Fresh rehmannia root exosome as well as preparation and application thereof

Abstract

The invention discloses a fresh rehmannia root exosome as well as preparation and application thereof. The fresh rehmannia root exosome comprises lipid vesicles filled with rehmannia root metabolites, the rehmannia root metabolites comprise phenylpropanoid and polyketone compounds, and the preparation method comprises the following steps: centrifuging fresh rehmannia root juice and taking supernate; carrying out ultracentrifugation on the supernate, taking precipitate, and resuspending the precipitate by adopting a buffer solution to obtain a first solution; adding the first solution to a concentrated solution of sucrose, and taking liquid at the interface of the concentrated solution of sucrose after ultracentrifugation to obtain a second solution; adding a buffer solution into the second solution, and taking the precipitate after ultracentrifugation to obtain the fresh rehmannia root exosome. Only the exosome extracted from the fresh rehmannia root is used as an active pharmaceutical ingredient and is matched with the modified bone targeting peptide, so that a good osteoporosis treatment effect is achieved, the problems of low active ingredient concentration, large medicinal ingredient extraction loss and poor medicinal effect of single use in the fresh rehmannia root are effectively solved, toxic and side effects of the medicine are avoided, and the curative effect is good. Good patent medicine prospects are realized.

Description

Technical Field

[0001] This invention relates to the preparation and application of plant exosomes, and more particularly to fresh Rehmannia glutinosa exosomes, their preparation, and their application in drug preparation. Background Technology

[0002] Bone tissue has limited blood supply and poor drug penetration, especially in avascular cartilage areas. Therefore, in the treatment of bone-related diseases, maintaining effective drug concentrations in bone tissue often relies on increasing the dosage, leading to systemic toxicity, drug resistance, and increased medical costs. Bone-targeting peptides, composed of multiple repeating aspartic-serine-serine (Asp-Ser-Ser, or DSS) sequences, denoted as (DSS)n, exhibit high affinity for calcium phosphate compounds. The binding affinity of DSS-containing peptides to substrates largely depends on peptide length; affinity increases proportionally with peptide length, but beyond a certain number of repetitions, affinity no longer increases further. When n=6, the six-repetition DSS6 sequence is the optimal choice. DSS6 has high affinity for low-crystallinity hydroxyapatite and calcium phosphate on bone-forming surfaces and can be used in research on orthopedic diseases such as osteoporosis, osteoarthritis, osteomyelitis, multiple myeloma, bone metastases, and skull defects.

[0003] Plant exosomes are miniature packages, approximately 30-150 nanometers in diameter, secreted by plant cells. They consist of proteins, RNA (such as miRNA), and small bioactive molecules encapsulated by a lipid bilayer. Although research on the composition, particle size characteristics, and functions of plant exosomes is limited, some studies have indicated that plant-derived exosomes may participate in intercellular communication and serve as a means of regulating plant functions. Existing research confirms that plant exosomes have various applications, including immunomodulation, improvement of gut microbiota, anti-inflammatory effects, and anti-tumor activity.

[0004] Traditional Chinese medicine theory holds that the kidneys store essence, govern bones, and generate bone marrow, thus nourishing bones with marrow. Rehmannia glutinosa, with its sweet and bitter taste and cold nature, enters the liver and kidney meridians, possessing the effects of nourishing blood and yin, replenishing marrow and essence. The description in the *Compendium of Materia Medica* of "filling bone marrow, building muscle, and generating essence and blood" affirms its beneficial effects on various symptoms of weakness and fatigue in the muscles and bones. Modern research has also confirmed that the active ingredients in Rehmannia glutinosa extract promote bone formation and inhibit bone resorption. However, these studies and theories all describe the efficacy of raw or processed Rehmannia glutinosa, without confirming the efficacy of fresh Rehmannia glutinosa and its exosomes. In other words, traditional theories and research do not consider the medicinal properties of fresh Rehmannia glutinosa, assuming that it must be processed to exert its medicinal effects. This is mainly because fresh Rehmannia glutinosa contains more water and impurities, resulting in a lower concentration of active ingredients and thus poorer efficacy. Drying and processing can significantly increase the content of active ingredients; therefore, raw and processed Rehmannia glutinosa have better medicinal effects.

[0005] However, even though raw and processed Rehmannia glutinosa have clearly defined medicinal properties, current technology typically combines them with other traditional Chinese medicine decoction pieces to treat osteoporosis. For example, Chinese patent CN106109695A discloses a formula called Tongluo Busui Tang for treating osteoporosis, which contains raw Rehmannia glutinosa, processed Rehmannia glutinosa, Taxillus chinensis, Spatholobus suberectus, Angelica sinensis (tail), Paeonia lactiflora, Salvia miltiorrhiza, Aucklandia lappa, Cornus officinalis, Eucommia ulmoides, Schisandra chinensis, and many other Chinese herbs. The therapeutic effect of Rehmannia glutinosa alone is extremely limited, and it is rarely used alone to treat bone-related diseases. This is closely related to the theory of compatibility in traditional Chinese medicine and the synergistic effects observed in modern pharmacodynamic studies. Without the assistance and enhancement of other drugs, Rehmannia glutinosa alone is difficult to exert its medicinal effect. Therefore, how to extract an active ingredient from fresh Rehmannia glutinosa that can effectively treat osteoporosis has become an urgent technical problem to be solved. Summary of the Invention

[0006] Objectives of this invention: The objective of this invention is to provide fresh Rehmannia glutinosa exosomes that can be used to treat osteoporosis. A second objective is to propose a method for preparing fresh Rehmannia glutinosa exosomes, solving the problem of how to extract exosomes from fresh Rehmannia glutinosa. A third objective is to propose the application of fresh Rehmannia glutinosa exosomes in the preparation of osteoporosis treatment drugs, solving the problem of how to prepare osteoporosis treatment drugs.

[0007] Technical solution: The present invention provides a fresh Rehmannia glutinosa exosome, comprising lipid vesicles filled with Rehmannia glutinosa metabolites, wherein the Rehmannia glutinosa metabolites include at least one of phenylpropanoid compounds and polyketide compounds.

[0008] The fresh Rehmannia glutinosa mentioned in this invention refers to fresh Rehmannia glutinosa tubers that have not been dried or processed, while raw Rehmannia glutinosa refers to Rehmannia glutinosa tubers that have been processed by drying methods such as sun-drying or oven-drying.

[0009] Preferably, the Rehmannia glutinosa metabolites include at least one of methyl caffeate, anhydrous andrographolide, neoandrographolide aglycone, and amyristol.

[0010] In some embodiments, the Rehmannia glutinosa metabolites further include miRNA.

[0011] Preferably, the surface of the lipid vesicle is modified with a bone-targeting peptide containing the amino acid sequence shown in SEQ ID No. 1.

[0012] The second aspect of this invention discloses a method for preparing the above-mentioned fresh Rehmannia glutinosa exosomes, comprising the following steps: (1) Fresh Rehmannia glutinosa juice is obtained by juicing fresh Rehmannia glutinosa juice, and the supernatant is collected by centrifugation. (2) After ultracentrifuging the supernatant, the precipitate was collected and resuspended in buffer solution to obtain the first solution; (3) Add the first solution to the concentrated sucrose solution, centrifuge at high speed, and take the liquid at the interface of the concentrated sucrose solution to obtain the second solution; (4) Add buffer solution to the second solution, centrifuge at ultraspeed and take the precipitate to obtain fresh Rehmannia exosomes.

[0013] Preferably, in step (1), the centrifugation conditions for fresh Rehmannia glutinosa juice are 9000-11000g for at least 30min; in steps (2)-(4), the conditions for ultracentrifugation are 80000-120000g for 50-90min, and the buffer solution is phosphate buffer or Tris buffer. Preferably, in step (3), the concentration of sucrose in the concentrated sucrose solution is 25-35%, and the solvent is water or phosphate buffer. Preferably, in step (4), after removing the precipitate, the following steps are also included: The precipitate was resuspended in buffer solution to obtain an exosecretory resuspension. DSPE-PEG-DSS6 was obtained by clicking reaction of 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol-N-maleimide and a bone-targeting peptide with thiol-terminal modification. DSPE-PEG-DSS6 was added to the exosome suspension, and the reaction was carried out in an ice bath with stirring to obtain fresh Rehmannia glutinosa exosomes modified with bone-targeting peptides.

[0014] Preferably, the concentration of exosomes in the exosome suspension is 0.5-1.5 mg / mL; The mass ratio of the 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol-N-maleimide to the bone-targeting peptide with thiol-terminal modification is 1-2:1-2. The average molecular weight of the polyethylene glycol segment in the 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol-N-maleimide is 2000-5000.

[0015] The conditions for the click reaction are: using an organic amine as a catalyst in a neutral buffer environment, stirring at 30-40°C in the dark for 2-6 hours; The volume ratio of DSPE-PEG-DSS6 to the exocrine body suspension is 1:10-30.

[0016] In some embodiments, the preparation method of the above-mentioned fresh Rehmannia exosomes further includes a freeze-drying step, wherein the modified or unmodified fresh Rehmannia exosomes with bone-targeting peptides are resuspended in a buffer or aqueous solution containing 0.2-0.4M glycine and then freeze-dried at -80°C.

[0017] The third aspect of this invention discloses the application of the above-mentioned fresh Rehmannia glutinosa exosomes in the preparation of osteoporosis treatment drugs.

[0018] This invention further discloses the application of the above-mentioned fresh Rehmannia glutinosa exosomes in any of the following: To prepare drugs that reduce the production of osteoclast differentiation factors; To prepare drugs that inhibit osteoclast formation and / or promote osteogenic differentiation; To prepare drugs that reduce reactive oxygen species levels in bone marrow mesenchymal stem cells; To prepare drugs to alleviate oxidative stress; To prepare drugs that promote bone repair or bone regeneration; Prepare a drug that increases the expression level of at least one of RUNX2, type I collagen, osteopontin, and osteocalcin in bone tissue; To prepare a drug that downregulates NF-κB and upregulates NRF2 expression in bone tissue; To prepare drugs that inhibit bone tissue inflammation; To prepare drugs that promote angiogenesis.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. This invention uses only exosomes extracted from fresh Rehmannia glutinosa as active pharmaceutical ingredients. When combined with modified bone-targeting peptides, it achieves good therapeutic effects on osteoporosis. It effectively overcomes the problems of low concentration of active ingredients in fresh Rehmannia glutinosa, large loss of medicinal ingredients during extraction, and poor efficacy when used alone. It does not require combination with other Chinese herbal medicines, avoids drug toxicity and side effects, reduces drug production costs, and has good prospects for drug development.

[0020] 2. Fresh Rehmannia glutinosa exosomes, as natural nanoscale vesicles, possess a lipid bimembrane structure that protects their carried active ingredients (such as nucleic acids, proteins, and small molecule compounds) from enzymatic degradation or oxidative damage, thus enhancing their biological stability. Simultaneously, their membrane structure allows for specific binding to target cells (such as osteoblasts and bone marrow mesenchymal stem cells), efficiently delivering active substances to bone tissue, reducing non-specific effects on other organs, and improving local drug concentration and bioavailability.

[0021] 3. Fresh Rehmannia glutinosa, as a plant raw material, can be used to extract its exosomes through relatively simple processes such as crushing, centrifugation, and purification. It does not require complicated processing of medicinal slices and is suitable for large-scale industrial production, which helps to reduce the cost of drug preparation. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating the preparation process of fresh Rehmannia glutinosa exosomes modified with bone-targeting peptides in this invention. Figure 2 Representative transmission electron microscope images of exosome samples prepared in Comparative Example 1, Example 1, and Example 2; Figure 3Particle size distribution and zeta potential analysis for DREV, FREV and FR-DSS6 samples; Figure 4 The results of nanoparticle tracking analysis for DREV, FREV, and FR-DSS6 samples; Figure 5 Electrophoretic analysis results of proteins in DREV, FREV, and FR-DSS6 samples; Figure 6 The classification of differential metabolites in DREV and FREV samples and the proportion of each category; Figure 7 KEGG classification maps of differentially metabolites in DREV and FREV samples; Figure 8 The results are HPLC analysis of metabolites contained in the exosomes of fresh Rehmannia glutinosa. Figure 9 The high performance liquid chromatography UV-Vis absorption spectra of methyl caffeate and exosomes from fresh Rehmannia glutinosa are shown. Figure 10 The high performance liquid chromatography UV-Vis absorption spectra of dehydrated andrographolide and fresh Rehmannia exosomes; Figure 11 The high performance liquid chromatography UV-Vis absorption spectra of neoandrographolide aglycone and fresh Rehmannia exosomes; Figure 12 High performance liquid chromatography UV-Vis absorption spectra of African myristicin and fresh Rehmannia glutinosa exosomes; Figure 13 The effects of DREV, FREV, and FR-DSS6 samples on endothelial cell migration ability; Figure 14 Results of angiogenesis potential testing for DREV, FREV, and FR-DSS6 samples; Figure 15 Results of the ability of DREV, FREV and FR-DSS6 samples to scavenge ROS in cells; Figure 16 The expression levels of NFAT2 and C-Fos in differentiated osteoclasts treated with different samples under hypoxic conditions; Figure 17 Immunofluorescence images of osteoclasts formed in different sample treatment groups after RANKL stimulation; Figure 18 The effects of DREV, FREV, and FR-DSS6 samples on the expression levels of osteogenic markers; Figure 19 The effects of DREV, FREV, and FR-DSS6 samples on the entire osteogenic differentiation process; Figure 20Volcano plot of differentially expressed miRNAs between DREV and FREV; Figure 21 A heatmap showing the differentially expressed miRNAs between DREV and FREV; Figure 22 Bar chart for GO enrichment analysis and enrichment pathway of target genes of differentially expressed miRNAs in the KEGG pathway. Figure 23 The effects of different Rehmannia glutinosa exosome samples on the expression levels of p-NF-κB, NF-κB, and NRF2 in RANKL-induced osteoclasts were investigated. Figure 24 The effects of different Rehmannia glutinosa exosome samples on the expression levels of p-NF-κB, NF-κB, and NRF2 in BMSCs differentiating into osteoblasts under hypoxic conditions; Figure 25 Fluorescence imaging of major organs in mice 24 hours after tail vein injection of DiR-labeled exosomes; Figure 26 Fluorescence imaging of the femur of mice 24 h after tail vein injection of DiR-labeled exosomes; Figure 27 Representative fluorescence images of the trabecular and cortical bone regions of the femur in mice from different drug administration groups; Figure 28 Three-dimensional microstructure images and quantitative analysis of the distal trabecular bone of mice in different drug administration groups; Figure 29 H&E staining and TRAP staining results of femurs in mice from different drug administration groups; Figure 30 Immunohistochemical staining results of femoral tissues from mice in different drug administration groups; Figure 31 Immunofluorescence staining results of the femurs of mice in different drug administration groups. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0024] Example 1: As Figure 1 As shown, a method for preparing fresh Rehmannia glutinosa exosomes is as follows: (1) Fresh Rehmannia root is juiced and then centrifuged at 10000g for 30min. The centrifugation is repeated twice to remove fibers, large particles and intact organelles. The supernatant is then collected. (2) After centrifuging the supernatant at 100,000g for 70 min, the precipitate was collected and resuspended in 1×PBS pre-cooled at 4℃ to obtain the first solution; (3) Slowly transfer the first solution to a 30% sucrose aqueous solution, centrifuge at 100000g for 70min, and then take the liquid at the interface of the 30% sucrose solution to obtain the second solution; (4) Add pre-cooled 1×PBS to the second solution, centrifuge at 100000g for 70min, and take the precipitate to obtain fresh Rehmannia exosomes (FREV).

[0025] Example 2: A method for preparing fresh Rehmannia glutinosa exosomes modified with bone-targeting peptides is as follows: (1) Fresh Rehmannia root is juiced and then centrifuged at 10000g for 30min. The centrifugation is repeated twice to remove fibers, large particles and intact organelles. The supernatant is then collected. (2) After centrifuging the supernatant at 100,000g for 70 min, the precipitate was collected and resuspended in 1×PBS pre-cooled at 4℃ to obtain the first solution; (3) Slowly transfer the first solution to a 30% sucrose aqueous solution, centrifuge at 100000g for 70min, and then take the liquid at the interface of the 30% sucrose solution to obtain the second solution; (4) Add pre-cooled 1×PBS to the second solution, centrifuge at 100000g for 70min, and then take the precipitate to obtain fresh Rehmannia exosomes; (5) 1 mg of 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol-N-maleimide (DSPE-PEG2000-Mal) and 1 mg of bone-targeting peptide (HS-DSS6) with thiol-terminal modification were dissolved in 1 mL of 1×PBS, and then 10 μL of triethylamine at pH 7.0 was added as a catalyst. The mixture was stirred in a water bath at 37 °C in the dark for 4 h to obtain DSPE-PEG2000-DSS6 through a thiol-maleimide click reaction.

[0026] (6) Fresh Rehmannia exosomes were suspended in 1×PBS at a concentration of 1 mg / mL to obtain an exosome suspension. Then, 50 μL LDSPE-PEG2000-DSS6 was added to 1 mL of the exosome suspension and stirred in an ice bath for 4 h to obtain fresh Rehmannia exosomes modified with bone-targeting peptides (FR-DSS6).

[0027] Example 3: A method for preparing fresh Rehmannia glutinosa exosomes modified with bone-targeting peptides is as follows: (1) Fresh Rehmannia root is juiced and then centrifuged at 11000g for 30min. The centrifugation is repeated twice to remove fibers, large particles and intact organelles. The supernatant is then collected. (2) After centrifuging the supernatant at 120000g for 50min, the precipitate was collected and resuspended in 1×PBS pre-cooled at 4℃ to obtain the first solution; (3) Slowly transfer the first solution to a 25% sucrose aqueous solution, centrifuge at 120000g for 50 min, and then take the liquid at the interface of the 25% sucrose solution to obtain the second solution; (4) Add pre-cooled 1×PBS to the second solution, centrifuge at 120000g for 50 min, and take the precipitate to obtain fresh Rehmannia exosomes; (5) 2 mg of 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol-N-maleimide (DSPE-PEG5000-Mal) and 1 mg of thiol-modified bone-targeting peptide (HS-DSS6) were dissolved in 1 mL of 1×PBS, and then 10 μL of triethylamine at pH 7.0 was added as a catalyst. The mixture was stirred in a water bath at 37 °C in the dark for 6 h, and DSPE-PEG5000-DSS6 was obtained by thiol-maleimide click reaction. DSS6 represents the Asp-Ser-Ser peptide repeated 6 times; (6) Fresh Rehmannia exosomes were suspended in 1×PBS at a concentration of 1 mg / mL to obtain an exosome suspension. Then, 50 μL LDSPE-PEG2000-DSS6 was added to 1.5 mL of the exosome suspension and stirred in an ice bath for 6 h to obtain fresh Rehmannia exosomes modified with bone-targeting peptides.

[0028] Example 4: A method for preparing fresh Rehmannia glutinosa exosomes modified with bone-targeting peptides is as follows: (1) Fresh Rehmannia root is juiced and then centrifuged at 9000g for 40min. The centrifugation is repeated twice to remove fibers, large particles and intact organelles. The supernatant is then collected. (2) After centrifuging the supernatant at 80000g for 90min, the precipitate was collected and resuspended in 1×PBS pre-cooled at 4℃ to obtain the first solution; (3) Slowly transfer the first solution to a 35% sucrose aqueous solution, centrifuge at 80000g for 90 min, and then take the liquid at the interface of the 35% sucrose solution to obtain the second solution; (4) Add pre-cooled 1×PBS to the second solution, centrifuge at 80000g for 90 min, and take the precipitate to obtain fresh Rehmannia exosomes; Comparative Example 1: Everything else is the same as in Example 1, except that: Fresh Rehmannia glutinosa is replaced with raw Rehmannia glutinosa, which is obtained by drying fresh Rehmannia glutinosa tubers until the moisture content is 10%.

[0029] Rehmannia glutinosa was added to 1×PBS at a material-to-liquid ratio of 1g:1mL, and after thorough mixing and juicing, Rehmannia glutinosa juice was obtained. This juice was then used for subsequent exosome extraction, and finally Rehmannia glutinosa exosomes (DREV) were obtained.

[0030] The exosome samples obtained in Comparative Example 1, Example 1, and Example 2 were analyzed by transmission electron microscopy, and the results are as follows: Figure 2 As shown (scale bar: 100nm). Figure 2 DREV, FREV, and FR-DSS6 all exhibited an elliptical morphology, indicating that modification with the bone-targeting peptide DSS6 did not affect the structure of the exosomes from fresh Rehmannia glutinosa. Furthermore, extracellular vesicles similar to those found in fresh Rehmannia glutinosa were also present in raw Rehmannia glutinosa.

[0031] The particle size and zeta potential of different exosome samples were determined using dynamic light scattering (DLS). The results are as follows: Figure 3 As shown, the average diameters of DREV, FREV, and FR-DSS6 are 160 nm, 136 nm, and 148 nm, respectively. Figure 3 The results showed that the exosomes derived from Rehmannia glutinosa had a relatively large particle size, which increased slightly after modification with DSS6. The average zeta potentials were -15 mV, -27 mV, and -25 mV, respectively, indicating that all exosomes carried a negative surface charge and had good biocompatibility. The zeta potential of FR-DSS6 was slightly increased due to DSS6 modification.

[0032] The particle size distribution was assessed using nanoparticle tracking analysis, and the results are as follows: Figure 4 As shown (scale bar: 600nm). Figure 4 The real-time graphs of DREV, FREV, and FR-DSS6 are shown. It can be seen that the highest peak wavelengths of DREV, FREV, and FR-DSS6 are approximately 167.6nm, 154.1nm, and 158.2nm, respectively. Figure 3 The results show the same trend.

[0033] After protein gel electrophoresis, DREV, FREV, and FR-DSS6 samples were stained with Coomassie Brilliant Blue. The results are as follows: Figure 5 As shown, Figure 5 The proteins in DREV, FREV, and FR-DSS6 were mainly concentrated in the 17-25 kDa and 35-45 kDa ranges. Modification with the bone-targeting peptide DSS6 did not alter the protein content and types in fresh Rehmannia glutinosa exosomes, suggesting that the functional activity of the exosomes remained unchanged. However, compared to fresh Rehmannia glutinosa exosomes (FREV), the protein content in the DREV sample extracted from Rehmannia glutinosa was significantly reduced.

[0034] Plant exosome non-target metabolomics sequencing analysis was performed on DREV and FREV samples, and the results are as follows: Figure 6 As shown, Figure 6The differences between DREV and FREV are shown in their metabolites. The basic composition of exosomes is mainly lipids, but they also contain a large amount of Rehmannia glutinosa metabolites and nucleic acid substances.

[0035] Differential metabolites from DREV and FREV samples were analyzed, and the results are as follows: Figure 7 As shown, a large number of compounds related to anti-inflammatory and antioxidant activity were found, among which phenylpropanoids and polyketides are associated with oxidative stress. Osteoporosis is accompanied by oxidative stress, and these compounds can alleviate oxidative stress caused by osteoporosis by affecting osteoblasts and osteoclasts to treat osteoporosis.

[0036] High-performance liquid chromatography (HPLC) analysis was performed on the metabolites in FREV that alleviate oxidative stress, providing a reference for further validation and quality control of the contained compounds. Furthermore, four metabolite standards—dehydroandrographolide, methyl caffeate, andrograpanin, and paradol—were selected from the metabolites related to oxidative stress alleviation and analyzed by HPLC, obtaining characteristic chromatograms for each compound. Results Figure 8 As shown, by comparison, it was found that FREV has a retention peak with the same retention time as the standard.

[0037] High-performance liquid chromatography (HPLC) UV-Vis absorption spectroscopy was performed on four metabolite standards (dehydrated andrographolide, methyl caffeate, African myristol, and neoandrographolide aglycone) and FREV samples, respectively. The results are as follows: Figure 9-12 As shown, Figure 9 Figure (A) shows the caffeic acid methyl ester reference standard, and Figure (B) shows the FREV group; Figure 10 Figure (A) shows the dehydrated andrographolide reference standard, and Figure (B) shows the FREV group; Figure 11 (A) New andrographolide aglycone standard; (B) FREV group. Figure 12 (A) African myristicin standard, (B) FREV group; comparison of these spectra shows that the compound retaining the peak is the same substance as the standard.

[0038] The effects of DREV, FREV, and FR-DSS6 samples on endothelial cell migration were investigated. The experiments were conducted according to the methods described in the literature (Xiang K, Hao M, Zhang Z, et al. Engineering 3D-BMSC exosome-based hydrogels that collaboratively regulate bone microenvironment and promote osteogenesis for enhanced cell‐free bone regeneration[J]. Materials Today Bio, 2025, 32.DOI:10.1016 / j.mtbio.2025.101881.). The final concentration of DREV, FREV, and FR-DSS6 was 1 mg / mL.

[0039] result Figure 13 As shown, Figure 13 The left-middle image shows representative images of human umbilical vein endothelial cells (HUVECs) at 4, 8, 12, and 24 hours under different treatment conditions (scale bar: 500 µm). The right-middle image shows the statistical results of cell migration area at each time point under different treatment methods. Data are expressed as mean ± standard deviation (n=3). Significant differences were determined by one-way ANOVA, **p<0.01, ***p<0.005, compared with the control group.

[0040] Depend on Figure 13 It is evident that the migration area of ​​HUVECs in the FR-DSS6 group increased continuously over time and had a similar migration effect to FREV. Both showed the best migration effect at 24h, when the scratch area of ​​HUVECs in the FR-DSS6 group was approximately 65% ​​of that in the control group (Con) and 85% of that in the DREV group. These results indicate that FR-DSS6 can enhance the migration ability of endothelial cells under hypoxic conditions.

[0041] Following the experimental methods described in the aforementioned literature, using Matrigel... ® Angiogenesis assays were performed to assess the angiogenic potential of DREV, FREV, and FR-DSS6 samples, with a final concentration of 1 mg / mL for all three. Results are as follows: Figure 14 As shown, Figure 14 Image A in the middle shows representative tubular network images of human umbilical vein endothelial cells (HUVECs) in different treatment groups (scale bar: 100 μm). Figure 14Figures B, C, D, and E in the figure represent, respectively, the number of HUVEC network structures, the total length of HUVEC main branches, the total length of HUVECs, and the total branch length of HUVECs in hypoxic environments under different treatment conditions. Data are expressed as mean ± standard deviation (n=3). Significant differences compared to the control group were determined by one-way ANOVA, ***p<0.005.

[0042] Depend on Figure 14 It is evident that the FR-DSS6 group exhibits a large number of primary vascular-like networks characterized by tubular structures. These structures extend outward from the cell aggregates and have a significantly higher number of pores than the Con group. Furthermore, the FR-DSS6 group outperforms other groups in terms of total number of connection points, total length of main segments, and branch length.

[0043] The ability of DREV, FREV, and FR-DSS6 samples to scavenge ROS in cells was investigated. Bone marrow mesenchymal stem cells (BMSCs) and FR-DSS6 were co-cultured under hypoxic conditions using the AnaeroPack® anaerobic culture system for 12 hours. After co-culture, BMSCs were stained with a 10 μM ROS dye and incubated at 37 °C for 20 minutes. The stained BMSCs were then thoroughly washed with serum-free medium to remove excess unbound dye. ROS levels within BMSCs under hypoxic conditions were imaged using fluorescence microscopy. Finally, ImageJ software was used to quantitatively analyze the fluorescence signals in the acquired images to assess the relative levels of intracellular ROS in different samples. Results are as follows: Figure 15 As shown, Figure 15 The upper and middle figures show representative images of reactive oxygen species (ROS) fluorescence staining in bone marrow mesenchymal stem cells (BMSCs) after different sample treatments. The lower figure shows the quantitative analysis results of ROS fluorescence staining. Data are expressed as mean ± standard deviation (n=3). Significant differences were determined by one-way ANOVA, *p<0.01, ***p<0.005, compared with the control group.

[0044] The fluorescence intensity of the FR-DSS6 group was significantly lower than that of the Con group, indicating that the ROS scavenging ability of FR-DSS6 is stronger than that of Con. Furthermore, the fluorescence intensity of DREV was slightly lower than that of the Con group, but still significantly lower than that of FR-DSS6. Quantitative analysis showed that the ROS level in the FR-DSS6 group was only 20% of that in the control group (Con group).

[0045] Bone marrow was extracted from 4-8 week old C57 mice. After centrifugation, the cells were resuspended in α-MEM medium and cultured in culture dishes for 1 day. Subsequently, 5 × 10⁶ cells were seeded per well. 5 Cells were collected. The wells were washed daily with α-MEM containing 30 ng / ml macrophage colony-stimulating factor (M-CSF) to remove floating cells for 3 days. DREV, FREV, and FR-DSS6 samples were added to the cells to a final concentration of 1 mg / mL, and the cells were induced with α-MEM medium containing 30 ng / ml M-CSF and 100 ng / ml nuclear factor κB receptor activator ligand (RANKL). The medium was changed every 2 days for 10 days. Differentiated osteoclasts cultured under hypoxic conditions were collected after 10 days for Western blotting (WB) to detect the expression levels of NFAT2, C-Fos, and β-actin. Results are shown below. Figure 16 As shown, NFAT2 levels in osteoclasts induced by different treatments (e.g., Figure 16 (as shown in Figure A) and C-Fos (as shown in Figure A) Figure 16 Quantitative analysis of relative expression levels (n=3, as shown in Figure B). Data are expressed as mean ± standard deviation (n=3). Significant differences compared to the control group were determined by one-way ANOVA, ***p<0.005. NFAT2 is a crucial transcription factor in osteoclast differentiation, highly expressed after RANKL stimulation, thus promoting osteoclast differentiation. C-Fos is also a protein related to osteoclast formation, and its expression increases after hypoxia and inflammation. Both showed high expression in Con, with slightly reduced expression in the DREV group, but almost no expression in FREV and FR-DSS6. These results indicate that FR-DSS6 can significantly reduce the production of osteoclast differentiation factors.

[0046] To further investigate the inhibitory effects of DREV, FREV, and FR-DSS6 samples on osteoclast formation, bone marrow was extracted from 4-8 week old C57 mice. After centrifugation, the cells were resuspended in α-MEM medium and cultured in culture dishes for 1 day. Subsequently, 5 × 10⁶ cells were seeded per well. 5Cells were cultured daily using α-MEM containing 30 ng / ml macrophage colony-stimulating factor (M-CSF) to remove floating cells for 3 days. DREV, FREV, and FR-DSS6 samples at a final concentration of 1 mg / mL were added to cell culture wells and induced with α-MEM containing 30 ng / ml M-CSF and 100 ng / ml receptor activator of nuclear factor κB (RANKL). The medium was changed every 2 days for 10 days. After osteoclast formation in the control group, F-actin and 4',6-diamidin-2-phenylindole (DAPI) staining were performed. The resulting immunofluorescence images are shown below. Figure 17 As shown (green fluorescence: actin cytoskeleton, blue fluorescence: cell nucleus), the scale bar represents 200 micrometers. Figure 17 The results showed that the number of fused osteoclasts in the FR-DSS6 group was almost zero, which was similar to the effect in the FREV group. DREV had a certain ability to inhibit osteoclast formation, but a small number of osteoclasts still fused, which was still inferior to FR-DSS6.

[0047] Bone marrow mesenchymal stem cells (BMSCs) were seeded and cultured in 6-well plates. When the cell confluence reached 60%, the medium was replaced with OriCell® osteogenic differentiation medium for BMSCs, FR-DSS6 was added, and the cells were co-cultured in a hypoxic microenvironment. The expression levels of type I collagen (Col 1), RUNX2, osteocalcin (OCN), and β-actin in the cells were then detected by Western blotting.

[0048] like Figure 18 As shown, the expression level of RUNX2, a key transcription factor in early osteogenic differentiation, was significantly higher in the FR-DSS6 group than in the DREV group (e.g., ...). Figure 18 (As shown in Figure B). The FR-DSS6 group also showed advantages in the expression of OCN (a marker of late osteogenic differentiation) and Col 1 (e.g., Figure 18 As shown in Figures C and A), OCN is the main non-collagenous protein in bone tissue, specifically expressed in osteoblasts. Col 1 directly reflects osteoblast activity and bone formation capacity. All three osteogenic markers showed the highest expression levels in the FR-DSS6 group, and FERV also exhibited the same trend, indicating that DSS6 modification does not alter the osteogenic role of FERV, and the osteogenic effect is far superior to that in the DREV group.

[0049] The effects of DREV, FREV, and FR-DSS6 samples on the entire osteogenic differentiation process were investigated. Bone marrow mesenchymal stem cells (BMSCs) were seeded in 6-well plates and cultured. When cell confluence reached 60%, the medium was replaced with OriCell® osteogenic differentiation medium, and DREV, FREV, and FR-DSS6 samples were added to a final concentration of 1 mg / mL and co-cultured in a hypoxic microenvironment. After 3 days of induction, cells were washed twice with PBS for 3 minutes each time; fixed with 1 mL of 4% paraformaldehyde (PFA) for 15 minutes, and then stained with alkaline phosphatase (ALP) using the BCIP / NBT alkaline phosphatase staining kit to determine ALP activity. After 10 days of induction, the cells were washed twice with PBS for 3 minutes each time; 1 mL of 4% PFA was added for fixation for 15 minutes, and then the cells were stained with Alizarin Red S (ALIR-10001, OriCell®) for 5 minutes to observe the formation of calcium nodules and to evaluate the osteogenic induction capacity of different exosome samples.

[0050] The results are as follows Figure 19 As shown, the ALP staining results in the FR-DSS6 group were superior to those in the DREV group, and the positive expression level was 7 times that of the Con group, indicating that FR-DSS6 can enhance ALP expression in the early stage of osteogenic differentiation. Alizarin red staining showed that FR-DSS6 has significant osteogenic induction ability, with obvious calcified nodules appearing as early as day 10 of induction. In addition, the alizarin red staining results 10 days after induction were consistent with the ALP staining trend: quantitative analysis showed that the alizarin red stained calcified nodule expression level in the FR-DSS6 group was 70 times that of the control group, while the FR-DSS6 group reached 3 times that of DREV.

[0051] The above results indicate that FR-DSS6 can reduce ROS levels in BMSCs, inhibiting osteoclast formation and promoting osteogenic differentiation in vitro.

[0052] To investigate the potential functions of exosome-derived miRNAs (F-miRs) from fresh Rehmannia glutinosa, miRNA sequencing was used to further explore the mechanism by which F-miRs translocate to osteoclasts and osteoblasts via FR-DSS6 and exert their potential effects. Sequencing revealed 1391 differentially expressed miRNAs between FREV and DREV, of which 625 showed significant differences. A differential miRNA volcano plot showed that compared to the DREV group, 314 miRNAs were upregulated and 277 miRNAs were downregulated in the FREV group (fold change ≥2, P < 0.05). Figure 20 As shown.

[0053] The primary function of miRNAs is to upregulate their expression; therefore, the focus is on significantly upregulated F-miRs. Within F-miRs, many smaller subgroups of miRNAs exist within the same major class. Sequencing results showed that miR166 had the highest proportion of miRNAs in the overall class. After filtering out miRNAs with relatively low abundance, seven upregulated and three downregulated differentially expressed genes (DEGs) between DREV and FREV were selected. The results are as follows... Figure 21 As shown.

[0054] Gene Ontology (GO) reveals that multiple pathways, such as the FoxO signaling pathway and the TGF-beta signaling pathway, are involved in alleviating oxidative stress and promoting osteogenic development. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment map shows that differentially expressed genes are enriched in pathways that promote osteogenic differentiation and alleviate oxidation, such as… Figure 22 As shown, Figure 22 The size of the midpoint indicates the number of differentially expressed genes in the pathway; the larger the enrichment factor value, the higher the degree of KEGG enrichment.

[0055] Further investigation using NRF2 and NF-κB revealed that NRF2 primarily regulates oxidative stress responses and cellular protection mechanisms, defends against xenobiotic metabolism, and provides an effective antioxidant system. The NRF2 and NF-κB pathways interact to regulate cellular redox homeostasis and responses to stress and inflammation. NRF2 plays a crucial role in counteracting NF-κB-driven inflammatory responses, and compounds that inhibit NF-κB to reduce inflammation can also activate the NRF2 pathway. Their synergistic effect alleviates oxidative stress and influences osteoblasts and osteoclasts. Figure 23 As shown, among osteoclast proteins, the Con group exhibited the highest expression of NF-κB and p-NF-κB, while NRF2 expression was the lowest. After drug administration, the expression levels of NF-κB and p-NF-κB proteins decreased significantly in the FREV and FR-DSS6 groups, while the expression level of NRF2 protein increased significantly. This indicates that FREV and FR-DSS6 influence osteoclast fusion by affecting oxidative stress. The same trend was observed in the pathway validation of osteoblasts.

[0056] Bone marrow was extracted from 4-8 week old C57 mice. After centrifugation, the cells were resuspended in α-MEM medium and cultured in culture dishes for 1 day. Subsequently, 5 × 10⁶ cells were seeded per well. 5Cells were collected. The wells were washed daily with α-MEM containing 30 ng / ml M-CSF to remove floating cells for 3 days. After treatment with FR-DSS6 to a final concentration of 1 mg / mL, cells were induced with α-MEM medium containing 30 ng / ml M-CSF and 100 ng / ml RANKL, with the medium changed every 2 days for 10 days. Proteins were extracted from osteoclasts using a KeyGen Biotech Total Protein Extraction Kit (China). Equal volumes of total protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, USA). After blocking, the membranes were incubated overnight with primary antibodies against β-actin (1:50,000), NF-κB (1:1,000), phosphorylated NF-κB (p-NF-κB, 1:1,000), and NRF2 (1:3,000, Abcam). Subsequently, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000, Proteintech) for 1.5 hours. Following FR-DSS6 treatment, the expression levels of p-NF-κB, NF-κB, NRF2, and β-actin in RANKL-induced osteoclasts were determined. Figure 23 It can be seen that the relative p-NF-κB in osteoclasts induced by different treatment conditions (e.g. Figure 23 As shown in Figure A), NF-κB (as shown in Figure A) Figure 23 (as shown in Figure B) and NRF2 (as shown in Figure B) Figure 23 The quantitative analysis results (n=3) are shown in Figure C. Data are expressed as mean ± standard deviation (n=3). Significant differences were determined by one-way ANOVA, with ***p<0.005 compared to the control group.

[0057] Bone marrow mesenchymal stem cells (BMSCs) were seeded and cultured in 6-well plates. When cell confluence reached 60%, the medium was replaced with OriCell® osteogenic differentiation medium, and DREV, FREV, and FR-DSS6 samples were added to a final concentration of 1 mg / mL and co-cultured in a hypoxic microenvironment. Proteins were extracted from BMSCs using a KeyGenBiotech Total Protein Extraction Kit (KeyGenBiotech, China), and Western blotting was performed to detect the expression levels of p-NF-κB, NF-κB, NRF2, and β-actin in BMSCs differentiating into osteoblasts under hypoxic conditions after FR-DSS6 treatment. Figure 24 It can be seen that after bone marrow mesenchymal stem cells (BMSCs) differentiate into differentially expressed osteoblasts under different treatment conditions, their relative proportions of p-NF-κB (such as...) are significantly higher. Figure 24 As shown in Figure A), NF-κB (as shown in Figure A) Figure 24 (as shown in Figure B) and NRF2 (as shown in Figure B) Figure 24 The quantitative analysis results (n=3) are shown in Figure C.

[0058] Six weeks after establishing an osteoporosis model in mice, DiR-labeled DREV, FREV, and FR-DSS6 samples were administered via tail vein injection. Mice were sacrificed 24 hours later, and their internal organs were harvested for in vivo imaging. The results are as follows: Figure 25 As shown, Figure 25 The right-middle figure shows the quantitative analysis of fluorescence intensity in major organs 24 hours after injection. Among the organs, the liver showed the strongest fluorescence intensity due to its metabolic function and rich blood flow, while the FR-DSS6 group showed the lowest fluorescence intensity among all organs.

[0059] In vivo imaging of the femurs of mice in each group was performed, and the results are as follows: Figure 26 As shown, Figure 26 The right-middle figure shows the quantitative analysis of fluorescence intensity in major organs 24 hours after injection. It can be seen that almost no fluorescence was observed in the Con group, while FR-DSS6 exhibited the strongest fluorescence intensity, three times that of the Con group. The fluorescence intensities of DREV and FREV were similar, indicating that FR-DSS6 has a clear bone-targeting ability. Data are expressed as mean ± standard deviation (n=3). One-way ANOVA was used to determine statistical significance; compared with the control group, ***p<0.005.

[0060] Frozen sections of the femurs of mice in each group were used to further verify the bone-targeting ability of FR-DSS6, such as... Figure 27 As shown, the fluorescence intensity of trabecular bone and cortical bone in the FR-DSS6 group was the strongest. The fluorescence intensity of DREV was similar to that of FREV and much smaller than that of the FR-DSS6 group, further validating the excellent bone-targeting ability of FR-DSS6. These results indicate that DSS6 can target and deliver FREV into bone tissue, increasing the concentration of FREV in bone tissue for better treatment of osteoporosis.

[0061] 3D reconstruction of micro-CT images revealed sparse trabeculae in the Con group. The trabeculae number in the DREV and FREV groups was higher than in the Con group. Due to the targeted nature of DSS6-modified FREV, the FR-DSS6 group had significantly more trabeculae than the Con group. Quantitative analysis of bone volume fraction (BV / TV) and trabeculae number (Tb.N) within the selected osteoporotic region further validated these findings. Figure 28 The results showed that Figure 28The top-middle image shows a representative micro-CT image displaying the three-dimensional microstructure of the distal femur trabecular bone; the bottom-left image shows a quantitative analysis of bone volume fraction (BV / TV); and the bottom-right image shows a quantitative analysis of trabecular bone number (Tb.N). Figure 28 It was observed that 4 weeks after tail vein administration of FR-DSS6, the BV / TV and Tb.N of the defect area were 4 times and 1.5 times larger, respectively, than those of the control group. The BV / TV and Tb.N of the DREV and FREV groups were similar and slightly larger than those of the control group.

[0062] Histological analysis using H&E and TRAP staining was performed to investigate the effects of FR-DSS6 on the micromorphology of the femur in osteoporosis. Results are as follows: Figure 29 As shown, Figure 29 The upper image shows a representative photograph of the femur stained with HE (hematoxylin and eosin), scale bar = 100 μm; the lower image shows a representative photograph of the femur stained with TRAP (transferase in chromatogram), scale bar = 100 μm. Figure 29 As can be seen, significant bone loss and thinning of trabeculae were observed in the Con group. Trabeculae formation was observed in the DREV, FREV, and FR-DSS6 groups, with the FR-DSS6 group showing intact trabecular structure and thicker, denser trabecular arrangement. The DREV group exhibited sparser trabecular arrangement and thinner trabecular structure, significantly different from the FR-DSS6 group. The FREV group also showed some trabecular formation, but it was slightly less than that of the FR-DSS6 group. Furthermore, TRAP staining revealed a significant increase in TRAP-positive osteoclasts in the Con group, a significant decrease in TRAP-positive osteoclasts in the DREV and FREV groups, and almost complete absence of TRAP-positive osteoclasts in the FR-DSS6 group. Overall, the DSS6-modified FREV demonstrated excellent bone regeneration capacity and the ability to inhibit osteoclast formation.

[0063] The expression of two inflammatory factors, TNF-α and IL-1β, in bone and femoral tissue was determined by immunohistochemistry. The results are as follows: Figure 30 As shown, Figure 30 The left image shows a representative photograph of TNF-α immunohistochemical staining in the femur, scale bar = 100 μm; the right image shows a representative photograph of IL-1β immunohistochemical staining in the femur, scale bar = 100 μm. Figure 30 As can be seen, the Con group contained a large number of dark brown inflammatory cells, while the DREV group showed a slight decrease in inflammatory factors but still maintained high expression. In contrast, inflammatory expression almost disappeared in the FR-DSS6 group, and IL-1β showed the same expression trend as TNF-α. This demonstrates that FR-DSS6 promotes bone repair by alleviating oxidative stress.

[0064] Immunofluorescence (IF) staining was used to further determine the osteogenic capacity and mechanism of FR-DSS6 in vivo. Results are as follows: Figure 31 The image shows representative immunofluorescence images of RUNX2 (red), OPN (green), CD31 (red), NF-κB (red), and NRF2 (green) in the femur. Scale bar = 100 μm. The relative gray values ​​of immunofluorescence staining for RUNX2, OPN, CD31, NF-κB, and NRF2 in ovariectomized mice from different treatment groups are also shown. Data are expressed as mean ± standard deviation (n=3). Significant differences compared to the control group were determined by one-way ANOVA, *p<0.01, ***p<0.005.

[0065] In bone tissue, Runt-associated transcription factor 2 (RUNX2) is an important transcription factor involved in osteogenic differentiation. In the control group, almost no RUNX2 expression was observed (red signal). RUNX2 expression in the DREV group was similar to that in the control group. However, RUNX2 fluorescence intensity was significantly enhanced in FR-DSS6 (e.g., Figure 31 (As shown in Figure A). These results indicate that FR-DSS6 can promote RUNX2 expression. Osteopontin (OPN) is one of the major components of the extracellular matrix. Figure 31 As shown in Figure B, OPN expression (green signal) was low in the newly formed bone of the control group (Con group) and the DREV group. OPN expression in the FREV group was upregulated compared to the Con group, but still lower than that in the FR-DSS6 group. The trend of these results is consistent with the in vitro experiments, indicating that FR-DSS6 can promote OPN formation during osteogenic differentiation in both in vivo and in vitro.

[0066] After initially validating that FR-DSS6 promotes lumen formation in human umbilical vein endothelial cells (HUVECs), further in vivo CD31 studies were conducted to evaluate the pro-angiogenic capacity of FR-DSS6. CD31 (red signal) is an endothelial cell marker, and its expression helps in assessing angiogenesis. Figure 31 As shown in Figure C, only a small amount of neovascularization was observed in the DREV group and the control group. However, the FR-DSS6 group showed significantly more neovascularization in bone tissue than the DREV and FREV groups. This result indicates that FR-DSS6 can promote angiogenesis in vivo. NF-κB and NRF2 have a bidirectional interaction with oxidative stress and play a central role in redox homeostasis. Under oxidative stress conditions, intracellular NF-κB can be activated, while NRF2 is inhibited. Figure 31As shown in Figures D and E, in the control group, significant NF-κB (red signal) and low NRF2 expression (green signal) were observed in bone tissue. In the DREV group, NF-κB expression decreased, but NRF2 expression showed an increasing trend. However, in the FR-DSS6 group, NF-κB was difficult to observe in bone, but NRF2 expression was the highest. These results indicate that FR-DSS6 can affect the expression of NF-κB and NRF2 in bone tissue by scavenging ROS, promoting bone formation and angiogenesis, and alleviating OVX.

Claims

1. A fresh Rehmannia glutinosa exosome, characterized in that, It includes lipid vesicles filled with Rehmannia glutinosa metabolites, said Rehmannia glutinosa metabolites including at least one of phenylpropanoid compounds and polyketide compounds.

2. The fresh Rehmannia glutinosa exosomes according to claim 1, characterized in that, The rehmannia metabolites include at least one of methyl caffeate, anhydrous andrographolide, neoandrographolide aglycone, and amyristol.

3. The fresh Rehmannia glutinosa exosomes according to claim 1, characterized in that, The surface of the lipid vesicles is modified with a bone-targeting peptide containing the amino acid sequence shown in SEQ ID No.

1.

4. The method for preparing fresh Rehmannia glutinosa exosomes according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Fresh Rehmannia glutinosa juice is obtained by juicing fresh Rehmannia glutinosa juice, and the supernatant is collected by centrifugation. (2) After ultracentrifuging the supernatant, the precipitate was collected and resuspended in buffer solution to obtain the first solution; (3) Add the first solution to the concentrated sucrose solution, centrifuge at high speed, and take the liquid at the interface of the concentrated sucrose solution to obtain the second solution; (4) Add buffer solution to the second solution, centrifuge at ultraspeed and take the precipitate to obtain fresh Rehmannia exosomes.

5. The method for preparing fresh Rehmannia glutinosa exosomes according to claim 4, characterized in that, In step (1), the centrifugation conditions for fresh Rehmannia glutinosa juice are 9000-11000g for at least 30min; in steps (2)-(4), the conditions for ultracentrifugation are 80000-120000g for 50-90min, and the buffer is phosphate buffer or Tris buffer.

6. The method for preparing fresh Rehmannia glutinosa exosomes according to claim 4, characterized in that, In step (3), the concentration of sucrose in the concentrated sucrose solution is 25-35%.

7. The method for preparing fresh Rehmannia glutinosa exosomes according to claim 4, characterized in that, In step (4), after removing the precipitate, the following steps are also included: The precipitate was resuspended in buffer solution to obtain an exosecretory resuspension. DSPE-PEG-DSS6 was obtained by clicking reaction of 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol-N-maleimide and a bone-targeting peptide with thiol-terminal modification. DSPE-PEG-DSS6 was added to the exosome suspension, and the reaction was carried out in an ice bath with stirring to obtain fresh Rehmannia glutinosa exosomes modified with bone-targeting peptides.

8. The method for preparing fresh Rehmannia glutinosa exosomes according to claim 4, characterized in that, The concentration of exosomes in the exosome suspension is 0.5-1.5 mg / mL; The mass ratio of the 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol-N-maleimide to the bone-targeting peptide with thiol-terminal modification is 1-2:1-2. The conditions for the click reaction are: using an organic amine as a catalyst in a neutral buffer environment, stirring at 30-40°C in the dark for 2-6 hours; The volume ratio of DSPE-PEG-DSS6 to the exocrine body suspension is 1:10-30.

9. The use of fresh Rehmannia glutinosa exosomes according to any one of claims 1-3 in the preparation of a drug for treating osteoporosis.

10. The use of the fresh Rehmannia glutinosa exosomes according to any one of claims 1-3 in any of the following: To prepare drugs that reduce the production of osteoclast differentiation factors; To prepare drugs that inhibit osteoclast formation and / or promote osteogenic differentiation; To prepare drugs that reduce reactive oxygen species levels in bone marrow mesenchymal stem cells; To prepare drugs to alleviate oxidative stress; To prepare drugs that promote bone repair or bone regeneration; Prepare a drug that increases the expression level of at least one of RUNX2, type I collagen, osteopontin, and osteocalcin in bone tissue; To prepare drugs that downregulate NF-κB and upregulate NRF2 expression in bone tissue; To prepare drugs that inhibit bone tissue inflammation; To prepare drugs that promote angiogenesis.

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