Preparation method and application of plant nanovesicles from pepper source

Abstract

This invention provides a method for preparing plant nanovesicles derived from pepper. The method involves: crushing washed pepper fruits, enzymatically hydrolyzing them, and then sequentially filtering the crude extract of pepper nanovesicles obtained from the enzymatic hydrolysis through a coarse filter, a microfiltration membrane, and a microporous membrane to obtain a filtrate. The filtrate is then centrifuged to obtain the plant nanovesicles derived from pepper. This invention also provides an application of the prepared plant nanovesicles derived from pepper for the preparation of drugs to improve peripheral nerve injury. The plant nanovesicles derived from pepper of this invention possess anti-inflammatory, analgesic, and nerve-repairing activities, easily penetrate tissue barriers, and promote the absorption and targeted delivery of active ingredients.

Description

Technical Field

[0001] This invention belongs to the field of plant nanovesicle technology, specifically relating to a method for preparing and applying plant nanovesicles derived from pepper. Background Technology

[0002] Peripheral nerve injury is a common peripheral neuropathy, often leading to sensory disturbances, loss of motor function, and chronic neuropathic pain, severely impacting patients' quality of life. Current clinical treatments mainly include surgical anastomosis, neurotrophic factor supplementation, and physical rehabilitation, but the overall treatment effect remains unsatisfactory. While surgical anastomosis can restore nerve continuity, nerve regeneration is slow, and functional recovery is often incomplete. Neurotrophic factors, although promoting nerve cell survival and axonal growth, suffer from short half-lives, require repeated administration, and are difficult to maintain effective concentrations. Furthermore, long-term use of neurotrophic factors may cause adverse reactions such as local tissue hyperplasia and allergies. Physical rehabilitation can improve some function, but the treatment course is long, the onset of action is slow, and the effect is limited for patients with severe injuries. Therefore, developing novel treatment strategies that can effectively promote nerve regeneration, repair myelin sheath structure, alleviate neuropathic pain, and have good safety profiles is of great significance for improving the prognosis of patients with peripheral nerve injury.

[0003] Plant-derived nanovesicles (PDNVs) have attracted widespread attention due to their natural origin, low immunogenicity, and good biocompatibility. Studies have shown that PDNVs can carry bioactive components such as proteins, nucleic acids, and lipids, penetrate tissue barriers, and regulate cell proliferation, differentiation, and inflammatory responses. In recent years, various plant PDNVs have been demonstrated to possess anti-inflammatory, antioxidant, and tissue repair potential, showing particular promise in the field of nerve regeneration.

[0004] Pepper (Piper nigrum L.) is a traditional plant used in both medicine and food, and its extracts are known to possess anti-inflammatory, analgesic, and neuroprotective activities. Pharmacological studies have shown that alkaloids (such as piperine) and terpenoids in pepper can inhibit the release of inflammatory factors, reduce oxidative stress, and participate in promoting axonal regeneration and myelin repair. However, its crude extract suffers from low bioavailability and poor stability, which to some extent limits its clinical application. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing and applying plant nanovesicles derived from pepper, which have anti-inflammatory, analgesic and nerve repair activities, and can be used to prepare drugs to improve peripheral nerve damage.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing plant nanovesicles derived from pepper, the method being as follows: S1. Raw material preparation and processing: After washing, the pepper fruits are crushed and soaked in a 70% ethanol aqueous solution. After rinsing, the processed pepper fruits are obtained. The processed pepper fruits are mixed with an enzymatic hydrolysate, enzymatically hydrolyzed, centrifuged, and the supernatant is collected to obtain a crude extract of pepper nanovesicles. S2. Multi-stage filtration purification: The crude extract of pepper nanovesicles obtained in S1 is sequentially filtered through a filter screen, a microfiltration membrane, and a microporous membrane to obtain the filtrate. S3. Ultracentrifugation extraction: Centrifuge the filtrate obtained in S2, take the supernatant, ultracentrifuge, collect the precipitate, resuspend the precipitate in pre-cooled phosphate buffer, ultracentrifuge again, collect the precipitate, and resuspend it in phosphate buffer to obtain plant nanovesicles derived from pepper.

[0007] Preferably, the soaking time in S1 is 1 minute; the mass ratio of the treated pepper fruit to the enzymatic hydrolysate is 1:5.

[0008] Preferably, the enzymatic hydrolysis conditions in S1 are: enzymatic hydrolysis for 5 hours at a temperature of 45°C and a pH of 5.5 to 6.0; and centrifugation conditions are: centrifugation for 30 to 60 minutes at a speed of 10000×g.

[0009] Preferably, in S2, the mesh size of the coarse filter is 200 mesh; the pore size of the microfiltration membrane is 200 nm; and the pore size of the microporous filter membrane is 0.45 μm.

[0010] Preferably, the centrifugation conditions in S3 are: centrifugation for 30 min at a temperature of 4℃ and a rotation speed of 10000×g; the ultracentrifugation conditions are: ultracentrifugation for 60 min at a temperature of 4℃ and a rotation speed of 100000×g; and the pre-cooling temperature is 4℃.

[0011] The present invention also provides the application of the pepper-derived plant nanovesicles prepared by the above preparation method, wherein the pepper-derived plant nanovesicles are used to prepare drugs to improve peripheral nerve injury.

[0012] Preferably, when the pepper-derived plant nanovesicles are used to prepare a drug for improving peripheral nerve injury, the pepper-derived plant nanovesicles are added as a drug ingredient.

[0013] Preferably, the dosage form of the drug is one or more of the following: injection, capsule, tablet, oral liquid, and granule.

[0014] Compared with the prior art, the present invention has the following advantages: The plant nanovesicles derived from pepper of the present invention have anti-inflammatory, analgesic, and nerve-repairing activities; and based on their nanoscale size characteristics, they can easily penetrate tissue barriers, promote the absorption and targeted delivery of active ingredients. Experimental results show that plant nanovesicles derived from pepper have a significant repair effect on sciatic nerve injury, and can be further developed into natural drug products for the treatment of sciatic nerve injury, providing a new approach to solving the problems of limited efficacy and obvious side effects of existing treatment methods.

[0015] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 These are electron microscope images of each group in Embodiment 2 of the present invention; wherein Figure A is PMPs; Figure B is PNPs; and Figure C is Lytic PNPs.

[0017] Figure 2 This is a particle size distribution diagram of plant nanovesicles derived from pepper in Example 2 of the present invention.

[0018] Figure 3 This is a Coomassie brilliant blue staining result of plant nanovesicles derived from pepper in Example 2 of the present invention.

[0019] Figure 4 This is a metabolomics analysis diagram of plant nanovesicles derived from pepper in Example 2 of the present invention.

[0020] Figure 5 This is a lipidomics analysis diagram of plant nanovesicles derived from pepper in Example 2 of the present invention.

[0021] Figure 6 This is a graph showing the changes in body weight of mice in each group in Example 2 of the present invention.

[0022] Figure 7 These are HE staining images of the major organs of mice in each group in Example 2 of the present invention.

[0023] Figure 8 This is an immunofluorescence and statistical graph of neurons from the dorsal root ganglia of L4-L6 on the injured side, taken from mice with sciatic nerve injury modeling in each group after three days of gavage.

[0024] Figure 9 This is an example of the SCG-10 immunofluorescence and statistical graph of the sciatic nerve on the injured side of mice after three days of gavage following the establishment of the sciatic nerve injury model in each group of mice in Example 2 of the present invention.

[0025] Figure 10This is a fluorescent gold retrograde tracing image of the dorsal root ganglia of L4-L6 on the injured side of mice three days after gavage in each group of mice with sciatic nerve injury modeling in Example 2 of the present invention.

[0026] Figure 11 This is a transcriptome of the intervention of sciatic nerve injury by plant nanovesicles derived from pepper, analyzed by RNA-seq three days after gavage in Example 2 of the present invention.

[0027] Figure 12 This is a graph showing the expression of pro-inflammatory and anti-inflammatory cytokines in serum and the dorsal root ganglia of L4-L6 on the injured side, measured by ELISA three days after gavage in Example 2 of the present invention.

[0028] Figure 13 Figure A shows the effect of sciatic nerve injury on the gastrocnemius muscle in each group of mice in Example 2 of the present invention; Figure B shows the relative wet weight of the gastrocnemius muscle in mice with the affected side (left) and the healthy side (right); Figure B shows the relative wet weight of the gastrocnemius muscle.

[0029] Figure 14 Figure A shows the effect of different treatments on the regenerated myelin sheath of the sciatic nerve in Embodiment 2 of the present invention; Figure B shows the transmission electron microscopy of the myelin sheath of the sciatic nerve and the scatter plot and linear regression fitting of the G-ratio as a function of axonal diameter.

[0030] Figure 15 Figure A shows the effect of different treatments on the recovery of sciatic nerve function in Example 2 of the present invention; Figure B shows the typical three-dimensional stress diagram and representative footprint diagram of the left hind limb at the time of modeling; Figure B shows the nerve function index of mice on day 28 under different treatments.

[0031] Figure 16 These are the electrophysiological and statistical graphs of each group in Embodiment 2 of the present invention. Detailed Implementation

[0032] Example 1

[0033] This embodiment describes a method for preparing plant nanovesicles derived from pepper. The method is as follows: S1. Raw material preparation and processing: Fresh peppercorns, after being washed with distilled water, are mechanically crushed to obtain pepper tissue blocks. The sample size after crushing is 2mm. 3To increase the contact area, the pepper fruits were soaked in a 70% ethanol aqueous solution for 1 minute. After rinsing with distilled water to remove ethanol residue and enhance cell wall permeability, the treated pepper fruits were obtained. The treated pepper fruits were then mixed with an enzymatic hydrolysate (composed of raw materials with the following final concentrations: 2 wt% pectinase, 3 wt% cellulase, and 0.6 mol / L mannitol) at a mass ratio of 1:5. The mixture was then enzymatically hydrolyzed for 5 hours at 45℃ and pH 5.7 to degrade the cell wall and promote the release of nanovesicles. After centrifugation at 10000×g for 45 minutes, the supernatant was collected to obtain a crude extract of pepper nanovesicles. S2. Multi-stage filtration purification: The crude extract of pepper nanovesicles obtained in S1 is first subjected to positive pressure coarse filtration through a 200-mesh filter to remove undigested tissue residues. Then, it is successively passed through a 200nm pore size microfiltration membrane for fine filtration to achieve preliminary purification and dialysis microfiltration. Finally, the dialysate is filtered through a 0.45μm microporous membrane to remove bacteria, subcellular debris and large-diameter vesicles, resulting in filtrate. S3. Ultracentrifugation extraction: The filtrate obtained in S2 was centrifuged at 4℃ and 10000×g for 30 min to remove residual large particles. The supernatant was collected and ultracentrifuged at 4℃ and 100000×g for 60 min. The supernatant was discarded and the precipitate was collected. The precipitate was then resuspended in phosphate-buffered saline (PBS) pre-cooled at 4℃ and ultracentrifuged again under the same conditions to remove soluble proteins and other impurities. Finally, the precipitate was resuspended in 200µL PBS to obtain plant nanovesicles derived from pepper.

[0034] Experimental parameters for this embodiment: The sample size after crushing in step S1 is 1 mm. 3 ~3mm 3 The pH value can be 5.5 to 6.0, and the centrifugation time can be 10000×g for 30 to 60 minutes.

[0035] In this embodiment, when pepper-derived plant nanovesicles are used to prepare a drug for improving peripheral nerve injury, pepper-derived plant nanovesicles are added as a drug raw material; the dosage form of the drug is one or more of the following: injection, capsule, tablet, oral liquid, and granules. Example 2

[0036] This embodiment demonstrates the application of plant nanovesicles derived from pepper in the preparation of drugs to improve peripheral nerve damage.

[0037] The preparation method of pepper pulp in this embodiment is as follows: fresh pepper fruits, after being washed with distilled water, are processed by a juicer to obtain pepper pulp (PMPs); the preparation method of hydrogen peroxide pyrolysis pepper-derived nanoparticles is as follows: after obtaining pepper-derived plant nanovesicles in Example 1, hydrogen peroxide is added and incubated for 48 hours to destroy their nanostructure. After treatment, the hydrogen peroxide is removed by evaporation to obtain the pyrolysis product, namely hydrogen peroxide pyrolysis pepper-derived nanoparticles (Lytic PNPs).

[0038] 1. Transmission electron microscopy observation: 10 μL of PMPs, Lytic PNPs, and pepper-derived plant nanovesicles (PNPs) suspensions prepared in Example 1 were respectively dropped onto a Formvar-carbon copper grid and allowed to stand at room temperature for 1 min. The liquid was carefully removed from the edge with filter paper. 10 μL of uranium acetate staining solution was dropped onto the copper grid and stained in the dark for 1 min. The staining solution was removed with filter paper and the grid was dried at room temperature for 5 min. The images were then observed and acquired using a Hitachi HT-7700 transmission electron microscope at an accelerating voltage of 100 kV.

[0039] Electron microscopy results as follows Figure 1 As shown in Figure A, with a scale bar of 1 μm, this proves that PMPs are micron-sized particles, such as... Figure 1 As shown in Figure B, with a scale bar of 100 nm, PNPs are nanoscale particles. The electron microscopy results of Lytic PNPs are as follows. Figure 1 As shown in Figure C, with a scale bar of 50 nm, the nanovesicle structure of Lytic PNPs is disrupted.

[0040] 2. Particle size analysis of nanovesicle samples: Take 10 μL of PNPs sample and dilute it to 30 μL with PBS. Before loading the sample, use standards to verify the performance of the nanoflow cytometer (NanoFCM N30E). To prevent needle clogging, the sample needs to be serially diluted with PBS to an appropriate concentration. After detection, the instrument automatically outputs the exosome particle size distribution and particle concentration results. Figure 2 As shown, the average particle size distribution of PNPs is 91.7 nm.

[0041] 3. Sample protein extraction and concentration determination: Protein extraction and concentration determination: After rapid thawing of the sample at 37℃, 5×RIPA lysis buffer was added at a ratio of 1:4, vortexed to mix, and lysed on ice for 30 min (vortexing once every 10 min). A standard solution of 0–0.5 mg / mL was prepared using BSA. 5 μL of sample or standard was taken, and 200 μL of BCA working solution was added, mixed, and incubated at 37℃ in the dark for 30 min, then cooled to room temperature. The absorbance was measured at 562 nm, and a standard curve was plotted (R0).2 (≥0.99), calculate the sample protein concentration based on the curve equation.

[0042] 4. Coomassie brilliant blue staining analysis: After PNP protein electrophoresis, the SDS-PAGE gel was placed in Coomassie Brilliant Blue staining solution and stained with shaking at room temperature for 15 min. The staining solution was discarded, and the gel was briefly and quickly rinsed with ultrapure water. Then, Coomassie Brilliant Blue rapid destaining solution was added, and the gel was destained with shaking at room temperature until the background was transparent and the protein bands were clearly visible (usually 30-60 min, during which the destaining solution can be changed 1-2 times to speed up the process). After destaining, the gel was stored in ultrapure water and scanned and analyzed using a gel imaging system. This staining method is used to verify the presence of proteins in the sample and their molecular weight distribution.

[0043] The results are as follows Figure 3 As shown, the PNPs sample mainly contains a protein component with a molecular weight of approximately 30 kDa.

[0044] 5. The pepper-derived plant nanovesicles (PNPs) prepared in Example 1 were subjected to metabolomics and lipidomics analysis at Lianchuan Biotechnology. The metabolomics results are as follows: Figure 4 As shown, the major chemical categories identified were lipids and lipid molecules (n=99), organic acids and derivatives (n=74), and a large number of undefined compounds (n=61); minor metabolites and other major categories of organic compounds were also well represented, including benzene compounds (n=48), organic heterocyclic compounds (n=46), organic oxygen compounds (n=59), phenylpropanoids and polyketides (n=28), and nucleosides, nucleotides and analogs (n=18); alkaloids and their derivatives (n=7) and lignans, neolignans and their related compounds (n=3) were present but present in smaller quantities; organohalogen compounds, metal / nonmetal mixed compounds, and organosulfur compounds had the fewest metabolites, with one metabolite of each, a distribution consistent with the expected chemical diversity in the metabolome of typical plant extracts. The results of lipidomics are as follows... Figure 5 As shown, PNPs mainly contain diacylglycerol (DAG, ~17.9%), N-acylethanolamines (NAE, ~12.8%), ceramide non-hydroxy fatty acid-sphingosine (~12.8%), and fatty acyls (~10.3%), demonstrating that PNPs are nanovesicles rich in active ingredients such as proteins, nucleic acids, and lipids.

[0045] 6. Weight monitoring of mice during drug administration: Eighty specific pathogen-free (SPF) 6-week-old male C57BL / 6 mice were randomly divided into three groups: PBS control group (PBS), pepper-derived microparticles (PMPs), pepper-derived nanoparticles (PNPs) prepared in Example 1, and lytic PNPs (hydrogen peroxide-cracked pepper-derived nanoparticles). Each group consisted of 20 mice. The mice were housed in an SPF-grade animal housing with a 12 / 12h light / dark cycle at 23±2℃ and had free access to water and food.

[0046] To assess the systemic safety of long-term administration in each group, this study employed random sampling for weight monitoring. Starting from the start of administration, six mice were randomly selected from each group at fixed time points every four days. Individual weights were measured and recorded using a calibrated electronic analytical balance (accurate to 0.01 g). This sampling procedure was repeated until the end of administration (day 28). By analyzing the inter-group differences in weight during each sampling and the weight growth trend of each group over time, the potential impact of the drug on the general growth and development of mice was assessed. This sampling method effectively obtains representative data reflecting the growth status of the population while reducing stress caused by frequent manipulation, serving as a key basis for systemic toxicity evaluation.

[0047] Results of the experiment over 28 consecutive days are as follows Figure 6 As shown, after oral administration of PBS, PMPs, PNPs and Lytic PNPs, the body weight of mice maintained a steady growth trend throughout the observation period, with no significant difference compared with the control group.

[0048] 7. HE staining to assess the effects of drugs on major organs in mice: Twenty-four hours after the last administration of the drug, six mice from each group were selected, anesthetized, and euthanized. The heart, liver, spleen, lung, and kidney were quickly dissected, washed with pre-cooled PBS, and blotted dry with filter paper. Each organ was fixed in 4% paraformaldehyde for 24–48 hours. After fixation, the tissues were dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and serially sectioned (4–5 μm thick) using a microtome. The sections were then mounted on glass slides. After dewaxing and rehydration, the sections were stained with hematoxylin and eosin (HE) as usual, and finally mounted with neutral resin. The organ sections from each group were observed blinded under an optical microscope. The focus was on whether the basic tissue structure and cell morphology of each organ were normal, and the presence of pathological changes such as congestion, edema, inflammatory cell infiltration, fatty degeneration, necrosis, and apoptosis was recorded in detail.

[0049] HE-stained pathological sections of major organs, as shown Figure 7 As shown in the figure, the scale bar is 250 μm. No obvious pathological changes such as inflammation, necrosis or structural abnormalities were observed in the liver, kidneys, intestines and other tissues of mice in each experimental group. These results comprehensively demonstrate that pepper-derived PMPs, PNPs and Lytic PNPs have good oral biocompatibility.

[0050] 8. Effects of PNP intervention on axonal regeneration of dorsal root ganglion neurons in mice with sciatic nerve injury: A standard squamous nerve compression injury model of the left hind limb in mice: 100 C57BL / 6J mice were used. The modeling process was performed under aseptic surgical conditions. After intraperitoneal anesthesia with 1% sodium pentobarbital, the mice were fixed in a prone position. A longitudinal skin incision of about 1 cm was made on the posterolateral aspect of the left hind limb thigh. The biceps femoris and semitendinosus muscles were bluntly dissected to expose the deep sciatic nerve trunk. A microsurgical atraumatic vascular clamp (Roboz RS-6462 model, USA) with a jaw width of 0.5 mm was used to clamp the nerve trunk vertically about 5 mm proximal to the bifurcation of the sciatic nerve (the bifurcation of the tibial and common peroneal nerves). Constant pressure was applied for 30 seconds. After releasing the clamp, a clear translucent indentation was visible on the nerve at the clamping site. The distal neurovascular bundle showed slight congestion due to re-perfusion. Subsequently, the surgical area was irrigated with sterile saline, and then 7-0 non-absorbable sutures (MANI) were used. (Japan) The muscles and fascia are sutured layer by layer, and the skin incision is closed with 5-0 sutures or skin clips. Finally, the surgical area is disinfected with povidone-iodine solution.

[0051] To evaluate the effects of different treatments on the axonal regeneration capacity of injured neurons, after sciatic nerve clamping modeling was completed, C57BL / 6J mice were randomly divided into PBS, PMPs, PNPs, and Lytic PNPs groups. These groups received continuous gavage intervention for three days starting from the first day after surgery. After the last administration, the dorsal root ganglia (DRGs) of the L4-L6 segment on the injured side of each group were aseptically isolated. Single-cell suspensions were obtained by sequential digestion with collagenase and trypsin, followed by mechanical pipetting. These suspensions were then seeded into poly-L-lysine / laminusoidal coated culture plates and cultured in Neurobasal complete medium containing B-27 and neurotrophic factors. After 24 hours of culture in a 37°C, 5% CO2 incubator, cell morphology (such as neurite length and density) and function were observed and analyzed.

[0052] like Figure 8 As shown in the figure, with a scale bar of 100 μm, the dorsal root ganglion neurons in the PNPs group showed the most significant neurite growth after in vitro culture.

[0053] 9. SCG-10 Immunofluorescence Assay Procedure: From the first day after modeling, mice were continuously intervened by gavage for three days. After the last administration, sciatic nerve tissue from the injured side of the mice was collected, fixed with 4% paraformaldehyde, dehydrated with a sucrose gradient, embedded in OCT, and frozen sectioned. The sections were washed with PBS, permeated with 0.3% Triton X-100, and blocked with 5% BSA. Rabbit-derived SCG-10 primary antibody was added and incubated overnight at 4°C. The next day, the sections were washed with PBS, and the corresponding species of fluorescently labeled secondary antibody was added and incubated at room temperature in the dark for 1 hour. After washing again, the sections were mounted with mounting medium containing DAPI. Finally, the images were observed and acquired under a fluorescence microscope, and the average fluorescence intensity of SCG-10 in each group was counted to evaluate axonal regeneration.

[0054] The results are as follows Figure 9 As shown in the figure, with a scale bar of 1000 μm, the PNPs group showed the most significant SCG-10 immunofluorescence expression in the sciatic nerve, indicating active axonal regeneration.

[0055] 10. Fluorescent gold retrograde tracer DRGs: After establishing a standard crush injury model of the sciatic nerve in the left hind limb of mice, six mice were selected from each group. Immediately afterward, 2 μL of 3% fluorogold solution was slowly injected subpericardium using a 1 μL Hamilton microsyringe at a distance of 2 mm distal to the injury site. After injection, the area was gently pressed with sterile gelatin sponge for 1 minute to prevent leakage. Postoperatively, the mice were routinely housed separately. 72 hours later, after cardiac perfusion fixation, the dorsal root ganglia (DRGs) of the ipsilateral L4-L6 lumbar vertebrae were dissected and removed, then fixed in 4% paraformaldehyde for 2 hours. Subsequently, they were dehydrated with graded sucrose, embedded in OCT, and frozen sections were prepared for observation and image acquisition under a fluorescence microscope. βIII Tubulin fluorogold, using immunofluorescence technology, simultaneously displays βIII Tubulin (labeling all neurons) and fluorogold (labeling neurons that still retain axonal transport function after axonal injury). The co-localized (overlapping) yellow signal indicates neurons that successfully received retrograde tracer signals, used to assess the recovery of axonal transport function.

[0056] The results are as follows Figure 10 As shown in the figure, with a scale bar of 100 μm, retrograde fluorescent gold tracing showed that the PNPs group had the highest number of labeled neurons in the dorsal root ganglion, indicating the most ideal recovery of axonal transport function. The PMPs group had the second highest number of indicators, while the Lytic PNPs group and the PBS control group had the worst results, suggesting that the intact structure of PNPs is crucial to its neural repair function.

[0057] 11. GO enrichment analysis and ELISA experiment: GO enrichment analysis: First, a mouse sciatic nerve clamp injury model was established, and the animals were divided into a PNPs gavage treatment group and a solvent control group. After 7 days of continuous intervention, dorsal root ganglion (DRG) tissue samples were collected. Then, total RNA of DRG was extracted and transcriptome sequencing was performed to obtain gene expression profile data. Differentially expressed genes were identified by bioinformatics analysis, and GO enrichment analysis was further used to focus on the "biological process" functional annotation and significance test of downregulated genes. Finally, a bar chart was plotted with -log10 (padjust) as the x-axis to visually show the biological processes and related genes that were significantly downregulated after PNPs treatment.

[0058] GO enrichment analysis results are as follows Figure 11 As shown, PNP treatment exhibited a clear dual-effect regulatory pattern of "anti-inflammatory and repair-promoting" on the gene expression profile of DRG after sciatic nerve injury. On the one hand, inflammation-related pathways such as "inflammatory response" and "acute inflammatory response," along with their key genes (e.g., Alox5, Cxcr2, Il18rap), were significantly downregulated, indicating that PNPs effectively inhibited acute and chronic inflammation following nerve injury. On the other hand, repair and protection-related pathways were significantly activated, including neuroprotective and regenerative pathways such as "negative regulation of neuronal apoptosis" and "regulation of neuroblast proliferation," as well as immunomodulatory pathways such as "negative regulation of IL-1β / TNF production" and "regulation of adaptive immune response," involving key genes such as Trem2 and Cx3cl1. This suggests that PNPs not only suppress harmful inflammation but also actively activate the neural regeneration program and guide the immune microenvironment towards a repair-oriented transformation, revealing at the genomic level the molecular mechanism by which they promote neural regeneration through the synergistic regulation of inflammation and repair processes.

[0059] ELISA Experiment: Based on the anti-inflammatory mechanism suggested by GO enrichment analysis, this experiment aimed to verify the inhibitory effect of PNPs on pro-inflammatory cytokine pathways at the protein level. Seven days after PNP gavage treatment, serum and dorsal root ganglion (DRG) tissue from the side of sciatic nerve injury were collected from mice. The concentrations of key cytokines in serum (representing systemic inflammation) and DRG homogenate (representing the local inflammatory microenvironment) were quantitatively detected using a commercially available ELISA kit. The detected indicators included pro-inflammatory factors (IL-1β, IL-6, TNF-α, IL-12) and anti-inflammatory factors (IL-10, TGF-β1). Finally, the effects of PNP treatment on local and systemic cytokine expression profiles were systematically compared by calculating the logarithmic transformation values ​​of the concentrations and using visualization methods such as 3D radar charts.

[0060] ELISA test results as follows Figure 12As shown, PNPs treatment significantly remodeled the local and systemic cytokine expression profiles after sciatic nerve injury. In the dorsal root ganglion (DRG) at the injury site, PNPs treatment significantly downregulated key pro-inflammatory cytokines, including IL-1β, TNF-α, and IL-6, while increasing the expression level of the anti-inflammatory factor IL-10, effectively reversing the inflammatory microenvironment. In serum, pro-inflammatory cytokines also showed a decreasing trend, while the expression of the anti-inflammatory factor TGF-β1 was relatively upregulated. These results indicate that PNPs gavage therapy can not only effectively inhibit the harmful inflammatory response at the nerve injury site but also promote a systemic anti-inflammatory and immune-regulating state, validating its clear anti-inflammatory effect at the protein level and providing key experimental evidence for the GO analysis results.

[0061] 12. Detect the wet weight of the gastrocnemius muscle: Mice were euthanized by overdose anesthesia on postoperative day 28. Six mice were selected from each group, and they were quickly dissected to fully expose and separate the gastrocnemius muscles of the left (affected side, model side) and right (healthy side, control side) hind limbs. The muscles were carefully dissected along the edges using fine instruments, taking care to preserve the integrity of the muscles and avoid traction damage. After removal, the muscles were immediately rinsed with PBS buffer pre-cooled at 4°C to remove surface blood, and then the surface liquid was gently blotted dry with sterile filter paper. The wet weight of the gastrocnemius muscles on both sides was measured and recorded using a calibrated electronic analytical balance (accurate to 0.01g). The wet weight percentage of the gastrocnemius muscles was calculated according to the formula "Gastrocnemius muscle wet weight percentage = experimental side wet weight / normal side wet weight × 100%" to assess the degree of muscle atrophy and recovery after nerve injury.

[0062] like Figure 13 As shown, A is a comparison of the affected side (left) and healthy side (right) of the gastrocnemius muscle in mice; B is a statistical chart of the relative wet weight of the gastrocnemius muscle in different groups, with the PNPs group having the highest relative wet weight of the gastrocnemius muscle.

[0063] 13. Observation of regenerated myelin sheath by transmission electron microscopy: After the last administration, five mice were randomly selected from each group, and the distal segment of the sciatic nerve on the injured side (approximately 5 mm from the clamping point) was harvested. The tissue sample was immediately placed in pre-cooled 2.5% glutaraldehyde (prepared with 0.1M PBS, pH 7.4) and fixed at 4°C for 24 hours. After thorough rinsing with 0.1M PBS, it was post-fixed with 1% osmium tetroxide solution for 2 hours. After fixation, the sample was dehydrated by gradient ethanol and acetone, impregnated with epoxy resin, embedded, and polymerized. Ultrathin sections with a thickness of 50–70 nm were cut using an ultramicrotome, stained with uranyl acetate and lead citrate, and observed and images were acquired under a transmission electron microscope. The effects of different doses of intervention on nerve myelin regeneration were evaluated quantitatively and qualitatively.

[0064] The results are as follows Figure 14As shown, A is a transmission electron microscope image of the sciatic nerve myelin sheath with a scale bar of 5 μm, and B is a scatter plot and linear regression fitting of the G-ratio as a function of axonal diameter. The PNPs group has the most regular sciatic nerve myelin sheath structure and the best G-ratio distribution, indicating that muscle atrophy inhibition and myelin sheath regeneration are optimal.

[0065] 14. An experiment using the Catwalk XT gait analysis system to evaluate functional recovery in mice with sciatic nerve injury: The Catwalk XT gait analysis system was used to assess the recovery of motor function in mice with sciatic nerve injury. Adaptation training was conducted and baseline data were collected before modeling, and formal testing was performed on day 28 post-surgery. The gait of mice spontaneously crossing the track was recorded, and footprint information of the left hind limb (affected limb) was obtained. The system software generated a three-dimensional dynamic stress map (with pressure distribution displayed in pseudo-color) of the affected limb's impact point and representative footprint sequences, visually demonstrating the plantar contact morphology and gait sequence. Simultaneously, the footprint length, footprint length, and footprint width of the affected and healthy limbs were measured. The Sciatic Functional Index (SFI) was calculated using the Bain-Mackinnon-Hunter formula; a closer SFI to 0 indicates better functional recovery. By comparing the three-dimensional stress maps, footprint maps, and SFI values ​​of each group at different time points, the recovery of nerve function was evaluated quantitatively and qualitatively.

[0066] In terms of function, such as Figure 15 As shown, A is a typical three-dimensional stress diagram and representative footprint diagram of the left hind limb at the time of modeling, and B is the SFI of mice on day 28 under different treatments. The PNPs group showed the closest recovery of gait and footprint to normal, and the highest neurological function index (SFI).

[0067] 15. Electrophysiological testing procedures for sciatic nerve injury in mice: On day 28 after continuous gavage intervention, mice in each group were anesthetized intraperitoneally, fixed in a prone position, and the skin was incised along the posterior thigh. The muscles were bluntly dissected to expose the sciatic nerve on the injured side. The stimulating electrode was placed proximally to the sciatic nerve, and the recording electrode was placed distally at the extensor digitorum brevis muscle. Square wave pulse stimulation was applied. The latency difference (Δt) of the compound muscle action potential (CMAP) evoked by the proximal and distal stimulation points and the distance (D) between the two stimulation points were recorded and calculated. The motor nerve conduction velocity was calculated according to the formula MNCV=D / Δt, and the CMAP amplitude was measured simultaneously. All data were exported and statistically analyzed to evaluate the effect of different treatments on the recovery of nerve conduction function.

[0068] like Figure 16As shown, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no statistically significant difference. Significant differences were observed among the groups in the neurophysiological function assessment. The PNPs group performed best in all key indicators, specifically: ① CAMP latency: The PNPs group had the shortest latency, significantly lower than other groups, indicating the fastest initiation of neural depolarization; ② CAMP peak amplitude: The PNPs group had the highest amplitude, significantly higher than other groups (p < 0.01), suggesting the largest number of functional axons or the best synchronicity; ③ Nerve conduction velocity: The PNPs group had the fastest conduction velocity, significantly better than other groups, reflecting the best recovery of myelination and axonal integrity.

[0069] The PMPs group performed second best in all the above indicators. Its latency, amplitude and conduction velocity were better than the LyticPNPs group and the PBS group, but not as good as the PNPs group. In contrast, the Lytic PNPs group and the PBS group had the worst performance in all electrophysiological indicators, and there was no statistical difference between the two (p>0.05).

[0070] In summary, pepper-derived plant nanovesicles (PNPs) demonstrate remarkable comprehensive advantages in treating peripheral nerve injury, with their intact nanovesicle structure being key to their function. In animal models, PNPs optimally promote axonal regeneration and myelin repair, significantly improve electrophysiological functions such as nerve conduction velocity and amplitude, and effectively slow down target muscle atrophy. Their mechanism of action is related to remodeling the local inflammatory microenvironment and activating the nerve repair program. Furthermore, oral administration of PNPs shows good safety, indicating that it is a highly promising natural nerve repair therapy strategy.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for the preparation of plant nanovesicles of Piper nigrum L. origin, characterized by, The method is as follows: S1. Raw material preparation and processing: After washing, the pepper fruits are crushed and soaked in a 70% ethanol aqueous solution. After rinsing, the processed pepper fruits are obtained. The processed pepper fruits are mixed with an enzymatic hydrolysate, enzymatically hydrolyzed, centrifuged, and the supernatant is collected to obtain a crude extract of pepper nanovesicles. S2. Multi-stage filtration purification: The crude extract of pepper nanovesicles obtained in S1 is sequentially filtered through a filter screen, a microfiltration membrane, and a microporous membrane to obtain the filtrate. S3. Ultracentrifugation extraction: Centrifuge the filtrate obtained in S2, take the supernatant, ultracentrifuge, collect the precipitate, resuspend the precipitate in pre-cooled phosphate buffer, ultracentrifuge again, collect the precipitate, and resuspend it in phosphate buffer to obtain plant nanovesicles derived from pepper.

2. The method for preparing plant nanovesicles derived from pepper according to claim 1, characterized in that, The soaking time in S1 is 1 minute; the mass ratio of the treated pepper fruit to the enzymatic hydrolysate is 1:

5.

3. The method for preparing plant nanovesicles derived from pepper according to claim 1, characterized in that, The enzymatic hydrolysis conditions described in S1 are: enzymatic hydrolysis for 5 hours at a temperature of 45℃ and a pH of 5.5 to 6.0; and centrifugation conditions are: centrifugation for 30 to 60 minutes at a speed of 10000×g.

4. The method for preparing plant nanovesicles derived from pepper according to claim 1, characterized in that, The filter screen used for coarse filtration in S2 has a mesh size of 200; the microfiltration membrane has a pore size of 200 nm; and the microporous membrane has a pore size of 0.45 μm.

5. The method for preparing plant nanovesicles derived from pepper according to claim 1, characterized in that, The centrifugation conditions in S3 are: centrifugation for 30 min at a temperature of 4℃ and a rotation speed of 10000×g; the ultracentrifugation conditions are: ultracentrifugation for 60 min at a temperature of 4℃ and a rotation speed of 100000×g; the pre-cooling temperature is 4℃.

6. An application of a pepper-derived plant nanovesicle prepared by the preparation method according to any one of claims 1-5, characterized in that, The plant nanovesicles derived from pepper are used to prepare drugs that improve peripheral nerve damage.

7. The application according to claim 6, characterized in that, When the pepper-derived plant nanovesicles are used to prepare a drug for improving peripheral nerve injury, the pepper-derived plant nanovesicles are added as a drug ingredient.

8. The application according to claim 7, characterized in that, The dosage form of the drug is one or more of the following: injection, capsule, tablet, oral liquid, and granule.

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