Application of nano vesicles from andrographis paniculata nees in preparation of drugs for treating postmenopausal osteoporosis

Abstract

This invention belongs to the fields of bone disease treatment and natural medicine, and provides the application of Andrographis paniculata-derived nanovesicles in the preparation of drugs for treating postmenopausal osteoporosis. The Andrographis paniculata-derived nanovesicles are extracted from Andrographis paniculata by differential centrifugation and PEG precipitation, with a particle size of 50-200 nm, and contain protein, RNA, and lipid components. The Andrographis paniculata-derived nanovesicles can significantly improve the bone microstructure of ovariectomized mice, increase bone volume fraction and trabecular bone number, reduce trabecular bone separation, inhibit osteoclast formation, and promote bone defect repair, with good in vivo safety. The Andrographis paniculata-derived nanovesicles contain extremely low levels of andrographolide, but their activity in promoting osteogenic differentiation and inhibiting osteoclast differentiation is significantly better than that of andrographolide monomer, indicating that their efficacy comes from the entire vesicle rather than a single component. This invention provides a safe and effective new drug regimen for the treatment of postmenopausal osteoporosis.

Description

Technical Field

[0001] This invention belongs to the fields of bone disease treatment and natural medicine, specifically involving the application of Andrographis paniculata-derived nanovesicles in the preparation of drugs for treating postmenopausal osteoporosis. Background Technology

[0002] Postmenopausal osteoporosis (PMOP) is a systemic bone metabolic disease caused by the decline in estrogen levels after menopause in women. It is mainly characterized by low bone mass, bone microarchitectural deterioration, and increased bone fragility, making fractures highly likely. Statistics show that the prevalence of osteoporosis in postmenopausal women over 40 years of age in my country is as high as 32.5%, which is 4 to 5 times higher than that in men of the same age group. Its complication, fragility fractures, has a high rate of disability and mortality, placing a heavy burden on patients' families and the social healthcare system.

[0003] Currently, commonly used drugs for treating postmenopausal osteoporosis include bisphosphonates, estrogen, calcitonin, and other bone resorption inhibitors and bone formation promoters. However, these drugs often face safety issues with long-term use. For example, bisphosphonates are frequently associated with atypical femoral fractures and osteonecrosis of the mandible; estrogen therapy carries risks of tumors and thrombotic diseases; and long-term use of calcitonin can lead to diminishing efficacy and gastrointestinal adverse reactions, all of which limit their application. Therefore, developing novel anti-postmenopausal osteoporosis drugs with significant efficacy and high safety remains a current research hotspot and challenge.

[0004] Traditional Chinese medicine (TCM) has a long history and unique advantages in treating postmenopausal osteoporosis. TCM theory holds that postmenopausal osteoporosis largely falls under the categories of "bone atrophy" and "bone decay," with kidney deficiency as the root cause and blood stasis as the manifestation. Kidney yin deficiency and internal heat can lead to malnourishment of the bones, while blood stasis exacerbates bone decay. Therefore, clinical treatment primarily focuses on nourishing yin, clearing heat, and promoting blood circulation. Unlike the single-target drugs of modern medicine, TCM, through the synergistic effects of multiple components, targets, and pathways, can simultaneously regulate the balance between bone formation and bone resorption, improve systemic symptoms, and demonstrate good long-term safety, thus attracting increasing attention from scholars both domestically and internationally. Andrographis paniculata ( Andrographis paniculata Andrographis paniculata, a plant of the Acanthaceae family, is a traditional southern Chinese medicine. It is bitter and cold in nature, effectively clearing deficiency heat and dispersing blood stasis. It possesses the effects of clearing heat and detoxifying, cooling blood and reducing swelling, which highly aligns with the pathogenesis of postmenopausal osteoporosis characterized by "yin deficiency and internal heat, with blood stasis." Modern pharmacological studies have shown that Andrographis paniculata and its main active ingredient, andrographolide, have multiple pharmacological activities, including anti-inflammatory, antioxidant, and bone metabolism-regulating effects, suggesting potential application value in the treatment of postmenopausal osteoporosis.

[0005] However, andrographolide, as a diterpenoid lactone monomer, has extremely poor water solubility, resulting in low oral bioavailability. Furthermore, andrographis paniculata is cold in nature, and long-term use can easily damage the spleen and stomach, potentially exacerbating the gastrointestinal discomfort already common in osteoporosis patients. Its raw material contains a complex system of multiple components, with varying pharmacological activities among the different components, making precise quality control difficult. These issues severely restrict the clinical translation and application of andrographis paniculata and its active ingredients in the field of osteoporosis treatment.

[0006] In recent years, extracellular vesicles (EVs) have attracted much attention in the biomedical field due to their advantages such as mediating intercellular communication, low immunogenicity, and low toxicity. Among them, plant-derived nanovesicles (PDVs) not only possess the aforementioned advantages of animal extracellular vesicles but also have the characteristics of mass production and low cost. As a whole delivery system for natural active substances, they may overcome the aforementioned shortcomings. However, whether they can be used to treat postmenopausal osteoporosis, and whether their efficacy comes from andrographolide, are currently unreported. Summary of the Invention

[0007] Based on this, the present invention extracts nanovesicles from Andrographis paniculata and systematically studies their efficacy and safety in treating postmenopausal osteoporosis, providing a foundation for the development of novel anti-postmenopausal osteoporosis drugs.

[0008] On the one hand, this application provides the application of Andrographis paniculata-derived nanovesicles in the preparation of drugs for treating postmenopausal osteoporosis.

[0009] On the other hand, this application provides a drug for treating postmenopausal osteoporosis, said drug comprising andrographis paniculata-derived nanovesicles.

[0010] Furthermore, the average particle size of the Andrographis paniculata-derived nanovesicles is 50~200 nm.

[0011] Furthermore, the andrographis paniculata-derived nanovesicles are prepared by a method including differential centrifugation and polyethylene glycol precipitation.

[0012] Furthermore, the preparation method of the Andrographis paniculata-derived nanovesicles includes: taking Andrographis paniculata medicinal material, washing, soaking, homogenizing, and filtering; removing impurities by graded centrifugation; precipitating with polyethylene glycol; resuspending and filtering for sterilization.

[0013] Furthermore, the preparation method of the Andrographis paniculata-derived nanovesicles includes: (1) Take dried Andrographis paniculata, wash it with water and soak it in water for 1-4 hours, then homogenize it mechanically and filter it to obtain the filtrate. (2) Centrifuge the filtrate obtained in step (1) at 800-1500×g for 5-15 min; take the supernatant and centrifuge at 2000-4000×g for 10-30 min; take the supernatant and centrifuge at 8000-12000×g for 20-40 min; take the supernatant and centrifuge at 14000-18000×g for 40-80 min; take the supernatant; filter and add PEG to a final concentration of 6-10%, incubate overnight; take the supernatant and centrifuge at 14000-18000×g for 30-40 min; discard the supernatant and take the precipitate; (3) Add PBS buffer to the precipitate obtained in step (2), blow the precipitate evenly to form a mixture, and filter to remove bacteria.

[0014] Furthermore, the preparation method of the Andrographis paniculata-derived nanovesicles includes: (1) Take dried Andrographis paniculata, wash it with water and soak it in water for 2 hours, then homogenize it mechanically for 1 minute and filter to obtain the filtrate. (2) Centrifuge the filtrate obtained in step (1) at 1000×g and 4℃ for 10 min; take the supernatant and centrifuge at 3000×g and 4℃ for 20 min; take the supernatant and centrifuge at 10000×g and 4℃ for 30 min; take the supernatant and centrifuge at 16000×g and 4℃ for 60 min; take the supernatant; filter with a 1 μm filter and add PEG8000 to a final concentration of 8%, incubate overnight at 40 rpm and 4℃; take the supernatant and centrifuge at 16000×g and 4℃ for 30 min; discard the supernatant and take the precipitate; (3) Add PBS buffer to the precipitate obtained in step (2), blow the precipitate evenly to form a mixture, and filter to remove bacteria.

[0015] Furthermore, the content of andrographolide in the andrographis-derived nanovesicles is less than 1 μM.

[0016] Furthermore, the drug is an oral preparation.

[0017] Furthermore, the drug has one or more of the following effects: (1) Reduce bone loss and bone defects; (2) Assists in bone tissue repair; (3) Promotes osteoblast differentiation and mineralization, as well as the expression of osteoblast-related genes; (4) Inhibit osteoclast differentiation and osteoclast-related gene expression.

[0018] The osteogenic genes include, but are not limited to, Alp. The osteoclast-related genes include, but are not limited to, Nfatc1, Trap, and C-fos.

[0019] The application documents mention Andrographis paniculata-derived nanovesicles, nanovesicles (abbreviated as APNVs). Andrographis paniculata -derived nanovesicles) have the same meaning and can be used interchangeably.

[0020] Beneficial effects This invention reveals for the first time the application of Andrographis paniculata-derived nanovesicles in the treatment of postmenopausal osteoporosis, broadening the application scope of plant-derived nanovesicles and providing a new treatment option for clinical practice.

[0021] The andrographis paniculata-derived nanovesicles provided by this invention have a multi-target regulatory effect on bone metabolism, which can simultaneously promote bone formation and inhibit bone resorption, overcoming the limitations of existing single-target drugs.

[0022] The preparation process of Andrographis paniculata-derived nanovesicles provided by this invention is simple, can be mass-produced, and has good in vivo safety, solving the problems of low bioavailability of Andrographis paniculata raw materials and monomer components, and the easy occurrence of adverse reactions with long-term use.

[0023] This invention demonstrates through experiments that the pharmacological effects of nanovesicles derived from Andrographis paniculata do not depend on the trace amounts of andrographolide they encapsulate, but rather on their therapeutic effects as a whole intact vesicle, providing a new theoretical basis for the development of this type of drug. Attached Figure Description

[0024] Figure 1A Transmission electron microscopy image of nanovesicles derived from Andrographis paniculata, scale bar indicates 100 nanometers.

[0025] Figure 1B The results show the size and concentration of nanovesicles derived from Andrographis paniculata determined using nanoflow cytometry.

[0026] Figure 1C The purity of the andrographis paniculata-derived nanovesicles was determined by mixing 0.1% TritonX-100 with the nanovesicles and using a membrane rupture method.

[0027] Figure 1D The image shows the SDS-PAGE gel electrophoresis pattern of proteins from Andrographis paniculata-derived nanovesicles. Proteins extracted from Andrographis paniculata-derived nanovesicles were separated by SDS-PAGE and stained with Coomassie blue dye.

[0028] Figure 1EThis image shows the agarose gel electrophoresis pattern of RNA from Andrographis paniculata nanovesicles. RNA extracted from Andrographis paniculata nanovesicles was divided into two portions. One portion was treated with RNase, and the remaining RNA was electrophoresed together with the untreated RNA on a 1.5% agarose gel. Markers ranging from 100 to 2000 base pairs were used.

[0029] Figure 1F Thin-layer chromatography (TLC) chromatograms of lipids in nanovesicles derived from Andrographis paniculata; lipids from nanovesicles derived from Andrographis paniculata were separated on silica gel plates for TLC and visualized using 10% ethanol-sulfuric acid solution.

[0030] Figure 2A This is a representative micro-CT image of the trabecular structure of the L2 lumbar spine.

[0031] Figure 2B The results of quantitative analysis of proximal tibial bone volume fraction, structural model index, trabecular thickness, trabecular number, and trabecular separation are presented.

[0032] Figure 2C Representative images of the tibia stained with HE, with scale bars of 500 μm and 100 μm, respectively.

[0033] Figure 2D Bar graphs showing the tibial trabecular bone volume of mice in each group. Values ​​for each group are expressed as mean ± standard deviation. Compared with the sham-operated group, # P <0.05, ## P <0.01, ### P <0.001; compared with the model group, * P <0.05, ** P <0.01, *** P <0.001.

[0034] Figure 2E Representative images of TRAP staining in the tibia, with scale bars of 500 μm and 100 μm.

[0035] Figure 2F Bar graphs showing the relative number of osteoclasts in the tibia of mice in each group. Values ​​for each group are expressed as mean ± standard deviation. Compared with the sham-operated group, # P <0.05, ## P <0.01, ### P <0.001; compared with the model group, * P <0.05,** P <0.01, *** P <0.001.

[0036] Figure 2G Bar graphs showing the relative surface area of ​​osteoclasts in the tibia of mice in each group. Values ​​for each group are expressed as mean ± standard deviation. Compared with the sham-operated group, # P <0.05, ## P <0.01, ### P <0.001; compared with the model group, * P <0.05, ** P <0.01, *** P <0.001.

[0037] Figure 3A These are representative micro-CT images of the bone microstructure in the proximal tibial bone defect area 1 week and 2 weeks after surgery.

[0038] Figure 3B This study quantitatively analyzed the cortical to cancellous bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) in the tibial bone defect area at 1 and 2 weeks post-operation. Values ​​for each group are expressed as mean ± standard deviation. Compared with the sham-operated group, # P <0.05, ## P <0.01, ### P <0.001; compared with the model group, * P <0.05, ** P <0.01, *** P <0.001.

[0039] Figure 4A shows representative HE-stained images of liver, kidney, brain, heart, and lung tissues from ovariectomized mice in the sham-operated group and those treated with Andrographis paniculata-derived nanovesicles. Scale bar: 100 μm.

[0040] Figure 4BThe levels of liver function-related parameters (serum ALT, total protein TP) and kidney function-related parameters (serum urea nitrogen BUN, serum creatinine Creatinine) in ovariectomized mice treated with Andrographis paniculata-derived nanovesicles were compared between the sham-operated group and the control group. Values ​​for each group are expressed as mean ± standard deviation. ns indicates no statistically significant difference compared to the sham-operated group. # P <0.05.

[0041] Figure 5 The content of andrographolide in nanovesicles derived from Andrographis paniculata was detected by HPLC-MS. The content of andrographolide in nanovesicles was only 19.91±4.12 ng / mL, which is far lower than the lowest effective dose reported in the literature (1 μM, about 350 ng / mL).

[0042] Figure 6 This section compares the osteogenic differentiation-promoting activities of andrographolide-derived nanovesicles and andrographolide. Part A compares the osteogenic differentiation-promoting activities of andrographolide-derived nanovesicles and andrographolide-promoting activities using ALP staining. Part B compares the bone marrow mesenchymal stem cell mineralization-promoting activities of andrographolide-derived nanovesicles and andrographolide-promoting activities using ARS staining. Part C compares the gene expression-promoting activities of andrographolide-derived nanovesicles and andrographolide-promoting activities using RT-qPCR. All values ​​are expressed as mean ± standard deviation. Compared with the model group, *** P <0.001.

[0043] Figure 7 This study compares the inhibitory activities of andrographolide on osteoclast differentiation using nanovesicles derived from Andrographis paniculata. Part A compares the inhibitory activities of the two nanovesicles on osteoclast differentiation using TRAP staining. Part B compares the inhibitory activities of the two nanovesicles on osteoclast marker genes Nfatc1, Trap, and C-fos using RT-qPCR. All values ​​are expressed as mean ± standard deviation. *** P <0.001. Detailed Implementation

[0044] Example 1: Preparation and characterization of Andrographis paniculata-derived nanovesicles (hereinafter referred to as "nanovesicles") Main materials used in preparation: Andrographis paniculata medicinal material, ddH2O, PEG8000, PBS buffer.

[0045] Preparation method: Weigh 80 g of dried Andrographis paniculata, wash once with ddH2O, soak in 500 mL of ddH2O for 2 h, homogenize mechanically for 1 min, and then filter to collect the filtrate. Centrifuge the collected Andrographis paniculata filtrate at 1000×g, 4℃ for 10 min; collect the supernatant and centrifuge at 3000×g, 4℃ for 20 min; collect the supernatant and centrifuge at 10000×g, 4℃ for 30 min; collect the supernatant and centrifuge at 16000×g, 4℃ for 60 min; collect the supernatant, filter through a 1 μm filter, add PEG8000 to a final concentration of 8%, and incubate overnight at 40 rpm, 4℃; collect the supernatant and centrifuge at 16000×g, 4℃ for 30 min; discard the supernatant, collect the precipitate, add an appropriate amount of PBS buffer, and evenly disperse the precipitate to form a mixture; filter through a 0.20 μm filter for sterilization to obtain Andrographis paniculata-derived nanovesicles.

[0046] Characterization of nanovesicles derived from Andrographis paniculata: Morphology of Andrographis paniculata-derived nanovesicles was examined using transmission electron microscopy: The sample was applied to a luminescent copper mesh coated with a continuous carbon film and stained with 0.75% uranyl formate. The mesh was observed and analyzed using a Krios G4 cryo-transmission electron microscope, and images were recorded at 22,000x magnification.

[0047] Flow cytometry determination of the size and purity of nanovesicles derived from Andrographis paniculata: A nanoflow cytometer was used, with a 250 nm quality control particle calibration laser as the particle concentration reference. A particle size distribution reference curve was generated using a mixture of particles with different sizes (68–155 nm). The particle concentration and particle size distribution of the samples were calculated using NF Profession 1.0 software. The samples were then mixed with 0.1% Triton and incubated at room temperature for 0.5 h. The particle concentration and particle size were measured again to calculate the purity.

[0048] SDS-PAGE gel electrophoresis was used to detect the protein profile of Andrographis paniculata-derived nanovesicles: Andrographis paniculata-derived nanovesicles were lysed using exosome-specific lysis buffer. A 10% SDS-PAGE gel electrophoresis plate was prepared. 20 μg of Andrographis paniculata-derived nanovesicle protein was added to each gel well. The gel was stained with Coomassie Brilliant Blue after electrophoresis, and finally imaged using a chemiluminescence imaging system.

[0049] RNA profiling of Andrographis paniculata nanovesicles by agarose gel electrophoresis: RNA was extracted from Andrographis paniculata nanovesicles using an RNA extraction kit and incubated with RNase at 37°C for 30 min. A 1.5% agarose gel was prepared, and the distribution and intensity of RNA bands before and after RNase treatment were compared.

[0050] Lipid profile analysis of *Andrographis paniculata*-derived nanovesicles by thin-layer chromatography: Lipids were extracted from *Andrographis paniculata*-derived nanovesicles using the Blight-Dyer method, and lipid profile analysis was performed using thin-layer chromatography. Preparation of thin-layer plates: 3–4 g of 200-mesh silica gel G was weighed, 5–6 mL of water was added, and the mixture was spread onto a plate. After natural drying, the plate was placed in an oven and activated at 110 °C for 30 min. Spotting: Lipid extract was spotted onto the activated silica gel plate 2 cm from the bottom using a capillary tube, ensuring the spot diameter was no greater than 3 cm. The plate was then dried with cold air. Development: Developing solvent (ethyl acetate:petroleum ether = 1:20, volume ratio) was added to the chromatography tank to a depth of approximately 1 cm. The spotted silica gel plate was placed in the tank for development. When the developing solvent front moved to approximately 2 cm from the top of the thin layer, the silica gel plate was removed, the developing solvent front position was marked, and the plate was dried with hot air. Color development: A 10% sulfuric acid ethanol solution was sprayed evenly for color development. The lipid spot patterns were observed and recorded.

[0051] Experimental results: Transmission electron microscopy revealed that the nanovesicles derived from Andrographis paniculata exhibited a typical circular vesicle shape formed by a lipid bilayer, consistent with the shape characteristics of exosomes. Figure 1A The particle size of nanovesicles derived from Andrographis paniculata was determined by nanoflow cytometry. The particles were found to be mainly concentrated between 50 and 200 nm, which meets the particle size requirements for exosomes, and the concentration reached 1.12 × 10¹¹ particles / mL. Figure 1B Andrographis paniculata-derived nanovesicles have a membrane-like structure. In this study, the purity of Andrographis paniculata-derived nanovesicles was determined using the Triton membrane disruption method. The results showed that the purity of Andrographis paniculata-derived nanovesicles could reach approximately 80%. Figure 1C After SDS-PAGE electrophoresis and Coomassie brilliant blue staining, it was observed that the nanovesicles derived from Andrographis paniculata contained proteins, with molecular weights mainly between 25 and 40 kDa. Figure 1D Agarose gel electrophoresis revealed RNA bands in the gel image, which disappeared after incubation with RNase. Therefore, RNA is present in nanovesicles derived from *Andrographis paniculata*, primarily small RNAs. Figure 1E Thin-layer chromatography was used to detect lipids in andrographis paniculata-derived nanovesicles, revealing that they contained a variety of lipid components. Figure 1F The above results indicate that nanovesicles containing proteins, RNA, and lipids derived from Andrographis paniculata have been successfully extracted.

[0052] Example 2: Therapeutic effect of Andrographis paniculata-derived nanovesicles on osteoporosis in ovariectomized mouse model. Laboratory animals and model establishment: Female C57BL / 6 mice (weighing 17-20 g) aged 6-8 weeks were used and acclimatized for 1 week. The mice were randomly divided into 5 groups (n=10): sham-operated group (Control), model group (OVX), positive control group (OVX+ALN), low-dose group of Andrographis paniculata-derived nanovesicles (OVX+Low APNVs), and high-dose group of Andrographis paniculata-derived nanovesicles (OVX+High APNVs).

[0053] After intraperitoneal anesthesia with 2% sodium pentobarbital, the OVX group underwent bilateral oophorectomy, while the sham surgery group only had a small amount of surrounding adipose tissue removed. Postoperatively, penicillin was administered intramuscularly for 3 consecutive days to prevent infection.

[0054] Dosage regimen: Gavage administration began 5 days post-surgery. The sham-operated group and model group received normal saline via gavage, the positive control group received alendronate sodium 1 mg / mL via gavage, and the experimental group received andrographis paniculata-derived nanovesicles at a low concentration (10 mg / mL). 10 particles / mL), high dose (10 11 Animals were administered 0.2 mL of particles / mL via gavage for 8 consecutive weeks, after which samples were collected.

[0055] Evaluation indicators: Micro-CT examination of the fine structure of mouse bone tissue: The L2 lumbar vertebra and proximal tibia of mice were scanned and reconstructed using Micro-CT. Each slice was 6 μm thick, and the voltage and current were set to 100 kV and 800 μA, respectively. After scanning, the built-in analysis software of Micro-CT was used to obtain the bone volume fraction, trabecular bone number, trabecular bone thickness, and trabecular bone separation in the cancellous bone region.

[0056] HE staining for mouse bone tissue morphology: Mouse tibias were decalcified with EDTA and then embedded in paraffin (4 μm thick). After dewaxing and rehydration, HE staining was performed. After dehydration with graded ethanol and clearing with xylene, the sections were mounted with neutral resin, and images were acquired using a digital pathological section scanning system.

[0057] TRAP staining was used to detect the number and distribution of osteoclasts in mouse bone tissue: Mouse tibias were decalcified with EDTA and then embedded in paraffin (4 μm thick). After dewaxing and rehydration, the sections were stained with TRAP. After dehydration with graded ethanol and clearing with xylene, the sections were mounted with neutral resin, observed under a microscope, and images were collected by scanning.

[0058] Statistical methods: The Shapiro-Wilk method was used to test the normality of each group of data. Data conforming to a normal distribution were represented by (…). ±s) represents the mean, and M(P25~P75) is used when the data does not conform to a normal distribution. When comparing two groups of data, if the data simultaneously meet the tests for normality and homogeneity of variance, an independent samples t-test is used; if the data meet the tests for normality but not homogeneity of variance, Welch's t-test is used. When comparing three groups of data, if the data simultaneously meet the tests for normality and homogeneity of variance, one-way ANOVA is used; if the data meet the tests for normality but not homogeneity of variance, Dunnett's T3 method is used for post-hoc pairwise comparisons. A p-value < 0.05 is considered statistically significant.

[0059] Experimental results: MicroCT was used to perform three-dimensional reconstruction of bone tissue. The results showed that compared to the sham surgery group, the model group experienced significant bone loss, while the positive control group and the nanovesicle intervention group showed significant increases in bone mass. Figure 2A Further analysis of bone microstructure revealed that, compared to the sham-operated group, the model group showed a significant decrease in bone volume fraction, trabecular thickness, and trabecular number, while trabecular separation and structural model index were significantly increased. However, in ovariectomized mice, after administration of alendronate sodium and nanovesicles, bone volume fraction, trabecular thickness, and trabecular number significantly increased, while trabecular separation and structural model index significantly decreased. Figure 2B HE staining revealed morphological changes in bone trabeculae in each group of mice. Compared with the sham-operated group, the model group showed increased intertrabecular spacing, thinner trabeculae, and increased trabecular fracture and continuity. In contrast, ovariectomized mice administered alendronate sodium and nanovesicles showed increased and thickened trabeculae with reduced fracture. Figure 2C , Figure 2D TRAP staining was performed on mouse bone tissue to observe osteoclast formation. Compared with the sham-operated group, the model group showed a significant increase in the wine-red area in bone tissue sections, indicating a greater number of osteoclasts. The relative number and surface area of ​​osteoclasts in the nanovesicle group were significantly decreased. Figures 2E to 2G These results indicate that gavage administration of Andrographis paniculata-derived nanovesicles can increase bone formation, inhibit osteoclast differentiation and resorption, and alleviate bone loss in ovariectomized mice.

[0060] Example 3: Therapeutic effect of Andrographis paniculata-derived nanovesicles on ovariectomized mouse model of bone defects Laboratory animals and model establishment: Female C57BL / 6 mice (weighing 17-20 g) aged 6-8 weeks were used and acclimatized for 1 week. The mice were randomly divided into 5 groups (n=10): sham-operated group (Control), model group (OVX), positive control group (OVX+ALN), low-dose group of Andrographis paniculata-derived nanovesicles (OVX+Low APNVs), and high-dose group of Andrographis paniculata-derived nanovesicles (OVX+High APNVs).

[0061] After intraperitoneal anesthesia with 2% sodium pentobarbital, the OVX group underwent bilateral ovariectomy, while the sham surgery group only had a small amount of surrounding adipose tissue removed. Postoperatively, penicillin was administered intramuscularly for 3 consecutive days to prevent infection. Six weeks postoperatively, a bone defect model was established by drilling a hole in the proximal tibia of the mice using a 1 mm diameter electric drill.

[0062] Dosage regimen: The administration method was the same as in Example 2. Administration began by gavage 5 days after oophorectomy. The sham-operated group and the model group received normal saline via gavage, the positive control group received alendronate sodium 1 mg / mL via gavage, and the experimental group received Andrographis paniculata-derived nanovesicles at a low concentration (10 mg / mL). 10 particles / mL), high dose (10 11 The samples were administered via gavage at a dose of 0.2 mL / animal / day, and samples were collected at 1 week and 2 weeks after the bone defect model was established.

[0063] Evaluation indicators: Micro-CT scanning of bone defect sites: Similar to Example 2, Micro-CT scanning and reconstruction were performed on the perforated surface of the proximal tibia of mice. Each slice was 6 μm thick, and the voltage and current were set to 100 kV and 800 μA, respectively, during scanning. After scanning, the built-in analysis software of Micro-CT was used to analyze the bone volume fraction, trabecular bone number, trabecular bone thickness, and trabecular bone separation in the cortical and cancellous bone regions.

[0064] Statistical methods: Same as in Example 2.

[0065] Experimental results: The healing status of bone defects in each group of mice was observed using Micro-CT three-dimensional reconstruction images. Figure 3A The results showed that the bone defect in the model group did not show significant improvement in the second week after modeling compared to the first week, while the other groups showed varying degrees of bone tissue repair. In the second week after modeling, compared with the model group, the high-dose nanovesicle group had significantly increased bone volume fraction and trabecular bone number (P<0.001), and significantly decreased trabecular bone separation (P<0.05).

[0066] Further quantitative analysis of bone microstructural parameters ( Figure 3BIn cortical bone, during the first week after modeling, the bone volume fraction and trabecular bone number in the model group mice were significantly lower than those in the sham-operated group. Compared with the model group, the bone volume fraction and trabecular bone number were significantly higher in the positive control group and the low-dose nanovesicle group, while the trabecular bone number was significantly higher in the high-dose nanovesicle group. During the second week after modeling, the bone volume fraction in the model group mice was significantly lower than that in the sham-operated group, while the trabecular bone separation was significantly higher. Compared with the model group, the bone volume fraction and trabecular bone number were significantly higher in the low-dose nanovesicle group, while the bone volume fraction and trabecular bone number were significantly higher in the high-dose nanovesicle group, and the trabecular bone separation was significantly lower.

[0067] In cancellous bone, during the first week after modeling, the bone volume fraction and trabecular bone number in the model group mice were significantly lower than those in the sham-operated group; compared with the model group, the bone volume fraction and trabecular bone number in the low-dose nanovesicle group were significantly higher. During the second week after modeling, the bone volume fraction and trabecular bone number in the model group mice were significantly lower than those in the sham-operated group, while trabecular bone separation was significantly higher; compared with the model group, the bone volume fraction, trabecular bone thickness, and trabecular bone number in the positive control group were significantly higher, while trabecular bone separation was significantly lower; the bone volume fraction and trabecular bone number in the low-dose nanovesicle group were significantly higher, while trabecular bone separation was significantly lower; and the bone volume fraction and trabecular bone number in the high-dose nanovesicle group were significantly higher, while trabecular bone separation was significantly lower.

[0068] The above results indicate that andrographis paniculata-derived nanovesicles can promote bone tissue repair in ovariectomized mice with bone defects and improve bone microstructure. Overall, the effect of andrographis paniculata-derived nanovesicles is better than that of alendronate sodium, showing a concentration gradient dependence.

[0069] Example 4: In vivo safety evaluation of Andrographis paniculata-derived nanovesicles Testing indicators: HE staining of major organs: Liver, kidney, brain, heart, and lung tissues from mice that had been continuously administered via gavage for 8 weeks as in Example 2 were fixed, embedded, sectioned, and then stained with HE. Except for the absence of decalcification, the other steps were the same as in Example 2.

[0070] Serum liver and kidney function indicators were detected: Blood was collected from the orbital sinus of mice that had been continuously administered the medication by gavage for 8 weeks as in Example 3, and serum was separated. The levels of alanine aminotransferase (ALT), total protein (TP), blood urea nitrogen (BUN), and creatinine (Crea) were detected using the corresponding kits (Nanjing Jiancheng). All operations were performed according to the kit instructions.

[0071] Statistical methods: Same as in Example 2.

[0072] Experimental results: Use concentration of 10 11After continuous gavage administration of Andrographis paniculata-derived nanovesicles at particle / mL for 8 weeks to mice, no damage was caused to the liver, kidneys, brain, heart, or lungs. Figure 4A Further testing of serum hepatotoxicity markers (ALT, TP) and nephrotoxicity markers (BUN, Crea) revealed no significant differences in TP, BUN, and Crea compared to mice not treated with nanovesicles via gavage. ALT showed a decreasing trend within the normal range, but this was not clinically significant. Figure 4B The above results indicate that Andrographis paniculata-derived nanovesicles administered via gavage are safe and non-toxic to mice.

[0073] Example 5: Determination of Andrographolide Content in Nanovesicles Derived from Andrographis paniculata Extraction of andrographolide from nanovesicles: Take the Andrographis paniculata-derived nanovesicle solution prepared in Example 1 (concentration 1.12 × 10⁻⁶). 11 Add 1 mL of ethyl acetate to the residue, vortex for 1 min, sonicate for 5 min, centrifuge at 4000 rpm for 5 min, and collect the supernatant. Add 1 mL of ethyl acetate to the residue, vortex for 1 min, and centrifuge at 4000 rpm for 5 min. Combine the supernatants. Dry the combined supernatant under a nitrogen stream in a 50℃ water bath. Dissolve the residue in 0.3 mL of acetonitrile, vortex for 3 min, sonicate for 10 min, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0074] Detection using high performance liquid chromatography-mass spectrometry (HPLC-MS): Liquid chromatography conditions: Waters HSS T3 column (1.8 μm, 2.1 mm × 100.0 mm); column temperature: room temperature; sample pan temperature: 4℃; flow rate: 0.40 mL / min; injection volume: 1.0 μL. The mobile phase consisted of water (A) and acetonitrile (B), with the following gradient elution program: 0–1 min, 35%–40% B; 1–10 min, 40%–40.5% B; 10–12 min, 40.5%–90% B.

[0075] Mass spectrometry conditions: Electrospray ionization (ESI) source, multiple reaction monitoring (MRM) mode; ion pair of andrographolide: m / z 349.2021→287.2014; collision energy: 15 V; cone voltage: 50 V; mass spectrometry acquisition time: 12 min.

[0076] Experimental results: See Figure 5HPLC-MS detected that the andrographolide content in the andrographolide-derived nanovesicles was only 19.91±4.12 ng / mL. According to literature reports (Wang Tao. Study on the effects and mechanisms of andrographolide on bone loss and osteoclasts in ovariectomized mice [D]. Guangxi Medical University, 2016), the minimum effective dose of andrographolide to exert its anti-osteoporosis effect is 1 μM (approximately 350 ng / mL). The andrographolide content (19.91±4.12 ng / mL) in the nanovesicles measured in this example is far lower than this minimum effective dose, and theoretically insufficient to exert a pharmacological effect on osteoporosis on its own. This indicates that the efficacy of the andrographolide-derived nanovesicles does not originate from the trace amount of andrographolide they contain.

[0077] Example 6: Comparison of the osteogenic differentiation and mineralization promotion activities of andrographolide-derived nanovesicles and andrographolide monomers. Experimental materials and grouping: Extraction of bone marrow mesenchymal stem cells (BMSCs): Six- to eight-week-old female C57BL / 6 mice (weighing 17-20g) were anesthetized and euthanized by cervical dislocation. Under aseptic conditions, the femur and tibia were separated, and the ends of the bone shafts were cut to expose the medullary cavity. The medullary cavity was repeatedly flushed with α-MEM medium containing 10% fetal bovine serum using a syringe. The flushing fluid was collected and pipetted to prepare a single-cell suspension. The cell suspension was transferred to a 25 cm² culture dish and placed in a 37°C, 5% CO2 incubator. Cells were passaged when the confluence reached 80%-90%. P4-P5 generation cells were used for subsequent experiments.

[0078] Preparation of andrographolide monomers: To fully demonstrate the pharmacological activity of andrographolide monomer, a comparative experiment was conducted using the higher effective dose (10 μM) reported in the literature. 0.35 mg of andrographolide was accurately weighed, dissolved in 150 μL of DMSO, and brought to a final volume of 10 mL with PBS to obtain a stock solution with a concentration of 35 μg / mL. This stock solution was then diluted 10-fold before use, resulting in a final concentration of 3.5 μg / mL (approximately 10 μM).

[0079] Preparation of osteogenic induction solution: Weigh 88.6 mg of vitamin C and dissolve it in 10 mL of PBS buffer to obtain a vitamin C solution; weigh 4.32 g of sodium β-glycerophosphate and dissolve it in 10 mL of PBS buffer to obtain a sodium β-glycerophosphate solution; weigh 19.623 mg of dexamethasone and dissolve it in 10 mL of anhydrous ethanol to obtain a dexamethasone solution. Take 0.05 mL of the above vitamin C solution, 0.5 mL of sodium β-glycerophosphate solution, and 1 μL of dexamethasone solution, add 5 mL of fetal bovine serum and 0.5 mL of antibiotic solution, and make up to 50 mL with α-MEM complete culture medium. Mix well to obtain the final solution.

[0080] Grouping and Intervention: Bone marrow mesenchymal stem cells were collected and divided into the following three groups: Osteogenic Induction Solution Group (OI): only osteogenic induction solution was added; OI+APNVs Group: osteogenic induction solution and nanovesicles (10 10 particles / mL); OI+andrographolide group: osteogenic induction solution and andrographolide (10 μM) were added.

[0081] Testing indicators: Alkaline phosphatase (ALP) staining: Bone marrow mesenchymal stem cells were stained with alkaline phosphatase (ALP) at a concentration of 2 × 10⁻⁶. 4 Seeds were planted at a density of 100 cells / well in 48-well plates, with 200 μL of culture medium added to each well. After overnight adhesion, the original culture medium was discarded. 200 μL of osteogenic induction solution or osteogenic induction solution containing the corresponding drug was added to each well according to the grouping. The medium was changed every 3 days. After 7 days of culture, the culture medium was discarded, and the plates were washed 3 times with PBS. The plates were then fixed with 4% paraformaldehyde for 30 minutes, followed by 3 washes with PBS to remove the fixative. ALP staining solution was added, and the plates were incubated in the dark for 1 hour. The staining solution was discarded, and the plates were washed 3 times with PBS. Images were then acquired using a scanner.

[0082] Alizarin Red S (ARS) staining: bone marrow mesenchymal stem cells were stained at 4 × 10⁻⁶. 3 Seeds were planted at a density of 100 cells / well in 48-well plates, with 200 μL of culture medium added to each well. After overnight adhesion, the original culture medium was discarded. Then, according to the groups, 200 μL of osteogenic induction solution or osteogenic induction solution containing the corresponding drug was added to each well. The medium was changed every 3 days. After 14 days of culture, the culture medium was discarded, and the plates were washed three times with PBS. The plates were then fixed with 4% paraformaldehyde for 30 minutes, followed by three washes with PBS to remove the fixative. ARS staining solution was added, and the plates were incubated in the dark for 30 minutes. The staining solution was discarded, and the plates were washed three times with PBS. Images were then acquired using a scanner.

[0083] RT-qPCR detection of osteogenic marker gene expression: Bone marrow mesenchymal stem cells were used in a assay of 2×10⁻⁶ m³ / h. 4 Cells were seeded at a density of 1 cell / well in 6-well plates. After overnight adhesion, the medium was changed according to the groups. Eight hours after drug intervention, the culture medium was discarded, and the cells were washed three times with PBS. Trizol reagent was added to lyse the cells, and total RNA was extracted. cDNA was synthesized according to the reverse transcription kit (TaKaRa, 2610A) instructions and amplified using a qPCR kit (TaKaRa, RR820A). β-actin was used as an internal control, and the relative expression level of the Alp gene was calculated using the 2-ΔΔCt method.

[0084] Statistical methods: Same as in Example 2.

[0085] Experimental results: See Figure 6 ALP staining results showed ( Figure 6(Part A) The number of ALP-positive cells and the staining depth in the OI+APNVs group were significantly higher than those in the OI+andrographolide group, indicating that the nanovesicles have a stronger effect on promoting early osteogenic differentiation. ARS staining results showed ( Figure 6 In Part B of the study, the number and area of ​​mineralized nodules in the OI+APNVs group were significantly greater than those in the OI+andrographolide group, indicating that the nanovesicles had a stronger effect on promoting mineralization. Alp is a marker of osteogenic development. RT-qPCR was used to detect the expression levels of the Alp gene after intervention with bone marrow mesenchymal stem cells by OI, OI+APNVs, and OI+andrographolide, respectively. The results showed that the expression level of Alp in the OI+APNVs group was higher than that in the OI+andrographolide group. Figure 6 (Part C of the text). It can be seen that the osteogenic differentiation-promoting activity of andrographolide-derived nanovesicles is stronger than that of andrographolide.

[0086] Example 7: Comparison of the inhibitory activity of andrographolide-derived nanovesicles and andrographolide monomers on osteoclast differentiation. Experimental materials and grouping: Raw264.7 cells were divided into the following four groups: control group: cultured normally without any inducing agent; RANKL group: osteoclast differentiation induced by adding 50 ng / mL RANKL and 25 ng / mL M-CSF; RANKL+APNVs group: RANKL, M-CSF and nanovesicles (10¹⁰ particles / mL) were added; RANKL+andrographolide group: RANKL, M-CSF and andrographolide (10 μM) were added.

[0087] Testing indicators: TRAP staining was used to detect osteoclastogenesis: Raw264.7 cells were stained at a concentration of 4 × 10⁻⁶ cells / mL. 3 Seeds were planted at a density of cells / well in 96-well plates and cultured overnight. After incubation, the appropriate drugs were added according to the groupings described above, and the medium was changed every 3 days. After 5 days of intervention, the culture medium was discarded, and the cells were washed three times with PBS. The cells were then fixed with 4% paraformaldehyde for 30 minutes, washed with PBS, and then incubated with TRAP staining solution at 37°C in the dark for 3 hours. After washing three times with PBS, the cells were observed and photographed under a microscope. TRAP+ cells with three or more nuclei were considered mature osteoclasts.

[0088] RT-qPCR detection of osteoclast marker gene expression: Raw264.7 cells were cultured at 2×10⁻⁶ cells / year. 4 Seeds were planted at a density of 100 cells / well in 6-well plates, and the mRNA expression levels of osteoclast marker genes Nfatc1, Trap, and C-fos were detected. The remaining procedures were the same as in Example 6.

[0089] Statistical methods: Same as in Example 2.

[0090] Experimental results: See Figure 7 TRAP staining was used to observe the number and area of ​​osteoclasts in Raw264.7 cells after intervention with nanovesicles and andrographolide. A large number of TRAP-positive osteoclasts were observed in the Rank1 group. The number of osteoclasts in the Rank1+APNVs and Rank1+andrographolide groups was significantly reduced compared with the Rank1 group, and the number of osteoclasts in the Rank1+APNVs group was even less than that in the Rank1+andrographolide group. Figure 7 Part A). The expression of osteoclast-related genes was detected by RT-qPCR. Compared with the Rank1 group, the expression levels of osteoclast-related genes Nfatc1, Trap, and C-fos were significantly reduced in the Rank1 + APNVs and Rank1 + andrographolide groups. Furthermore, the expression of osteoclast-related genes was even lower in the Rank1 + APNVs group than in the Rank1 + andrographolide group. Figure 7 (Part B of the study). The above results indicate that the activity of andrographis paniculata-derived nanovesicles in inhibiting osteoclast differentiation is stronger than that of andrographolide.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of Andrographis paniculata-derived nanovesicles in the preparation of drugs for treating postmenopausal osteoporosis.

2. A drug for treating postmenopausal osteoporosis, characterized in that, The drug contains nanovesicles derived from Andrographis paniculata.

3. The application according to claim 1 or the drug according to claim 2, wherein the average particle size of the Andrographis paniculata-derived nanovesicles is 50~200 nm.

4. The application according to claim 1 or the drug according to claim 2, wherein the andrographis paniculata-derived nanovesicles are prepared by a method including differential centrifugation and polyethylene glycol precipitation.

5. The application or drug according to claim 4, wherein the preparation method of the andrographis paniculata-derived nanovesicles comprises: The Andrographis paniculata herb is cleaned, soaked, homogenized, and filtered; impurities are removed by graded centrifugation. Polyethylene glycol precipitation; Resuspension and filtration for sterilization.

6. The application or drug according to claim 4, wherein the preparation method of the andrographis paniculata-derived nanovesicles comprises: (1) Take dried Andrographis paniculata, wash it with water and soak it in water for 1-4 hours, then homogenize it mechanically and filter it to obtain the filtrate. (2) Centrifuge the filtrate obtained in step (1) at 800-1500×g for 5-15 min; take the supernatant and centrifuge at 2000-4000×g for 10-30 min; take the supernatant and centrifuge at 8000-12000×g for 20-40 min; take the supernatant and centrifuge at 14000-18000×g for 40-80 min; take the supernatant; filter and add PEG to a final concentration of 6-10%, incubate overnight; take the supernatant and centrifuge at 14000-18000×g for 30-40 min; discard the supernatant and take the precipitate; (3) Add PBS buffer to the precipitate obtained in step (2), blow the precipitate evenly to form a mixture, and filter to remove bacteria.

7. The application or drug according to claim 4, wherein the preparation method of the andrographis paniculata-derived nanovesicles comprises: (1) Take dried Andrographis paniculata, wash it with water and soak it in water for 2 hours, then homogenize it mechanically for 1 minute and filter to obtain the filtrate. (2) Centrifuge the filtrate obtained in step (1) at 1000×g and 4℃ for 10 min; take the supernatant and centrifuge at 3000×g and 4℃ for 20 min; take the supernatant and centrifuge at 10000×g and 4℃ for 30 min; take the supernatant and centrifuge at 16000×g and 4℃ for 60 min; take the supernatant; filter with a 1 μm filter and add PEG8000 to a final concentration of 8%, incubate overnight at 40 rpm and 4℃; take the supernatant and centrifuge at 16000×g and 4℃ for 30 min; discard the supernatant and take the precipitate; (3) Add PBS buffer to the precipitate obtained in step (2), blow the precipitate evenly to form a mixture, and filter to remove bacteria.

8. The application according to claim 1 or the drug according to claim 2, wherein the content of andrographolide in the andrographis paniculata-derived nanovesicles is less than 1 μM.

9. The application according to claim 1 or the medicament according to claim 2, wherein the medicament is an oral preparation.

10. The application according to claim 1 or the medicament according to claim 2, wherein the medicament has one or more of the following effects: (1) Reduce bone loss and bone defects; (2) Assists in bone tissue repair; (3) Promotes osteoblast differentiation and mineralization, as well as the expression of osteoblast-related genes; (4) Inhibit osteoclast differentiation and osteoclast-related gene expression.

Images