Traditional Chinese medicine fermentation composition for treating vascular inflammation and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a traditional Chinese medicine fermentation composition for treating vascular inflammation and a preparation method thereof. The traditional Chinese medicine fermented composition for treating vascular inflammation comprises the following components in parts by mass: 1-10 parts of folium mori, 5-15 parts of bitter gourd, 5-15 parts of radix polygonati officinalis, 1-10 parts of earthworm protein, 10-20 parts of rhizoma polygonati and 5-20 parts of corn stigma. The traditional Chinese medicine fermentation composition has the effect of reducing and treating vascular inflammation, can improve the content of LDL-C and HDL-C in serum, and has good clinical guiding significance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a traditional Chinese medicine fermentation composition for treating vascular inflammation and its preparation method. Background Technology

[0002] Vasculitis is an inflammatory pathological change occurring in the blood vessel wall and surrounding tissues. It is the common pathological basis and key driving factor for atherosclerosis, plaque formation, thrombosis, and even acute cardiovascular and cerebrovascular events (such as myocardial infarction and stroke). Its occurrence and development are closely related to multiple factors, including lipid metabolism disorders, oxidative stress, immune cell infiltration, and abnormal release of cytokines. Currently, clinical treatment for vasculitis mainly relies on statins, antiplatelet drugs, and nonsteroidal anti-inflammatory drugs (NSAIDs). While these drugs can control inflammation and thrombosis progression to some extent, long-term use can easily lead to drug resistance, gastrointestinal damage, and abnormal liver and kidney function. Furthermore, existing treatment plans usually only focus on the local blood vessels and fail to effectively intervene in the "gut-vascular axis," which is closely related to vascular health, making it difficult to achieve systemic vascular protection.

[0003] Traditional Chinese medicine (TCM) exhibits unique advantages in preventing and treating vascular inflammation due to its multi-component, multi-pathway, and multi-target nature. Chinese patent CN103566184B describes a TCM composition for treating vascular endothelial inflammation and its preparation method, which reduces vascular inflammatory factors. Pharmacological experiments have confirmed its effective inhibitory effect on inflammatory factors and its protective effect on vascular endothelial cells. However, traditional TCM decoctions or ordinary extracts generally suffer from low dissolution rates of active ingredients, difficulty in direct absorption of large molecules, and low bioavailability, significantly limiting their efficacy.

[0004] In recent years, probiotic intervention has received widespread attention as a novel strategy for regulating the gut-vascular axis. Specific probiotics can indirectly exert anti-vascular inflammation effects by improving gut microbiota structure, enhancing intestinal barrier function, and reducing the production of pro-inflammatory / pro-atherosclerotic metabolites such as TMAO. However, the scope and efficacy of single probiotic strains are limited.

[0005] Combining probiotic fermentation technology with traditional Chinese medicine (TCM) theoretically allows for the degradation of large TCM molecules into more easily absorbed small-molecule active ingredients using microbial enzymes, improving bioavailability. It also generates new prebiotics and post-biotics, synergistically regulating the gut microbiota. However, existing TCM-probiotic fermentation preparations often lack a systematic design targeting the pathological processes of vascular inflammation, leading to insufficient targeted conversion of key active ingredients and unstable metabolite profiles. Ultimately, this results in insufficient anti-vascular inflammation activity and poor reproducibility of therapeutic effects.

[0006] The evaluation of the therapeutic effects on vascular inflammation relies on objective and quantifiable pathophysiological indicators. Among these, plasma low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels are key blood biochemical indicators reflecting lipid metabolism and inflammatory status. Pathological observation of the morphology, size, and vessel wall structure of aortic root plaques, and precise calculation of the plaque area to vessel lumen area, is one of the "gold standards" for evaluating the degree of vascular inflammation, plaque burden, and the effectiveness of treatment interventions. Developing a fermented traditional Chinese medicine product that can significantly improve these core indicators has clear clinical guiding significance. Summary of the Invention

[0007] The purpose of this invention is to provide a traditional Chinese medicine fermentation composition for treating vascular inflammation and its preparation method.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A traditional Chinese medicine fermentation composition for treating vascular inflammation comprises the following components in parts by weight: 1-10 parts mulberry leaves, 5-15 parts bitter melon, 5-15 parts Solomon's seal rhizome, 1-10 parts earthworm protein, 10-20 parts Polygonatum rhizome, and 5-20 parts corn silk.

[0009] Preferably, the components include the following parts by weight: 6-9 parts mulberry leaves, 8-12 parts bitter melon, 7-13 parts Solomon's seal rhizome, 3-7 parts earthworm protein, 10-15 parts Polygonatum rhizome, and 6-11 parts corn silk.

[0010] The preparation method of the traditional Chinese medicine fermentation composition for treating vascular inflammation includes the following steps: (1) Mix mulberry leaves, bitter melon, Solomon's seal, Polygonatum sibiricum and corn silk and then crush them. Mix them evenly with earthworm protein powder to obtain Chinese medicine mixed powder; (2) Add deionized water to the mixed powder of Chinese medicine, stir evenly, adjust the pH, cool, and obtain the fermentation substrate; (3) Inoculate Lactobacillus acidophilus into the fermentation substrate for anaerobic fermentation; inoculate Lactobacillus rhamnosus for anaerobic fermentation; inoculate Lactobacillus plantarum for anaerobic fermentation to obtain fermentation broth; (4) The fermentation liquid is concentrated under reduced pressure to obtain a clear extract. The clear extract is freeze-dried and pulverized to obtain a traditional Chinese medicine fermentation composition.

[0011] Preferably, the strain of Lactobacillus acidophilus is numbered JYLA-191, and the dosage of Lactobacillus acidophilus is (3-5) × 10⁻⁶. 9 CFU / mL fermentation substrate.

[0012] Preferably, the strain of *Lactobacillus rhamnosus* is numbered JYLR-005, and the dosage of *Lactobacillus rhamnosus* is (6-8) × 10⁻⁶. 9 CFU / mL fermentation substrate.

[0013] Preferably, the strain of *Lactobacillus plantarum* is numbered JYLP-326, and the dosage of *Lactobacillus plantarum* is (1-3) × 10⁻⁶. 9 CFU / mL fermentation substrate.

[0014] The preferred anaerobic fermentation conditions for Lactobacillus acidophilus are: a temperature of 36-37℃ and a fermentation time of 10-15 hours.

[0015] The preferred anaerobic fermentation conditions for Lactobacillus rhamnosus are: temperature 38-39℃, fermentation time 15-20h.

[0016] The preferred conditions for anaerobic fermentation of Lactobacillus plantarum are: temperature 30-32℃, fermentation time 10-15h.

[0017] Preferably, the total fermentation time for Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus plantarum is 40-45 hours.

[0018] This invention is based on the design concept of "traditional Chinese medicine synergistic with probiotics, fermentation enhances efficacy and protects blood vessels". It precisely selects traditional Chinese medicine combinations and probiotic strains: The traditional Chinese medicine combination consists of mulberry leaves (dispersing wind-heat, clearing the liver and improving eyesight), bitter melon (clearing heat and relieving summer heat, improving eyesight and detoxifying), Solomon's seal (nourishing yin and moistening dryness, promoting body fluid and quenching thirst), earthworm protein (activating blood circulation and removing blood stasis, unblocking meridians and collaterals), polygonatum (tonifying qi and nourishing yin, strengthening the spleen and benefiting the kidneys), and corn silk (promoting diuresis and reducing swelling, promoting diuresis and relieving jaundice). After fermentation, the various components obtained have a synergistic effect on multiple targets such as anti-inflammation, improving circulation, and protecting vascular endothelium, which can specifically relieve vascular inflammation. You can optionally add wolfberry (nourishing the liver and kidneys, benefiting essence and improving eyesight) or salvia miltiorrhiza (activating blood circulation and removing blood stasis, unblocking meridians and relieving pain) to further enhance the blood-activating and anti-inflammatory effects.

[0019] This invention screens Lactobacillus acidophilus JYLA-191, Lactobacillus rhamnosus JYLR-005, and Lactobacillus plantarum JYLP-326, which play a dual role. They are not only used for fermenting Chinese medicine raw materials, but this combination can also tolerate the acid-base environment of the gastrointestinal tract. During the fermentation process, it efficiently metabolizes the macromolecular components of Chinese medicine (such as polysaccharides and saponins) and converts them into small molecule active substances (such as oligosaccharides and aglycones). At the same time, it regulates the balance of intestinal flora and forms a synergistic anti-inflammatory effect with the components of Chinese medicine.

[0020] The preparation method of this invention is optimized based on the characteristics of traditional Chinese medicine components and the metabolic rules of probiotics: a gradient fermentation method is adopted to ensure the rapid proliferation of probiotics and promote the generation of active metabolites; vacuum freeze-drying technology maximizes the preservation of the number of live probiotics and active components of traditional Chinese medicine, avoids high temperature damage, and can also form formulations; mannitol and microcrystalline cellulose are selected as carriers to improve the stability and oral compliance of the formulation.

[0021] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. This invention uses gradient probiotic fermentation to transform large molecular components in traditional Chinese medicine that are not easily absorbed into small molecule active substances, thereby improving the absorption and utilization rate by the human body and solving the problem of low bioavailability of traditional Chinese medicine.

[0022] 2. The raw materials of this invention are all natural Chinese herbal medicines and probiotics, with no chemically synthesized components. Long-term toxicity tests show that there are no abnormalities in the liver and kidney function and blood routine indicators of rats, good gastrointestinal tolerance, and no obvious side effects. 3. The traditional Chinese medicine fermentation composition of the present invention can be used for the prevention and treatment of diseases such as vascular inflammation, arteriosclerosis, and hyperlipidemia, and can also be used as a health care preparation for cardiovascular healthy people, adapting to different needs and scenarios. Attached Figure Description

[0023] Figure 1 The image shows the results of Oil Red O staining of the aorta in three mice from the model group.

[0024] Figure 2 The image shows the results of Oil Red O staining of the aorta in three mice from Example 1. Figure 3 HE staining images of three mice in the model group.

[0025] Figure 4 HE staining images of three mice in the model group. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0027] All raw materials used in the following embodiments of the present invention are commercially available products: The strain of Lactobacillus acidophilus, with the accession number JYLA-191, was purchased from Shandong Zhongke Jiayi Biotechnology Co., Ltd., and its depositary is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. Its accession number is CGMCC No. 21371.

[0028] The strain of *Lactobacillus rhamnosus*, numbered JYLR-005, was purchased from Shandong Zhongke Jiayi Biotechnology Co., Ltd., and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 16103.

[0029] The strain of *Lactobacillus plantarum*, numbered JYLP-326, was purchased from Shandong Zhongke Jiayi Biotechnology Co., Ltd., and its depositary address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.18038.

[0030] Example 1 This embodiment provides a capsule formulation of a traditional Chinese medicine fermentation composition for treating vascular inflammation, the preparation method of which includes the following steps: 1. Preparation of materials: Weigh out 8 parts of mulberry leaves, 10 parts of bitter melon, 9 parts of Solomon's seal, 5 parts of earthworm protein, 12 parts of Polygonatum, and 8 parts of corn silk according to the weight. Mix the mulberry leaves, bitter melon, Solomon's seal, Polygonatum, and corn silk and grind them to less than 70 mesh. Mix them evenly with the earthworm protein powder to obtain a mixed powder of Chinese medicine. 2. Preparation of fermentation substrate: Take 50 parts of mixed Chinese medicine powder, add 125 parts of deionized water, stir evenly, adjust the pH to 5.8 with citrate-sodium dihydrogen phosphate buffer, sterilize at 115℃ for 30 min, cool to 28℃ to obtain fermentation substrate; 3. Inoculation and Fermentation: Inoculate the fermentation substrate with Lactobacillus acidophilus at a dosage of 4 × 10⁻⁶. 9 The fermentation substrate was prepared at CFU / mL, and nitrogen gas was introduced to maintain an anaerobic environment (oxygen content 0.1 (v / v)%). The temperature was controlled at 37℃, and fermentation was carried out for 12 hours. To maintain the anaerobic environment, the temperature was adjusted to 39℃, and then *Lactobacillus rhamnosus* was inoculated at a concentration of 7 × 10⁻⁶ CFU / mL. 9 Fermentation substrate: CFU / mL, temperature 39℃, fermentation time 18h, with slow stirring for 10min every 6 hours during this stage; then, lower the fermentation broth temperature to 32℃ and inoculate with *Lactobacillus plantarum* at a dosage of 1×10⁻⁶. 9 The fermentation substrate was prepared at CFU / mL, maintaining an anaerobic environment at 32℃ for 12 hours. The mixture was stirred for 10 minutes at the 6th hour of fermentation to obtain the fermentation broth. 4. Formulation: The fermentation broth is concentrated under reduced pressure to a clear extract with a relative density of 1.15 (measured at 60℃). The clear extract is freeze-dried at -40℃ and a vacuum of 12Pa for 18 hours and pulverized to less than 100 mesh to obtain a traditional Chinese medicine fermentation composition. 7 parts mannitol and 5 parts microcrystalline cellulose are added to the traditional Chinese medicine fermentation composition and mixed evenly to obtain the active ingredient. The active ingredient is used to fill capsules to make a capsule formulation containing 250mg of active ingredient per capsule.

[0031] Example 2 This embodiment provides a capsule formulation of a traditional Chinese medicine fermentation composition for treating vascular inflammation, the preparation method of which includes the following steps: 1. Preparation of materials: Weigh out 6 parts of mulberry leaves, 12 parts of bitter melon, 13 parts of Solomon's seal, 3 parts of earthworm protein, 15 parts of Polygonatum, and 6 parts of corn silk according to the weight. Mix the mulberry leaves, bitter melon, Solomon's seal, Polygonatum, and corn silk and grind them to less than 70 mesh. Mix them evenly with the earthworm protein powder to obtain a mixed powder of traditional Chinese medicine. 2. Preparation of fermentation substrate: Take 50 parts of mixed Chinese medicine powder, add 125 parts of deionized water, stir evenly, adjust the pH to 5.8 with citrate-sodium dihydrogen phosphate buffer, sterilize at 115℃ for 30 min, cool to 28℃ to obtain fermentation substrate; 3. Inoculation and Fermentation: Inoculate the fermentation substrate with Lactobacillus acidophilus at a dosage of 3 × 10⁻⁶. 9 The fermentation substrate was prepared at CFU / mL, and nitrogen gas was introduced to maintain an anaerobic environment (oxygen content 0.1 (v / v)%). The temperature was controlled at 37℃, and fermentation was carried out for 11 hours. To maintain the anaerobic environment, the temperature was adjusted to 39℃, and then *Lactobacillus rhamnosus* was inoculated at a concentration of 8 × 10⁻⁶ CFU / mL. 9 Fermentation substrate: CFU / mL, temperature 39℃, fermentation time 17h, with slow stirring for 10min every 6 hours during this stage; then, lower the fermentation broth temperature to 32℃ and inoculate with *Lactobacillus plantarum* at a dosage of 3×10⁻⁶. 9 The fermentation substrate was prepared at CFU / mL, maintaining an anaerobic environment at 32℃ for 14 hours. The mixture was stirred for 10 minutes at the 6th hour of fermentation to obtain the fermentation broth. 4. Formulation: The fermentation broth is concentrated under reduced pressure to a clear extract with a relative density of 1.15 (measured at 60℃). The clear extract is freeze-dried at -40℃ and a vacuum of 12Pa for 18 hours and pulverized to a particle size of less than 100 mesh. 7 parts mannitol and 5 parts microcrystalline cellulose are added to the fermentation composition of traditional Chinese medicine and mixed evenly to obtain the active ingredient. The active ingredient is used to fill capsules to make a capsule formulation containing 250mg of active ingredient per capsule.

[0032] Comparative Example 1 The difference between this comparative example and Example 1 is that gradient fermentation is not performed.

[0033] A capsule formulation of a traditional Chinese medicine fermentation composition for treating vascular inflammation, the preparation method of which includes the following steps: 1. Preparation of materials: Weigh out 8 parts of mulberry leaves, 10 parts of bitter melon, 9 parts of Solomon's seal, 5 parts of earthworm protein, 12 parts of Polygonatum, and 8 parts of corn silk according to the weight. Mix the mulberry leaves, bitter melon, Solomon's seal, Polygonatum, and corn silk and grind them to less than 70 mesh. Mix them evenly with the earthworm protein powder to obtain a mixed powder of Chinese medicine. 2. Preparation of fermentation substrate: Take 50 parts of mixed Chinese medicine powder, add 125 parts of deionized water, stir evenly, adjust the pH to 5.8 with citrate-sodium dihydrogen phosphate buffer, sterilize at 115℃ for 30 min, cool to 28℃ to obtain fermentation substrate; 3. Inoculation and Fermentation: Inoculate the fermentation substrate with a mixed probiotic strain, including Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus plantarum. The amount of Lactobacillus acidophilus used is 4 × 10⁻⁶. 9 The amount of CFU / mL fermentation substrate and Lactobacillus rhamnosus was 7 × 10⁻⁶. 9 The amount of CFU / mL fermentation substrate and Lactobacillus plantarum was 1×10⁻⁶. 9 The fermentation substrate was prepared with CFU / mL, and nitrogen gas was introduced to maintain an anaerobic environment (oxygen content 0.1 (v / v)%). The temperature was controlled at 37℃, and the fermentation was carried out for 42 hours. The mixture was slowly stirred for 10 minutes every 12 hours to obtain the fermentation broth. 4. Formulation: The fermentation broth is concentrated under reduced pressure to a clear extract with a relative density of 1.15 (measured at 60℃). The clear extract is freeze-dried at -40℃ and a vacuum of 12Pa for 18 hours and pulverized to less than 100 mesh to obtain a traditional Chinese medicine fermentation composition. 7 parts mannitol and 5 parts microcrystalline cellulose are added to the traditional Chinese medicine fermentation composition and mixed evenly to obtain the active ingredient. The active ingredient is used to fill capsules to make a capsule formulation containing 250mg of active ingredient per capsule.

[0034] Comparative Example 2 The difference between this comparative example and Example 1 is that the order in which the bacterial strains were used is different.

[0035] A capsule formulation of a traditional Chinese medicine fermentation composition for treating vascular inflammation, the preparation method of which includes the following steps: 1. Preparation of materials: Weigh out 8 parts of mulberry leaves, 10 parts of bitter melon, 9 parts of Solomon's seal, 5 parts of earthworm protein, 12 parts of Polygonatum, and 8 parts of corn silk according to the weight. Mix the mulberry leaves, bitter melon, Solomon's seal, Polygonatum, and corn silk and grind them to less than 70 mesh. Mix them evenly with the earthworm protein powder to obtain a mixed powder of Chinese medicine. 2. Preparation of fermentation substrate: Take 50 parts of mixed Chinese medicine powder, add 125 parts of deionized water, stir evenly, adjust the pH to 5.8 with citrate-sodium dihydrogen phosphate buffer, sterilize at 115℃ for 30 min, cool to 28℃ to obtain fermentation substrate; 3. Inoculation and Fermentation: Inoculate the fermentation substrate with *Lactobacillus rhamnosus* and purge with nitrogen to maintain an anaerobic environment (oxygen content 0.1 (v / v)%). The amount of *Lactobacillus rhamnosus* used is 7 × 10⁻⁶. 9 Fermentation substrate: CFU / mL, temperature 39℃, fermentation time 18h, with slow stirring for 10min every 6 hours during this stage; then adjust the temperature to 32℃ and inoculate with *Lactobacillus plantarum* at a dosage of 1×10⁻⁶. 9 Fermentation substrate was prepared at CFU / mL, maintaining an anaerobic environment at 32℃ for 12 hours, with stirring for 10 minutes at the 6th hour. The fermentation broth temperature was maintained at 37℃, and Lactobacillus acidophilus was inoculated at a concentration of 4 × 10⁻⁶ CFU / mL. 9A CFU / mL fermentation substrate was used, and nitrogen gas was introduced to maintain an anaerobic environment (oxygen content ≤1%). The temperature was controlled at 37℃, and fermentation was carried out for 12 hours to obtain the fermentation broth. 4. Formulation: The fermentation broth is concentrated under reduced pressure to a clear extract with a relative density of 1.15 (measured at 60℃). The clear extract is freeze-dried at -40℃ and a vacuum of 12Pa for 18 hours and pulverized to less than 100 mesh to obtain a traditional Chinese medicine fermentation composition. 7 parts mannitol and 5 parts microcrystalline cellulose are added to the traditional Chinese medicine fermentation composition and mixed evenly to obtain the active ingredient. The active ingredient is used to fill capsules to make a capsule formulation containing 250mg of active ingredient per capsule.

[0036] Comparative Example 3 The difference between this comparative example and Example 1 is that the order in which the bacterial strains were used is different.

[0037] A capsule formulation of a traditional Chinese medicine fermentation composition for treating vascular inflammation, the preparation method of which includes the following steps: 1. Preparation of materials: Weigh out 8 parts of mulberry leaves, 10 parts of bitter melon, 9 parts of Solomon's seal, 5 parts of earthworm protein, 12 parts of Polygonatum, and 8 parts of corn silk according to the weight. Mix the mulberry leaves, bitter melon, Solomon's seal, Polygonatum, and corn silk and grind them to less than 70 mesh. Mix them evenly with the earthworm protein powder to obtain a mixed powder of Chinese medicine. 2. Preparation of fermentation substrate: Take 50 parts of mixed Chinese medicine powder, add 125 parts of deionized water, stir evenly, adjust the pH to 5.8 with citrate-sodium dihydrogen phosphate buffer, sterilize at 115℃ for 30 min, cool to 28℃ to obtain fermentation substrate; 3. Inoculation and Fermentation: Inoculate the fermentation substrate with Lactobacillus plantarum at a dosage of 1×10⁻⁶. 9 The fermentation substrate was prepared at CFU / mL, and nitrogen gas was introduced to maintain an anaerobic environment (oxygen content ≤0.1%). The anaerobic environment was maintained at 32℃ for 12 hours. At the 6th hour of fermentation, the mixture was stirred for 10 minutes, and Lactobacillus acidophilus was inoculated at a concentration of 4 × 10⁻⁶ CFU / mL. 9 Fermentation substrate was prepared at CFU / mL, with the temperature controlled at 37℃ for 12 hours. An anaerobic environment was maintained, and the temperature was adjusted to 39℃ before inoculation with *Lactobacillus rhamnosus* at a concentration of 7 × 10⁻⁶ CFU / mL. 9 CFU / mL fermentation substrate, temperature 39℃, fermentation for 18h, during this stage, slowly stir for 10min every 6 hours; 4. Formulation: The fermentation broth is concentrated under reduced pressure to a clear extract with a relative density of 1.15 (measured at 60℃). The clear extract is freeze-dried at -40℃ and a vacuum of 12Pa for 18 hours and pulverized to less than 100 mesh to obtain a traditional Chinese medicine fermentation composition. 7 parts mannitol and 5 parts microcrystalline cellulose are added to the traditional Chinese medicine fermentation composition and mixed evenly to obtain the active ingredient. The active ingredient is used to fill capsules to make a capsule formulation containing 250mg of active ingredient per capsule.

[0038] Comparative Example 4 The difference between this comparative example and Example 1 is that the total fermentation time of the strains is the same, but the fermentation times of the three strains are different.

[0039] A capsule formulation of a traditional Chinese medicine fermentation composition for treating vascular inflammation, the preparation method of which includes the following steps: 1. Preparation of materials: Weigh out 8 parts of mulberry leaves, 10 parts of bitter melon, 9 parts of Solomon's seal, 5 parts of earthworm protein, 12 parts of Polygonatum, and 8 parts of corn silk according to the weight. Mix the mulberry leaves, bitter melon, Solomon's seal, Polygonatum, and corn silk and grind them to less than 70 mesh. Mix them evenly with the earthworm protein powder to obtain a mixed powder of Chinese medicine. 2. Preparation of fermentation substrate: Take 50 parts of mixed Chinese medicine powder, add 125 parts of deionized water, stir evenly, adjust the pH to 5.8 with citrate-sodium dihydrogen phosphate buffer, sterilize at 115℃ for 30 min, cool to 28℃ to obtain fermentation substrate; 3. Inoculation and Fermentation: Inoculate the fermentation substrate with Lactobacillus acidophilus at a dosage of 4 × 10⁻⁶. 9 The fermentation substrate was prepared at CFU / mL, and nitrogen gas was introduced to maintain an anaerobic environment (oxygen content 0.1 (v / v)%). The temperature was controlled at 37℃, and fermentation was carried out for 10 hours. To maintain the anaerobic environment, the temperature was adjusted to 39℃, and then *Lactobacillus rhamnosus* was inoculated at a concentration of 7 × 10⁻⁶. 9 Fermentation substrate: CFU / mL, temperature 39℃, fermentation time 14h, with slow stirring for 10min every 6 hours during this stage; then, lower the fermentation broth temperature to 32℃ and inoculate with *Lactobacillus plantarum* at a dosage of 1×10⁻⁶. 9 The fermentation substrate was prepared at CFU / mL, maintaining an anaerobic environment at 32℃ for 18 hours. The mixture was stirred for 10 minutes at the 6th hour of fermentation to obtain the fermentation broth. 4. Formulation: The fermentation broth is concentrated under reduced pressure to a clear extract with a relative density of 1.15 (measured at 60℃). The clear extract is freeze-dried at -40℃ and a vacuum of 12Pa for 18 hours and pulverized to less than 100 mesh to obtain a traditional Chinese medicine fermentation composition. 7 parts mannitol and 5 parts microcrystalline cellulose are added to the traditional Chinese medicine fermentation composition and mixed evenly to obtain the active ingredient. The active ingredient is used to fill capsules to make a capsule formulation containing 250mg of active ingredient per capsule.

[0040] Comparative Example 5 The difference between this comparative example and Example 1 is that the total fermentation time of the strains is different.

[0041] A capsule formulation of a traditional Chinese medicine fermentation composition for treating vascular inflammation, the preparation method of which includes the following steps: 1. Preparation of materials: Weigh out 8 parts of mulberry leaves, 10 parts of bitter melon, 9 parts of Solomon's seal, 5 parts of earthworm protein, 12 parts of Polygonatum, and 8 parts of corn silk according to the weight. Mix the mulberry leaves, bitter melon, Solomon's seal, Polygonatum, and corn silk and grind them to less than 70 mesh. Mix them evenly with the earthworm protein powder to obtain a mixed powder of Chinese medicine. 2. Preparation of fermentation substrate: Take 50 parts of mixed Chinese medicine powder, add 125 parts of deionized water, stir evenly, adjust the pH to 5.8 with citrate-sodium dihydrogen phosphate buffer, sterilize at 115℃ for 30 min, cool to 28℃ to obtain fermentation substrate; 3. Inoculation and Fermentation: Inoculate the fermentation substrate with Lactobacillus acidophilus at a dosage of 4 × 10⁻⁶. 9 The fermentation substrate was prepared at CFU / mL, and nitrogen gas was introduced to maintain an anaerobic environment (oxygen content ≤0.1%). The temperature was controlled at 37℃, and fermentation was carried out for 15 hours. To maintain the anaerobic environment, the temperature was adjusted to 39℃, and then *Lactobacillus rhamnosus* was inoculated at a concentration of 7 × 10⁻⁶ CFU / mL. 9 Fermentation substrate: CFU / mL, temperature 39℃, fermentation time 20h, with slow stirring for 10min every 6 hours during this stage; then, lower the fermentation broth temperature to 32℃ and inoculate with *Lactobacillus plantarum* at a dosage of 1×10⁻⁶. 9 The fermentation substrate was prepared at CFU / mL, maintaining an anaerobic environment at 32℃ for 15 hours. The mixture was stirred for 10 minutes at the 6th hour of fermentation to obtain the fermentation broth. 4. Formulation: The fermentation broth is concentrated under reduced pressure to a clear extract with a relative density of 1.15 (measured at 60℃). The clear extract is freeze-dried at -40℃ and a vacuum of 12Pa for 18 hours and pulverized to less than 100 mesh to obtain a traditional Chinese medicine fermentation composition. 7 parts mannitol and 5 parts microcrystalline cellulose are added to the traditional Chinese medicine fermentation composition and mixed evenly to obtain the active ingredient. The active ingredient is used to fill capsules to make a capsule formulation containing 250mg of active ingredient per capsule.

[0042] Comparative Example 6 The difference between this comparative example and Example 1 is that the dosage of the three bacterial strains is different.

[0043] A capsule formulation of a traditional Chinese medicine fermentation composition for treating vascular inflammation, the preparation method of which includes the following steps: 1. Preparation of materials: Weigh out 8 parts of mulberry leaves, 10 parts of bitter melon, 9 parts of Solomon's seal, 5 parts of earthworm protein, 12 parts of Polygonatum, and 8 parts of corn silk according to the weight. Mix the mulberry leaves, bitter melon, Solomon's seal, Polygonatum, and corn silk and grind them to less than 70 mesh. Mix them evenly with the earthworm protein powder to obtain a mixed powder of Chinese medicine. 2. Preparation of fermentation substrate: Take 50 parts of mixed Chinese medicine powder, add 125 parts of deionized water, stir evenly, adjust the pH to 5.8 with citrate-sodium dihydrogen phosphate buffer, sterilize at 115℃ for 30 min, cool to 28℃ to obtain fermentation substrate; 3. Inoculation and Fermentation: Inoculate the fermentation substrate with Lactobacillus acidophilus at a dosage of 7 × 10⁻⁶. 9 The fermentation substrate was prepared at CFU / mL, and nitrogen gas was introduced to maintain an anaerobic environment (oxygen content ≤0.1%). The temperature was controlled at 37℃, and fermentation was carried out for 12 hours. Maintaining the anaerobic environment, the temperature was adjusted to 39℃, and then *Lactobacillus rhamnosus* was inoculated at a concentration of 1×10⁻⁶. 9 Fermentation substrate: CFU / mL, temperature 39℃, fermentation time 18h, with slow stirring for 10min every 6 hours during this stage; then, lower the fermentation broth temperature to 32℃ and inoculate with *Lactobacillus plantarum* at a dosage of 4×10⁻⁶. 9 The fermentation substrate was prepared at CFU / mL, maintaining an anaerobic environment at 32℃ for 12 hours. The mixture was stirred for 10 minutes at the 6th hour of fermentation to obtain the fermentation broth. 4. Formulation: The fermentation broth is concentrated under reduced pressure to a clear extract with a relative density of 1.15 (measured at 60℃). The clear extract is freeze-dried at -40℃ and a vacuum of 12Pa for 18 hours and pulverized to less than 100 mesh to obtain a traditional Chinese medicine fermentation composition. 7 parts mannitol and 5 parts microcrystalline cellulose are added to the traditional Chinese medicine fermentation composition and mixed evenly to obtain the active ingredient. The active ingredient is used to fill capsules to make a capsule formulation containing 250mg of active ingredient per capsule.

[0044] Comparative Example 7 The difference between this comparative example and Example 1 is that 10 parts of mulberry leaves, 7 parts of bitter melon, 14 parts of Solomon's seal, 9 parts of earthworm protein, 9 parts of Polygonatum sibiricum, and 5 parts of corn silk were weighed out according to weight.

[0045] Comparative Example 8 The comparative example is the extract from Example 1 of CN103566184B, a traditional Chinese medicine composition for treating vascular endothelial inflammation and its preparation method. The extract was freeze-dried at -40°C and a vacuum of 12 Pa for 18 hours and then pulverized to a particle size of less than 100 mesh to obtain the composition.

[0046] Performance testing The performance of the fermented Chinese medicine compositions of Examples 1-2 and Comparative Examples 1-7, as well as the composition of Comparative Example 8, was tested.

[0047] (a) Finished atherosclerotic mice Adult mice (20-25g, purchased from Beijing Weishang Lide Biotechnology Co., Ltd., strain number VSM40001, application field: atherosclerosis) were purchased and acclimatized for 3 days. They were then divided into a model group and drug treatment groups (Examples 1-9). The model group consisted of 8 mice, and each drug treatment group consisted of 8 mice. The drug treatment groups were fed a high-fat diet throughout the treatment and were administered the fermented traditional Chinese medicine composition by gavage once daily for two weeks. The solid-liquid ratio of the fermented traditional Chinese medicine composition and purified water was diluted to 1g:18mL, and then administered by gavage at a volume of 8mL / kg. The model group was administered an equal volume of physiological saline by gavage.

[0048] (II) General Observation and Sample Collection General monitoring: During the intervention period, the weight changes of mice in each group were recorded daily, and the mice's mental state, activity, food intake and fecal characteristics were observed to ensure that there were no abnormal stress responses.

[0049] Sample collection: Two weeks after intervention, mice were fasted but allowed free access to water for 12 hours. Blood was collected and serum was separated.

[0050] (III) Detection of serum lipid metabolism indicators The levels of LDL-C (low-density lipoprotein cholesterol) and HDL-C (high-density lipoprotein cholesterol) in the serum of mice in each group were measured to analyze the regulatory effect of the traditional Chinese medicine fermentation composition on lipid metabolism in mice. The results are shown in Table 1.

[0051] (iv) Pathological morphological examination of the aorta and aortic root Oil Red O staining of the aorta: The separated aortic tissue was longitudinally cut, laid flat on a glass slide, fixed with 4% paraformaldehyde for 30 min, rinsed with distilled water, stained with Oil Red O staining solution for 30 min, differentiated with 70% ethanol until the background was colorless, rinsed with distilled water, mounted with glycerol gelatin, and the lipid deposition of the aortic intima was observed under an optical microscope. The percentage of lipid-positive area to the total aortic area was calculated using Image-ProPlus software.

[0052] HE staining of the aortic root: Fixed aortic root tissue was dehydrated in a gradient, embedded in paraffin, and serially sectioned (5 μm thick) for HE staining. The steps were as follows: sections were dewaxed to water, stained with hematoxylin for 5 min, differentiated with hydrochloric acid and ethanol for 30 s, blued with ammonia for 1 min, stained with eosin for 2 min, dehydrated in a gradient, cleared, and mounted with neutral resin. The morphology, size, and vessel wall structure of the aortic root plaques were observed under an optical microscope, and the ratio of plaque area to vessel lumen area was measured.

[0053] The results are shown in Table 1.

[0054] Table 1 Test Results

[0055] pass Figure 1-4 It can be seen that differences in lipid droplet deposition were observed in the aortic tissue sections of the model group and Experimental Example 1 after Oil Red O and HE staining. The model group showed a large amount of lipid deposition in the vascular region, while the lipid deposition in the aorta of Experimental Example 1 was significantly improved. Table 1 shows that the sequential inoculation and gradient fermentation process used in Examples 1-2 is an optimized synergistic system. Any change in the strain, sequence, time, ratio, or raw materials will disrupt this synergy, leading to a decrease in the efficacy of the final product in regulating blood lipids and inhibiting atherosclerotic plaques.

[0056] In Comparative Example 1, no gradient fermentation was performed, and the three strains were simultaneously inoculated. In the gradient fermentation used in this invention, the metabolites of the preceding strains become the substrates of the subsequent strains, thereby generating more complex and active secondary metabolites. Mixed fermentation cannot achieve this kind of targeted and deep biotransformation, resulting in insufficient types and contents of active ingredients, which weakens the core lipid-regulating and anti-plaque effects of Comparative Example 1.

[0057] The different order of bacterial strains used in Comparative Examples 2 and 3 is analyzed to be because the order in Example 1 was to start fermentation with Lactobacillus acidophilus under mild conditions to degrade some substances; then add Lactobacillus rhamnosus, and the two bacteria carried out the main transformation at a slightly higher temperature; finally, inoculate Lactobacillus plantarum, and the three bacteria carried out the finishing and stabilization at a lower temperature. The plaque ratio of Comparative Examples 2 and 3 was significantly higher than that of Example 1, and the LDL-C level was also higher, indicating that changing the order seriously weakened the core effect of anti-atherosclerosis.

[0058] In Comparative Example 4, the three strains had different fermentation time allocations but the total time was the same, resulting in insufficient fermentation during the critical stages and failure to fully accumulate their core metabolites. Excessively extending the third stage constitutes over-fermentation, which can lead to the degradation of beneficial products.

[0059] The fact that the total fermentation time of the strains in Comparative Example 5 was longer than that of the strains indicates that longer fermentation time is not necessarily better. Excessive fermentation time may cause the beneficial active ingredients that have been generated to be decomposed by the bacteria themselves or undergo chemical changes, producing some ineffective or potentially harmful metabolites.

[0060] The different proportions of the three bacterial strains in Comparative Example 6 disrupted the balance of the microbial community. Different strains contribute different metabolites, and changing the inoculation ratio directly alters the composition and relative content of various metabolites in the final fermentation broth, leading to an imbalance in the optimized active ingredient formulation. The extremely high LDL-C in Comparative Example 6 indicates that altering the strain ratio severely impairs the efficacy.

[0061] The different proportions of Chinese herbal raw materials in Comparative Example 7 disrupted the compatibility principle of the Chinese herbal compound and altered the fermentation substrate. This directly affected the growth and metabolic behavior of the microbial community, resulting in a completely different metabolite profile.

[0062] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fermented traditional Chinese medicine composition for treating vascular inflammation, characterized in that, The product contains the following components in parts by weight: 1-10 parts mulberry leaves, 5-15 parts bitter melon, 5-15 parts Solomon's seal rhizome, 1-10 parts earthworm protein, 10-20 parts Polygonatum rhizome, and 5-20 parts corn silk.

2. The traditional Chinese medicine fermentation composition for treating vascular inflammation according to claim 1, characterized in that, The components include the following parts by weight: 6-9 parts mulberry leaves, 8-12 parts bitter melon, 7-13 parts Solomon's seal rhizome, 3-7 parts earthworm protein, 10-15 parts polygonatum rhizome, and 6-11 parts corn silk.

3. A method for preparing a traditional Chinese medicine fermentation composition for treating vasculitis as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Mix mulberry leaves, bitter melon, Solomon's seal, Polygonatum sibiricum and corn silk and then crush them. Mix them evenly with earthworm protein powder to obtain Chinese medicine mixed powder; (2) Add deionized water to the mixed powder of Chinese medicine, stir evenly, adjust the pH, cool, and obtain the fermentation substrate; (3) Inoculate Lactobacillus acidophilus into the fermentation substrate for anaerobic fermentation; inoculate Lactobacillus rhamnosus for anaerobic fermentation; inoculate Lactobacillus plantarum for anaerobic fermentation to obtain fermentation broth; (4) The fermentation liquid is concentrated under reduced pressure to obtain a clear extract. The clear extract is freeze-dried and pulverized to obtain a traditional Chinese medicine fermentation composition.

4. The method for preparing the traditional Chinese medicine fermentation composition for treating vascular inflammation according to claim 3, characterized in that, The strain number of Lactobacillus acidophilus is JYLA-191, and the dosage of Lactobacillus acidophilus is (3-5) × 10. 9 CFU / mL fermentation substrate.

5. The method for preparing the traditional Chinese medicine fermentation composition for treating vascular inflammation according to claim 3, characterized in that, The strain number of *Lactobacillus rhamnosus* is JYLR-005, and the dosage of *Lactobacillus rhamnosus* is (6-8) × 10⁻⁶. 9 CFU / mL fermentation substrate.

6. The method for preparing the traditional Chinese medicine fermentation composition for treating vascular inflammation according to claim 3, characterized in that, The strain number of *Lactobacillus plantarum* is JYLP-326, and the dosage of *Lactobacillus plantarum* is (1-3) × 10⁻⁶. 9 CFU / mL fermentation substrate.

7. The method for preparing the traditional Chinese medicine fermentation composition for treating vascular inflammation according to claim 3, characterized in that, The anaerobic fermentation conditions for Lactobacillus acidophilus are: temperature controlled at 36-37℃, fermentation time 10-15h.

8. The method for preparing the traditional Chinese medicine fermentation composition for treating vascular inflammation according to claim 3, characterized in that, The anaerobic fermentation conditions for Lactobacillus rhamnosus are: temperature 38-39℃, fermentation time 15-20h.

9. The method for preparing the traditional Chinese medicine fermentation composition for treating vascular inflammation according to claim 3, characterized in that, The conditions for anaerobic fermentation of Lactobacillus plantarum are: temperature 30-32℃, fermentation time 10-15h.

10. The method for preparing the traditional Chinese medicine fermentation composition for treating vascular inflammation according to claim 3, characterized in that, The total fermentation time for Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus plantarum is 40-45 hours.

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