Fresh gynostemma pentaphyllum exosome and preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a fresh product gynostemma pentaphyllum exosome and a preparation method and application thereof. The preparation method comprises the following steps: crushing the fresh product gynostemma pentaphyllum, adding a buffer solution to homogenate, filtering, and obtaining a primary extract; performing multi-stage differential centrifugation on the primary extract, removing impurities, and collecting supernatant; performing ultracentrifugation enrichment on the supernatant, collecting the precipitate and resuspending, and obtaining the fresh product gynostemma pentaphyllum exosome. The nematode experiment shows that the fresh product gynostemma pentaphyllum exosome can significantly prolong the lifespan of Caenorhabditis elegans, enhance the movement ability, reduce the accumulation of lipofuscin related to aging, and improve the heat stress survival rate. The fresh product gynostemma pentaphyllum exosome provided by the present application has clear components, good uniformity, and significant anti-aging activity, can provide material evidence for the scientific connotation of the "different use of raw and cooked" of gynostemma pentaphyllum, and be applied to the preparation of drugs for delaying aging, enhancing stress resistance, or preventing and treating neurodegenerative diseases, and has high scientific research value and clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a fresh Gynostemma pentaphyllum exosome, its preparation method, and its application. Background Technology

[0002] Gynostemma pentaphyllum ( Gynostemma pentaphyllum Gynostemma pentaphyllum is a perennial herb belonging to the genus Gynostemma of the Cucurbitaceae family. Its main active ingredient is dammarane-type triterpenoid saponins. Modern pharmacological studies have shown that Gynostemma pentaphyllum possesses various biological activities, including antioxidant, anti-inflammatory, immunomodulatory, and anti-aging effects. Existing literature reports that oral administration of Gynostemma pentaphyllum to rats can scavenge oxygen free radicals in hypothalamic tissue, thereby enhancing the body's antioxidant capacity and counteracting D-galactose-induced subacute aging in rats. Gynostemma pentaphyllum saponins can reduce oxidative damage to the skin of aging mice, thus delaying skin aging in mice. Gynostemma pentaphyllum extract improves mitochondrial function by upregulating the expression of mitochondrial-associated proteins SIRT3 and TOM20. Of the 44 dammarane-type saponins isolated from it, 13 can activate these two proteins, providing a new molecular mechanism and active substance basis for the anti-aging effect of Gynostemma pentaphyllum.

[0003] However, existing research largely focuses on traditional extracts of Gynostemma pentaphyllum, such as decoctions and alcohol extracts, or single saponin monomers, and the raw materials used are mostly dried products. Traditional Chinese medicine theory holds that "raw and processed products have different uses," suggesting significant differences in chemical composition and biological activity between fresh and dried products. However, the forms in which active ingredients exist and their biological functions in fresh Gynostemma pentaphyllum have not been fully elucidated. In recent years, plant-derived nanovesicles (PDNVs) have gradually become a hot topic in drug delivery systems and natural active carriers due to their natural nanoscale particle size, good biocompatibility, and ability to carry proteins, nucleic acids, lipids, and secondary metabolites. However, research on the extraction, identification, and anti-aging applications of Gynostemma pentaphyllum exosomes, especially those from fresh Gynostemma pentaphyllum, remains largely unexplored.

[0004] Therefore, there is an urgent need to develop a method for preparing exosomes that can preserve the complete active ingredients in fresh Gynostemma pentaphyllum and to systematically verify its anti-aging function. This will not only help to explain the scientific connotation of the "different uses of raw and processed Gynostemma pentaphyllum" but also provide technical support for the development of new anti-aging drugs and functional products. Summary of the Invention

[0005] In view of the above-mentioned shortcomings in the prior art, the purpose of this invention is to provide a fresh Gynostemma pentaphyllum exosome, its preparation method, and its application in anti-aging.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: In a first aspect, a method for preparing exosomes from fresh Gynostemma pentaphyllum is provided, comprising the following steps: (1) Crush the fresh Gynostemma pentaphyllum, add buffer solution, homogenize, juice, and filter to obtain the initial extract; (2) Perform multi-stage differential centrifugation on the initial extract to remove impurities and collect the supernatant; (3) The supernatant was enriched by ultracentrifugation, the precipitate was collected and resuspended to obtain fresh Gynostemma pentaphyllum exosomes.

[0007] Preferably, the Gynostemma pentaphyllum is Gynostemma pentaphyllum seven-leafed from Dayao Mountain in Jinxiu County, Guangxi.

[0008] Preferably, the buffer solution in step (1) is a pre-cooled phosphate buffer solution with pH 6.0-8.0 and temperature 0-10℃; the ratio of the fresh Gynostemma pentaphyllum to the buffer solution is 1:0.1-1:10 (g:mL).

[0009] Preferably, the multi-stage differential centrifugation in step (2) is carried out at 0-10℃, and the centrifugation sequence and parameters are as follows: (1000-1500)×g centrifugation for (10-30) min, and supernatant is collected; (2500-3500)×g centrifugation for (30-50) min, and supernatant is collected; (7500-12500)×g centrifugation for (70-110) min, and supernatant is collected.

[0010] Preferably, the ultracentrifugation conditions in step (3) are: centrifugal force 80000-200000×g, time 1-4 h, and temperature 0-10℃.

[0011] Secondly, a fresh Gynostemma pentaphyllum exosome is provided, wherein the exosome has a particle size of 30-200 nm, exhibits a bilayer vesicle structure, and is enriched with one or more Gynostemma pentaphyllum saponins.

[0012] Preferably, the Gynostemma pentaphyllum saponins include at least one of Gypenoside XXVIII, Gypenoside XIII, Gypenoside LII, and Ginsenoside Rg3.

[0013] Thirdly, the application of the fresh Gynostemma pentaphyllum exosomes in the preparation of anti-aging related products is provided.

[0014] Preferably, the anti-aging related products are used for at least one of the following purposes: prolonging lifespan, enhancing athletic performance, reducing lipofuscin accumulation, improving heat stress resistance, improving oxidative stress resistance, scavenging oxygen free radicals, and improving mitochondrial function.

[0015] Fourthly, a pharmaceutical composition is provided, comprising the fresh Gynostemma pentaphyllum exosomes and a pharmaceutically acceptable carrier or excipient; the dosage form of the pharmaceutical composition is any one of an injection, an oral preparation, or a topical preparation.

[0016] The beneficial effects of this invention are as follows: (1) Raw material innovation: This invention is the first to use fresh Gynostemma pentaphyllum produced in Jinxiu Dayao Mountain as raw material, which is different from the dried product or cell culture used in traditional research. It preserves the heat-sensitive and structurally intact native saponin components in Gynostemma pentaphyllum to the greatest extent, providing material evidence for explaining the scientific connotation of "different uses for raw and cooked Gynostemma pentaphyllum".

[0017] (2) Simple and efficient process: The present invention adopts a physical extraction method of differential centrifugation combined with ultracentrifugation, which does not require complex inducing agents or affinity purification steps. The operation is simple and the cost is controllable. It can obtain Gynostemma pentaphyllum exosomes with a highly uniform population (particle size 108.1 nm, single peak distribution).

[0018] (3) Significant advantages of components: LC-MS / MS analysis showed that the fresh Gynostemma pentaphyllum exosomes obtained in this invention were significantly enriched with native saponins such as Gypenoside XXVIII, XIII, LII and Ginsenoside Rg3. Among them, the content of Gypenoside XIII in the fresh product was 39.6 times that in the dried product. These native glycosides have stronger biological activity and signal regulation potential.

[0019] (4) The efficacy verification is sufficient: This invention has confirmed through the nematode model that the exosomes of fresh Gynostemma pentaphyllum have the following effects: prolonging life and enhancing motility (nematode model, P≤0.001); reducing the accumulation of aging-related lipofuscin (the effect is close to that of rapamycin); and enhancing the resistance to heat stress (the survival rate is significantly improved, P<0.05).

[0020] (5) Broad application prospects: The fresh Gynostemma pentaphyllum exosomes provided by this invention have clear components, good uniformity, and significant anti-aging activity. They can be applied to the development of drugs to delay aging, enhance stress resistance, and prevent and treat neurodegenerative diseases. They have high scientific research value and industrial application prospects. Attached Figure Description

[0021] Figure 1 Photographs of fresh Gynostemma pentaphyllum from Dayao Mountain, Jinxiu County, Guangxi Province, used in this invention.

[0022] Figure 2 : Flowchart of the extraction process of exosomes from fresh Gynostemma pentaphyllum in this invention.

[0023] Figure 3Characterization results of exosomes from fresh / dried Gynostemma pentaphyllum. A: Particle size distribution of exosomes from fresh Gynostemma pentaphyllum (NTA determination), median particle size 108.1 nm, showing a unimodal distribution; B: Particle size distribution of exosomes from dried Gynostemma pentaphyllum (NTA determination), median particle size 125.3 nm, showing a bimodal distribution; C: Transmission electron microscope (TEM) image of nanoparticles from exosome samples of fresh Gynostemma pentaphyllum; D: Transmission electron microscope (TEM) image of nanoparticles from exosome samples of dried Gynostemma pentaphyllum.

[0024] Figure 4 Total ion chromatogram (TIC) of exosomes from fresh Gynostemma pentaphyllum. A: Positive ion mode; B: Negative ion mode.

[0025] Figure 5 Total ion chromatogram (TIC) of exosomes from dried Gynostemma pentaphyllum. C: Positive ion mode; D: Negative ion mode.

[0026] Figure 6 Effects of fresh Gynostemma pentaphyllum exosomes on the motility of Caenorhabditis elegans. Compared with the control group, ***P≤0.001.

[0027] Figure 7 Effects of fresh Gynostemma pentaphyllum exosomes on the lifespan of Caenorhabditis elegans.

[0028] Figure 8 Effects of fresh Gynostemma pentaphyllum exosomes on lipofuscin accumulation in Caenorhabditis elegans. A: Representative fluorescence image; B: Quantitative analysis results.

[0029] Figure 9 Effects of fresh Gynostemma pentaphyllum exosomes on the stress resistance of *C. elegans*. A: The survival rate of the 50 μg / mL exosome group after heat shock was significantly higher than that of the control group, **P<0.01. B: Effects of fresh Gynostemma pentaphyllum exosomes on the survival time of *C. elegans* under oxidative stress.

[0030] Figure 10 Comparative figure showing the effects of fresh and dried Gynostemma pentaphyllum exosomes (50 μg / mL) on the motility of Caenorhabditis elegans. **P<0.01 compared to the control group; no statistically significant difference was found between the fresh and dried groups. Detailed Implementation

[0031] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0032] The preparation method of fresh Gynostemma pentaphyllum exosomes according to the present invention is described in detail below, mainly including the following parts: raw material selection and pretreatment, differential centrifugation for impurity removal, ultracentrifugation for enrichment, exosome characterization, chemical composition analysis, and pharmacodynamic verification. Each step is described in detail below.

[0033] Example 1: Preparation of exosomes from fresh Gynostemma pentaphyllum 1. Raw material selection and pretreatment The Gynostemma pentaphyllum raw material used in this embodiment comes from the core production area of ​​Jinxiu Dayao Mountain, which has obtained national geographical indication protection. Relying on the unique local ecological environment and the patented variety 'Jinxuan No. 1' (Guangxi Approval No. 2012011), its content of core components such as saponins is higher than that of general commercially available products (the total saponin content is ≥5.2% as tested), thus having a significant advantage in efficacy and quality.

[0034] Fresh Gynostemma pentaphyllum leaves (characteristics: palmate compound leaves composed of 7 leaflets, young stems and leaves have a sweet taste, and saponin content is significantly higher than in other producing areas) were selected from the Dayao Mountains of Jinxiu County, Guangxi. 200g of fresh leaves were taken, washed clean with deionized water, and chopped for later use.

[0035] The fresh product in this embodiment was mailed from Guangxi to Beijing two days after harvesting and placed in a refrigerator at 4°C. The above-ground parts of Gynostemma pentaphyllum (mainly tender stems and leaves) that have not undergone drying or blanching treatment have a moisture content of 75%~85% and remain in an active state.

[0036] 2. Juicing and Filtering The chopped fresh Gynostemma pentaphyllum was added to a juicer in portions, along with 100 mL of pre-cooled phosphate-buffered saline (PBS, pH 7.4, 4°C) for homogenization and juicing. The resulting homogenate was filtered through four layers of gauze, and the filtrate was collected to obtain approximately 100 mL of the initial Gynostemma pentaphyllum extract. The entire process was carried out under ice bath conditions, maintaining a low temperature of 4°C.

[0037] 3. Differential centrifugation for impurity removal Transfer the initial extract to a 50 mL centrifuge tube and centrifuge sequentially at 4°C: centrifuge at 1200×g for 20 min, carefully collect the supernatant and discard the precipitate (to remove cell debris); centrifuge at 3000×g for 40 min, carefully collect the supernatant and discard the precipitate (to remove large vesicles and organelles); centrifuge at 10000×g for 90 min, carefully collect the supernatant and discard the precipitate (to remove microvesicles and large protein complexes).

[0038] 4. Ultracentrifugation enrichment Transfer the supernatant obtained in step 3 to an ultracentrifuge bottle, balance the liquid, and centrifuge at 4°C and 150,000 × g for 2 hours. After centrifugation, carefully discard the supernatant; a green gelatinous precipitate will be visible at the bottom of the tube. Add 5–10 mL of pre-chilled PBS to the precipitate and gently resuspend it using a pipette to obtain the Gynostemma pentaphyllum exosome suspension. Aliquot the sample and store it at -80°C for later use.

[0039] 5. Preparation of control samples of dried Gynostemma pentaphyllum exosomes Take 200g of fresh Gynostemma pentaphyllum from the same batch as in Example 1, and dry it naturally or at low temperature until constant weight to obtain approximately 10g of dried product. Cut the dried product into small pieces, add 100mL of pre-cooled PBS and soak for 30 minutes. Follow the same steps as in Example 1 to obtain dried Gynostemma pentaphyllum exosomes for subsequent comparative studies.

[0040] Example 2: Physical characterization of Gynostemma pentaphyllum exosomes 1. Particle size distribution and concentration determination (NTA method) Fresh and dried Gynostemma pentaphyllum exosome samples prepared in Example 1 were diluted with PBS to an appropriate concentration (approximately 1 × 10⁻⁶). 6 -1×10 8 Particle size distribution and concentration were determined using a ZetaView nanoparticle tracking analyzer (particles / mL).

[0041] Table 1. Comparison of particle size distribution of exosomes from fresh and dried Gynostemma pentaphyllum. As shown in Table 1, the median particle size of exosomes from fresh Gynostemma pentaphyllum was 108.1 nm, exhibiting a unimodal distribution and uniform population height; while the median particle size of exosomes from dried Gynostemma pentaphyllum was 125.3 nm, exhibiting a bimodal distribution, suggesting that the drying process may have led to exosome fusion or subpopulation differentiation.

[0042] 2. Morphological observation (TEM method) 10 μL of exosome suspension was dropped onto a copper grid and allowed to stand for 10 min to adsorb. Excess liquid was then absorbed from the edges with filter paper. 2% phosphotungstic acid solution (pH 6.8) was added for negative staining for 5 min, the solution was blotted dry with filter paper, and the mixture was air-dried at room temperature before being observed and photographed under a transmission electron microscope. The results showed that both fresh and dried Gynostemma pentaphyllum exosomes exhibited typical saucer-shaped or cup-shaped bilayered vesicle structures with intact morphology and clearly visible membrane structures.

[0043] Example 3: Chemical composition analysis of Gynostemma pentaphyllum exosome contents 1. Sample pretreatment Take 50 μL each of the fresh and dried Gynostemma pentaphyllum exosome samples prepared in Example 1, add 200 μL of 80% methanol that has been cooled to -20°C in advance, and extract by sonication in an ice bath for 10 min. Remove the sample, centrifuge at 14000 rpm at 4°C, take 200 μL of the supernatant, dry it with nitrogen, redissolve it with 30 μL of 80% methanol, centrifuge again at 14000 rpm at 4°C, and take 20 μL of the supernatant to add to the sample vial for UPLC-MS / MS analysis.

[0044] 2. UPLC-MS / MS Analysis Conditions Liquid phase conditions: Chromatographic column: ACQUITY UPLC HSS T3 column (2.1×100mm, 1.8μm); column temperature: 35℃; injection volume: 10μL; flow rate: 0.25 mL / min; mobile phase: A (deionized water, containing 0.1% formic acid); B (acetonitrile, containing 0.1% formic acid), gradient elution. Specific gradient elution conditions are as follows: Mass spectrometry conditions: Mass spectrometry data acquisition was performed using Q-TOF high-resolution mass spectrometry. The detection mode was MSe, with separate scans for positive and negative ions. The scan range was 100-1300 m / z. The ion source voltage was 2.0 kV, the cone voltage was 20 V, the ion source temperature was 120 °C, the desolvation temperature was 360 °C, the cone gas flow rate was 50 L / min, and the desolvation gas flow rate was 600 L / min. The collision energy used to trigger the MSe scan was the stepped fragmentation voltage NCE, set to 20-50 V.

[0045] 3. Component identification and comparative analysis The detected compounds were identified by comparison with reference standards and a self-built database. In fresh exosomes, 122 components were detected in positive ion mode and 57 in negative ion mode, for a total of 118 components. In dried exosomes, 150 components were detected in positive ion mode and 109 in negative ion mode, for a total of 145 components.

[0046] Table 2. Comparison of peak areas of representative compounds in exosomes of fresh and dried Gynostemma pentaphyllum. 4. Analysis of unique components of fresh products Further analysis revealed that several Gynostemma pentaphyllum saponin variants were detected only in the exosomes of the fresh product, and were almost undetectable in the dried product (peak area <1000).

[0047] Table 3. Saponin components unique to fresh Gynostemma pentaphyllum exosomes As shown in Tables 2 and 3, the exosomes of fresh Gynostemma pentaphyllum were significantly enriched with structurally intact native saponins such as Gypenoside XXVIII, XIII, LII, and Ginsenoside Rg3. The content of Gypenoside XXVIII in fresh Gynostemma pentaphyllum was 43.4 times that of dried Gynostemma pentaphyllum, and Gypenoside XIII was 39.6 times higher. In contrast, the exosomes of dried Gynostemma pentaphyllum were significantly enriched with palmitic acid, gypsogenin, and sterols (such as cholesterol and campesterol). This result reveals the material basis for the difference in efficacy between raw and processed Gynostemma pentaphyllum and provides a component-based basis for the subsequent differences in its medicinal effects.

[0048] Example 4: Experiment on the anti-aging efficacy of nematodes 1. Nematode strains and culture conditions Caenorhabditis elegans N2 wild-type was used, with Escherichia coli OP50 as its food source. The nematodes were cultured at 20°C on NGM medium containing OP50 bacterial culture. All experiments were conducted in a 20°C incubator.

[0049] 2. Nematode synchronization treatment Wash the plate containing oviposition-stage nematodes with M9 buffer, collect the suspension into a 15 mL centrifuge tube, centrifuge at 2500 rpm for 2 min, and discard the supernatant. Add 1 mL of nematode lysis buffer (1M NaOH: 84 disinfectant = 1:1), vortex to mix for 5 min, centrifuge at 2500 rpm for 2 min, and discard the supernatant. Add 1 mL of M9 buffer to wash the eggs, centrifuge at 2500 rpm for 2 min, and repeat the washing 3 times. Add 10 mL of M9 buffer, shake well to distribute the eggs evenly, and incubate at 20℃ for 12-24 h. The eggs hatch and arrest at the L1 larval stage, obtaining synchronized offspring.

[0050] 3. Dosing grouping and concentration preparation The protein concentration of fresh Gynostemma pentaphyllum exosomes (GELNs) prepared in Example 1 was determined using the BCA method. The exosomes were mixed with OP50 bacterial suspension to prepare drug delivery systems with protein concentrations of 2.5, 5, 10, and 50 μg / mL. The positive control group (RAP) received 100 μM rapamycin, and the blank control group (Con) received an equal volume of OP50 bacterial suspension. Three parallel plates were prepared for each group.

[0051] 4. Motor ability test Nematodes synchronized to the L4 stage were transferred to NGM medium with or without the drug (each plate contained 80 μM FUdR to inhibit oviposition) and cultured at 20°C. This was recorded as day 0 of nematode survival. On days 4, 6, and 8 after drug administration, the number of times the nematodes completed a sinusoidal movement within 30 seconds was observed. One head swing plus one body bend was counted as one sinusoidal movement. Ten nematodes were randomly counted from each plate, and 30 nematodes were counted from each group.

[0052] Table 4. Effects of fresh Gynostemma pentaphyllum exosomes on the motility of nematodes (number of sinusoidal movements within 30 seconds). Note: Compared with group Con, *P<0.05, **P<0.01.

[0053] As shown in Table 4, motor function naturally declined with age in all groups, but the decline was significantly smaller in the GELNs-treated group than in the control group. This indicates that fresh Gynostemma pentaphyllum exosomes can effectively delay the age-related decline in motor function in nematodes and enhance the vitality of muscles and the nervous system.

[0054] 5. Lifetime test Based on the motility test, the GELNs group selected 2.5 and 50 μg / mL dosing systems for subsequent experiments. Nematodes synchronized to the L4 stage were transferred to NGM medium with or without the drug (each plate contained 80 μM FUdR to inhibit oviposition) and cultured at 20°C. This was recorded as day 0 of nematode survival. The number of dead nematodes was observed and recorded daily thereafter; nematodes were considered dead if they did not respond to gentle touching with a needle. The plates were transferred and the drug was re-administered every two days until all nematodes died. Survival curves were plotted using the Kaplan-Meier method, and statistical analysis was performed using the Log-rank test.

[0055] Table 5. Effects of fresh Gynostemma pentaphyllum exosomes on nematode lifespan. Note: Compared with group Con, *P<0.05, **P<0.01.

[0056] As shown in Table 5, fresh Gynostemma pentaphyllum exosomes can significantly prolong the lifespan of nematodes, and the effect at high doses is superior to that of the commonly used anti-aging positive control drug RAP.

[0057] The exosomes derived from fresh Gynostemma pentaphyllum from the Dayao Mountains of Jinxiu County used in this application exhibited clear anti-aging activity in the model organism Caenorhabditis elegans: they improved motor function (delaying muscle / nerve aging) and extended overall lifespan. This result provides preliminary experimental evidence for the application of this raw material in the development of anti-aging functional foods or drugs, especially with outstanding effects at high doses (50 μg / mL), warranting further mechanistic research.

[0058] 6. Lipofuscin Accumulation Assay Twelve days after drug intervention, nematodes from each group were collected and washed three times with M9 buffer. They were then fixed with 500 μL of 4% paraformaldehyde for 20 min, centrifuged, and the supernatant was discarded. The nematodes were washed three times with M9 buffer. The nematodes were transferred to a 2% agarose gel, covered with a coverslip, and photographed under a fluorescence microscope using the DAPI channel (excitation wavelength 358 nm, emission wavelength 525 nm). The average fluorescence intensity of the intestinal region was measured using ImageJ software to reflect the level of lipofuscin accumulation.

[0059] Table 6. Effects of fresh Gynostemma pentaphyllum exosomes on lipofuscin accumulation in nematodes. Note: Compared with group Con, *P<0.05, **P<0.01.

[0060] As shown in Table 6, the autofluorescence intensity of the nematode intestine was significantly reduced in the 50 μg / mL Gynostemma pentaphyllum exosome group, and the lipofuscin accumulation inhibition rate reached 16.1%, which was close to that of the positive drug rapamycin group (12.6%).

[0061] 7. Heat stress experiment Seven days after drug administration and culture, 30 nematodes from each group were transferred to a new culture dish, sealed with sealing film, and placed in a 35°C incubator for heat shock for 4 hours. After that, the sealing film was removed, and the dish was placed in a 20°C incubator for recovery for 12 hours. The survival rate of Caenorhabditis elegans was then calculated.

[0062] Table 7. Effects of fresh Gynostemma pentaphyllum exosomes on the heat stress resistance of nematodes. Note: Compared with group Con, *P<0.05, **P<0.01.

[0063] As shown in Table 7, pretreatment with Gynostemma pentaphyllum exosomes can significantly enhance the heat stress resistance of nematodes. The survival rate of the 50 μg / mL concentration group after heat shock reached 80%, which was significantly higher than that of the control group (58.89%), P<0.01.

[0064] 8. Oxidative stress experiment To evaluate the resistance of fresh Gynostemma pentaphyllum exosomes to oxidative stress, an acute oxidative stress model was established using 3% hydrogen peroxide. Nematodes were synchronized and cultured for 7 days after administration (group settings were the same as in Example 4), with 30 nematodes per group and three replicates. After administration, the nematodes were transferred to NGM medium coated with 3% hydrogen peroxide solution, and survival was observed every 30 minutes, recording the survival time of each nematode. Death was defined as no response upon gentle needle touch.

[0065] Table 8. Effects of fresh Gynostemma pentaphyllum exosomes on the oxidative stress resistance of nematodes. Note: Compared with group Con, *P<0.05, **P<0.01.

[0066] As shown in Table 8, compared with the control group, both the 2.5 μg / mL and 50 μg / mL groups of fresh Gynostemma pentaphyllum exosomes significantly prolonged the survival time of nematodes under oxidative stress (P<0.05), and the 50 μg / mL group was more effective than the 2.5 μg / mL group, showing a dose-dependent effect. The effect of the 50 μg / mL group was comparable to that of the 100 μM rapamycin positive control group, indicating that fresh Gynostemma pentaphyllum exosomes can significantly enhance the nematode's ability to resist oxidative stress.

[0067] Example 5: Comparative Study on the Motility of Gynostemma pentaphyllum Exosomes between Fresh and Dried Products on Nematode Motility To compare the differences in improving motility between fresh and dried Gynostemma pentaphyllum exosomes, fresh Gynostemma pentaphyllum exosomes (GELNs-F) and dried Gynostemma pentaphyllum exosomes (GELNs-D) prepared in Example 1 were administered at a concentration of 50 μg / mL. The methods for nematode synchronization, culture, and motility measurement were the same as in Example 4. The number of sinusoidal movements within 30 seconds in the nematodes was recorded on days 4, 6, and 8 after administration.

[0068] Table 9. Effects of fresh and dried Gynostemma pentaphyllum exosomes on the motility of nematodes (number of sinusoidal movements within 30 seconds). Note: Compared with group Con, *P<0.05, **P<0.01.

[0069] The results are shown in Table 9. Compared with the control group, both the fresh exosome group and the dried exosome group significantly improved the motility of nematodes on days 4, 6, and 8 after administration (P<0.01), but there were no statistically significant differences between the two groups at any time point (P>0.05). This suggests that although the dried exosomes differ from the fresh ones in composition, they still have significant activity in improving the motility of nematodes.

[0070] Example 7: Preparation of the pharmaceutical composition 1. Injectables Fresh Gynostemma pentaphyllum exosomes prepared in Example 1 were diluted with sterile PBS to a concentration of 0.5 mg / mL, filtered through a 0.22 μm microporous membrane for sterilization, and aseptically dispensed into 2 mL vials (1 mL per vial). After freeze-drying, the vials were sealed to obtain lyophilized Gynostemma pentaphyllum exosome powder for injection. The powder was reconstituted with sterile PBS immediately before use.

[0071] 2. Oral liquid Take Gynostemma pentaphyllum exosomes, add appropriate amounts of deionized water, mannitol (lyophilization protectant) and potassium sorbate (preservative), prepare an oral solution with a concentration of 0.1 mg / mL, fill into 10 mL brown glass bottles, seal, and the product is ready.

[0072] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0073] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing exosomes from fresh Gynostemma pentaphyllum, characterized in that, Includes the following steps: (1) Crush the fresh Gynostemma pentaphyllum, add buffer solution, homogenize, juice, and filter to obtain the initial extract; (2) Perform multi-stage differential centrifugation on the initial extract to remove impurities and collect the supernatant; (3) The supernatant was enriched by ultracentrifugation, the precipitate was collected and resuspended to obtain fresh Gynostemma pentaphyllum exosomes.

2. The preparation method according to claim 1, characterized in that, The Gynostemma pentaphyllum mentioned is the seven-leaf Gynostemma pentaphyllum from Dayao Mountain in Jinxiu, Guangxi.

3. The preparation method according to claim 1, characterized in that, The buffer solution mentioned in step (1) is a pre-cooled phosphate buffer solution with pH 6.0-8.0 and temperature 0-10℃; the ratio of fresh Gynostemma pentaphyllum to the buffer solution is 1:0.1-1:10 (g:mL).

4. The preparation method according to claim 1, characterized in that, The multi-stage differential centrifugation described in step (2) is carried out at 0-10℃. The centrifugation sequence and parameters are as follows: (1000-1500)×g centrifugation for (10-30) min, and supernatant is collected; (2500-3500)×g centrifugation for (30-50) min, and supernatant is collected; (7500-12500)×g centrifugation for (70-110) min, and supernatant is collected.

5. The preparation method according to claim 1, characterized in that, The ultracentrifugation conditions described in step (3) are: centrifugal force 80000-200000×g, time 1-4 h, and temperature 0-10℃.

6. A type of exosome from fresh Gynostemma pentaphyllum, characterized in that, The exosomes are prepared by the method described in any one of claims 1-5; the exosomes have a particle size of 30-200 nm, have a bilayer vesicle structure, and are enriched with one or more Gynostemma pentaphyllum saponins.

7. The Gynostemma pentaphyllum exosomes according to claim 6, characterized in that, The gypsum saponins include at least one of Gypenoside XXVIII, Gypenoside XIII, Gypenoside LII, and Ginsenoside Rg3.

8. The use of the fresh Gynostemma pentaphyllum exosomes as described in claim 6 or 7 in the preparation of anti-aging related products.

9. The application according to claim 8, characterized in that, The anti-aging related products are used for at least one of the following purposes: extending lifespan, enhancing athletic performance, reducing lipofuscin accumulation, improving heat stress resistance, improving oxidative stress resistance, scavenging oxygen free radicals, and improving mitochondrial function.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the fresh Gynostemma pentaphyllum exosomes as described in claim 6 or 7, and a pharmaceutically acceptable carrier or excipient; the dosage form of the pharmaceutical composition is any one of injection, oral preparation, or topical preparation.

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