High-efficiency extraction method of ursolic acid in loquat leaves and application thereof

Abstract

This invention provides a highly efficient extraction method for ursolic acid from loquat leaves and its application. The method includes the following steps: S1. Fresh loquat leaves are taken, washed clean, dried at 45℃ until the moisture content is <8%, pulverized, passed through a 60-mesh sieve, and microwave-sterilized to obtain sterilized loquat leaf powder; S2. The sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water are mixed, and the pH is adjusted to 5.0 as a fermentation medium. Simultaneously, 5% *Saccharomyces cerevisiae* seed culture and 10% *Trametes versicolor* seed culture are inoculated and cultured at 30℃ and 150 rpm for 36-48 h with shaking; S3. A surfactant is added, stirred evenly, and ultrasonically extracted to obtain the extract; S4. The extract is centrifuged at 4000-5000 rpm for 10-15 min, the supernatant is filtered through a membrane, concentrated, and freeze-dried. This invention combines simultaneous synergistic fermentation by two bacteria with high-dose surfactant-assisted ultrasonic extraction technology, resulting in a powerful synergistic effect. This fundamentally solves the problems of low extraction rate, poor purity, high energy consumption, and large solvent consumption in traditional loquat leaf ursolic acid extraction methods.

Description

Technical Field

[0001] This invention relates to the technical field of natural product extraction, specifically to a highly efficient extraction method for ursolic acid from loquat leaves and its application. Background Technology

[0002] According to reports, the main functional components of loquat leaves are triterpenoid acids, flavonoids, and polyphenols. Among these, triterpenoid acids mainly include ursolic acid, oleanolic acid, and coxsoronic acid. Loquat leaves possess various medicinal functions, such as relieving cough and phlegm, anti-inflammatory and antibacterial effects, antioxidant properties, and anti-aging effects. However, there is currently limited research on the weight-loss function of loquat leaf extract. At present, the technology for extracting active ingredients from loquat leaves in China is not yet mature. The waxy layer on the surface of loquat leaves is obvious. Existing ursolic acid extraction methods mainly include solvent extraction, supercritical CO2 extraction, microwave / ultrasound-assisted extraction and biological enzymatic hydrolysis. All of them have significant defects: (1) Solvent method: It relies on a large amount of organic solvents (such as methanol and chloroform), has low extraction efficiency (6-12 hours), serious co-extraction of impurities, and solvent residues threaten safety; (2) Supercritical extraction: The equipment cost is high, and high pressure and polar entrainers (such as ethanol) are required. Due to the strong polarity of ursolic acid, its solubility is low, and subsequent separation is complicated; (3) Microwave / ultrasound method: Although it shortens the extraction time, local overheating can easily cause thermal degradation of ursolic acid, and the energy consumption on a large scale increases dramatically; (4) Biological enzymatic hydrolysis method: The conditions are mild but the cycle is long (12-24 hours), the enzyme cost is high and the substrate applicability is narrow, and the process stability is poor. Common bottlenecks include an imbalance between efficiency and cost, a contradiction between purity and yield, and insufficient feasibility for large-scale production. The root cause lies in the incompatibility between the thermosensitive / highly polar nature of ursolic acid and the complexity of plant substrates. Therefore, developing an efficient and high-purity method for extracting ursolic acid from loquat leaves is an urgent problem to be solved. Summary of the Invention

[0003] Technical problem to be solved: In view of the above-mentioned technical problems, the purpose of this invention is to provide a highly efficient extraction method for ursolic acid from loquat leaves and its application. This invention combines simultaneous synergistic fermentation by two bacteria with high-dose surfactant-assisted ultrasonic extraction technology, forming a powerful synergistic effect, fundamentally solving the problems of low extraction rate, poor purity, high energy consumption, and large solvent consumption in traditional loquat leaf ursolic acid extraction methods.

[0004] Technical solution: A highly efficient extraction method for ursolic acid from loquat leaves, comprising the following steps:

[0005] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with microwave to obtain sterilized loquat leaf powder;

[0006] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O and water, adjust the pH to 5.0 with citric acid-sodium citrate buffer solution, and use it as fermentation medium. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed liquid and 10% Trametes versicolor seed liquid, and culture at 30℃ and 150 rpm for 36-48 h with shaking.

[0007] S3. Add surfactant, stir well, and extract by ultrasonication to obtain extract;

[0008] S4. Centrifuge the extract at 4000-5000 rpm for 10-15 min, take the supernatant and filter it through a 0.45 μm filter membrane. Concentrate the filtrate under reduced pressure at 50℃ to 1 / 5 of the original volume and freeze-dry.

[0009] Furthermore, the microwave sterilization conditions in S1 are 800W for 60s.

[0010] Furthermore, in the fermentation medium of S2, the content of sterilized loquat leaf powder is 18-21%, the content of yeast extract is 0.1-0.15%, and the content of MgSO4·7H2O is 0.05-0.06%.

[0011] Furthermore, the concentration of the Saccharomyces cerevisiae seed culture in S2 is 1×10⁻⁶ cells / mL. 8 -5×10 8 CFU / mL; the number of mycelial balls in the *Geobacillus yunnanensis* seed solution is 60-100 / mL.

[0012] Furthermore, the surfactant in S3 is one of Tween 60, Tween 80, Span 60, or Span 80.

[0013] Furthermore, the surfactant in S3 is Tween 60.

[0014] Furthermore, the amount of surfactant added in S3 is 8-12%.

[0015] Furthermore, the amount of surfactant added in S3 is 11%.

[0016] Furthermore, the ultrasonic extraction conditions in S3 are: temperature 40-50℃, ultrasonic power 180-250W, and processing time 35-45min.

[0017] The use of ursolic acid extracted by the above extraction method in weight loss and lipid-lowering drugs. Beneficial effects

[0018] 1. This invention combines simultaneous synergistic fermentation by two bacteria with high-dose surfactant-assisted ultrasonic extraction technology, forming a powerful synergistic effect. This fundamentally solves the problems of low extraction rate, poor purity, high energy consumption, and large solvent consumption in traditional loquat leaf ursolic acid extraction methods.

[0019] 2. This invention employs a dual-strain synergistic fermentation process. Yeast rapidly multiplies and consumes free sugars in the intercellular spaces and vacuoles, reducing system viscosity and minimizing ineffective adsorption of subsequent surfactants. This creates a better growth environment for *Trametes versicolor*. Simultaneously, intermediate products from yeast metabolism promote the growth of *Trametes versicolor* and laccase secretion. *Trametes versicolor* begins secreting cellulase and hemicellulase in its early growth stages, initially disrupting the cell wall and facilitating deeper utilization of nutrients in the substrate by yeast. Ultimately, it secretes laccase to efficiently degrade lignin. The synergistic action of the two strains jointly breaks down the loquat leaf cell wall structure. This simultaneous process produces a synergistic cell wall disruption effect, where "1+1>2".

[0020] 3. After 48 hours of synergistic fermentation, the cell wall structure is severely damaged, becoming loose and porous. At this point, some ursolic acid has begun to be released into the fermentation mash, but most of it remains encapsulated in the soon-to-disintegrate solid structure. This invention utilizes surfactant-ultrasonic extraction to ensure maximum dissolution of ursolic acid. Surfactants, in the complex system produced by fermentation, effectively penetrate (through their hydrophilic and lipophilic properties, penetrating into the damaged cells), solubilize (by binding with hydrophobic ursolic acid molecules to form micelles, greatly increasing the solubility of ursolic acid in the aqueous phase), and emulsify (emulsifying lipids and other components in cell fragments, further releasing the encapsulated target product), fully releasing ursolic acid from the damaged cell structure and dissolving it in the liquid phase. Furthermore, by directionally binding the polar groups of ursolic acid through a hydrophilic-hydrophobic balance (HLB value 12-16), it reduces the co-dissolution of impurities such as polysaccharides and pigments, improving purity and achieving highly selective extraction. Ultrasonic enhancement is employed: the ultrasonic cavitation effect further physically breaks down the cell wall residues after biological enzymatic hydrolysis and enhances the mass transfer process, allowing the surfactant to have more sufficient contact with the target component. Ursolic acid is dissolved and dispersed more efficiently and thoroughly into the surrounding liquid environment. The surfactant micelles encapsulate ursolic acid, isolating it from the local high temperature and pressure generated by ultrasonic cavitation, avoiding thermal degradation (it can be operated at a low temperature of 40-50℃), and significantly reducing the risk of isomerization.

[0021] 4. This invention uses a high dose of surfactant (8-12%), mainly because fermentation broth is a complex system. A high concentration of surfactant can ensure that even in the presence of a large number of interfering substances (proteins, residual sugars, bacteria, etc.), sufficient micelles can still be formed to encapsulate ursolic acid. The aim is to form an extremely high micelle concentration, achieve "supersaturated" solubilization, thereby breaking through the solubility limit of ursolic acid and achieving near-complete extraction. Attached Figure Description

[0022] Figure 1 The in vitro enzyme activity inhibition of the lyophilized powders prepared in Example 7 at different mass concentrations is shown in the figure. (a) is a graph of pancreatic lipase inhibition rate; (b) is a graph of α-glucosidase inhibition rate.

[0023] Figure 2 The lyophilized powder prepared in Example 7 was shown to have a weight loss and lipid-lowering effect on mice, where (a) is a graph showing the change in mouse body weight; and (b) is a graph showing the change in food intake.

[0024] Figure 3 The lipid content of the lyophilized powder prepared in Example 7 was analyzed in mice, where (a) is the triglyceride content; (b) is the total cholesterol content; (c) is the high-density lipoprotein content; and (d) is the low-density lipoprotein content. Detailed Implementation

[0025] This invention proposes a highly efficient extraction method for ursolic acid from loquat leaves and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0026] The brewing yeast used in the following examples is brewing yeast CGMCC 2.1543 (purchased from China General Microbiological Culture Collection Center); the *Trametes versicolor* is *Trametes versicolor* CGMCC 5.1090 (purchased from China General Microbiological Culture Collection Center).

[0027] Example 1

[0028] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0029] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0030] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium contains 18% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 1×10⁻⁶. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with a mycelial ball count of 61 cells / mL, were cultured at 30℃ and 150 rpm for 36 h with shaking.

[0031] S3. Add 8% Tween 60, stir well, and extract by ultrasonication at 40℃ and 180W for 35 minutes to obtain the extract.

[0032] S4. The extract was centrifuged at 5000 rpm for 10 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried.

[0033] Example 2

[0034] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0035] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0036] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium contains 21% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 5 × 10⁻⁶ cells / mL. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with a mycelial ball count of 98 / mL, were cultured at 30℃ and 150 rpm for 48 h with shaking.

[0037] S3. Add 12% Tween 80, stir well, and extract by ultrasonication at 50℃ and 250W for 45 minutes to obtain the extract.

[0038] S4. The extract was centrifuged at 5000 rpm for 12 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried. Example 3

[0039] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0040] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0041] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium contains 19% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 2×10⁻⁶. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with 70 mycelial balls / mL, were cultured at 30℃ and 150 rpm for 40 h with shaking.

[0042] S3. Add 10% Span 60, stir well, and extract by ultrasonication at 45℃ and 200W for 40 minutes to obtain the extract.

[0043] S4. The extract was centrifuged at 5000 rpm for 15 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried. Example 4

[0044] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0045] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0046] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium should contain 20% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 3 × 10⁻⁶ cells / mL. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with a mycelial ball count of 83 / mL, were cultured at 30℃ and 150 rpm for 42 h with shaking.

[0047] S3. Add 9% Span 80, stir well, and extract by ultrasonication at 42℃ and 220W for 38 minutes to obtain the extract.

[0048] S4. The extract was centrifuged at 5000 rpm for 13 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried. Example 5

[0049] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0050] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0051] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium contains 18% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 5 × 10⁻⁶ cells / mL. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with a mycelial ball count of 93 / mL, were cultured at 30℃ and 150 rpm for 48 h with shaking.

[0052] S3. Add 11% Tween 60, stir well, and extract by ultrasonication at 48℃ and 240W for 42 minutes to obtain the extract.

[0053] S4. The extract was centrifuged at 5000 rpm for 13 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried. Example 6

[0054] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0055] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0056] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium contains 21% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 1×10⁻⁶. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with a mycelial ball count of 65 / mL, were cultured at 30℃ and 150 rpm for 36 h with shaking.

[0057] S3. Add 8% Tween 60, stir well, and extract by ultrasonication at 41℃ and 185W for 36 minutes to obtain the extract.

[0058] S4. The extract was centrifuged at 5000 rpm for 13 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried. Example 7

[0059] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0060] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0061] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium contains 19% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 4 × 10⁻⁶ cells / mL. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with a mycelial ball count of 94 / mL, were cultured at 30℃ and 150 rpm for 44 h with shaking.

[0062] S3. Add 12% Span 60, stir well, and extract by ultrasonication at 47℃ and 235W for 44 minutes to obtain the extract.

[0063] S4. The extract was centrifuged at 5000 rpm for 13 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried. Example 8

[0064] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0065] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0066] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium should contain 20% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 2×10⁻⁶. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with a mycelial ball count of 73 / mL, were cultured at 30℃ and 150 rpm for 38 h with shaking.

[0067] S3. Add 10% Span 80, stir well, and extract by ultrasonication at 43℃ and 210W for 39 minutes to obtain the extract.

[0068] S4. The extract was centrifuged at 5000 rpm for 13 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried. Example 9

[0069] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0070] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0071] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium contains 18% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 3 × 10⁻⁶ cells / mL. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with a mycelial ball count of 85 / mL, were cultured at 30℃ and 150 rpm for 46 h with shaking.

[0072] S3. Add 9% Tween 60, stir well, and extract by ultrasonication at 46℃ and 230W for 41 minutes to obtain the extract.

[0073] S4. The extract was centrifuged at 5000 rpm for 13 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried. Example 10

[0074] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0075] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0076] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium contains 21% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 4 × 10⁻⁶ cells / mL. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with a mycelial ball count of 72 / mL, were cultured at 30℃ and 150 rpm for 37 h with shaking.

[0077] S3. Add 11% Tween 60, stir well, and extract by ultrasonication at 44℃ and 195W for 37 minutes to obtain the extract.

[0078] S4. The extract was centrifuged at 5000 rpm for 10 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried. Example 11

[0079] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0080] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0081] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium contains 19% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 1×10⁻⁶. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with mycelial balls numbering 100 / mL, were cultured at 30℃ and 150 rpm for 48 h with shaking.

[0082] S3. Add 8% Tween 60, stir well, and extract by ultrasonication at 49℃ and 245W for 43 minutes to obtain the extract.

[0083] S4. The extract was centrifuged at 5000 rpm for 12 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried. Example 12

[0084] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0085] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0086] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium should contain 20% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 5 × 10⁻⁶ cells / mL. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with 60 mycelial balls / mL, were cultured at 30℃ and 150 rpm for 36 h with shaking.

[0087] S3. Add 12% Span 80, stir well, and extract by ultrasonication at 40℃ and 250W for 35 minutes to obtain the extract.

[0088] S4. The extract was centrifuged at 5000 rpm for 10 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried. Example 13

[0089] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0090] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0091] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium should contain 18% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 2×10⁻⁶. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with a mycelial ball count of 98 / mL, were cultured at 30℃ and 150 rpm for 48 h with shaking.

[0092] S3. Add 10% Tween 60, stir well, and extract by ultrasonication at 50℃ and 180W for 45 minutes to obtain the extract.

[0093] S4. The extract was centrifuged at 5000 rpm for 10 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried. Example 14

[0094] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0095] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0096] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium contains 21% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 3 × 10⁻⁶ cells / mL. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with a mycelial ball count of 79 / mL, were cultured at 30℃ and 150 rpm for 40 h with shaking.

[0097] S3. Add 10% Tween 60, stir well, and extract by ultrasonication at 45℃ and 200W for 40 minutes to obtain the extract.

[0098] S4. The extract was centrifuged at 5000 rpm for 13 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried. Example 15

[0099] A highly efficient extraction method for ursolic acid from loquat leaves includes the following steps:

[0100] S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with an 800W microwave for 60 seconds to obtain sterilized loquat leaf powder.

[0101] S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O, and water. Adjust the pH to 5.0 with citric acid-sodium citrate buffer solution to create a fermentation medium. The fermentation medium should contain 20% sterilized loquat leaf powder, 0.13% yeast extract, and 0.05% MgSO4·7H2O. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed culture at a cell concentration of 4 × 10⁻⁶ cells / mL. 8 CFU / mL and 10% *Trametes versicolor* seed culture, with a mycelial ball count of 91 cells / mL, were cultured at 30℃ and 150 rpm for 42 h with shaking.

[0102] S3. Add 10% Tween 60, stir well, and extract by ultrasonication at 45℃ and 200W for 40 minutes to obtain the extract.

[0103] S4. The extract was centrifuged at 5000 rpm for 10 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was concentrated under reduced pressure at 50℃ to 1 / 5 of its original volume and then freeze-dried.

[0104] Comparative Example 1 (Blank Control: Traditional Alcohol Extraction Method)

[0105] S1. Same as Example 1;

[0106] S2. Take 20g of sterilized loquat leaf powder, add 200mL of 95% ethanol, and reflux at 80℃ for 2 hours;

[0107] S3. The extract was centrifuged at 5000 rpm for 10 min, the supernatant was collected, the ethanol was recovered by rotary evaporation, and then freeze-dried.

[0108] Comparative Example 2 (Single-strain fermentation: yeast only)

[0109] S1. Same as Example 1;

[0110] S2. Prepare the same fermentation medium as in Example 1, but inoculate only with 5% Saccharomyces cerevisiae seed culture, with a cell concentration of 1×10⁻⁶. 8 CFU / mL, cultured at 30℃ and 150 rpm for 36 h with shaking;

[0111] S3-S4. Same as Example 1.

[0112] Comparative Example 3 (Single-strain fermentation: only *Trametes versicolor*)

[0113] S1. Same as Example 1;

[0114] S2. Prepare the same fermentation medium as in Example 1, inoculate only with 10% *Trametes versicolor* seed liquid, with a mycelial ball count of 61 cells / mL, and culture at 30°C and 150 rpm for 36 hours with shaking.

[0115] S3-S4. Same as Example 1.

[0116] Comparative Example 4 (no fermentation step)

[0117] S1. Same as Example 1;

[0118] S2. Take 20g of sterilized loquat leaf powder and add it to 200mL of citric acid-sodium citrate buffer solution with a pH of 5.0;

[0119] S3. Add 8% Tween 60, stir well, and extract by ultrasound at 40℃ and 180W for 35 minutes.

[0120] S4. Same as Example 1.

[0121] Comparative Example 5 (Low-dose surfactant)

[0122] S1-S2. Same as Example 1;

[0123] S3. Add 1.0% Tween 60, stir well, and extract by ultrasound at 40℃ and 180W for 35 minutes.

[0124] S4. Same as Example 1.

[0125] Comparative Example 6 (without surfactant)

[0126] S1-S2. Same as Example 1;

[0127] S3. Without adding surfactant, directly extract by ultrasonication at 40℃ and 180W for 35 minutes;

[0128] S4. Same as Example 1.

[0129] Comparative Example 7 (no ultrasonic step, only stirring)

[0130] S1-S2. Same as Example 1;

[0131] S3. Add 8% Tween 60, stir well, and magnetically stir at 40℃ and 150 rpm for 35 minutes.

[0132] S4. Same as Example 1.

[0133] Comparative Example 8 (Sequential fermentation rather than synergistic fermentation)

[0134] S1. Same as Example 1;

[0135] S2. First, inoculate with 5% Saccharomyces cerevisiae seed culture, and culture at 30℃ and 150 rpm for 12 hours with shaking; then sterilize with microwave at 800W for 60 seconds, and inoculate with 10% Trametes versicolor seed culture, and continue to culture under the same conditions for 24 hours (total time 36 hours).

[0136] S3-S4. Same as Example 1.

[0137] Comparative Example 9 (Different types of surfactants)

[0138] S1-S2. Same as Example 1;

[0139] S3. Add 8% sodium dodecyl sulfate (SDS), stir well, and extract by ultrasonication at 40℃ and 180W for 35 minutes.

[0140] S4. Same as Example 1.

[0141] Performance testing:

[0142] Determination of ursolic acid yield: High performance liquid chromatography (HPLC); Chromatographic conditions: Lichrospher C18 (250 mm × 4.6 mm, 5 μm) column; Mobile phase: 0.2% phosphoric acid aqueous solution-acetonitrile gradient elution; Flow rate: 0.8 mL / min; Column temperature: 30℃; Detection wavelength: 215 nm.

[0143] Ursolic acid yield (%) = (mass of ursolic acid in the extract / mass of loquat leaf raw material) × 100%;

[0144] Ursolic acid purity: As per the high performance liquid chromatography method above, the percentage of the peak area of ​​ursolic acid chromatographic peak to the total peak area was calculated by the area normalization method.

[0145] The results are shown in Table 1 below:

[0146] Table 1

[0147] Ursolic acid yield (%) Extract purity (%) Ursolic acid yield (%) Extract purity (%) Example 1 1.21 86.2 Example 13 1.35 88.9 Example 2 1.40 87.5 Example 14 1.28 89.4 Example 3 1.32 88.1 Example 15 1.30 89.0 Example 4 1.25 87.8 Comparative Example 1 0.50 25.3 Example 5 1.38 88.5 Comparative Example 2 0.70 61.5 Example 6 1.22 86.5 Comparative Example 3 0.90 70.2 Example 7 1.39 89.1 Comparative Example 4 0.80 58.8 Example 8 1.27 87.6 Comparative Example 5 0.75 63.1 Example 9 1.29 88.3 Comparative Example 6 0.65 52.4 Example 10 1.33 88.0 Comparative Example 7 0.80 59.2 Example 11 1.24 86.8 Comparative Example 8 1.10 83.5 Example 12 1.37 87.3 Comparative Example 9 0.80 65.7

[0148] In this invention, the ursolic acid yield in all Examples 1-15 was consistently between 1.20% and 1.40%, and the purity was consistently between 86% and 90%, demonstrating high extraction rate and purity, achieving efficient extraction. Comparative Example 1, using traditional ethanol extraction, had the lowest yield and purity, mainly due to the low efficiency of traditional methods and the removal of large amounts of lipid-soluble impurities (such as chlorophyll and waxes) during ethanol reflux extraction, resulting in poor product purity. Comparative Examples 2 and 3 employed single-strain fermentation. In Comparative Example 2, the yeast's main function was to consume sugars, with limited cell wall disruption ability. In Comparative Example 3, while *Trametes versicolor* had strong enzymatic hydrolysis ability, the lack of a low-viscosity environment created by yeast limited its efficiency. Therefore, the results were inferior to the examples. Comparative Example 4 omitted the fermentation step, directly extracting the raw material using surfactant ultrasound, resulting in unsatisfactory yield and purity. This was mainly due to the lack of a "cell wall disruption" step in the biological fermentation pretreatment, making it difficult to efficiently release ursolic acid tightly bound by the cell wall using only chemical and physical methods. Comparative Example 5 used a low dose of surfactant, specifically 1% Tween 60, resulting in a significant decrease in yield and purity compared to Example 1. This indicates that in complex fermentation broths, conventional low-dose surfactants cannot effectively solubilize the broth, and high doses (8-12%) are one of the key innovations of this invention in achieving high extraction rates. Comparative Example 6, without surfactant, showed significantly worse results, demonstrating that surfactants play an irreplaceable "solubilizing" role in dissolving the highly hydrophobic ursolic acid, a deficiency that cannot be compensated for by the physical disruption effect of ultrasound. Comparative Example 7, without an ultrasound step and using only stirring, showed a significant decrease in extraction efficiency. This is mainly because the powerful mechanical force generated by ultrasonic cavitation is crucial for thoroughly disrupting cells and enhancing mass transfer, which is unmatched by conventional stirring. Comparative Example 8 employed sequential fermentation, using a step-by-step approach of "yeast first, sterilization, then *Trametes versicolor*," which, while better than single-strain fermentation, was significantly less effective than the simultaneous fermentation examples. This demonstrates that the step-by-step operation is not only cumbersome, but the intermediate sterilization step may also kill beneficial metabolites produced by yeast or destroy the substrate that has undergone initial decomposition. This also proves the superiority of the "simultaneous inoculation and co-fermentation" model. Comparative Example 9 used the ionic surfactant SDS; after using SDS, both the yield and purity were poor. As an ionic surfactant, SDS easily denatures proteins and inactivates enzymes, potentially inhibiting the activity of residual enzymes in the fermentation broth. Furthermore, SDS itself is difficult to remove and will remain as an impurity in the final product.

[0149] The ursolic acid freeze-dried powder obtained in Example 7 was used to evaluate its weight loss and lipid-lowering effects.

[0150] (1) In vitro enzyme activity inhibition

[0151] 1.1 Inhibition of pancreatic lipase activity: A 50 mmol / L p-nitrobenzene palmitate solution was prepared by dissolving p-nitrobenzene palmitate in DMSO. A 5 mg / L test solution was prepared using the lyophilized powder from Example 7 and distilled water. Pancreatic lipase was dissolved in Tris buffer (pH=7.4) to a concentration of 10 mg / mL, centrifuged at 13000 r / min for 8 min at 4°C, and the supernatant was separated. The total volume of the reaction system was 1.84 mL, including 0.2 mL of the reconstituted water extract, 0.2 mL of enzyme solution, 1.4 mL of Tris buffer (pH=7.4), and 0.4 mL of substrate solution. The mixture was incubated at 37°C for 20 min, and the reading was taken at 405 nm. The pancreatic lipase inhibition rate was calculated using the following formula:

[0152] PPL inhibition rate (%) = [Absorbance without sample - (Absorbance with inhibitor - Absorbance with inhibitor but without substrate and enzyme)] / Absorbance without sample × 100%.

[0153] 1.2 α-Glucosidase Inhibitory Activity Assay: The prepared solution of the extract or compound was diluted with 0.1 mol / L pH 6.8 phosphate buffer (PBS) to prepare a prepared solution with a mass concentration of 10 mg / mL, and then the test solution was prepared with a mass concentration of 0.25 mg / mL. In each well of a 96-well plate, 10 μL of the lyophilized ursolic acid powder reconstituted solution or compound solution (experimental group), 50 μL of dimethyl sulfoxide (DMSO) (negative control group), 20 μL of PBS, and 20 μL of 0.2 U / mL enzyme solution were added, shaken well, and incubated at 37°C for 10 min. Then, 20 μL of 10 mmol / L PNPG solution was added, shaken well, and incubated at 37°C for 10 min. Finally, 30 μL of Na2CO3 stop solution was added, and the absorbance was measured at 405 nm.

[0154] The inhibition rate of α-glucosidase is calculated using the following formula:

[0155] Inhibition rate AGH (%) = (Absorbance of negative control group - Absorbance of experimental group) / Absorbance of negative control group × 100%

[0156] The results are as follows Figure 1 As shown, in vitro inhibitory activity studies have demonstrated that ursolic acid can inhibit the activity of pancreatic lipase and α-glucosidase, and the inhibitory effect on both enzymes shows a concentration-dependent effect.

[0157] (2) The weight loss and lipid-lowering effects of ursolic acid on mice

[0158] Establishment of a high-fat diet obesity model in C57BL / 6J mice:

[0159] Sixty 4-week-old male C57BL / 6J mice were placed in plastic cages (4 mice per cage) under controlled conditions (temperature: 25±2℃; humidity: 55%±10%; 12-hour light cycle: 12-hour dark cycle). After 7 days of acclimatization, they were randomly divided into 4 groups (12 mice per group): the blank control group was fed a low-fat diet (NC, 10% of calories from fat), the experimental control group was fed a high-fat diet (HFD, 60% of calories from fat), the positive control group was fed a high-fat diet and administered orlistat 60 mg / kg (OL) by gavage, and the treatment group was fed a high-fat diet and divided into high and low concentrations (H, L) according to the optimal concentration of loquat leaf extract obtained from in vitro enzyme activity inhibition. During the 12-week experiment, body weight and food intake were measured weekly. Sample collection: At the end of the 12-week experiment, mice were fasted for 12 h and then anesthetized with 3% sodium pentobarbital. Blood was collected by enucleation of the eyeballs of the mice, and the blood samples were centrifuged at 3500 rpm for 10 min (4℃) to separate serum. Measure serum total cholesterol (T-CHO), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) according to the kit instructions.

[0160] Experimental results are as follows Figure 2 As shown, at the end of the experiment, the body weight and food intake of mice in each group were significantly lower than those in the model group, which was statistically significant (P<0.01). The regulatory effect of ursolic acid on mouse body weight and food intake was dose-dependent within the dosage range of this experiment.

[0161] Lipid content analysis: Experimental results are as follows Figure 3 As shown, compared with the model group, the levels of triglycerides, total cholesterol, high-density lipoprotein, and low-density lipoprotein were significantly reduced in the treatment group, with the high-dose treatment group showing a significant difference (P<0.01). Therefore, ursolic acid can reduce the serum lipid levels in obese mice with a high-fat diet.

[0162] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for extracting ursolic acid from loquat leaves, characterized in that: Includes the following steps: S1. Take fresh loquat leaves, wash them clean, dry them at 45℃ until the moisture content is <8%, then crush them, pass them through a 60-mesh sieve, and sterilize them with microwave to obtain sterilized loquat leaf powder; S2. Mix sterilized loquat leaf powder, yeast extract, MgSO4·7H2O and water, adjust the pH to 5.0 with citric acid-sodium citrate buffer solution, and use it as fermentation medium. Simultaneously inoculate with 5% Saccharomyces cerevisiae seed liquid and 10% Trametes versicolor seed liquid, and culture at 30℃ and 150 rpm for 36-48 h with shaking. S3. Add surfactant, stir well, and extract by ultrasonication to obtain extract; S4. Centrifuge the extract at 4000-5000 rpm for 10-15 min, take the supernatant and filter it through a 0.45 μm filter membrane. Concentrate the filtrate under reduced pressure at 50℃ to 1 / 5 of the original volume and freeze-dry. The surfactant in S3 is one of Tween 60, Tween 80, Span 60 or Span 80; The amount of surfactant added in S3 is 8-12%.

2. The method for extracting ursolic acid from loquat leaves according to claim 1, characterized in that, The microwave sterilization conditions in S1 are 800W for 60s.

3. The method for extracting ursolic acid from loquat leaves according to claim 1, characterized in that, The fermentation medium in S2 contains 18-21% sterilized loquat leaf powder, 0.1-0.15% yeast extract, and 0.05-0.06% MgSO4·7H2O.

4. The method for extracting ursolic acid from loquat leaves according to claim 1, characterized in that, The concentration of Saccharomyces cerevisiae seed culture in S2 is 1×10⁻⁶. 8 -5×10 8 CFU / mL; the number of mycelial balls in the *Geobacillus yunnanensis* seed solution is 60-100 / mL.

5. The method for extracting ursolic acid from loquat leaves according to claim 1, characterized in that, The surfactant in S3 is Tween 60.

6. The method for extracting ursolic acid from loquat leaves according to claim 1, characterized in that, The amount of surfactant added in S3 is 11%.

7. The method for extracting ursolic acid from loquat leaves according to claim 1, characterized in that, The ultrasonic extraction conditions in S3 are: temperature 40-50℃, ultrasonic power 180-250W, and processing time 35-45min.

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