Dry / fresh mulberry root bark-derived extracellular vesicle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a dried / fresh mulberry root bark-derived extracellular vesicle as well as a preparation method and application thereof. According to the preparation method of the extracellular vesicles from the dry / fresh mulberry root bark, through optimization and purification steps, tissue homogenate is obtained through step-by-step filtration with multiple layers of filter screens, impurities are removed through differential centrifugation, supernatant is taken and centrifuged to remove subcell debris, vesicle precipitates are enriched through ultracentrifugation, sucrose density gradient centrifugal purification and size exclusion chromatography are carried out, and the extracellular vesicles from the dry / fresh mulberry root bark are obtained. The extracellular vesicles are successfully extracted from dry mulberry root bark and fresh mulberry root bark respectively, and the treatment effect of the dry mulberry root bark extracellular vesicles is verified to be the same as that of the fresh mulberry root bark extracellular vesicles. The method technically solves the problem of extracting the vesicles from the dry Chinese herbal medicines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a dry / fresh mulberry root bark-derived extracellular vesicle and a preparation method and application thereof. BACKGROUND

[0002] The intestine and the liver are important components of the digestive system, each having extremely complex physiological activities. The intestine and the liver originate from the same germ layer, and have many anatomical and functional connections between them. Intestinal diseases can cause liver damage, and liver diseases can also cause intestinal function disorders. The gut-liver axis refers to an interactive pathway formed between the intestine and the liver through the intestinal mucosal barrier, intestinal microbiota, intestinal immune system, portal vein system and other factors, which is integrated by signals from diet, genetics and environmental factors. The liver portal vein collects blood from the small intestine and large intestine, and intestinal-derived nutrients and other signals are transported to the liver through the portal vein circulation. The liver, as the largest immune organ, recruits and activates immune cells to respond to the significant ability of intestinal-derived metabolic substances or pathogenic signals. The gut-liver axis is a wide and close information exchange between the liver and the intestine, which not only affects the medium of interaction between the environment and the host, but also controls the health of the gastrointestinal tract and the liver. The "dialogue" between the intestine and the liver is the key to maintaining the homeostatic balance of the intestine and liver metabolism, and the two interact and cause each other, which has important physiological significance.

[0003] Intestinal barrier is a high-efficiency selective barrier system in the intestinal tract, which is composed of intestinal mucosa non-specific immune barrier (including mechanical barrier, chemical barrier, biological barrier, etc.) and intestinal mucosa specific immune barrier (immune barrier). Each of them has different structure, molecular regulation mechanism and biological function, and is organically combined together through their own signal pathways to jointly defend against the invasion of foreign antigenic substances. The impairment of intestinal barrier function is a prerequisite for the dysfunction of intestinal-liver axis and the induction of related diseases. Ulcerative colitis (UC) is a chronic nonspecific intestinal inflammatory disease. Although the pathogenesis of UC is not very clear, the damage of intestinal mucosa and the change of barrier function caused by external factors are the key factors in the pathogenesis of UC. Non-alcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver injury closely related to insulin resistance and genetic susceptibility. NAFLD includes three disease grades and stages, namely non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH) and its related cirrhosis and hepatocellular carcinoma. The impairment of intestinal barrier function plays an important role in the development of intestinal inflammation and NAFLD. It is of great clinical significance to strengthen the understanding of the pathophysiological mechanism of intestinal barrier dysfunction and carry out effective targeted intervention treatment.

[0004] The process of mulberry root bark regulating the liver-gut axis involves multiple levels such as intestinal flora, immune regulation, metabolic products and signal pathways. 1) Anti-inflammatory and immune regulation: flavonoids (such as Sanggenone H, Moracin) and alkaloids in mulberry root bark have significant anti-inflammatory effects, can inhibit the release of pro-inflammatory factors (such as TNF-α, IL-6), and reduce inflammation in the intestine and liver. For example: mulberry root bark extract can inhibit the activation of the NF-κB pathway in intestinal epithelial cells, reduce intestinal mucosal barrier damage, thereby reducing intestinal permeability, preventing endotoxins (such as LPS) from entering the liver through the portal vein, and reducing inflammation in the liver. In liver injury models, Sanggenone H reduces hepatocyte apoptosis by blocking endoplasmic reticulum stress and regulating autophagy, improving liver function. 2) Regulation of intestinal flora and metabolic products: polysaccharides and polyphenols in mulberry root bark can selectively promote the growth of probiotics (such as lactobacillus, bifidobacterium) and inhibit the proliferation of conditional pathogenic bacteria (such as escherichia coli), thereby maintaining the balance of intestinal flora. This regulatory effect regulates the liver-gut axis by regulating short-chain fatty acid (SCFA) production and bile acid metabolism. 3) Antioxidant and anti-fibrosis: Moracin and Sanggenone H in mulberry root bark have strong antioxidant activity, can scavenge free radicals and inhibit lipid peroxidation. 4) Intestinal-brain-liver axis neural regulation: Certain components in mulberry root bark may affect the liver-gut axis through the vagus nerve and neurotransmitters. 5) Diuresis and metabolic regulation: The diuretic effect of mulberry root bark (by promoting aquaporin expression) can reduce liver ascites and systemic edema, indirectly improving the liver microenvironment. In addition, mulberry root bark can also regulate blood glucose and lipid metabolism and relieve cholestasis to regulate the liver microenvironment. In summary, the mechanism of mulberry root bark on the liver-gut axis involves multi-target and multi-level regulation, including anti-inflammatory, flora regulation, antioxidant and neural-endocrine network intervention.

[0005] There are some bottlenecks in the application of Chinese herbal medicine, such as low efficacy, slow effect, and limited dosage form. Chinese Herbal Medicine-derived extracellular vesicles (CHM-EVs) not only retain the original effective components of Chinese herbal medicine, but also inherit the drug delivery capacity of extracellular vesicles, thereby significantly improving the delivery efficiency and in vitro and in vivo stability of drugs. CHM-EVs have unique properties such as stability, specificity, and safety, and have attracted widespread attention. Using CHM-EVs as therapeutic agents and drug delivery carriers can take advantage of Chinese herbal medicine in disease treatment and has a very broad application prospect. Therefore, the development of Chinese herbal medicine extracellular vesicles with intestinal barrier repair function to intervene in the 'liver-gut dialogue' is expected to become a new strategy for the intervention of intestinal and liver inflammation.

[0006] Previous studies on CHM-EVs mainly obtained vesicles from fresh Chinese herbal medicines by juicing. However, the storage and transportation costs of fresh Chinese herbal medicines are high, and the outdated technology for preserving medicinal materials has limited the clinical application of extracellular vesicles from fresh Chinese herbal medicines. Summary of the Invention

[0007] In clinical applications, dried products are the primary form of traditional Chinese medicine. If vesicles could be obtained from dried traditional Chinese medicine, it would not only broaden the source of extracellular vesicles in traditional Chinese medicine but also better suit its practical applications. Therefore, this invention provides extracellular vesicles from mulberry root bark, their preparation method, and their applications.

[0008] In a first aspect, the present invention provides a method for preparing extracellular vesicles derived from dried / fresh mulberry root bark, comprising the following steps: S1, White Mulberry ( Morus alba L. Fresh or dried mulberry root bark from the source was added to phosphate buffer for homogenization, followed by extraction and multi-layer filtration to obtain tissue homogenate. S2. Impurities in the tissue homogenate were removed sequentially by differential centrifugation. The supernatant was then centrifuged at 120,000~140,000×g and 3~5℃ for 80~100 min to enrich the vesicle precipitate. S3. Resuspend the vesicle precipitate, purify it by sucrose density gradient centrifugation, and collect the target band; S4. Size exclusion chromatography was performed on the target band to obtain extracellular vesicles of mulberry root bark with uniform size distribution.

[0009] This invention successfully extracted extracellular vesicles from dried and fresh mulberry root bark by optimizing the purification process (multi-layer filtration to obtain tissue homogenate, differential centrifugation to remove impurities, centrifugation of the supernatant to remove subcellular debris, ultracentrifugation to enrich vesicle precipitate, and sucrose density gradient centrifugation for purification). This significantly improved purity (≥95%) and yield (0.04 mg EVs / g). Furthermore, after final sucrose purification, size exclusion chromatography was used to ensure uniform EV size distribution, and the therapeutic effects of dried and fresh mulberry root bark extracellular vesicles were verified. This invention technically solves the problem of vesicle extraction from dried traditional Chinese medicine.

[0010] Furthermore, in S1, the extraction conditions are 3~5℃ for 10~12 h.

[0011] Furthermore, in S2, the conditions for differential centrifugation are: 200xg, 8~10 min; 400~500xg, 8~10 min; 800~1,000xg, 8~10 min; 1500~2,000xg, 15~20 min; 2500~3,000xg, 25~30 min; 4000~5,000xg, 25~30 min; 10,000~11,000xg, 55~60 min.

[0012] Further, in S3, the sucrose density gradient centrifugation is performed under the condition that the mass concentration of the sucrose solution from bottom to top is 8% to 60%, the centrifugal condition is 120,000~140,000xg, 3~5℃, and the target band is the sucrose solution with a mass concentration of 30%~45%.

[0013] In a second aspect, the present application provides the mulberry root bark extracellular vesicle prepared by the preparation method.

[0014] In a third aspect, the present application provides the use of the mulberry root bark extracellular vesicle in the preparation of an inflammation treatment drug.

[0015] Further, the inflammation includes ulcerative colitis and non-alcoholic fatty liver disease.

[0016] In a fourth aspect, the present application provides an inflammation treatment drug, wherein the inflammation treatment drug takes the mulberry root bark extracellular vesicle as an effective component.

[0017] Further, the inflammation treatment drug further includes a pharmaceutically acceptable excipient.

[0018] Further, in the drug, the content of the mulberry root bark extracellular vesicle is 1w%~99.9w%.

[0019] Compared with the prior art, the present application has the following beneficial effects: The storage and transportation cost of fresh Chinese herbal medicines is relatively high, and the preservation technology of medicinal materials is backward, so that the clinical application of the extracellular vesicles extracted from fresh Chinese herbal medicines by juicing is limited. Dry products are the main form of Chinese herbal medicines, and the present application obtains vesicles from dry mulberry root bark, and realizes the goal that the structure, material basis and treatment effect of dry mulberry root bark extracellular vesicles are almost the same as those of fresh mulberry root bark extracellular vesicles through quality control, so that the source of Chinese herbal medicine extracellular vesicles is more extensive, and the practical application of Chinese herbal medicine extracellular vesicles is laid a foundation.

[0020] Based on the current research status, whether the transformation of the same Chinese herbal medicine from fresh medicine to dry medicine has an impact on the pharmacodynamic material basis of extracellular vesicles and the prognosis of diseases needs to be solved. The present application explores the efficient separation and purification method of extracellular vesicles of dry and fresh mulberry root bark Chinese herbal medicine, compares the differences in pharmacodynamic material basis between fresh Chinese herbal medicine vesicles and dry Chinese herbal medicine vesicles, and explores their biological functions from the aspects of disease treatment, prognosis analysis, monitoring and tracking, etc. It is proved that the purified dry and fresh mulberry root bark extracellular vesicles have similar material basis and treatment effect. It has important significance for realizing the wider disease intervention and clinical transformation of Chinese herbal medicine extracellular vesicles.

[0021] The present application observes the ultrastructure of dry and fresh mulberry root bark extracellular vesicles before and after freeze-drying by transmission electron microscopy, quantifies the hydrodynamic diameter and surface Zeta potential before and after freeze-drying by dynamic light scattering, and proves that the freeze-drying strategy adopted in the present application does not change the structure, diameter and potential of dry and fresh mulberry root bark extracellular vesicles. The present application further evaluates the free radical scavenging capacity of mulberry root bark extracellular vesicles before and after freeze-drying by electron spin resonance spectroscopy (ESR), and observes the intracellular reactive oxygen species (ROS) scavenging capacity of mulberry root bark extracellular vesicles before and after freeze-drying by DCFH-DA fluorescent probe method. It is proved that the free radical scavenging capacity and ROS scavenging efficiency of dry and fresh mulberry root bark extracellular vesicles preserved by the present application have a slight decay trend, but still retain high activity.

[0022] Chinese herbal medicine vesicles not only retain the pharmacologically active substances of Chinese herbal medicine, but also have the unique drug delivery capacity of vesicles. Chinese herbal medicine vesicles are expected to become an important breakthrough in the inheritance and innovative application of traditional Chinese medicine. Mulberry root bark, a traditional Chinese medicine, is widely used in clinical practice, and has the effects of purging lung and relieving asthma, promoting water and reducing swelling, as well as immune regulation, antioxidant and anti-inflammatory effects. The present application comprehensively analyzes the contents and metabolites of mulberry root bark extracellular vesicles by multi-omics technology, identifies the bioactive molecules by characteristic atlas, mass spectrometry and high performance liquid chromatography, and comprehensively analyzes the similarities and differences between FMB-EVs and DMB-EVs by combining the prognosis effect of mulberry root bark extracellular vesicles in the intervention of inflammation, and analyzes the pharmacodynamic material basis of FMB-EVs and DMB-EVs, which provides inspiration for elucidating the pharmacodynamic material basis of traditional Chinese medicine and creating new traditional Chinese medicine drug carriers.

[0023] Intestinal barrier damage is a prerequisite for the alteration of the gut-liver axis and exacerbates intestinal and liver inflammation and injury. Targeted intervention of the gut-liver axis is expected to achieve effective relief of intestinal and liver diseases. By establishing in vitro and in vivo intestinal barrier damage models, the intervention of mulberry root bark cell extracellular vesicles on the intestinal barrier function from the aspects of mucosal barrier, physical barrier and immune barrier is evaluated, and the repair effect of mulberry root bark cell extracellular vesicles on the intestinal barrier function is determined. By exploring the regulatory effect of mulberry root bark cell extracellular vesicles on the intestinal barrier function in pathological conditions such as colitis and NAFLD, combined with multi-omics analysis, the therapeutic effect is revealed. The project effectively intervenes the gut-liver axis, which is expected to provide a new intervention strategy for intestinal / liver inflammation relief, and has important clinical significance. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Particle size distribution and TEM images of mulberry root bark cell extracellular vesicles obtained under different extraction conditions, wherein: A: Particle size distribution of mulberry root bark cell extracellular vesicles obtained under initial conditions.

[0025] B: TEM image of mulberry root bark cell extracellular vesicles obtained under initial conditions.

[0026] C: Particle size distribution of mulberry root bark cell extracellular vesicles obtained under improved condition 1.

[0027] D: TEM image of mulberry root bark cell extracellular vesicles obtained under improved condition 1.

[0028] E: Particle size distribution of mulberry root bark cell extracellular vesicles obtained under improved condition 2.

[0029] F: TEM image of mulberry root bark cell extracellular vesicles obtained under improved condition 2.

[0030] G: Particle size distribution of mulberry root bark cell extracellular vesicles obtained under improved condition 3.

[0031] H: TEM image of mulberry root bark cell extracellular vesicles obtained under improved condition 3.

[0032] Figure 2 Raw material quality conversion rule of fresh / dry mulberry root bark, wherein: A: Typical pictures of fresh mulberry roots, fresh root bark and dried root bark.

[0033] B: Fresh mulberry root bark extracellular vesicles were determined and quantified by BCA protein detection.

[0034] C: Dry mulberry root bark extracellular vesicles were determined and quantified by BCA protein detection.

[0035] D: Particle concentration of fresh mulberry root bark cell vesicle solution detected by electrical resistance pulse sensing technology.

[0036] E: Particle concentration of dried mulberry root bark cell vesicle solution detected by resistance pulse sensing technology.

[0037] Figure 3 The ability of extracellular vesicles in fresh / dried mulberry root bark to scavenge reactive oxygen species before and after freeze-drying storage, including: A: Morphological and structural changes of DMB-EVs before and after freeze-drying under transmission electron microscopy.

[0038] B: Particle size distribution of DMB-EVs before freeze-drying.

[0039] C: Zeta potential of DMB-EVs before freeze-drying.

[0040] D: Particle size distribution of DMB-EVs after freeze-drying.

[0041] E: Zeta potential of DMB-EVs after lyophilization.

[0042] F: •OH radicals and O2 before and after freeze-drying of DMB-EVs •⁻ Comparison of free radical scavenging capabilities.

[0043] G: ABTS before and after DMB-EVs freeze-drying + • Comparison of free radical scavenging capabilities.

[0044] H: Comparison of •OH free radical scavenging ability of DMB-EVs before and after freeze-drying.

[0045] I: Comparison of DPPH free radical scavenging ability before and after DMB-EVs lyophilization.

[0046] J: Morphological and structural changes of FMB-EVs before and after freeze-drying under transmission electron microscopy.

[0047] K: Particle size distribution of FMB-EVs before freeze-drying.

[0048] L: Zeta potential of FMB-EVs before freeze-drying.

[0049] M: Particle size distribution of FMB-EVs after freeze-drying.

[0050] N: Zeta potential of FMB-EVs after lyophilization.

[0051] O: OH radicals and O2 before and after freeze-drying of FMB-EVs •⁻ Comparison of free radical scavenging capabilities.

[0052] P: ABTS before and after freeze-drying of FMB-EVs + • Comparison of free radical scavenging capabilities.

[0053] Q: Comparison of •OH free radical scavenging ability of FMB-EVs before and after freeze-drying.

[0054] R: Comparison of DPPH radical scavenging ability of FMB-EVs before and after lyophilization.

[0055] Figure 4 To verify the effect of oral mulberry root cell extracellular vesicles in relieving DSS-induced acute colitis, wherein: A: Schematic diagram of DSS-induced acute colitis and oral MB-EVs strategy.

[0056] B: Comparison of body weight between the control group and the intervention group within 12 days.

[0057] C: Comparison of disease activity index between the control group and the intervention group.

[0058] D: Comparison of spleen index between the control group and the intervention group.

[0059] E: Comparison of colon length between the control group and the intervention group.

[0060] F: Typical picture of colon length difference between the control group and the intervention group.

[0061] G: Endoscopic image of mice on the 11th day between the control group and the intervention group.

[0062] H: Results of histological evaluation of colon tissue by H&E staining.

[0063] I: Results of mucus layer damage assessment by Alcian blue staining of colon tissue.

[0064] Figure 5 is the verification result of oral mulberry root cell extracellular vesicles in relieving liver inflammation and colon inflammation in mouse NASH model, wherein: A: Schematic diagram of in vitro establishment of NASH model.

[0065] B: Oral FITC-dextran detection of intestinal barrier permeability.

[0066] C: Assessment of the effect of MB-EVs on the liver by liver specific gravity.

[0067] D: Schematic diagram of colon and liver dissection.

[0068] E: Histological evaluation of the effect of MB-EVs on the liver by H&E staining, oil red O staining, and Masson staining.

[0069] F: Evaluation of whether liver inflammation affects intestinal structure by intestinal H&E staining. DETAILED DESCRIPTION

[0070] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0071] Example 1: Extraction and characterization of extracellular vesicles from dried and fresh mulberry root bark.

[0072] I. Experimental Methods 1. Extraction of extracellular vesicles from dried and fresh mulberry root bark. Fresh white mulberry (Morus alba) Morus alba L. The cortical tissue was obtained by dissecting and separating the roots. After removing the xylem, some fresh mulberry root bark was dried in a 60℃ hot air drying oven for 48 hours.

[0073] Equal volumes of fresh and dried mulberry root bark samples were added to pre-cooled phosphate-buffered saline (PBS, pH 7.4) (material-to-liquid ratio: fresh mulberry root bark 1:4 (g / mL), dried mulberry root bark 1:6 (g / mL)) for mechanical homogenization (using a Polytron homogenizer), followed by extraction at 4℃ for 12 hours. The homogenate was obtained by filtration through three layers of filters: 50 mesh → 100 mesh → 200 mesh (280 μm → 150 μm → 74 μm).

[0074] Impurities in the tissue homogenate were removed sequentially using a differential centrifugation program. The differential centrifugation program was as follows: 200×g, 10 min, 4℃; 500×g, 10 min, 4℃; 1,000×g, 10 min, 4℃; 2,000×g, 20 min, 4℃; 3,000×g, 30 min, 4℃; to remove dead cells and large debris; 5,000 × g, 30 min, 4℃; 10,000×g, 60 min, 4℃; further removal of subcellular debris; 130,000×g, 90 min, 4℃, ultracentrifugation to enrich vesicle precipitates.

[0075] After resuspending in 1 mL of sterile PBS, the cells were purified by centrifugation (130,000×g, 90 min, 4℃) using a sucrose density gradient (8% / 30% / 45% / 60%). 30%-45% of the target bands were collected and then centrifuged again to obtain preliminarily purified mulberry root bark extracellular vesicles (EVs).

[0076] Further purification by size exclusion chromatography, the specific steps are as follows: Take a size exclusion column (Exosupur®sEV separation kit-1 ml exclusion column, ES911), place it vertically, equilibrate at room temperature, add 20 mL of phosphate buffer solution for flushing, and when there is no liquid flowing out, add 1 mL of mulberry root bark extracellular vesicle solution, and when the liquid enters the column. Add 500 μL of phosphate buffer solution each time, and repeat the above steps 7 times, discard the first 3 fractions totaling 1500 μL, collect the 4th-6th fractions totaling 1500 μL. The 4th-6th fractions are obtained as ultra-high-purity mulberry root bark extracellular vesicles with uniform size. Store in a -80°C deep freezer.

[0077] 2. Characterization and functional analysis Transmission electron microscopy sample preparation: Take 10 μL of EV suspension and drop it on a Formvar carbon film copper mesh, and observe the ultrastructure of the vesicles after negative staining with 2% phosphotungstic acid. Protein analysis: After SDS-PAGE electrophoresis, total protein composition was detected by Coomassie Brilliant Blue R-250 staining. Particle size and surface potential: The dynamic light scattering analysis system (Malvern Zetasizer Nano ZS) was used to determine the hydrodynamic diameter distribution and Zeta potential of nanoparticles. Active oxygen scavenging capacity evaluation: The ROS scavenging efficiency of EVs was quantified by detecting the change in absorbance at 734 nm, 645 nm and 520 nm wavelengths. Free radical scavenging properties: Electron spin resonance spectrometer (Electron Spin Resonance, ESR) was used to detect the scavenging capacity of the vesicles for hydroxyl radicals (•OH) and superoxide anions (O 2•⁻ ).

[0078] 3. Gastrointestinal stability evaluation EV suspension was incubated with simulated gastric fluid (pH 1.5, 37°C, 120 min) and simulated intestinal fluid (pH 6.8, 37°C, 60 min) respectively. Dynamic light scattering was used to monitor the change in particle size before and after digestion, and transmission electron microscopy was used to observe the integrity of the vesicle membrane structure, to comprehensively evaluate its stability in the gastrointestinal environment. (Note: Experimental parameters and detection methods are based on the International Society for Extracellular Vesicles (ISEV) guidelines for standardized operation).

[0079] 4. DSS animal model establishment and experimental design To evaluate the anti-inflammatory effect of MB-EVs in vivo, C57BL / 6 male mice (8 weeks old, 20-22 g) were used to establish the experimental colitis model. Experimental animals were randomly assigned into four groups (n=6 / group): (1) Control group (normal drinking water + phosphate buffer intervention); (2) DSS group (2.5% Dextran Sulfate Sodium (DSS) free intake to induce colitis + phosphate buffer intervention); (3) DMB-EVs group (DSS induction + DMB-EVs intervention); (4) FMB-EVs group (DSS induction + FMB-EVs intervention). From day 0 to day 6, DSS group and intervention group mice were continuously exposed to 2.5% DSS (molecular weight 36-50 kDa, MP Biomedicals) through drinking water, and the Control group maintained normal drinking water. From day 7, the DMB-EVs group and the FMB-EVs group were given 5 mg / kg MB-EVs (dissolved in 200 μL PBS) by oral gavage, and the Control group and the DSS group received the same volume of PBS intervention once a day until the end of the experiment.

[0080] The body weight change, stool character (Bristol score), occult blood level and activity state of mice were recorded daily. After 12 hours of fasting on day 11, isoflurane inhalation anesthesia was performed, and the degree of colonic mucosal injury was observed and the endoscopic score (0-12 points) was quantified by a miniature endoscope system (Karl Storz, Germany). On day 12, euthanasia was performed by cervical dislocation, and whole colon (cecum to rectum), liver, spleen and fresh fecal samples were collected. After fixation with 4% paraformaldehyde, the colon tissue was sectioned for subsequent analysis.

[0081] After paraffin embedding, 4 μm sections of the colon specimen were prepared for hematoxylin-eosin (H&E) staining to evaluate inflammatory cell infiltration, crypt structure destruction and histopathological score (0-12 points), and alcian blue staining (pH 2.5) to quantify goblet cell density. Real-time quantitative PCR (qPCR) was used to detect the mRNA expression levels of pro-inflammatory factors (IL-6, IL-1β, TNF-α) in colon and liver tissues: total RNA was extracted by kit method, reverse transcribed into cDNA by Biyun Tian RT kit, and SYBR Green Premix (Roche) was used for amplification in LightCycler 480 system (Roche), with GAPDH as the internal reference gene, and the relative expression was calculated by 2−ΔΔCt method.

[0082] 5. NASH animal model establishment and experimental design (intestinal-liver axis injury model) The present embodiment adopts methionine-choline-deficient (MCD) diet to induce the establishment of a non-alcoholic steatohepatitis (NASH) mouse model to explore the mechanism of MB-EVs regulating liver inflammation through the gut-liver axis. Experimental animals (C57BL / 6J male mice, n=40) were randomly divided into four groups (n=10 / group): ① normal control group (Control group) maintained standard rodent feed; ② NASH model group (NASH group); ③ DMB-EVs intervention group (DMB-EVs group); ④ FMB-EVs intervention group (FMB-EVs group). During the modeling stage (weeks 1-4), the model group and the intervention group were given MCD feed to establish an intestinal-liver injury model, and the control group was continuously routinely fed.

[0083] From the 5th week, the DMB-EVs group and the FMB-EVs group were given corresponding preparations (5 mg / kg, administered once every 48 hours) by oral gavage, and the control group and the model group were given the same volume of phosphate buffered saline (PBS) at the same time. During the experiment, the animal activity, food intake and body weight changes were monitored daily. After 4 weeks of intervention, euthanasia was performed, and intestinal, liver tissue, feces and whole blood samples were systematically collected for endotoxin quantification (limulus reagent colorimetric method) and histopathological evaluation (HE staining, immunohistochemical analysis).

[0084] At the 7th week of the experiment, the intestinal barrier function was evaluated. Fasting was performed 12 hours before the experiment, and then fluorescein isothiocyanate-dextran (FITC-dextran, molecular weight 4 kDa) was given by oral gavage at a dose of 0.5 mg / kg. After 4 hours of administration, whole blood samples were collected, and after standing at room temperature for 1 hour, the serum was separated by centrifugation at 3500 × g for 10 minutes (4°C). The concentration of FITC-dextran in the serum was quantitatively detected by ultraviolet spectrophotometry (excitation wavelength 485 nm, emission wavelength 528 nm), and the fluorescence intensity value per unit volume was calculated to evaluate the changes in intestinal mucosal permeability.

[0085] II. Experimental results 1. Preparation and purification of mulberry root cell extracellular vesicles The present embodiment discloses a white mulberry (Morus alba) plant of the genus Morus, Morus alba L.High-efficiency purification process of mulberry root bark extracellular vesicles (EVs). Taking FMB-EVs extracted from fresh mulberry root bark as an example, the specific implementation steps include: taking 100 g of fresh mulberry root bark tissue and mechanically crushing it, and adding phosphate buffer solution (PBS, pH 7.4) for low-temperature extraction (4°C, 12 h). After removing large particle impurities by primary centrifugation (3,000×g, 30 min; 10,000×g, 60 min; 130,000×g, 90 min), transmission electron microscopy (TEM) analysis shows that there are a large number of non-vesicular impurities (such as cell fragments and free protein aggregates) in the sample (initial condition), which makes it difficult to effectively distinguish functional EVs from other particles (Figs. 1A and 1B). Figure 1 A and Figure 1 B). To improve the purity of EVs, the centrifugation parameters were systematically optimized: the first attempt to increase the high-speed centrifugation step (improvement 1), TEM observation still exists heterogeneous impurities, and the BCA method detects protein concentration up to 4.0 mg / mL, it is speculated that high-speed centrifugation may cause EVs to co-precipitate with high-density protein impurities, with lower purity (EVs count less than 50% per field of view), and the real yield cannot be determined by protein quantification (Figs. 2A and 2B). Figure 1 C and Figure 1 D). Based on the difference in sedimentation coefficient, the improved scheme adopts multi-stage low-speed gradient centrifugation (improvement 2, 200×g→ 500×g→1,000×g→2,000×g→3,000×g→5,000×g→10,000×g, gradually increasing the time) (improvement 2), the results show that the density of EVs in the TEM image is significantly improved (EVs count per field of view increased to 90%), and the BCA protein concentration decreased to 2.0 mg / mL, indicating that protein impurities were selectively removed (Figs. 3A and 3B). Figure 1 E and Figure 1 F). Further fine purification by size exclusion chromatography (SEC) (improvement 3): load the EVs suspension after optimization of centrifugation into the chromatography column, use PBS as the elution buffer, and collect 1.5 mL of elution components. The final product verified by TEM shows uniform vesicle morphology (no impurities in the whole field of view) (particle size 180-220 nm), the coefficient of variation (CV) of particle size distribution is less than 15%, and the BCA protein concentration is stable at 1.0 mg / mL, and the final yield is improved to 0.04 mg / g (TEM whole field of view, protein quantification can determine the final real yield), confirming that SEC can effectively separate residual low molecular weight impurities and control the particle size distribution (Figs. 4A and 4B). Figure 1 G and Figure 1 H).

[0086] 2. Raw material quality conversion rule of fresh / dried mulberry root bark Fresh mulberry root bark was treated by hot air drying at 60°C to prepare dried samples (Figure 2 A) Mass balance analysis showed a raw material conversion rate of 50% (fresh weight / dry weight = 2:1). To verify the reliability of this mass ratio, this embodiment adopted a controlled experimental design: under strictly equal mass feeding conditions (fresh / dry samples), extracellular vesicles (EVs) were extracted through a standardized mechanical crushing, differential centrifugation, and sucrose density gradient centrifugation process. Quantitative analysis was performed using the BCA colorimetric method. Figure 2 B and Figure 2 C) It was found that the protein concentration of dried mulberry root bark EVs (2.15±0.23 mg / mL) was significantly higher than that of fresh samples (1.2±0.15 mg / mL) (p<0.01), and the two showed a quantitative relationship of 1:2. Further analysis using tunable resistive pulse sensing (TRPS) for absolute nanoparticle counting showed that the particle concentration of dried mulberry root bark EVs was 2.19×10⁻⁶. 13 The particle count (particles / mL) was approximately 9.81 × 10⁻⁶ for the fresh mulberry root bark sample. 12 2 times (particles / mL) Figure 2 D and Figure 2 E). The reverse verification of the above proteomics and nanoparticle quantitative data, together with the biomolecule content and the number of physical particles, confirmed the 2:1 raw material quality conversion law during the drying process of fresh mulberry root bark.

[0087] 3. Freeze-drying of mulberry root bark extracellular vesicles does not affect its medicinal efficacy. Transmission electron microscopy (TEM) analysis showed that the freeze-drying process did not significantly affect the ultrastructure of DMB-EVs; both the freeze-dried and unfrozen samples maintained the typical cup-shaped depression morphology and the intact bilayer membrane structure. Figure 3 A). Dynamic light scattering (DLS) quantitative results showed that lyophilization did not change the hydrodynamic diameter of DMB-EVs (before lyophilization: 215.9±65.11 nm, after lyophilization: 214.1±73.04 nm; p>0.05) or the surface Zeta potential (before lyophilization: -30.6±5.42 mV, after lyophilization: -33.1±3.15 mV; p>0.05), but its polydispersity index (PDI) increased from 0.190 to 0.346 (p<0.05), suggesting that the lyophilization process may have reduced the uniformity of particle dispersion due to the aggregation of free proteins. Figure 3 B- Figure 3 E). Further evaluation of free radical scavenging ability using electron spin resonance spectroscopy (ESR) revealed that DMB-EVs, before and after lyophilization, effectively scavenged hydroxyl radicals (•OH) and superoxide anions (O2). •⁻There was no significant difference in the half-maximal scavenging concentration (IC50) between the two groups (49.8 μg / mL vs. 52.1 μg / mL; Δ<6%, p>0.05). Figure 3 F). However, the reactive oxygen species (ROS) scavenging experiment based on the DCFH-DA fluorescent probe method showed that the scavenging efficiency of DMB-EVs at a concentration of 100 μg / mL after lyophilization was 76.3±5.1%, which was slightly lower than 82.4±4.8% before lyophilization (p<0.05), but still maintained high antioxidant activity. Figure 3 G- Figure 3 I). It is worth noting that FMB-EVs exhibited behavior consistent with DMB-EVs during freeze-drying: their morphological integrity ( Figure 3 J), Particle size and potential stability ( Figure 3 K- Figure 3 N), free radical scavenging ability ( Figure 3 O) and a slight decreasing trend in ROS scavenging efficacy ( Figure 4 The PRs were highly similar to DMB-EVs, indicating that the freeze-drying process has a universal effect on the physicochemical properties and biological activity of extracellular vesicles from different sources of mulberry plants.

[0088] 4. Application of extracellular vesicles from mulberry root bark: Oral administration of extracellular vesicles from mulberry root bark can alleviate DSS-induced acute colitis. This embodiment successfully constructed a DSS-induced mouse model of acute colitis and verified the intervention effect of MB-EVs. Figure 4 A). Euthanasia analysis at the experimental endpoint (day 12) showed that the control group mice maintained stable body weight (Δ<3%), while the DSS group experienced continuous weight loss from day 6. Although the DMB-EVs and FMB-EVs intervention groups showed a similar weight loss trend to the DSS group on days 6-7, they entered a recovery period from day 8, and their body weight recovered to near initial levels by day 12. Figure 4 B). The Disease Activity Index (DAI), calculated based on weight changes, stool characteristics, and the degree of rectal bleeding, showed that the DAI score in the DSS group continued to rise (final value 8.5±0.3), while the DAI score in the intervention group peaked on day 8 and then plateaued (final values ​​4.2±0.3 in the DMB-EVs group and 4.3±0.3 in the FMB-EVs group, p<0.01 vs. the DSS group). Figure 4 C). Anatomical analysis showed that the spleen index in the DSS group was significantly higher than that in the control group (1.02±0.18% vs 0.48±0.1%, p<0.001) and the intervention group (DMB-EVs group 0.48±0.25%, FMB-EVs group 0.51±0.16%, p<0.01 vs DSS group). Figure 4D). Colonic morphological measurements showed that the colon length of DSS group was significantly shorter than that of control group (5.9 ± 0.4 cm vs 7.6 ± 0.5 cm, p < 0.001), while the colon length of intervention groups was maintained at 7.0 ± 0.5 cm (DMB-EVs) and 7.1 ± 0.4 cm (FMB-EVs), which was significantly different from that of DSS group (p < 0.01) Figure 4 E- Figure 4 F). Endoscopy further confirmed that the colonic mucosa of DSS group had multiple ulcers and diffuse bleeding spots, while the intervention group only had local congestion Figure 4 G). Histopathological analysis showed that H&E staining of DSS group showed a large number of neutrophil infiltration and crypt structure destruction, while the inflammatory cell infiltration of the intervention group was significantly reduced, and the crypt integrity was preserved Figure 4 H). Alcian blue staining showed that the goblet cell density of DSS group was lower than that of control group, and the goblet cell density of intervention group recovered close to the control group Figure 5 I). The above results suggest that MB-EVs may play a synergistic anti-inflammatory role in the colon-liver axis by maintaining intestinal barrier integrity and systemically inhibiting the pro-inflammatory factor cascade.

[0089] 5. Application of mulberry root cell extracellular vesicles: oral administration of mulberry root cell extracellular vesicles relieves liver inflammation and colon inflammation in mouse NASH model A non-alcoholic steatohepatitis (NASH) mouse model was successfully induced by methionine-choline-deficient (MCD) diet Figure 5 A). At week 7 of the experiment, the intestinal barrier function was evaluated by fluorescein isothiocyanate-dextran (FITC-dextran) oral loading test, and the results showed that the fluorescence intensity of serum FITC-dextran in NASH model group was significantly higher than that in control group (p < 0.01), while the fluorescence intensity of DMB-EVs and FMB-EVs intervention groups decreased, indicating that the intestinal permeability increased in NASH model, and the treatment group could alleviate the intestinal permeability Figure 5 B). Liver phenotype analysis showed that the liver-body ratio (liver weight / body weight) of NASH group was significantly increased compared with the control group (p < 0.001), while the liver-body ratio of DMB-EVs and FMB-EVs intervention groups showed a downward trend Figure 5 C). Macroscopic anatomical observation showed that the liver surface of NASH group was rough and yellow, and the colon length was significantly shorter than that of the control group (p < 0.01), while the liver surface of DMB-EVs and FMB-EVs intervention groups was smooth, and the colon length was recovered compared with NASH group, indicating that mulberry root vesicles could effectively relieve liver inflammation and intestinal inflammation in NASH modelFigure 5 D). Liver histological analysis showed that H&E staining showed a large number of inflammatory cell infiltration in the liver lobule of the NASH group, and the inflammatory cell infiltration of the intervention group was significantly reduced; Oil red O staining showed that the lipid deposition area of the NASH group was significantly higher than that of the control group, and the intervention group could alleviate the liver lipid deposition of the NASH model; Masson trichrome staining showed that the liver fibrosis area of the NASH group was significantly higher than that of the control group, and the intervention group could alleviate the liver fibrosis caused by NASH Figure 5 E). Intestinal tissue H&E staining showed that the NASH group had colonic mucosal structure disorder accompanied by crypt abnormal morphology, suggesting that liver inflammation caused by NASH could affect intestinal homeostasis through the intestinal-liver axis, and the mulberry root bark vesicle treatment group could effectively alleviate the intestinal tissue structure disorder in the NASH model ​ F). In summary, the NASH model can affect intestinal homeostasis through the intestinal-liver axis, causing intestinal inflammation, while the mulberry root bark vesicle can effectively alleviate the symptoms of inflammatory cell infiltration, lipid deposition and liver fibrosis in the liver, and can also alleviate the intestinal mucosal disorder caused by NASH, thereby restoring the integrity of the intestinal tract.

[0090] It should be noted that when the present application claims involve numerical ranges, both endpoints of each numerical range and any number between the two endpoints can be selected. In order to prevent repetition, the present application describes preferred embodiments.

[0091] Although preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

Claims

1. A method for preparing extracellular vesicles derived from dried / fresh mulberry root bark, characterized in that, Includes the following steps: S1. Add fresh or dried mulberry root bark to phosphate buffer solution for homogenization, followed by extraction and multi-layer filtration to obtain tissue homogenate. S2. Impurities in the tissue homogenate were removed sequentially by differential centrifugation. The supernatant was then centrifuged at 120,000~140,000×g and 3~5℃ for 80~100 min to enrich the vesicle precipitate. S3. Resuspend the vesicle precipitate, purify it by sucrose density gradient centrifugation, and collect the target band; S4. Size exclusion chromatography was performed on the target band to obtain extracellular vesicles of mulberry root bark with uniform size distribution.

2. The method for preparing extracellular vesicles derived from dried / fresh mulberry root bark according to claim 1, characterized in that, In S1, the extraction conditions are 3~5℃ for 10~12 h.

3. The method for preparing extracellular vesicles derived from dried / fresh mulberry root bark according to claim 1, characterized in that, In S2, the conditions for differential centrifugation are: 200×g, 8~10 min; 400~500×g, 8~10 min; 800~1,000×g, 8~10 min; 1500~2,000×g, 15~20 min; 2500~3,000×g, 25~30 min; 4000~5,000×g, 25~30 min; 10,000~11,000×g, 55~60 min.

4. The method for preparing extracellular vesicles derived from dried / fresh mulberry root bark according to claim 1, characterized in that, In S3, the mass concentration of the sucrose solution in the sucrose density gradient centrifugation from bottom to top is 8% to 60%, the centrifugation conditions are 120,000 to 140,000 × g, 80 to 100 min, 3 to 5 °C, and the target band is a sucrose solution with a mass concentration of 30% to 45%.

5. Extracellular vesicles of mulberry root bark prepared by the preparation method according to any one of claims 1 to 4.

6. The use of the extracellular vesicles of mulberry root bark as described in claim 5 in the preparation of anti-inflammatory drugs.

7. The application of the extracellular vesicles of mulberry root bark according to claim 6 in the preparation of anti-inflammatory drugs, characterized in that, The inflammations include ulcerative colitis and non-alcoholic steatohepatitis.

8. An anti-inflammatory drug, characterized in that, The inflammatory treatment drug uses the extracellular vesicles of mulberry root bark as the active ingredient as described in claim 5.

9. The anti-inflammatory drug according to claim 8, characterized in that, It also includes pharmaceutically acceptable excipients.

10. The anti-inflammatory drug according to claim 9, characterized in that, The drug contains 1% to 99.9% extracellular vesicles from mulberry root bark.

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