory effect. The inhibitory effect of the Centella asiatica MY-9 fermentation broth at a 25% concentration was significantly better than that of 0.01 nM ilomastartrazine (p<0.05).

[0087] Example 5: Soothing effect of Centella Asiatica MY-9 fermented liquid

[0088] Kallikrein-associated enzyme-7 (KLK7) belongs to the secretory serine protease family and is expressed in various tissues. KLK7 is initially found in the skin and plays an important role in stratum corneum formation and desquamation. KLK7 can degrade intracellular linker molecules such as deltaminin, desmosome core protein 1, and desmosome glialin 1, leading to desquamation. Abnormal regulation of KLK7 can cause pathophysiological inflammation of the skin, thereby triggering diseases such as psoriasis, chronic skin diseases, and Netherton's syndrome. In this embodiment, a fluorescent substrate method was used to detect the inhibitory activity of Centella asiatica MY-9 fermentation broth on KLK7.

[0089] The 24-hour fermentation broth from the unfermented group and the 1 g / L NH4Cl group in Example 2 were diluted to 25% and 2.5% of their original concentrations, respectively, and used as test samples. The reaction system was prepared according to Table 3 and the reaction was carried out according to the steps. In step S1, the components of the detection buffer in the reaction system were: 50 mM Tris-HCl, 0.01% Tween-20, 150 mM NaCl, and pH adjusted to 8.0; the substrate was the polypeptide sequence Mca-RPKPVE-Nval-WRK(Dnp)-NH2 with the fluorescent group Dnp. KLK7 was treated before being added to the reaction system by mixing equal volumes of 200 μg / mL rhKLK7 and 20 μg / mL Thermolysin, and incubating at 37°C for 3 hours to activate the enzyme.

[0090] Table 3: KLK7 Inhibitory Activity Reaction System and Procedures

[0091]

[0092]

[0093] Calculate the KLK7 inhibition rate using the following formula.

[0094] Fluorescence slope S = (F1 - F0) / (T1 - T0)

[0095] Where F1 is the fluorescence intensity at the reaction endpoint, F0 is the fluorescence intensity at the reaction initiation point, and T1-T0 is the reaction time.

[0096]

[0097] The inhibition rate calculation results are as follows Figure 6 As shown, there was no significant difference in the inhibition rate of KLK7 between unfermented Centella asiatica and Centella asiatica MY-9 fermentation broth at a concentration of 25%, while the inhibition rate of Centella asiatica MY-9 fermentation broth at a concentration of 2.5% was significantly higher than that of unfermented Centella asiatica (p<0.05). This indicates that the increased content of active substances after fermentation makes Centella asiatica MY-9 fermentation broth exhibit more significant soothing biological activity than unfermented Centella asiatica at lower dilution concentrations.

[0098] Example 6: Moisturizing effect of Centella Asiatica MY-9 fermented liquid

[0099] In this embodiment, qPCR was used to detect the effect of Centella asiatica MY-9 fermentation broth on the expression of moisturizing-related genes. The expression of moisturizing-related genes (HAS1 and HAS2) was detected by qPCR, and the relative mRNA expression levels of HAS1 and HAS2 genes were calculated using YWHAZ as an internal reference gene.

[0100] The 24-hour fermentation broths of the unfermented group and the 1 g / L NH4Cl group from Example 2 were diluted with serum-free medium (DMEM) to 2.5% of their original concentration and used as test samples.

[0101] Fibroblasts were placed in cell culture flasks, and 5 mL of cell culture medium (84% DMEM medium + 15% fetal bovine serum + 1% penicillin-streptomycin solution, v / v) was added. The flasks were incubated at 37°C in a 5% CO2 incubator. Cell growth was observed under an inverted microscope. When the cells reached 80%–90% confluence, they were seeded. Cells were then seeded into 6-well plates at a density of 25 × 10⁶ cells / well. 4 Cells / mL, 1 mL per well, cultured for 24 h. Discard the culture medium, wash once with PBS, add the test samples, positive control group (0.5 g / L ascorbic acid and hyaluronic acid prepared with serum-free medium), blank group and model group (added with serum-free medium), cultured in a CO2 incubator for 24 h, washed once with PBS, then added TRIzol Reagent to extract total RNA from cells, and detected the expression of related genes by qPCR.

[0102] The detection and calculation results are as follows Figure 7As shown, for HAS1, both unfermented Centella asiatica and Centella asiatica MY-9 fermentation broth significantly upregulated HAS1 expression, and the expression level was superior to that of ascorbic acid and hyaluronic acid (p<0.05); for HAS2, Centella asiatica MY-9 fermentation broth also significantly upregulated HAS2 expression (p<0.05), and the expression level was superior to that of hyaluronic acid and unfermented Centella asiatica (p<0.05).

[0103] Example 7: In vitro antioxidant effect of Centella asiatica MY-9 fermentation broth

[0104] Take the 24h fermentation broth from the unfermented group and the 1g / L NH4Cl group in Example 2, and serially dilute them with LB medium to 50.0%, 25.0%, 12.5%, 6.25%, 3.13%, and 1.56% of the original fermentation broth concentration, respectively, or do not dilute them, and use them as test samples.

[0105] Accurately weigh 0.020 g K₂S₂O₈ and 0.1152 g ABTS, and dissolve them in 30 mL of 0.1 M PBS (pH 7.4, 0.15 M NaCl). React at room temperature in the dark for 16 h to produce ABTS. + Free radical scavenging. Before the experiment, the above ABTS solution was diluted with anhydrous ethanol to a wavelength of 734 nm with an absorbance of 0.70 ± 0.02. 200 μL of the diluted ABTS solution was added to a 96-well plate, followed by 2 μL of the test sample. The plate was shaken to mix thoroughly in a microplate reader, and the reaction was carried out in the dark for 6 min. The absorbance at 734 nm was measured immediately afterward. A blank control group was established by adding 2 μL of deionized water instead of the test sample; a positive control group was established by adding 100 μL of ascorbic acid (0.5 g / L) instead of the test sample. The ABTS free radical scavenging capacity was calculated using the following formula.

[0106]

[0107] Add 100 μL of the test sample and 100 μL of 200 μM DPPH solution to a 96-well plate, mix thoroughly by shaking, and react at 37°C in the dark for 30 min. A blank control group was established by adding 100 μL of deionized water instead of the test sample; a control group was established by adding 100 μL of anhydrous ethanol instead of the test sample; and a positive control group was established by adding 100 μL of ascorbic acid (0.5 g / L) instead of the test sample. After the reaction was terminated, the absorbance was measured at 517 nm. The DPPH radical scavenging capacity was calculated using the following formula.

[0108]

[0109] like Figure 8As shown in Figure A, at a 100% concentration, the ABTS free radical scavenging ability of Centella asiatica MY-9 fermented broth and unfermented Centella asiatica was not significantly different from that of the positive control group (p>0.05), demonstrating superior antioxidant potential. Figure 8 As shown in Figure B, in the DPPH free radical scavenging experiment, the scavenging ability of 100% concentration of Centella asiatica MY-9 fermentation broth was significantly better than that of unfermented Centella asiatica and the positive control group (p<0.05), indicating that fermentation significantly enhances the antioxidant capacity of Centella asiatica. Furthermore, at most dilution concentrations, the antioxidant capacity of Centella asiatica MY-9 fermentation broth was significantly better than that of unfermented Centella asiatica (p<0.05). The antioxidant capacity of Centella asiatica MY-9 fermentation broth and unfermented Centella asiatica showed a certain non-linear dose-dependent relationship; both exhibited strong antioxidant capacity, especially in DPPH scavenging, even at low concentrations.

[0110] Example 8: Determination of the cellular antioxidant effect of Centella asiatica MY-9 fermentation broth using CAA method

[0111] In this embodiment, the in vivo antioxidant effect of Centella asiatica MY-9 fermentation broth was determined using the CAA (Cellular Antioxidant Activity) method.

[0112] HaCaT cells were cultured in cell culture flasks. When the cells grew to 80%–90% of the flask's capacity, they were seeded into 96-well plates at a density of 10-1. 5 100 μL per well, cultured for 24 h. Take the 24 h fermentation broth from the unfermented group and the 1 g / L NH4Cl group in Example 2, dilute with culture medium to 2.5% of the original concentration, and use as the test samples. Set up blank groups, oxidation groups, sample groups, and positive control groups, and perform the procedures S1-S4 in Table 4 for each group. Maintain a constant temperature of 37℃ using a microplate reader. After starting the reaction in step S3, continuously measure fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 538 nm, measuring the fluorescence intensity every 5 min for 1 h.

[0113] Table 4: CAA Experimental Protocol

[0114] Calculate the CAA value using the following formula.

[0115]

[0116] Among them, AUC A Area under the fluorescence curve of the sample group; AUC B Area under the fluorescence curve (AUC) of the blank control group C Area under the fluorescence curve of the oxidation control group.

[0117] The measurement results are as followsFigure 9 As shown, unfermented Centella asiatica at a concentration of 2.5% has a certain antioxidant capacity, but Centella asiatica fermented with strain MY-9 is more effective in reducing ROS levels (p<0.05).

[0118] Example 9: Cellular antioxidant stress effect of Centella asiatica MY-9 fermentation broth

[0119] HaCaT cells were cultured in cell culture flasks. When the cells reached 80%–90% confluence, they were seeded into 6-well plates at a density of 25 × 10⁶ cells / well. 4 Cells / mL, 100 μL per well, cultured for 24 h. The 24 h fermentation broth from the unfermented group and the 1 g / L NH4Cl group in Example 2 was diluted to 2.5% of the original concentration with serum-free medium as the test sample. An oxidative stress cell model was constructed by UVB irradiation, and the 2.5% concentration of the test sample was added after UVB irradiation. A blank group was set up using unirradiated cells from the same batch, with serum-free medium added instead of the test sample; a model group was set up with serum-free medium added instead of the test sample; and a positive control group was set up with serum-free medium diluted with ascorbic acid solution added instead of the test sample. After culturing in a CO2 incubator for 24 h, GSH, GSH-Px, and other indicators were detected and analyzed according to the test kit requirements. The results are as follows: Figure 10 As shown.

[0120] Figure 10 The results showed that, due to UVB irradiation, HaCaT cells in the model group exhibited significantly decreased GSH-Px activity, reduced GSH content, and significantly increased MDA content, indicating a significant oxidative stress response in the cells. Compared to the model group, both unfermented Centella asiatica and Centella asiatica MY-9 fermentation broth significantly increased SOD and GSH-Px activities and GSH content (p<0.05), and decreased MDA levels (p<0.05). Specifically, in terms of enhancing SOD activity, Centella asiatica MY-9 fermentation broth was significantly superior to unfermented Centella asiatica, reaching levels close to ascorbic acid; regarding the reduction of MDA levels, the performance of unfermented Centella asiatica MY-9 fermentation broth was significantly superior to unfermented Centella asiatica, exceeding that of the positive control, demonstrating its unique advantage in alleviating lipid peroxidation damage.

[0121] Example 10: Anti-inflammatory effect of Centella Asiatica MY-9 fermentation broth

[0122] Cell culture and UVB induction were performed according to the method in Example 9. The positive control group received 0.5 mg / mL dexamethasone or 0.5 mg / mL asiaticoside solution diluted in serum-free medium. After culturing in a CO2 incubator for 24 h, the cell supernatant was collected and the cytokine levels in the supernatant were detected using an ELISA kit. Cells were harvested, total RNA was extracted, and reverse transcription was performed to obtain cDNA. The expression of cytokine-related genes was then detected by qPCR. Cytokines were categorized into pro-inflammatory and anti-inflammatory factors. Pro-inflammatory factors included TNF-α, IL-6, IL-8, IL-1α, IL-1β, PGE2, and COX-2, while anti-inflammatory factors included IL-10 and IL-37.

[0123] Effects on cytokine mRNA expression levels, such as Figure 11 As shown in the figure. Regarding pro-inflammatory factors, compared with the model group, the Centella asiatica MY-9 fermentation broth significantly downregulated the mRNA expression levels of TNF-α, IL-6, IL-8, IL-1α, IL-1β, and COX-2, while unfermented Centella asiatica only significantly downregulated the mRNA expression levels of IL-1α, IL-1β, and COX-2. Although both significantly upregulated the mRNA expression levels of anti-inflammatory factors IL-10 and IL-37, the upregulation effect of Centella asiatica MY-9 fermentation broth on IL-10 was significantly better than that of unfermented Centella asiatica (p<0.5). In addition, the regulatory effect of Centella asiatica MY-9 fermentation broth on the expression of IL-8 and IL-10 was comparable to that of the positive control dexamethasone and better than that of the positive control asiaticoside; its regulatory effect on the expression of TNF-α was better than that of the positive control asiaticoside.

[0124] Effects on cytokine secretion levels, such as Figure 12 As shown. Regarding pro-inflammatory factors, the fermentation broth of Centella asiatica strain MY-9 significantly affected the secretion levels of TNF-α, IL-8, IL-β, and PGE2 compared to unfermented Centella asiatica (p<0.05), with its effect on IL-β secretion levels comparable to the positive control. Regarding anti-inflammatory factors, the fermentation broth of Centella asiatica strain MY-9 significantly affected the secretion levels of IL-10 and IL-37 compared to unfermented Centella asiatica (p<0.05). The regulatory effects of Centella asiatica fermentation broth MY-9 on TNF-α, IL-6, IL-8, IL-1β, and IL-10 were superior to the positive controls dexamethasone and asiaticoside; its regulatory effect on IL-1β was comparable to the positive control dexamethasone and superior to the positive control asiaticoside.

[0125] The combined effects on the mRNA expression and secretion levels of cytokines demonstrate that Centella asiatica MY-9 fermentation broth has excellent anti-inflammatory effects.

[0126] In summary, the total content of characteristic active ingredients in Centella asiatica culture medium supplemented with 1 g / L NH4Cl after fermentation with *Lactobacillus plantarum* strain MY-9 increased by 88.5%. Compared with unfermented Centella asiatica, the Centella asiatica MY-9 fermented broth showed significantly enhanced inhibitory activity against the growth and film formation of *Staphylococcus aureus* and *Propionibacterium acnes*, inhibitory activity against key skin aging targets MMP-1 and MMP-9, inhibitory activity against the soothing target KLK7, and other bioactivities such as moisturizing, antioxidant, and anti-inflammatory effects. Therefore, Centella asiatica MY-9 fermented broth has broad application prospects in skincare products, daily necessities, food, health products, and pharmaceuticals.

Claims

1. A Centella asiatica fermentation product, characterized in that, The Centella asiatica fermentation product is obtained by fermenting raw materials containing Centella asiatica using Lactiplantibacillus plantarum.

2. The Centella asiatica fermentation product as described in claim 1, characterized in that, The fermentation process uses NH4Cl as the nitrogen source.

3. The Centella asiatica fermentation product as described in claim 2, characterized in that, The fermentation process uses NH4Cl as the nitrogen source at a concentration of 0.5–5.0 g / L.

4. The application of the Centella asiatica ferment product as described in any one of claims 1 to 3 in health products, food, cosmetics, or daily necessities, characterized in that, The Centella asiatica ferment product is used as one or more components selected from antibacterial, anti-inflammatory, antioxidant, anti-wrinkle, anti-aging, soothing, and moisturizing components.

5. The use of the Centella asiatica ferment product as described in any one of claims 1 to 3 in the preparation of a medicament for inhibiting the activity of MMP-1, MMP-9 or KLK7, or for treating abnormal expression of HAS1, HAS2, TNF-α, IL-6, IL-8, IL-1α, IL-1β, PGE2, COX-2, IL-10 or IL-37 or abnormal secretion of TNF-α, IL-6, IL-8, IL-1α, IL-1β, PGE2, COX-2, IL-10 or IL-37 proteins.

6. A composition, characterized in that, The composition comprises the Centella asiatica fermentation product as described in any one of claims 1 to 3 and an appropriate amount of excipients; the composition is a pharmaceutical composition, a health product composition, a food composition, a cosmetic composition, or a daily necessities composition.

7. The use of the composition according to claim 6 in health products, food, cosmetics or daily necessities, characterized in that, The composition is used as one or more ingredients selected from antibacterial ingredients, anti-inflammatory ingredients, antioxidant ingredients, anti-wrinkle ingredients, anti-aging ingredients, soothing ingredients, and moisturizing ingredients.

8. The use of the composition of claim 6 in the preparation of a medicament for inhibiting the activity of MMP-1, MMP-9 or KLK7, or for treating abnormal expression of HAS1, HAS2, TNF-α, IL-6, IL-8, IL-1α, IL-1β, PGE2, COX-2, IL-10 or IL-37 or abnormal secretion of TNF-α, IL-6, IL-8, IL-1α, IL-1β, PGE2, COX-2, IL-10 or IL-37 proteins.

9. A method for fermenting Centella asiatica, characterized in that, The method includes fermenting raw materials containing Centella asiatica using Lactiplantibacillus plantarum.

10. The method as described in claim 9, characterized in that, The fermentation process uses NH4Cl as the nitrogen source.

11. A *Lactiplantibacillus plantarum* MY-9, characterized in that, The preservation number of the *Lactobacillus plantarum* MY-9 is CCTCC M 20251284.

12. The application of Lactiplantibacillus plantarum MY-9 as described in claim 11 in the fermentation of Centella asiatica.

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