Skin anti-aging and firming composition, oil cream and preparation method of skin anti-aging and firming composition and oil cream

Abstract

The invention provides a skin anti-aging and firming composition, oil cream and a preparation method thereof, and belongs to the field of medical, dental or cosmetic preparations. The composition comprises a palmaria palmata and inula flower extract, a vaccinium macrocarpum fruit extract, a medicine root extract and the palmaria palmata and inula flower extract, palmaria palmata and inula flower serve as fermentation substrates, aerobic fermentation is conducted through aspergillus niger, and anaerobic fermentation is conducted through pediococcus pentosaceus; and purifying to obtain the product. According to the palmaria palmata inula flower fermentation extract provided by the invention, through sequential fermentation of aspergillus niger and pediococcus pentosaceus, deep biotransformation of a plant matrix is realized. The extract has the beneficial effects that the moisturizing and repairing characteristics of palmaria palmata and the anti-inflammatory and anti-oxidation characteristics of inula flower are fused, new active matters such as lactic acid and small molecule peptide are generated through microbial conversion, and the comprehensive effect is obviously superior to that of a traditional extract; macromolecules are converted into micromolecules easier to absorb through fermentation, bioavailability is improved, the pH value of the product is close to the skin, and the product is mild and low in irritation.

Description

Technical Field

[0001] This invention belongs to the field of medical, dental or cosmetic formulations, and particularly relates to a skin anti-aging and firming composition, cream and its preparation method. Background Technology

[0002] With the rapid development of science and technology and the continuous improvement of living standards, people's demand for health and wellness is increasing. Oxidative stress, as a mechanism closely related to various diseases and the aging process, has received widespread attention in recent years. The research and application of antioxidants has become a hot field, with natural plant extracts becoming an important direction in antioxidant research due to their rich diversity, low toxicity, and wide availability. In-depth research into the chemical components of natural plants has revealed that many plant extracts are rich in antioxidant active substances, such as polyphenols and flavonoids, which have the ability to scavenge free radicals and inhibit oxidation reactions.

[0003] There is a direct and close causal relationship between antioxidation and skin anti-aging. Oxidative stress is a core driver of skin aging, while antioxidant mechanisms are a key defense against this process. Skin aging is the result of multiple factors, including oxidative damage, collagen loss, and inflammatory responses. Therefore, achieving effective skin anti-aging requires a combination of approaches. Summary of the Invention

[0004] The first objective of this invention is to provide an extract of Inula japonica from the palm-leaf tree, which is obtained by using palm-leaf tree and Inula japonica as fermentation substrates, followed by aerobic fermentation by Aspergillus niger and then anaerobic fermentation by Pediococcus pentosus, and finally purification.

[0005] Preferably, the preparation method includes the following steps: S1: A fermentation medium is prepared by using palm-leaved tree and Inula japonica as fermentation substrates and adding inorganic salts; the inorganic salts include phosphorus, potassium, magnesium and manganese; S2: The activated Aspergillus niger spore suspension was inoculated into the culture medium and aerobic fermentation was carried out to obtain the primary fermentation product; S3: Inoculate the activated Pediococcus pentosaceus seed liquid into the primary fermentation product, and then ferment anaerobically to obtain the secondary fermentation product; S4: After the secondary fermentation product is inactivated, the filtrate is filtered and collected; the filtrate is concentrated to obtain the extract of Inula japonica.

[0006] In step S1: Of the fermentation substrate, *Palmeria palmatum* by mass ≥70%; The amount of inorganic salts added is calculated based on the fermentation substrate and includes: At least 0.2% potassium dihydrogen phosphate; At least 0.1% magnesium sulfate heptahydrate; At least 0.1% ammonium sulfate; At least 0.04% manganese sulfate monohydrate; Furthermore, it is sterilized after being prepared into a fermentation medium.

[0007] In step S2: The inoculum amount of the Aspergillus niger spore suspension is at least 5% of the mass of the fermentation medium; The conditions for the aerobic fermentation are: temperature 30±1℃, aeration rate of at least 1.0 vvm; fermentation time of at least 48 hours. Aerobic fermentation is complete when the pH of the fermentation product drops to ≤3.5.

[0008] In step S3: After aerobic fermentation is complete, the air in the fermentation environment is replaced to create an anaerobic environment. The inoculum size of the *Pediococcus pentosaceus* seed culture is at least 5% of the mass of the fermentation medium; The conditions for the anaerobic fermentation are: temperature 37±1℃; anaerobic environment; fermentation time of at least 96 hours. Fermentation is complete when the pH of the fermentation product changes by ≤0.1 within 6 hours.

[0009] In step S4: The inactivation is performed by treating at 70-75°C for at least 30 minutes; The filtration process involves centrifuging the secondary fermentation product to separate the solid and liquid components, and then taking the liquid portion. The liquid portion is then filtered through a microporous membrane to obtain a sterile filtrate. The concentration is achieved by vacuum low-temperature concentration of the filtrate at ≤50°C to ≤20% of the original volume.

[0010] After concentration in step S4, the concentrate is further freeze-dried.

[0011] A second object of the present invention is to provide a skin composition comprising, by weight, the following components: Inula japonica extract 10-15 Large blueberry fruit extract 5-10 Peony root extract 2-4; The extract of Inula japonica from the palm-leaf tree was obtained by using palm-leaf tree and Inula japonica as fermentation substrates, followed by aerobic fermentation by Aspergillus niger and then anaerobic fermentation by Pediococcus pentosus; the result was purified.

[0012] A third objective of this invention is to provide the application of the aforementioned skin composition in the preparation of cosmetics, primarily for the preparation of cosmetics with anti-aging and firming effects on the skin.

[0013] A fourth object of the present invention is to provide an oil cream comprising a base and an active ingredient, wherein the active ingredient comprises, by weight, the following components: Inula japonica extract 10-15 Large blueberry fruit extract 5-10 Peony root extract 2-4; The extract of Inula japonica from the palm-leaf tree was obtained by using palm-leaf tree and Inula japonica as fermentation substrates, followed by aerobic fermentation by Aspergillus niger and then anaerobic fermentation by Pediococcus pentosus; the result was purified.

[0014] This invention designs a cosmetic active extract prepared through a specific two-stage sequential microbial fermentation process using Palmaria palmata (also known as palmate red algae), a plant with both medicinal and edible properties, and Inula japonica, a traditional medicinal plant, as a composite substrate.

[0015] The principle of this invention is as follows: The ingredient combination avoids the limitation of using a single plant ingredient, which may have a narrower efficacy spectrum. Palmleaf algae, a type of red algae, is rich in sulfated polysaccharides (such as agar precursors), phycoerythrin, minerals, and vitamins; its extracts are known to have excellent moisturizing, film-forming, and soothing potential. Inula japonica, a member of the Asteraceae family, is rich in terpenoids (such as inula lactones), flavonoids (such as quercetin and kaempferol derivatives), and polysaccharides such as inulin; it is commonly used in traditional medicine for anti-inflammatory, antioxidant, and skin repair purposes. The combination of these two ingredients aims to integrate the moisturizing and repairing properties of seaweed with the anti-inflammatory and antioxidant properties of Asteraceae plants, resulting in comprehensive skincare benefits encompassing moisturizing, soothing, brightening, and anti-aging.

[0016] This invention abandons the common single extraction (water extraction, alcohol extraction) or single fermentation mode in the preparation of plant raw materials for cosmetics; it adopts a combination of Aspergillus niger and Pediococcus pentosaceus, and follows the sequential process of "aerobic fermentation first, followed by anaerobic fermentation". This method simulates the ecological process of organic matter degradation and transformation in nature, enabling the two microorganisms to complete the decomposition, transformation and enhancement of active substances in complex plant matrices in stages under different oxygen environments and pH conditions.

[0017] To ensure the controllability of the fermentation target, this invention sets clear quantitative parameters throughout the entire process, which not only ensures the reproducibility of the process and the batch stability of the product, but also enables the targeted enrichment and generation of target active molecules through a controllable biotransformation process.

[0018] The reasons for selecting the fermentation microorganisms in this invention are as follows: (a) The irreplaceable advantages of Aspergillus niger as the dominant strain in aerobic fermentation Among the many filamentous fungi that can be used for plant fermentation (such as Aspergillus oryzae, Trichoderma, Rhizopus, etc.), Aspergillus niger was chosen as the "cell wall disruptor" and "conversion engine" for the first stage, mainly based on the following considerations: Aspergillus niger is recognized as a "cell factory" in industrial microbiology, its most prominent feature being its ability to secrete a wide variety of highly active hydrolytic enzymes into the extracellular space. This perfectly matches the core requirement of the first stage of fermentation: to completely break down the tough cell walls of *Inula japonica* (rich in cellulose, hemicellulose, and complex polysaccharides) and *Inula japonica* (rich in inulin and cellulose).

[0019] Compared to *Aspergillus oryzae*, which is primarily used for producing proteases and amylases, *Aspergillus niger* typically exhibits a greater capacity for producing cellulase, pectinase, and hemicellulase. Compared to *Trichoderma*, *Aspergillus niger* has a broader enzyme spectrum, greater adaptability to complex substrates, and its fermentation broth is generally easier to process later. The inulinase secreted by *Aspergillus niger* can efficiently degrade inulin in *Inula japonica*, converting it into prebiotic fructooligosaccharides and directly usable monosaccharides. This step provides the initial, readily available carbon source for the entire fermentation system and is crucial for initiating efficient fermentation.

[0020] The first stage of this process requires a final pH of ≤3.5, which is a highly acidic environment. *Aspergillus niger* exhibits strong tolerance to low pH and relatively low temperatures, allowing it to grow vigorously and continuously produce enzymes even under conditions where the growth of many other microorganisms is inhibited. This tolerance ensures that *Aspergillus niger* maintains absolute dominance throughout the 48-72 hour fermentation process, effectively suppressing contamination and guaranteeing the purity of the fermentation. Its mycelial growth pattern also helps form a network in the fermentation broth, increasing the contact area with the solid substrate and improving decomposition efficiency.

[0021] During its metabolism, Aspergillus niger primarily produces organic acids such as citric acid and gluconic acid. These acids have a pure taste and are themselves mild chelating agents and pH adjusters commonly used in skincare products. More importantly, the formation of these organic acids is gradual and controllable, allowing the pH of the fermentation system to steadily decrease to the target value rather than changing drastically. This helps protect some pH-sensitive plant active ingredients. In contrast, some fungi may produce bitter peptides or volatile substances with unpleasant odors, affecting the sensory quality of the final product.

[0022] (II) Strategic selection of Pediococcus pentosaceus as the dominant strain in anaerobic fermentation In the second stage of anaerobic fermentation, Pediococcus pentosaceus was chosen instead of the more common Saccharomyces cerevisiae or Lactobacillus plantarum because of considerations for the fermentation objectives: the core task of this stage is not to produce ethanol or pursue a single acid production rate, but to stabilize the environment, transform precursors, and produce a metabolic profile with specific skin benefits.

[0023] Saccharomyces cerevisiae is a typical ethanol-fermenting yeast that converts the vast majority of sugars into ethanol and carbon dioxide under anaerobic conditions. Although ethanol itself can be used as a solvent, high levels of ethanol in cosmetic ingredients can cause skin dryness and irritation, requiring additional removal processes.

[0024] Pediococcus pentosaceus, a homofermentative lactic acid bacterium, efficiently converts sugars into lactic acid. Lactic acid is an important component of the skin's natural moisturizing factor (NMF), possessing multiple benefits including excellent moisturizing, gentle keratin renewal, and regulation of the skin's microecology (serving as an ideal component of the skin's slightly acidic sebum film).

[0025] Pediococcus pentosaceus continuously produces acid, which can stabilize the pH at a low level (about 3.8-4.2). This provides a natural and mild acidic preservation environment for the final product, which is conducive to inhibiting miscellaneous bacteria, stabilizing active ingredients, and making its pH value closer to the pH of healthy skin, thereby improving the skin-friendliness of the formula.

[0026] Although belonging to the same lactic acid bacteria family, *Pediococcus pentosaceus* possesses some unique physiological characteristics: it typically has a shorter lag phase, allowing it to adapt to the environment more quickly and initiate vigorous fermentation. It exhibits extremely high tolerance to acidic environments (created by the *Aspergillus niger* stage), seamlessly transitioning to a dominant microbial population even under harsh conditions of pH ≤ 3.5.

[0027] In addition to producing lactic acid, *Pediococcus pentosaceus* strains can also produce antimicrobial peptides such as bacteriocins, which provides a weak, natural auxiliary preservative potential for the final product. Their metabolic profile is relatively pure, producing fewer complex gases or byproducts, which is beneficial for obtaining fermentation broths with pure flavor and stable properties.

[0028] The clear division of ecological niche between Aspergillus niger and the fungi, which decompose first and the bacteria transform later, avoids the carbon source competition or metabolic inhibition that may occur when lactic acid bacteria and yeast co-ferment, making the whole process more linear, controllable and efficient.

[0029] (III) Synergistic effect of strain combinations Combining Aspergillus niger with Pediococcus pentosaceus and applying a controlled aerobic-to-anaerobic sequence produced a series of synergistic effects that went beyond those of single-species or conventional co-fermentation: Aspergillus niger's powerful enzymatic system performs the most challenging physical and chemical decomposition work, converting macromolecules into "pre-made" nutrient substrates (small sugars, peptides, and amino acids) that lactic acid bacteria can directly utilize. Pediococcus pentosus, on the other hand, focuses on converting these precursors into target active molecules (such as lactic acid and modified phenolic compounds). This division of labor allows both microorganisms to maximize their effectiveness under their respective optimal conditions.

[0030] Self-programming and driving force of pH curves: The pH change curve of the fermentation system is designed as an intrinsic driving force and indicator of the process. *Aspergillus niger* lowers the pH from near neutral to strongly acidic (≤3.5). This process not only fulfills its own purpose but also creates a "selective pressure" environment for the inoculation of *Pediococcus pentosaceus*—inhibiting most acid-intolerant bacteria. Subsequent fermentation by *Pediococcus pentosaceus* leads to a new dynamic equilibrium at an even lower pH level.

[0031] The final product not only contains the original active ingredients (such as small molecule polysaccharides, flavonoid aglycones, and terpenoids) released and modified after biotransformation of *Inula japonica* and *Inula rotundifolia*, but also includes contributions from two microorganisms: various organic acids, enzymatically produced functional peptides, and oligosaccharides from *Aspergillus niger*; and lactic acid, potential bacteriocins, and other metabolites from *Pediococcus pentosaceus*. These components together constitute a complex and synergistic matrix of natural active substances, whose efficacy potential far exceeds that of simple extraction or single microbial fermentation products.

[0032] In addition to providing a fermented extract of Inula japonica, this invention also provides a plant composition; the purpose is to systematically integrate active ingredients with different mechanisms of action and targeting different skin aging pathways to achieve a comprehensive anti-aging effect with multiple levels, multiple targets, and synergistic effects.

[0033] 1. Fermented extract of Inula japonica (10-15 parts) This component forms the technical basis of this composition; polysaccharides (such as sulfated polysaccharides) contained in *Inula japonica* help maintain the hydration of the stratum corneum by absorbing moisture and forming a thin film on the skin surface. Algal polysaccharides can affect skin fibroblasts and participate in the regulation of collagen synthesis. Flavonoids in *Inula japonica* undergo changes in molecular weight and polarity after microbial fermentation, increasing their skin permeability.

[0034] Flavonoids (such as quercetin) contained in Inula japonica have known in vitro antioxidant activity. The fermentation process converts some polyphenols into more bioavailable forms, thereby affecting their ability to scavenge free radicals.

[0035] The fermentation end products contain lactic acid and various organic acids, which help maintain the system and the skin surface at a slightly acidic pH range after application. This environment is similar to the physiological pH of the skin and is unfavorable for the colonization of some pathogenic microorganisms. The oligosaccharides produced during fermentation may affect the skin's symbiotic microbiota.

[0036] Blueberry fruit extract, with its high content of proanthocyanidins, anthocyanins, and other polyphenols, has been shown in multiple in vitro and model studies to possess significant free radical scavenging capabilities and inhibit lipid peroxidation. This mechanism helps protect skin cells and structural proteins (such as collagen and elastin) from damage by reactive oxygen species.

[0037] Paeonia lactiflora root extract, whose main active ingredient is paeoniflorin, has been shown in pharmacological studies to inhibit the production of inflammatory mediators (such as prostaglandin E2 and tumor necrosis factor-α) by regulating NF-κB and MAPK signaling pathways.

[0038] 1. Skin aging is the result of multiple factors, including oxidative damage, glycation, chronic inflammation, decreased barrier function, and extracellular matrix degradation. The three components mentioned above each focus on different but interconnected pathways: The fermented extract of Inula japonica focuses on basic barrier support and microenvironment balance, and provides potentially better skin condition for the action of subsequent active ingredients.

[0039] Large blueberry fruit extract focuses on combating oxidation and glycation, two direct biochemical reactions that cause protein damage.

[0040] Peony root extract focuses on inhibiting inflammatory pathways and breaking the vicious cycle between inflammation and oxidative stress.

[0041] The interactions between components can produce the following synergistic effects: The potential strengthening effect of Inula japonica fermentation extract on skin barrier function may create a more favorable physical and biochemical environment for the transdermal penetration and efficacy of other active ingredients.

[0042] Oxidative stress can trigger inflammation, and the inflammatory response can generate a large number of free radicals. The antioxidant effect of blueberry fruit extract can reduce oxidative stressors, while the anti-inflammatory effect of peony root extract can alleviate the inflammatory response. The two may form a bidirectional inhibitory loop, which theoretically can more effectively delay the aging process.

[0043] The Inula japonica fermentation extract provided by this invention achieves deep biotransformation of the plant matrix through sequential fermentation by Aspergillus niger and Pediococcus pentosaceus. Its beneficial effects lie in combining the moisturizing and repairing properties of Inula japonica with the anti-inflammatory and antioxidant properties of Inula japonica, and generating new active substances such as lactic acid and small molecule peptides through microbial transformation. The overall efficacy is significantly superior to traditional extracts. Fermentation transforms large molecules into more easily absorbed small molecules, improving bioavailability, and the product's pH is close to that of the skin, making it gentle and low-irritant. Detailed Implementation

[0044] To better understand the present invention, the present invention will be further described below with reference to specific serial numbers. The terminology used in the serial numbers is for describing specific embodiments and does not constitute a limitation on the scope of protection of the present invention.

[0045] In the specific implementation methods, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0046] Unless otherwise specified, percentages, % and other terms in the specific implementation method are assumed to be mass percentages.

[0047] The Aspergillus niger used in this invention is CCTCC AF 93027.

[0048] The Pediococcus pentosaceus used in this invention is CICC 22734.

[0049] The palm-leaved tree used in this invention has the Latin name Palmaria palmata; all parts are used.

[0050] The Latin name of the Inula japonica used in this invention is Inula japonica; the part used is the flower.

[0051] The INCI name of the vaccinium macrocarpon fruit extract used in this invention is vaccinium macrocarpon fruit extract. The INCI name of the peony root extract used in this invention is peony (PAEONIA LACTIFLORA) root extract.

[0052] The Inula japonica flower extract used in this invention has the INCI name Inula japonica flower extract.

[0053] The palmaria extract used in this invention has the INCI name of palmaria palmaria extract.

[0054] Example 1 The preparation method of Inula japonica complex fermentation product includes the following steps: S1: Dry the palm-leaf tree and Inula japonica separately, then pulverize them through a 40-mesh sieve; then mix them together at a mass ratio of 7:3. Then, based on the total mass of the palm-leaved tree and the Inula japonica, add the following percentages of inorganic salts by mass; 0.2% potassium dihydrogen phosphate; 0.1% magnesium sulfate heptahydrate; 0.1% ammonium sulfate; 0.04% manganese sulfate monohydrate; After adding the ingredients, add 15 times the total weight of water to the palm-leaf tree and Inula japonica, stir well, autoclave at 121°C for 20 minutes, and then cool to obtain the culture medium.

[0055] S2: Inoculate Aspergillus niger spores onto PDA slant medium and culture at 28-30°C for at least 3 days until the spores mature.

[0056] The spores were washed off with sterile saline to prepare a spore suspension, and the spore concentration was adjusted to ≥1×10⁻⁶. 6 per mL.

[0057] The spore suspension was inoculated into the culture medium at an inoculum rate of 5%.

[0058] Ferment under the following conditions: Temperature 30±1℃ Aeration rate of at least 1.0 vvm; slow stirring at 50 rpm.

[0059] Fermentation should last at least 48 hours; When the pH of the fermentation product drops to ≤3.5, and the fermentation liquid gradually thickens, turns yellowish-brown, and has the characteristic aroma of Aspergillus niger without any unpleasant odor, the aerobic fermentation is complete, and the primary fermentation product is obtained.

[0060] S3: Turn off the ventilation system of the fermentation vessel and switch to introducing sterile nitrogen for 5 minutes to displace the headspace air and establish an anaerobic environment.

[0061] The lyophilized powder of *Pediococcus pentosaceus* was inoculated into MRS liquid medium and incubated statically at 37°C for 18 hours until the bacterial culture became turbid (viable count ≥ 1 × 10⁻⁶). 8 CFU / mL); to obtain Pediococcus pentosaceus seed culture.

[0062] Inoculate the primary fermentation material, which has been switched to an anaerobic environment, with a 5% inoculum of Pediococcus pentosaceus seed culture; no sterilization is required during the process.

[0063] Ferment under the following conditions: Temperature 37±1℃; In an anaerobic environment, leave it to stand without stirring.

[0064] Fermentation for at least 96 hours; Fermentation is complete when the pH of the fermentation product changes by ≤0.1 within 6 hours; a secondary fermentation product is obtained.

[0065] S4: The secondary fermentation product was inactivated by treating it at 70-75℃ for at least 30 minutes; then the filtrate was coarsely filtered through a 100-mesh filter cloth. The filtrate was centrifuged at 6000 rpm for 15 min, and the supernatant was collected. The supernatant was filtered through three stages using filter membranes of 1.0 μm, 0.45 μm, and 0.22 μm to obtain a clear liquid. The clarified liquid is concentrated under vacuum at ≤50℃ to one-fifth of its original volume; then freeze-dried into powder to obtain the target product.

[0066] Example 2 The method for preparing the independent fermentation product of Inula japonica differs from that in Example 1 in that: In step S1, the palm-leaf tree and the Inula japonica are dried separately, and then inorganic salts of the same mass ratio as in Example 1 are added separately. After sterilization, separate palm-leaf tree culture media and Inula japonica culture media are obtained. Then, according to steps S2, S3, and S4 of Example 1, palm-leaf tree ferment and Inula japonica ferment are prepared; the palm-leaf tree ferment and Inula japonica ferment are mixed at a mass ratio of 7:3 to obtain palm-leaf tree and Inula japonica independent ferment.

[0067] Example 3 The method for preparing the fermented product of *Phyllostachys palmatus* differs from that in Example 1 in that: In step S1, only palm-leaf tree and inorganic salt mixture is used as fermentation medium; the amount of inorganic salt added is the same as in Example 1. Steps S2, S3, and S4 are the same as in Example 1.

[0068] Example 4 The preparation method of Inula japonica fermentate differs from that in Example 1 in that; In step S1, only palm-leaf tree and inorganic salt mixture is used as fermentation medium; the amount of inorganic salt added is the same as in Example 1. Steps S2, S3, and S4 are the same as in Example 1.

[0069] Example 5 The method for preparing aerobic fermentation products of Inula japonica differs from that in Example 1 in that: After step S2, step S3 is skipped, and step S4 is performed directly.

[0070] Example 6 The method for preparing the anaerobic ferment of Inula japonica differs from that in Example 1 in that: Step S2 is modified as follows: Add 1% of the compound enzyme by dry weight of the culture medium to the culture medium, and let it stand for 4 hours under the conditions of 50℃ and pH 4.5±0.2 (adjusted with citric acid); after enzymatic hydrolysis, adjust the pH to 3.5 with lactic acid; and then proceed with steps S3 and S4 of Example 1.

[0071] The complex enzyme contains cellulase, hemicellulase, pectinase, protease, and amylase in a mass ratio of 8:3:5:3; 1.

[0072] Experimental Example 1: Antioxidant Capacity Test Prepare the test samples according to the proportions in Table 1 below; use the DPPH free radical method to test their antioxidant capacity.

[0073] Table 1 ; The oils from the combined fermentation of *Inula japonica*, *Inula japonica*, *Inula japonica*, *Inula japonica* fermentation, *Inula japonica* fermentation, aerobic fermentation of *Inula japonica*, and anaerobic fermentation of *Inula japonica* in Table 1 were prepared in Examples 1-6. *Inula japonica* extract, *Inula japonica* flower extract, *Vaccinium bracteatum* fruit extract, and *Paeonia lactiflora* root extract were commercially available finished products.

[0074] The specific method is as follows: (1) Take the test sample of each serial number, mix it with sterile water, and prepare an equal volume (2 mL) of test solution with a concentration of 1.0% and 2 × 10 -4 Mix the mol / L DPPH solution thoroughly (Al); (2) Take equal volumes of anhydrous ethanol (the solvent for the analyte) and... Mix the DPPH solution thoroughly (A2); (3) Take an equal volume of anhydrous ethanol and mix it with the test solution (A3); (4) After reacting for 40 min, the absorbance values ​​of tubes A1, A2 and A3 were measured at 517 nm.

[0075] The formula for calculating the clearance rate is: Clearance rate (%) = [1 - (A1 - A3) / A2] × 100% The results are shown in Table 2 below.

[0076] Table 2 ; Experimental Example 2: Test for Promoting Type I Collagen Elastic fibers are composed of elastin and microfibrils. Elastase degrades elastin, leading to skin aging, wrinkles, and photoaging.

[0077] Using porcine pancreatic elastase as the research subject, N-succinyl-alanine-alanine-p-nitroaniline (AAAPVN) was used as the substrate. Porcine pancreatic elastase can hydrolyze AAAPVN, and its hydrolysis products can cause an increase in absorbance at a wavelength of 420 nm. The absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) reader to evaluate the firming and anti-wrinkle effects of the test samples.

[0078] 1. Solution preparation (1) Sample concentration setting: Select each composition in Table 1 and test concentration of 1.0%.

[0079] (2) Prepare Tris-HCl buffer (0.1M pH=8.0): Weigh 2.42g Tris into a beaker, add 200mL of ultrapure water, dissolve completely, and then adjust the pH to 8.0 with concentrated HCl.

[0080] (3) Preparation of positive control tea polyphenol solution (1 mg / ml, 0.1%): Weigh 5 mg of tea polyphenol and dissolve it in 5 mL of Tris-HCl buffer.

[0081] (4) Prepare substrate solution AAAPVN (2mM): Weigh 4.51mg N-succinyl-alanine-alanine-alanine-p-nitroaniline and dissolve it in 5ml Tris-HCl buffer.

[0082] (5) Prepare porcine pancreatic elastase solution (0.171 U / mL): Dissolve 280 μL in 10 mL Tris-HCl buffer.

[0083] 2. Sample addition The experiment was divided into four groups: sample group, positive control group, blank control group, and model control group. Each group had four replicates at the same concentration. The amounts of each solution added are shown in Table 3.

[0084] Table 3 ; The units in Table 3 are in μL.

[0085] 3. Measurement The reaction was allowed to proceed at room temperature for 15 minutes, and the absorbance was measured at 420 nm using an ELISA reader.

[0086] 4. Result Calculation

[0087] Where: A0 - the average absorbance of the blank control wells; A1 - The average absorbance of the sample well; The average absorbance of the A2 model control well.

[0088] 5. Data Statistics Statistical analysis was performed using SPSS 22.0 statistical software to conduct descriptive statistics on the measurements in the test area. Changes in the analyzed values ​​and differences between the control and sample groups were calculated. If the test data were normally distributed, the independent t-test was used for statistical analysis; if the test data were not normally distributed, the rank-sum test was used for statistical analysis.

[0089] 6. Result Determination Compared with the model control group, the absorbance of the sample group showed a significant positive difference, indicating that the tested sample had an inhibitory effect on porcine pancreatic elastase. Conversely, if the absorbance was significantly lower, the sample group had no such effect.

[0090] 7. The experimental results are shown in Table 4.

[0091] Table 4 Results of detection of porcine pancreatic elastase inhibition rate ; The serial numbers in Table 4 follow those in Table 1.

[0092] According to the results in Tables 2 and 1, the *Inula japonica*-*Inula japonica* composite ferment prepared by the two-stage composite fermentation process of the present invention (Example 1) exhibits significantly superior overall biological activity compared to other process controls. Specifically: The antioxidant activity (84.12%) and elastase inhibitory activity (91.09%) of this product were both higher than those of the product obtained by physical mixing after independent fermentation (Example 2, 78.66% and 88.41%, respectively), demonstrating that the synergistic transformation of microorganisms during co-fermentation is superior to simple physical mixing.

[0093] The two activities of this product are significantly higher than those of the products from aerobic fermentation only (Example 5) or anaerobic fermentation only (Example 6), proving that the aerobic fermentation stage of Aspergillus niger and the anaerobic fermentation stage of Pediococcus pentosus have an irreplaceable sequential synergistic effect in this process, and the biotransformation function of Aspergillus niger cannot be completely replaced by simple enzymatic hydrolysis and cell wall disruption (Example 6).

[0094] The activity of this product is also higher than that of fermentation products using a single plant as a substrate (Examples 3 and 4), demonstrating that *Inula japonica* and *Inula japonica*, as composite substrates, have complementary components and synergistic effects during fermentation.

[0095] The core product of this invention, the Inula japonica and Inula davidii complex fermentation product, exhibits remarkable fundamental efficacy. Test results for sample number 11 show that it, when used alone, possesses excellent DPPH free radical scavenging and elastase inhibition rates. This data is significantly superior to a traditional physical mixture of Inula japonica and Inula japonica flower extracts in the same mass ratio (sample number 12). This invention represents a substantial improvement and outstanding effect compared to traditional extraction methods.

[0096] The Inula japonica complex ferment of the present invention has good compatibility and can be used as a core active ingredient to construct compositions with specific efficacy.

[0097] In terms of antioxidant properties, the compound ferment itself exhibits high activity (serial number 11), and its activity can be optimized by adjusting the proportion of other auxiliary components in the composition.

[0098] In terms of anti-wrinkle effects, this compound fermentation product, when combined with specific plant extracts, produces a significant synergistic effect. For example, the elastase inhibition rate (91.09%) of sample number 1 (containing compound fermentation product, blueberry fruit extract, and peony root extract) is higher than that of any single component group or partial combination (such as numbers 9, 10, and 11), demonstrating that this specific combination has an unexpected synergistic enhancement effect in inhibiting elastase and potential anti-wrinkle effects.

[0099] Among the differences in the data in Tables 2 and 4, the clearance rate of complex 1 was lower than that of pure Inula japonica ferment 11; while the elastase inhibition rate of complex 1 was better than that of complex 11. This is due to the specificity of the action mechanism of different active ingredients and the synergistic effect of the compound.

[0100] In the elastase inhibition test, sample number 1 performed best, indicating that specific components in the compound fermentation product (possibly including certain small molecule peptides and organic acids produced by microbial transformation) and active substances in the blueberry fruit extract and peony root extract produced an unexpected synergistic enhancement effect on the pathway interfering with enzyme activity. This synergistic effect is directional and mechanism-dependent, and therefore was not simultaneously reflected in the DPPH test. This precisely demonstrates that compounding with the compound fermentation product of this invention as the core can specifically enhance a particular biological activity (such as anti-wrinkle), which is the advantage of this invention.

[0101] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. An extract of Inula japonica, characterized in that, The product was obtained by using palm-leaf tree and Inula japonica as fermentation substrates, followed by aerobic fermentation by Aspergillus niger and anaerobic fermentation by Pediococcus pentosus; and then purification.

2. The extract of Inula japonica according to claim 1, characterized in that, Its preparation method includes the following steps: S1: A fermentation medium is prepared by using palm-leaved tree and Inula japonica as fermentation substrates and adding inorganic salts; the inorganic salts include phosphorus, potassium, magnesium and manganese; S2: The activated Aspergillus niger spore suspension was inoculated into the culture medium and aerobic fermentation was carried out to obtain the primary fermentation product; S3: Inoculate the activated Pediococcus pentosaceus seed liquid into the primary fermentation product, and then ferment anaerobically to obtain the secondary fermentation product; S4: After the secondary fermentation product is inactivated, the filtrate is filtered and collected; the filtrate is concentrated to obtain the extract of Inula japonica.

3. The extract of Inula japonica according to claim 2, characterized in that, In step S1: Of the fermentation substrate, *Palmeria palmatum* by mass ≥70%; The amount of inorganic salts added is calculated based on the fermentation substrate and includes: At least 0.2% potassium dihydrogen phosphate; At least 0.1% magnesium sulfate heptahydrate; At least 0.1% ammonium sulfate; At least 0.04% manganese sulfate monohydrate; Furthermore, it is sterilized after being prepared into a fermentation medium.

4. The extract of Inula japonica according to claim 2, characterized in that, In step S2: The inoculum amount of the Aspergillus niger spore suspension is at least 5% of the mass of the fermentation medium; The conditions for the aerobic fermentation are: temperature 30±1℃, aeration rate of at least 1.0 vvm; fermentation time of at least 48 hours. Aerobic fermentation is complete when the pH of the fermentation product drops to ≤3.

5.

5. The extract of Inula japonica according to claim 2, characterized in that, In step S3: After aerobic fermentation is complete, the air in the fermentation environment is replaced to create an anaerobic environment. The inoculum size of the *Pediococcus pentosaceus* seed culture is at least 5% of the mass of the fermentation medium; The conditions for the anaerobic fermentation are: temperature 37±1℃; anaerobic environment; fermentation time of at least 96 hours. Fermentation is complete when the pH of the fermentation product changes by ≤0.1 within 6 hours.

6. The extract of Inula japonica according to claim 2, characterized in that, In step S4: The inactivation is performed by treating at 70-75°C for at least 30 minutes; The filtration process involves centrifuging the secondary fermentation product to separate the solid and liquid components, and then taking the liquid portion. The liquid portion is then filtered through a microporous membrane to obtain a sterile filtrate. The concentration is achieved by vacuum low-temperature concentration of the filtrate at ≤50°C to ≤20% of the original volume.

7. The extract of Inula japonica according to claim 2, characterized in that, After concentration in step S4, the concentrate is further freeze-dried.

8. The use of the extract of Inula japonica according to any one of claims 1-7 in the preparation of cosmetics.

9. A skin composition, characterized in that, The ingredients, calculated in parts by weight, include the following: Inula japonica extract 10-15 Large blueberry fruit extract 5-10 Peony root extract 2-4; The extract of Inula japonica from the palm-leaf tree was obtained by using palm-leaf tree and Inula japonica as fermentation substrates, followed by aerobic fermentation by Aspergillus niger and then anaerobic fermentation by Pediococcus pentosus; the result was purified.

10. An oil-based cream, characterized in that, It includes a matrix and active ingredients, wherein the active ingredients are calculated by mass fraction including the following components: Inula japonica extract 10-15 Large blueberry fruit extract 5-10 Peony root extract 2-4; The extract of Inula japonica from the palm-leaf tree was obtained by using palm-leaf tree and Inula japonica as fermentation substrates, followed by aerobic fermentation by Aspergillus niger and then anaerobic fermentation by Pediococcus pentosus; the result was purified.

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