Preparation method and application of folium cortex eucommiae fermentation extract for promoting cartilage growth

Abstract

The invention relates to the technical field of bioengineering, in particular to a preparation method and application of a folium cortex eucommiae fermentation extract for promoting cartilage growth. According to the method, a four-stage dynamic regulation and control process of low-temperature plasma synergistic enzymolysis pretreatment, aerobic wall breaking, aerobic and anaerobic transition and anaerobic conversion is adopted; four specific strains with complementary functions, namely bacillus subtilis, aspergillus niger, saccharomyces cerevisiae and enterococcus faecalis, are selected, and precise temperature control and pH regulation are combined, so that efficient release and conversion of active ingredients of the folium cortex eucommiae are realized. The active ingredients of the extract are rich in variety and sufficient in content, cartilage cell proliferation and osteogenic differentiation can be remarkably promoted, and the extract has full-scene cartilage repair adaptability and is good in safety through toxicology verification. The preparation process is stable and repeatable, reaches the pilot test effect, can be adapted to various dosage forms such as oral preparations, injections and the like, and is suitable for preparing cartilage repair related medicines, functional foods and cartilage tissue engineering scaffold composite materials.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a preparation method of a Eucommia ulmoides leaf fermented extract for promoting cartilage growth and joint injury repair, and application of the extract in preparation of a drug for promoting cartilage cell proliferation and osteogenic differentiation, a drug or functional food for treating osteoarthritis and postoperative repair of traumatic cartilage injury, and a cartilage tissue engineering scaffold composite material. BACKGROUND

[0002] The active ingredients such as chlorogenic acid, polysaccharide and geniposidic acid contained in Eucommia ulmoides leaves have potential cartilage repair-related biological activities, but these active ingredients are wrapped by a dense plant cell wall composed of cellulose and pectin, resulting in a dissolution rate of the active ingredients of only 30%-50% under traditional water extraction and alcohol extraction processes, and a blood drug concentration peak time of >4h and a half-life of <2h after oral administration, with extremely low bioavailability.

[0003] Although there are related reports on improving the activity of Eucommia ulmoides leaves by microbial fermentation in the prior art, such as some patents disclosing schemes for extracting Eucommia ulmoides gum and improving polysaccharide content by single bacterial fermentation, there are still many core defects.

[0004] The strain combination is not targeted: only the dissolution of active ingredients is taken as a single target, the synergistic effect of ingredients required for cartilage repair is not considered, and the necessity of multi-strain synergy is not verified;

[0005] The process design is not scientific: single-stage aerobic or anaerobic fermentation is used without a dynamic control mechanism, which cannot simultaneously achieve efficient cell wall breaking and accurate active ingredient conversion, and lacks effective pretreatment process assistance, with a breaking efficiency of less than 60%;

[0006] The effect verification is incomplete: only the content of active ingredients is detected, the cartilage cell proliferation / osteogenic differentiation activity is not verified by cell experiments, and the scenarios such as traumatic cartilage injury repair are not covered, which cannot meet the application requirements of full-scenario cartilage repair;

[0007] The industrialization feasibility is insufficient: long-term toxicity data and specific dosage form process parameters are not provided, which makes it difficult to meet the requirements of actual production and clinical application.

[0008] In view of the above defects of the prior art, the present application designs a multi-stage synergistic process, selects specific strains with complementary functions, and aims to achieve the synergy of active ingredient content improvement, cartilage repair function strengthening and industrialization feasibility, and solve the core problems of low bioavailability and single cartilage repair function of traditional Eucommia ulmoides leaf extract. SUMMARY

[0009] The purpose of this invention is to overcome the shortcomings of existing technologies, such as low dissolution rate of active ingredients from Eucommia ulmoides leaves, poor targeting of fermentation processes, insufficient cartilage repair effects, and high difficulty in industrialization. This invention provides a method for preparing a fermented extract of Eucommia ulmoides leaves that promotes cartilage growth and joint injury repair. Furthermore, this invention discloses the characteristics and applications of this extract to achieve efficient release of active ingredients from Eucommia ulmoides leaves, comprehensive enhancement of cartilage repair function, while ensuring process stability, safety standards, and compliance with industrial production requirements.

[0010] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a fermented extract of Eucommia ulmoides leaves that promotes cartilage growth, comprising the following steps:

[0011] S1 Raw Material Processing and Pretreatment: Select mature Eucommia ulmoides leaves, sun-dry until moisture content ≤8%, pulverize to 30-60 mesh, and mix with wheat bran at a mass ratio of (3-5):1 to obtain a mixture, wherein the wheat bran moisture content ≤12% and crude protein content ≥15%; place the mixture in a low-temperature plasma treatment instrument and treat for 5-10 minutes under air atmosphere, pressure 10-20 Pa, and power 30-50 W; then add 0.1%-0.3% cellulase and 0.05%-0.1% pectinase to the treated mixture, wherein the cellulase activity ≥5000 U / g and the pectinase activity ≥3000 U / g, using 0.1 mol / L... Adjust the pH to 4.5-5.0 with HCl, and enzymatically hydrolyze the material in a 45℃ constant temperature water bath for 1-2 hours. After enzymatic hydrolysis, control the moisture content of the material to 50%-55%. Sterilize the enzymatically hydrolyzed material at 115-125℃ and a gauge pressure of 0.12-0.15MPa for 15-25 minutes, and then cool it to ≤35℃.

[0012] S2. Strain preparation

[0013] Bacillus subtilis was activated on LB medium, Aspergillus niger on PDA medium, Saccharomyces cerevisiae on YPD medium, and Enterococcus faecalis on MRS medium, respectively, to prepare formulations with a concentration ≥1×10⁻⁶. 8 CFU / mL bacterial suspension or spore suspension;

[0014] S3. Staged dynamic regulation of fermentation

[0015] Aerobic cell-wall breaking fermentation: A suspension of Bacillus subtilis and Aspergillus niger is inoculated into the sterilized material at a volume ratio of 1:1, with a total inoculum amount of 3%-10% of the total material mass; the temperature is controlled at 28-35℃, and the ventilation rate is 0.5-1.0 m³ / h. 3 / (m 3• min), ferment for 36-60 hours; during fermentation, add 0.1 mol / L NaOH to maintain pH 6.5-7.0, and take samples every 12 hours and spray sterile water to maintain material moisture 45%-50%;

[0016] Aerobic-anaerobic transitional fermentation: After aerobic fermentation is completed, reduce the ventilation rate to 0.2-0.3 m³ / h. 3 / (m 3 Continue fermentation for 12 hours (min); then gradually reduce the ventilation rate to 0, and introduce sterile nitrogen gas with a purity of ≥99.99% for 45 minutes until the oxygen concentration in the fermenter is ≤0.3%;

[0017] Anaerobic fermentation: Inoculate with a suspension of Saccharomyces cerevisiae and Enterococcus faecalis at a volume ratio of 2:1, with a total inoculum of 2%-8% of the total material mass; control the temperature at 35-38℃ and allow static fermentation for 18-30 hours; during fermentation, add 0.1mol / L HCl to maintain pH 6.0-6.2; after fermentation, dry the material at 60℃ until the moisture content is ≤8% to obtain fermented dry material;

[0018] S4 Active ingredient extraction: Mix the fermented dry material with deionized water at a material-to-liquid ratio of 1:8-1:12, extract in a water bath at 70-90℃ for 1-3 hours, filter and collect the filtrate; repeat the extraction of the residue 1-3 times under the same conditions, combine all the filtrates, and filter through a 0.22μm microporous membrane to remove impurities.

[0019] S5 Concentration and Drying: The filtered filtrate is concentrated to 1 / 4-1 / 6 of its original volume under conditions of 50-65℃ and vacuum degree of -0.08 to -0.09MPa to obtain a concentrated solution; the concentrated solution is then subjected to spray drying treatment with an inlet air temperature of 180±5℃, an outlet air temperature of 85±5℃, and a feed rate of 15-20mL / min. After drying, a powdered Eucommia ulmoides leaf fermentation extract with a water content ≤3% is obtained.

[0020] Preferably, in step S1, the parameters for the low-temperature plasma treatment are: power 40W, pressure 15Pa, and treatment time 8 minutes; the parameters for the enzymatic hydrolysis are: cellulase addition 0.2%, pectinase addition 0.08%, and hydrolysis time 1.5 hours.

[0021] Preferably, in step S3, the temperature of the aerobic cell-wall breaking fermentation is 30°C, the fermentation time is 48 hours, and the inoculum amount is 6% of the total mass of the material; the temperature of the anaerobic conversion fermentation is 37°C, the fermentation time is 24 hours, and the inoculum amount is 2% of the total mass of the material; the total inoculum amount of the aerobic cell-wall breaking fermentation and the anaerobic conversion fermentation is 8% of the total mass of the material.

[0022] Preferably, in step S4, the material-to-liquid ratio is 1:10, the extraction time is 2 hours, and the extraction is repeated twice.

[0023] Preferably, the total content of chlorogenic acid, polysaccharide, and geniposide in the extract is ≥9.0%; at a concentration of 200 μg / mL, the extract has a relative proliferation rate of chondrocytes ≥190%, alkaline phosphatase activity ≥2.2 times that of the unfermented Eucommia ulmoides leaf extract, and a relative expression level of SOX9 gene ≥3.0 times.

[0024] This invention provides a method for preparing and applying a fermented extract of Eucommia ulmoides leaves that promotes cartilage growth. It has the following beneficial effects:

[0025] 1. This invention achieves efficient cell wall disruption of Eucommia ulmoides leaves through a multi-stage synergistic process, fully releasing the active ingredients encapsulated by the dense cell wall; the total content of chlorogenic acid, polysaccharides, and geniposide in the fermented extract obtained is ≥9.0%, which is more than 70% higher than that of unfermented Eucommia ulmoides leaves; at the same time, the cell wall disruption rate after pretreatment is increased from 60% in the traditional process to more than 85%, and the dissolution rate of active ingredients is increased to more than 90%.

[0026] 2. The Eucommia ulmoides leaf fermentation extract prepared by this invention can exert cartilage repair effects from multiple dimensions such as cell proliferation and osteogenic differentiation, and can cover multiple cartilage injury scenarios such as osteoarthritis treatment and postoperative repair of traumatic cartilage damage, meeting the application needs of cartilage repair in all scenarios.

[0027] 3. The extract of this invention has undergone systematic toxicological verification and has good safety for both long-term and short-term use; at the same time, the preparation process is stable and reproducible, the pilot production results meet the standards, and it can be adapted to the preparation of various dosage forms, which can meet the requirements of actual production transformation and clinical application. Attached Figure Description

[0028] Figure 1 This is a process flow diagram of the preparation method of the present invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1:

[0031] like Figure 1As shown in the figure, a multi-factor orthogonal experiment was designed to determine the optimal process conditions for the preparation method of the present invention. Using the total active ingredient content (the sum of chlorogenic acid, polysaccharides, and genipin acid) as the main evaluation index, key parameters such as aerobic fermentation temperature, anaerobic fermentation temperature, total inoculum size, material-to-liquid ratio, and fermentation time at each stage were systematically optimized. The results are shown in the table below:

[0032] Table 1. Effects of different parameters on the total active ingredient content

[0033] Key parameters Parameter 1 Parameter 2 Parameter 3 Aerobic fermentation temperature 28℃ 30℃ 35℃ Anaerobic fermentation temperature 35℃ 37℃ 38℃ Total inoculum (aerobic + anaerobic) 5%（3%+2%） 8%（6%+2%） 12%（8%+4%） Solid-liquid ratio 1:8 1:10 1:12 Fermentation time (aerobic / transition / anaerobic) 36h / 10h / 18h 48h / 12h / 24h 60h / 15h / 30h Verification effect (total active ingredient content) 9.48% 9.53% 9.51%

[0034] Experimental results showed that when the process parameters were combined as follows: low-temperature plasma treatment for 8 minutes (40W power, 15Pa pressure), followed by enzymatic hydrolysis at 45℃ for 1.5 hours using 0.2% cellulase and 0.08% pectinase; aerobic fermentation stage with a controlled temperature of 30℃ for 48 hours, an inoculum of 6% of the total material mass (a 1:1 mixture of Bacillus subtilis and Aspergillus niger), and pH maintained at 6.5-7.0 through dynamic alkali supplementation; and a transitional fermentation stage maintained for 12 hours with ventilation reduced to 0.25 m³ / h, the optimal fermentation method was found to be effective. 3 / (m 3 The anaerobic fermentation process involved 45 minutes of nitrogen purging, followed by anaerobic fermentation at 37°C for 24 hours. The inoculum size was 2% of the total material mass (a 2:1 mixture of brewer's yeast and Enterococcus faecalis), and the pH was maintained at 6.0-6.2. Finally, water extraction was performed at a material-to-liquid ratio of 1:10, for 2 hours each time, for a total of two extractions. Under this optimal parameter combination, the total active ingredient content in the obtained Eucommia ulmoides leaf fermentation extract reached 9.53%, the highest among all experimental groups.

[0035] Example 2:

[0036] To evaluate the advantages of the method of this invention, high-performance liquid chromatography (HPLC) was used to determine chlorogenic acid and geniposide, and the phenol-sulfuric acid method was used to determine polysaccharides. The contents of the three active ingredients in unfermented Eucommia ulmoides leaf raw material, extracts obtained by traditional single-stage aerobic fermentation process (without pretreatment steps), and extracts obtained by the optimal process of the method of this invention were compared. The results are shown in the table below:

[0037] Table 2 Comparison of the content of active ingredients in the extract of the present invention compared with unfermented Eucommia ulmoides leaves and traditionally fermented extracts.

[0038] Active ingredient Unfermented eucommia leaf (%) Traditional fermented extract (%) Invention extract (%) Increase rate compared to unfermented (%) Increase rate compared to traditional fermentation (%) Standard deviation Chlorogenic acid 1.21±0.05 1.75±0.06 2.18±0.08 79.3 24.6 ±0.08 Polysaccharide 3.82±0.12 5.18±0.14 6.54±0.15 71.2 26.3 ±0.15 Geniposidic acid 0.45±0.02 0.62±0.03 0.79±0.03 75.6 27.4 ±0.03 Total content 5.48±0.18 7.55±0.20 9.51±0.23 73.5 25.9 -

[0039] The results showed that the average contents of chlorogenic acid, polysaccharides, and geniposide in unfermented Eucommia ulmoides leaves were 1.21%, 3.82%, and 0.45%, respectively, with a total content of 5.48%. In traditional fermentation extracts, the average contents of these three components increased to 1.75%, 5.18%, and 0.62%, respectively, with a total content of 7.55%. In the extract of this invention, the average contents of these three components further increased to 2.18%, 6.54%, and 0.79%, with a total content as high as 9.51%. Calculations show that the total active ingredient content of the extract of this invention increased by 73.5% compared to unfermented raw materials and by 25.9% compared to traditional fermentation processes, with a balanced increase in each component.

[0040] Example 3:

[0041] To demonstrate the necessity of the synergistic effect of the four selected bacterial strains (Bacillus subtilis, Aspergillus niger, Saccharomyces cerevisiae, and Enterococcus faecalis), five comparative experiments were conducted in this embodiment of the invention: a group with all four strains intact, a group lacking Bacillus subtilis, a group lacking Aspergillus niger, a group lacking Saccharomyces cerevisiae, and a group lacking Enterococcus faecalis, respectively. Except for the changes in bacterial strains, all samples were prepared using the optimal process determined in Example 1. Subsequently, the total active ingredient content of the extracts in each group and their relative proliferation rate on chondrocytes at a concentration of 200 μg / mL were measured. The results are shown in the table below:

[0042] Table 3 Comparison of the effects of different bacterial strain combinations on total active ingredient content and chondrocyte proliferation rate

[0043] Group Total active ingredient content (%) Relative proliferation rate of chondrocytes (%), 200 μg / mL Difference from complete group (content) Difference from complete group (proliferation rate) Complete 4-bacteria group 9.53±0.23 195±8 - - Bacillus subtilis -deficient group 7.21±0.19 132±6 -24.3% -32.3% Aspergillus niger -deficient group 6.85±0.17 125±5 -28.1% -35.9% Saccharomyces cerevisiae -deficient group 7.58±0.20 140±7 -20.5% -28.2% Enterococcus faecalis -deficient group 6.92±0.18 128±6 -27.4% -34.4%

[0044] The results showed that the total active ingredient content of the extract from the complete four-strain group was 9.53%, and the relative proliferation rate of chondrocytes was 195%. The absence of any one strain significantly reduced both indicators. For example, the absence of *Bacillus subtilis* reduced the total content to 7.21% and the proliferation rate to 132%; the absence of *Aspergillus niger* reduced the total content to 6.85% and the proliferation rate to 125%; the absence of *Saccharomyces cerevisiae* reduced the total content to 7.58% and the proliferation rate to 140%; and the absence of *Enterococcus faecalis* reduced the total content to 6.92% and the proliferation rate to 128%. These results fully demonstrate that the four strains are functionally complementary and synergistic (*Bacillus subtilis* and *Aspergillus niger* mainly work together to efficiently break cell walls, while *Saccharomyces cerevisiae* and *Enterococcus faecalis* mainly work together to transform and modify active ingredients), and are indispensable for achieving high active ingredient content and outstanding chondrocyte repair function.

[0045] Example 4:

[0046] The embodiments of the present invention evaluate the effects of the extract of the present invention on the biological behavior of chondrocytes through in vitro cell experiments.

[0047] 1. Chondrocyte proliferation experiment

[0048] The effects of different concentrations (0, 50, 100, 200, 400 μg / mL) of extracts on the proliferation of chondrocytes in the knee joint of SD rats were detected using the CCK-8 assay. Cells treated with PBS were used as a control (proliferation rate set at 100%). The results are shown in the table below:

[0049] Table 4. Comparison of the proliferation rate of knee joint chondrocytes in SD rats with different concentrations of extracts.

[0050] Extract concentration (μg / mL) OD 450 (CCK-8) Relative proliferation rate (vs. PBS group) Standard deviation 0 (PBS group) 1.00±0.05 100% ±0.05 50 1.42±0.10 142% ±0.10 100 1.82±0.12 182% ±0.12 200 1.95±0.15 195% ±0.15 400 1.98±0.16 198% ±0.16

[0051] The experimental results showed that the extract's proliferative effect on chondrocytes was concentration-dependent. At 50 μg / mL, the relative proliferation rate reached 142%; at 100 μg / mL, it reached 182%; and at 200 μg / mL, it peaked at 195%. Further increasing the concentration to 400 μg / mL did not significantly increase the proliferation rate (198%). This indicates that 200 μg / mL is the optimal concentration of this extract for promoting chondrocyte proliferation, and its effect is significantly better than that reported in existing technologies (usually ≤145%).

[0052] 2. Detection of osteogenic differentiation-related indicators

[0053] At a concentration of 200 μg / mL, the effects of the extract on alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and SOX9 gene expression in chondrocytes were examined. The results are shown in the table below:

[0054] Table 5. Effects of the extract on ALP activity, OC secretion, and SOX9 gene expression in chondrocytes.

[0055] Detection index Blank control group (no extract added) Unfermented extract group Traditional fermented extract group Invention extract group P value (vs. blank group) ALP activity (U / L) 125.3±8.5 228.6±10.2 256.3±11.5 288.2±12.3 <0.001 OC secretion amount (ng / mL) 5.2±0.4 9.5±0.6 11.8±0.7 14.5±0.8 <0.01 SOX9 gene relative expression amount 1.00±0.08 1.85±0.12 2.42±0.15 3.12±0.18 <0.001

[0056] Compared with the blank control group, the extract of this invention significantly increased ALP activity to 288.2 U / L (p<0.001), significantly increased OC secretion to 14.5 ng / mL (p<0.01), and upregulated the relative expression of the SOX9 gene by 3.12-fold (p<0.001). Compared with the unfermented extract group and the conventionally fermented extract group, the extract of this invention showed superior ability to promote osteogenic differentiation in all indicators.

[0057] Example 5:

[0058] The embodiments of the present invention verify the effect of the extract of the present invention on repairing joint damage in vivo using a rat model.

[0059] 1. Osteoarthritis Treatment Experiment: An anterior cruciate ligament transection was used to establish an osteoarthritis model in SD rats. Rats with successful modeling were randomly divided into a model group (administered with saline), a positive control group (administered with diacerein, 10 mg / kg / d), and an experimental group (administered with the extract of this invention, 200 mg / kg / d). A sham-operated group was also included as a control. After 8 weeks of continuous gavage administration, the results were evaluated and are shown in the table below:

[0060] Table 6. Damage images of articular cartilage caused by different substances.

[0061] Detection index Sham operation group Model group Positive control group Experimental group P value (vs. model group) Mankin score 0.8±0.3 7.8±1.1 3.4±0.6 3.2±0.5 <0.001 BV / TV (%) 72.5±4.3 42.3±3.2 59.8±3.9 60.0±4.1 <0.01 Tb.Th (mm) 0.21±0.02 0.12±0.01 0.16±0.01 0.16±0.02 <0.01

[0062] The results showed that, compared with the model group (Mankin score 7.8), the experimental group rats exhibited significantly reduced articular cartilage damage, with the Mankin score decreasing to 3.2 (a reduction of approximately 64%), demonstrating an improvement comparable to the positive control group (score 3.4). Simultaneously, the bone volume fraction (BV / TV) in the experimental group significantly increased from 42.3% in the model group to 60.0%. Histological observation also confirmed that the cartilage surface in the experimental group was smoother, and inflammatory cell infiltration was significantly reduced.

[0063] 2. Traumatic Cartilage Injury Repair Experiment: Standardized cartilage defects were created in the knee joints of rats, who were randomly divided into a model group (physiological saline) and an experimental group (extract of this invention, 200 mg / kg / d). The drugs were administered by gavage for 8 weeks post-surgery. Results showed that the healing rate of cartilage defects in the experimental group was as high as 89%, while that in the model group was only 35%. Immunohistochemical staining showed that the positive expression rate of type II collagen in the newly formed tissue of the defect area in the experimental group exceeded 85%, significantly higher than the 42% in the model group, indicating that the newly formed tissue was mature cartilage-like tissue.

[0064] Example 6:

[0065] To assess the safety of the extract of this invention, systematic toxicological experiments were conducted in this embodiment of the invention.

[0066] 1. Acute toxicity test: ICR mice were administered a single oral gavage dose of up to 5000 mg / kg of the extract of this invention, and observed continuously for 14 days. During this period, all mice were healthy, exhibited normal behavior and activity, and showed no significant difference in weight gain compared to the saline control group. At the end of the experiment, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) levels were measured in mice. The experimental results are shown in the table below:

[0067] Table 7. Effects of different substances on liver and kidney function indicators

[0068] Liver and kidney function indicators Experimental group Control group P value ALT (U / L) 25.3±3.1 24.8±2.9 >0.05 AST (U / L) 45.2±4.2 44.7±3.8 >0.05 BUN (mmol / L) 5.1±0.4 5.0±0.3 >0.05 Cr (μmol / L) 35.2±2.8 34.8±2.5 >0.05

[0069] There were no statistically significant differences between the experimental group and the control group, indicating that no acute toxic reactions were observed at this dose.

[0070] 2. Long-term toxicity experiment: ICR mice were divided into a blank control group, a low-dose group (100 mg / kg / d, equivalent to the recommended adult dose), and a high-dose group (400 mg / kg / d, equivalent to 6 times the recommended adult dose). The mice were administered the drugs by gavage for 90 consecutive days. The results are shown in the table below:

[0071] Table 8 Effects of different dosages on organisms

[0072] Item Experimental dose Experimental results Acute toxicity experiment: SD rats single gavage maximum tolerance amount ≥5000 mg / kg No abnormalities were found Long-term toxicity experiment: Continuous administration for 90 days (400 mg / kg / d), Blood routine, liver and kidney function indicators were normal Mutagenicity experiment: Ames test (TA97a, TA98, TA100, TA102 strains) No mutagenicity was shown Teratogenicity experiment: Pregnancy administration (400 mg / kg / d) No malformations were found

[0073] The results showed no abnormalities in weight gain, food and water intake, or behavioral activity in mice across all dosage groups. Hematological parameters, blood biochemical parameters (liver and kidney function), and urinalysis were all within normal ranges. After the experiment, dissection of major organs (joints, liver, kidneys, spleen, etc.) for pathological examination revealed no drug-related organic lesions. Furthermore, the Ames test and teratogenicity pretest results were negative. Overall, the extract of this invention demonstrates good safety with long-term use.

[0074] Example 7:

[0075] The embodiments of this invention verify the process stability of the extract and conduct pilot-scale amplification experiments.

[0076] 1. Batch Stability Experiment: Under the same optimal process conditions, three batches (batch numbers: 202501, 202502, 202503) of Eucommia ulmoides leaf fermentation extract were prepared independently. The content of active ingredients and chondrocyte proliferation rate of each batch were tested. The chlorogenic acid content of the three batches ranged from 2.16% to 2.19%, the polysaccharide content from 6.52% to 6.55%, the geniposide content from 0.78% to 0.80%, the total active ingredient content from 9.46% to 9.54%, and the chondrocyte proliferation rate from 192% to 195%. The deviation rate of key quality indicators among the batches was less than 5%, demonstrating that the preparation process is stable and has good reproducibility.

[0077] 2. Pilot-scale industrialization experiment: Pilot-scale production was conducted on a 1000L fermenter system, operated entirely according to optimal process parameters, with a total of 5 batches (ZS-01 to ZS-05). Each batch used 500kg of Eucommia ulmoides leaf mixture, and the final yield of the extracted dry powder ranged from 42.2 kg to 43.5 kg, with a calculated yield consistently between 84.4% and 87.0%, and a product purity of no less than 98.0%. The pilot-scale results verified the feasibility of this invention for industrial-scale production.

[0078] Example 8:

[0079] The embodiments of the present invention provide the preparation of two different typical dosage forms based on the extract of the present invention.

[0080] 1. Capsule Preparation: 100g of the extract of this invention, 30g of microcrystalline cellulose, and 5g of magnesium stearate were mixed evenly. An appropriate amount of 5% povidone K30 ethanol solution was added as a binder to form a soft mass. The mass was sieved, granulated, dried, and sized. Magnesium stearate was then added and mixed thoroughly. The mixture was filled into No. 0 empty capsules to obtain capsules containing 100mg of the extract per capsule. The finished product was tested and found to have a disintegration time of less than 25 minutes, and the content uniformity met the requirements of the Chinese Pharmacopoeia.

[0081] 2. Preparation of intra-articular injection: Take 20g of the extract of this invention and 6.7g of sodium hyaluronate, add water for injection, stir to dissolve, and make up to 1000mL. Adjust the pH to 6.8-7.2 with 0.1mol / L NaOH, add activated carbon for adsorption treatment, filter to remove carbon, and then filter through a 0.22μm microporous membrane for sterilization. Dispense into 2mL ampoules and sterilize at 121℃ for 15 minutes to obtain an intra-articular injection with a concentration of approximately 20mg / mL. This preparation has good clarity, is sterile and pyrogen-free, and accelerated stability tests show that its content is stable within 6 months.

[0082] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a fermented extract of Eucommia ulmoides leaves that promotes cartilage growth, characterized in that, Includes the following steps: S1 Raw Material Processing and Pretreatment: Select mature Eucommia ulmoides leaves, sun-dry until moisture content ≤8%, pulverize to 30-60 mesh, and mix with wheat bran at a mass ratio of (3-5):1 to obtain a mixture, wherein the wheat bran moisture content ≤12% and crude protein content ≥15%; place the mixture in a low-temperature plasma treatment instrument and treat for 5-10 minutes under air atmosphere, pressure 10-20 Pa, and power 30-50 W; then add 0.1%-0.3% cellulase and 0.05%-0.1% pectinase to the treated mixture, wherein the cellulase activity ≥5000 U / g and the pectinase activity ≥3000 U / g, using 0.1 mol / L... Adjust the pH to 4.5-5.0 with HCl, and enzymatically hydrolyze the material in a 45℃ constant temperature water bath for 1-2 hours. After enzymatic hydrolysis, control the moisture content of the material to 50%-55%. Sterilize the enzymatically hydrolyzed material at 115-125℃ and a gauge pressure of 0.12-0.15MPa for 15-25 minutes, and then cool it to ≤35℃. S2. Strain preparation Bacillus subtilis was activated on LB medium, Aspergillus niger on PDA medium, Saccharomyces cerevisiae on YPD medium, and Enterococcus faecalis on MRS medium, respectively, to prepare formulations with a concentration ≥1×10⁻⁶. 8 CFU / mL bacterial suspension or spore suspension; S3. Staged dynamic regulation of fermentation Aerobic cell-wall breaking fermentation: A suspension of Bacillus subtilis and Aspergillus niger is inoculated into the sterilized material at a volume ratio of 1:1, with a total inoculum amount of 3%-10% of the total material mass; the temperature is controlled at 28-35℃, and the ventilation rate is 0.5-1.0 m³ / h. 3 / (m 3 • min), ferment for 36-60 hours; during fermentation, add 0.1 mol / L NaOH to maintain pH 6.5-7.0, and take samples every 12 hours and spray sterile water to maintain material moisture 45%-50%; Aerobic-anaerobic transitional fermentation: After aerobic fermentation is completed, reduce the ventilation rate to 0.2-0.3 m³ / h. 3 / (m 3 Continue fermentation for 12 hours (min); then gradually reduce the ventilation rate to 0, and introduce sterile nitrogen gas with a purity of ≥99.99% for 45 minutes until the oxygen concentration in the fermenter is ≤0.3%; Anaerobic fermentation: Inoculate with a suspension of Saccharomyces cerevisiae and Enterococcus faecalis at a volume ratio of 2:1, with a total inoculum of 2%-8% of the total material mass; control the temperature at 35-38℃ and allow static fermentation for 18-30 hours; during fermentation, add 0.1mol / L HCl to maintain pH 6.0-6.2; after fermentation, dry the material at 60℃ until the moisture content is ≤8% to obtain fermented dry material; S4 Active ingredient extraction: Mix the fermented dry material with deionized water at a material-to-liquid ratio of 1:8-1:12, extract in a water bath at 70-90℃ for 1-3 hours, filter and collect the filtrate; repeat the extraction of the residue 1-3 times under the same conditions, combine all the filtrates, and filter through a 0.22μm microporous membrane to remove impurities. S5 Concentration and Drying: The filtered filtrate is concentrated to 1 / 4-1 / 6 of its original volume under conditions of 50-65℃ and vacuum degree of -0.08 to -0.09MPa to obtain a concentrated solution; the concentrated solution is then subjected to spray drying treatment with an inlet air temperature of 180±5℃, an outlet air temperature of 85±5℃, and a feed rate of 15-20mL / min. After drying, a powdered Eucommia ulmoides leaf fermentation extract with a water content ≤3% is obtained.

2. The method for preparing a fermented extract of Eucommia ulmoides leaves that promotes cartilage growth according to claim 1, characterized in that: In step S1, the parameters for the low-temperature plasma treatment are: power 40W, pressure 15Pa, and treatment time 8 minutes; the parameters for the enzymatic hydrolysis are: cellulase addition 0.2%, pectinase addition 0.08%, and hydrolysis time 1.5 hours.

3. The method for preparing a fermented extract of Eucommia ulmoides leaves that promotes cartilage growth according to claim 1, characterized in that: In step S3, the temperature of the aerobic cell-wall breaking fermentation is 30℃, the fermentation time is 48 hours, and the inoculation amount is 6% of the total mass of the material; the temperature of the anaerobic conversion fermentation is 37℃, the fermentation time is 24 hours, and the inoculation amount is 2% of the total mass of the material; the total inoculation amount of aerobic cell-wall breaking fermentation and anaerobic conversion fermentation is 8% of the total mass of the material.

4. The method for preparing a fermented extract of Eucommia ulmoides leaves that promotes cartilage growth according to claim 1, characterized in that: In step S4, the material-to-liquid ratio is 1:10, the extraction time is 2 hours, and the extraction is repeated twice.

5. The Eucommia ulmoides leaf fermentation extract for promoting cartilage growth and joint injury repair prepared by the preparation method according to any one of claims 1-4, characterized in that: The extract contains a total content of chlorogenic acid, polysaccharides, and genipin glycosides ≥9.0%; at a concentration of 200 μg / mL, the extract exhibits a relative proliferation rate of ≥190% for chondrocytes, alkaline phosphatase activity ≥2.2 times that of the unfermented Eucommia ulmoides leaf extract, and a relative expression level of SOX9 gene ≥3.0 times.

6. The use of the Eucommia ulmoides leaf fermentation extract according to claim 5 in the preparation of a drug that promotes chondrocyte proliferation and osteogenic differentiation.

7. The use of the Eucommia ulmoides leaf fermentation extract according to claim 5 in the preparation of drugs or functional foods for repairing joint damage, treating osteoarthritis, and repairing traumatic cartilage damage after surgery.

8. The application according to claim 7, characterized in that: The drug is an oral preparation or an intra-articular injection; the oral preparation includes capsules or tablets, wherein the capsules are made by mixing the extract, microcrystalline cellulose, and magnesium stearate in a mass ratio of 1:0.3:0.05, and each capsule contains 100mg of the extract; the tablets have a weight of 250mg and a disintegration time of ≤30 minutes; the intra-articular injection is made by mixing the extract and sodium hyaluronate in a mass ratio of 3:1, and the extract concentration is 20mg / mL; the recommended daily dose of the oral preparation for an adult weighing 60kg is 100-400mg.

9. The application according to claim 7, characterized in that: The functional food is a solid beverage or compressed candy, and each 100g of the functional food contains 5-10g of the Eucommia ulmoides leaf fermentation extract.

10. The application of the Eucommia ulmoides leaf fermentation extract according to claim 5 in the preparation of cartilage tissue engineering scaffold composite materials, characterized in that: The extract was mixed with a polylactic acid scaffold at a mass ratio of 1:10 for the repair of cartilage defects.

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