Nanometer phase-state penthorum chinense pursh extract as well as preparation method and application thereof

Abstract

The invention provides a nano-phase penthorum chinense pursh extract, which is prepared by the following steps: soaking a penthorum chinense pursh medicinal material, decocting, filtering, collecting filtrate, carrying out layering centrifugation to remove precipitate, dialyzing supernate by adopting a dialysis bag with the molecular weight cutoff of 3.5 kDa, and freeze-drying dialysate to obtain the nano-phase penthorum chinense pursh extract. The invention further provides a preparation method and application of the nanometer phase-state penthorum chinense pursh extract. The invention provides an extraction method capable of accurately separating 50-200nm phase-state active components in a nanometer phase-state penthorum chinense pursh extract traditional Chinese medicine. Organic solvents are prevented from being used, and the safety of extracted products is improved; the purity of the target component is improved; the structural integrity of a natural nanometer phase state is kept; the obtained nanometer phase-state extract has the effects of remarkably improving metabolic dysfunction related steatohepatitis and resisting coagulation, is high in separation efficiency, can be completed in 2-3 days, and can be applied on a large scale; the equipment is simple and low in cost.

Description

Technical Field

[0001] This invention relates to a nano-phase extract of Gynostemma pentaphyllum. Background Technology

[0002] Traditional research on traditional Chinese medicine (TCM) compound decoctions has primarily focused on chemical components (such as monomers like puerarin and glycyrrhizic acid) and their pharmacological activities, but it has failed to fully explain the mechanisms of synergistic effects within these decoctions. Studies have shown that nanophases in TCM decoctions (such as colloidal self-assembled nanoparticles) may be the key physical structural basis for pharmacodynamic efficacy. For example, puerarin and glycyrrhizic acid in Ge Gen Qin Lian Tang significantly improve bioavailability by forming micellar structures, while the nanophases in Bai Hu Tang exhibit better antipyretic effects and lung-brain targeting compared to other phases. However, systematic research on TCM nanophases remains lacking, particularly in the theoretical framework for elucidating the mechanism of pharmacodynamic substance delivery from a phase perspective.

[0003] In the processing of traditional Chinese medicine (TCM) preparations, the high shear forces or high temperatures of traditional nanotechnology (such as high-pressure homogenization and ball milling) easily disrupt the structural integrity of natural nanophases. This disruption manifests as physical structural collapse, breakage of molecular forces, and loss of components and reduced activity. The destruction of the structural integrity of TCM nanophases significantly affects drug stability, bioavailability, and therapeutic efficacy, potentially leading to degradation of active ingredients, abnormal release behavior, and loss of targeting. This process involves complex physicochemical changes, such as particle size growth, phase separation, and alterations in surface properties, posing significant challenges to preparation processes, storage conditions, and in vivo delivery. Current technical challenges lie in how to precisely control the stability of nanostructures in large-scale production, while balancing the relationship between drug loading, encapsulation efficiency, and structural integrity, and developing reliable evaluation systems to monitor dynamic changes. Furthermore, the synergistic regulation of structure maintenance and controlled release when overcoming biological barriers, as well as batch consistency control in industrial production, are all critical scientific issues that urgently need to be addressed.

[0004] The hypercoagulable state and microthrombus formation in the early stages of metabolic-associated steatohepatitis (MASH) are core pathological mechanisms driving the progression of liver fibrosis. While existing anticoagulants (such as warfarin and DOACs) can inhibit coagulation, they suffer from high bleeding risk, narrow therapeutic window, and insufficient regulation of MASH-specific pathological changes. Furthermore, long-term use may exacerbate liver function burden, limiting their application in chronic liver diseases. Therefore, there is an urgent need to develop anticoagulants that combine multi-target intervention, high safety, and liver-targeting properties.

[0005] Traditional Chinese medicine nanoparticles, due to their unique physicochemical properties and multi-target synergistic effects, have shown significant advantages in early anticoagulation therapy for MASH (metastatic hypercoagulability). Nanoparticles (1-100 nm in particle size) in the decoction of *Gynostemma pentaphyllum* can achieve multi-level intervention in hypercoagulable states by inhibiting coagulation factor Xa and thrombin activity, blocking fibrin formation, and regulating inflammatory responses. Preliminary experiments show that its nanophase exhibits higher liver targeting and pharmacodynamic activity compared to other phases, which is closely related to the ultrastructure and high specific surface area of ​​the nanoparticles.

[0006] In summary, existing technologies have the following shortcomings: (1) Traditional Chinese medicine research relies excessively on chemical component analysis, neglecting the efficacy contribution of nanophases; (2) it damages the structural integrity of natural nanophases, resulting in the loss of target components; and (3) MASH anticoagulation therapy lacks specific drugs targeting hepatic microcirculation disorders. This invention fills the above-mentioned technological gaps by analyzing the phase characteristics and anticoagulation mechanism of *Gynostemma pentaphyllum* nanocomponents.

[0007] Currently, the main techniques used in the phase separation of traditional Chinese medicine include ultracentrifugation, microporous membrane filtration, dialysis, ultrafiltration, and size exclusion chromatography. Each of these methods has its limitations.

[0008] While ultracentrifugation (e.g., 6,000-12,000 rpm) can initially separate precipitate and colloidal phases, a single rotation speed is insufficient to accurately distinguish nanoscale phase groups of 50-200 nm, and high rotation speed can lead to the destruction of nanostructures.

[0009] Microporous membrane filtration (such as 0.45 / 0.22 μm filtration) can separate true solution phases, but it cannot effectively retain colloidal phases (1-100 nm), and the membrane pores are easily clogged, affecting reproducibility.

[0010] Dialysis (such as 3.5kDa dialysis bags) is suitable for the separation of small molecules, but it is time-consuming (requiring several hours to several days) and has low retention efficiency for nanophases.

[0011] Although ultrafiltration (such as 3kDa ultrafiltration tubes) can separate colloidal phases, it is complicated to operate, requires multiple centrifugations, and has a fixed molecular weight cutoff range, which lacks flexibility.

[0012] Size exclusion chromatography (such as dextran gel chromatography) has high separation efficiency, but the equipment cost is high and the sample processing capacity is small, making it difficult to apply on a large scale.

[0013] (1) It is impossible to accurately separate nanoscale phases within a specific particle size range (such as 50-200 nm);

[0014] (2) The separation process may cause loss or damage to the target components;

[0015] (3) It is difficult to maintain the structural integrity of the natural nanophase state;

[0016] (4) Currently, no studies have shown that nanophase extracts have significant effects in improving metabolic dysfunction-related fatty liver disease and its anticoagulant effects;

[0017] (5) Low separation efficiency, making it difficult to scale up applications;

[0018] (6) The equipment is complex and costly.

[0019]

[0020] References:

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[0029] *Penthorum chinense* Pursh, also known as *Chegencai*, *Shuiyangliu*, and *Shuizelan*, is a famous "liver-protecting herb" in Miao medicine. It is mainly produced in the wetlands of the Wumeng Mountains at an altitude of about 1000m in Gulin, Sichuan and Bijie, Guizhou. The whole plant is used medicinally; it is green in color and has a slightly bitter-sweet taste. Traditionally, it is classified as "sweet and cold, entering the liver and gallbladder meridians." Modern research has isolated flavonoids (quercetin, kaempferol), gallic acid, β-sitosterol, saponins, and polyphenols. The *Sichuan Provincial Standard for Traditional Chinese Medicine* stipulates that the dried product contains ≥0.10% quercetin and the extract contains ≥14.0%, which are quality evaluation indicators. Application number: CN201410273347.3, invention title: Provides the use of *Penthorum chinense* or its extract in the preparation of health products or medicines for the prevention or treatment of non-alcoholic fatty liver disease. This invention demonstrates that *Gynostemma pentaphyllum* and its extracts have good preventive or therapeutic effects on non-alcoholic fatty liver disease (NAFLD). They can effectively reduce serum ALT and TBIL levels, decrease serum TG levels, increase serum HDL-C levels, reduce TG and NEAF content in liver tissue, increase GSH-PX activity, and alleviate hepatic steatosis, providing a new option for clinical treatment of NAFLD. The literature discloses that the extract of *Gynostemma pentaphyllum* is an aqueous and / or ethanol extract.

[0030] Current research on Gynostemma pentaphyllum mainly focuses on liver protection (such as lowering enzymes, reducing jaundice, and antiviral activity) and antifibrosis. For example, clinical studies have shown that its ALT seroconversion rate in acute icteric hepatitis reaches 92%, but its anticoagulation mechanism in fatty liver disease has not been mentioned.

[0031] Research on extracts of Gynostemma pentaphyllum mainly focuses on the following types of extracts and their preparation methods. Each type of extract has different limitations in terms of processing or application:

[0032] 1. Water extract and alcohol extract

[0033] The crude extracts of *Gynostemma pentaphyllum* obtained by traditional water extraction or alcohol extraction (such as 70% ethanol extraction) mainly contain flavonoids (such as quercetin and kaempferol) and organic acids (such as gallic acid). The drawbacks are: in water extraction, flavonoid glycosides have poor water solubility and low bioavailability due to their glycoside structure; while alcohol extracts have higher activity, they suffer from a pronounced medicinal and raw taste, are prone to precipitation during long-term storage, and have low extraction efficiency, making it difficult to selectively enrich specific active ingredients. Furthermore, traditional methods cause significant damage to heat-sensitive components (such as some flavonoid glycosides), affecting the stability of the final product.

[0034] 2. Enzymatic extract

[0035] In recent years, enzymatic hydrolysis extracts (such as the complex enzymatic hydrolysis products of hemicellulase and β-glucosidase mentioned in the patent) have improved the conversion rate and extraction efficiency of flavonoid aglycones by degrading cell walls and glycosidic bonds. However, such methods have obvious drawbacks: the enzymatic hydrolysis conditions (such as pH and temperature) need to be precisely controlled, otherwise enzyme inactivation is likely to occur; the ratio of complex enzymes needs to be optimized (such as the ratio of hemicellulase to β-glucosidase), otherwise the synergistic effect will be insufficient; in addition, the enzymes need to be inactivated at high temperature (90°C) after enzymatic hydrolysis, which may affect the activity of heat-labile components.

[0036] 3. Ultra-high pressure extraction technology

[0037] Ultra-high pressure extraction (UHPE) technology is mainly used for enriching saponins from *Gynostemma pentaphyllum*, characterized by low temperature and high efficiency. However, its drawbacks include high equipment costs, difficulty in large-scale production, and its current application only to saponins; its applicability to other active ingredients (such as flavonoids) has not been fully verified. Furthermore, UHPE treatment may alter the physical properties of some components (such as hygroscopicity), affecting the stability of the formulation.

[0038] 4. Compound formula extract

[0039] While the *Gynostemma pentaphyllum* complex (such as when combined with purple glutinous wheat bran and phlorizin extract) can synergistically enhance lipid-lowering effects for non-alcoholic fatty liver disease, its drawback lies in the increased complexity of its components, which may lead to greater difficulties in quality control (such as fluctuations in the content of active ingredients between different batches). Furthermore, other components in the complex (such as phlorizin) may introduce new side effects risks, requiring additional safety assessments.

[0040] 5. Concentrates of single active ingredients

[0041] Some studies have obtained high-purity monomers (such as ellagic tannin-type flavonoids G2 and G3) through column chromatography, which exhibit significant antioxidant and anti-hepatocellular carcinoma activities. However, the drawbacks of these extracts are the cumbersome separation and purification steps, low yield (e.g., G3 was only isolated from *Gynostemma pentaphyllum* for the first time, with a limited yield), and the high-purity monomers may lose the multi-target synergistic effects of traditional extracts. Their clinical application value requires further verification. Summary of the Invention

[0042] This invention provides a nano-phase extract of *Gynostemma pentaphyllum*, its preparation method, and its uses.

[0043] This invention provides a nano-phase extract of Gynostemma pentaphyllum, which is made by soaking and decocting Gynostemma pentaphyllum, filtering, collecting the filtrate, centrifuging in stages to remove the precipitate, dialyzing the supernatant using a dialysis bag with a molecular weight cutoff of 3.5 kDa, and freeze-drying the dialysate to obtain the nano-phase extract of Gynostemma pentaphyllum.

[0044] The particle size of the nano-phase extract of *Gynostemma pentaphyllum* is 50-200 nm.

[0045] The particle size of the nano-phase extract of *Gynostemma pentaphyllum* is 100-150 nm, preferably 120 nm.

[0046] The nano-phase extract of *Gynostemma pentaphyllum* has a PDI of 0.25 and a potential between -15 and -20 mV.

[0047] This invention provides a method for preparing nano-phase extract of *Gynostemma pentaphyllum*, which includes the following steps:

[0048] a. Soaking: Mix the herbs with purified water and soak at room temperature;

[0049] b. Decoction: Heat and decoct the soaked medicinal materials;

[0050] c. Filtration: After decoction, filter the liquid while it is still hot using a filter cloth or filter paper, collect the filtrate and discard the dregs;

[0051] d. Centrifugation in stages: Place the filtered extract in a centrifuge and centrifuge to obtain the supernatant;

[0052] e. Collection of supernatant: After each centrifugation, discard the precipitate and retain the supernatant;

[0053] f. Dialysis bag selection: Use a dialysis bag with a molecular weight cutoff of 3.5kDa (MWCO 3.5kDa). Fill the dialysis bag with the supernatant and place the dialysis bag in deionized water for dialysis.

[0054] g. Freeze-drying preservation: After dialysis, take the dialysate from the dialysis bag and freeze-dry it to obtain the nano-phase extract.

[0055] In step d, centrifugation is carried out at different speeds: 4000 rpm, 6000 rpm, 8000 rpm and 10000 rpm, for a total of 30 minutes.

[0056] In step f, the dialysis time is 12-48 hours, and the water is changed every 3 hours.

[0057] This invention provides the use of the aforementioned nano-phase extract of *Gynostemma pentaphyllum* in the preparation of a medicament for treating fatty liver disease.

[0058] The drug mentioned is one that improves the anticoagulation effect of metabolic dysfunction-related steatohepatitis.

[0059] High-speed hierarchical centrifugation combined with dialysis significantly improves separation accuracy and efficiency by progressively separating different particle sizes using gradient centrifugation. Its advantages include: (1) precise separation: gradient centrifugation can target and capture nano-active components of 50-200 nm, avoiding the limitations of single-speed centrifugation; (2) structural protection: gentle centrifugation parameters (e.g., 30 min / step) reduce shear stress damage to nanostructures; (3) no solvent residue: no organic solvents are required throughout the process, meeting the safety requirements of traditional Chinese medicine; (4) activity retention: the obtained nano-phase extracts show significant effects in improving metabolic dysfunction-related fatty liver disease. This method optimizes the phase separation process through physical means, solving problems such as inaccurate separation, low efficiency, and structural damage associated with traditional techniques, providing a reliable solution for the study of nano-phases in traditional Chinese medicine.

[0060] This invention's self-assembled nano-components of *Gynostemma pentaphyllum* significantly overcome several shortcomings of traditional extracts through their unique nanostructure characteristics: In terms of bioavailability, nanoparticles (around 125 nm) enhance intestinal absorption and liver targeting; for example, baicalin-berberine self-assembled nanoparticles promote absorption by regulating tight junctions in the intestinal epithelium, while traditional water extracts suffer from low utilization rates due to poor water solubility of flavonoid glycosides. Regarding stability, self-assembly forces such as hydrogen bonding and electrostatic interactions protect heat-sensitive components like quercetin, delaying degradation, whereas alcohol extracts are prone to precipitation and high-temperature processing destroys their activity. In terms of synergistic effects, the nano-components retain the natural complex structure of polysaccharides, proteins, and flavonoids, exerting multi-target effects, while single-component enrichments (such as total lignans) may lose their synergistic effect. Furthermore, the nano-components require no organic solvents and are encapsulated by natural carriers, reducing gastrointestinal irritation, while complex enzymatic hydrolysates require high-temperature enzyme inactivation, introducing impurities.

[0061] The effective effects of this invention are:

[0062] This invention provides a method for precisely separating 50-200nm active components from the nano-phase extract of *Gynostemma pentaphyllum*. This method avoids the use of organic solvents, improving the safety of the extracted product; increases the purity of the target components; maintains the structural integrity of the natural nano-phase; and the obtained nano-phase extract exhibits significant effects in improving metabolic dysfunction-related fatty liver disease and its anticoagulant properties. The method boasts high separation efficiency, completing the process in 2-3 days, and is suitable for large-scale application. The equipment is simple and low-cost. Attached Figure Description

[0063] Figure 1 Transmission electron microscope image;

[0064] Figure 2 Characterization data of nano-phase extract of *Gynostemma pentaphyllum*;

[0065] Figure 3 Figure showing the experimental results of the anticoagulant effect of the nano-phase extract of *Gynostemma pentaphyllum* in this invention;

[0066] Figure 4 The safety of the heart, spleen, lungs and kidneys was evaluated using HE staining;

[0067] Figure 5 Three pathological staining methods for liver tissue were used to evaluate the progression of fibrosis.

[0068] Figure 6 Changes in immunofluorescence of Fibronectin (FN) and α-SMA in each group after drug administration;

[0069] Figure 7 Changes in Collagen-I and Collagen-III immunofluorescence in each group after drug administration;

[0070] Figure 8 Transcriptome results;

[0071] Figure 9 Changes in CD61 / CD31 and CD62 / CD31 immunofluorescence in each group after drug administration;

[0072] Figure 10 Results of coagulation panel tests;

[0073] Figure 11 Liver function (AST, ALT) results;

[0074] Figure 12 Western blotting in vitro validated that the nano-phase extract of Gynostemma pentaphyllum exerts its therapeutic effect through the PI3K-AKT-mTOR pathway.

[0075] Figure 13 Liver tissue pathological staining results;

[0076] Figure 14 Changes in Collagen-I and Collagen-III immunofluorescence in each group after gene knockout and drug administration;

[0077] Figure 15 Changes in Fibronectin (FN) and α-SMA immunofluorescence in each group after gene knockout and drug administration;

[0078] Figure 16 Changes in CD61 / CD31 and CD62 / CD31 immunofluorescence in each group after gene knockout and drug administration;

[0079] Figure 17 Western blotting in vitro validated that the nano-phase extract of *Gynostemma pentaphyllum* exerts its therapeutic effect through the PI3K-AKT-mTOR pathway. Detailed Implementation

[0080] Example 1: Preparation of the nano-phase extract of *Gynostemma pentaphyllum* according to the present invention

[0081] (1) Soaking: Mix the medicinal materials of Ganhuang with purified water at a ratio of 1:10 (mass ratio), soak at room temperature for 30 minutes to allow the medicinal materials to fully absorb water, and then soak in RO water.

[0082] (2) Decoction: Heat the soaked herbs to a boil, then reduce to a simmer and continue decocting for 60 minutes.

[0083] (3) Filtration: After decoction, filter the liquid while it is still hot using a filter cloth or filter paper, collect the filtrate and discard the dregs (coarse filtration).

[0084] (4) Layered centrifugation: Place the filtered extract in a centrifuge and centrifuge at different speeds (4000 rpm, 6000 rpm, 8000 rpm and 10000 rpm) for 30 minutes each. The purpose is to gradually remove larger particles and precipitates to obtain a purer extract.

[0085] (5) Collection of supernatant: After each centrifugation, discard the precipitate and retain the supernatant to ensure that the obtained liquid contains the target nanostructure.

[0086] (6) Dialysis bag selection: Use a dialysis bag with a molecular weight cutoff of 3.5 kDa (MWCO 3.5 kDa). Fill the dialysis bag with the supernatant and place it in deionized water for dialysis to remove low molecular weight impurities, such as small molecules and non-nanostructured components, ensuring the purity of the obtained nanostructured sample. Dialysis time is 12 hours, with water changed every 3 hours. When the 12-hour dialysis is complete and the sample is ready to be collected, observe the color of the water. If the water is not particularly pure, extend the dialysis time, but the total time should not exceed 48 hours.

[0087] (7) Freeze-drying preservation: After dialysis, take the dialysate from the dialysis bag and freeze-dry it to obtain the nano-phase extract.

[0088] The reasons for gradient centrifugation are as follows:

[0089] ① Separation of nano-components with different particle sizes: By gradually increasing the rotation speed, large particles or high-density components are preferentially settled, and then small particles are separated by a higher rotation speed.

[0090] ② Avoid structural damage to nano-components: Low-temperature centrifugation (such as high-speed refrigerated centrifuges) can reduce the risk of denaturation of heat-sensitive components (such as protein nanoparticles), achieving the purpose of gentle centrifugation and preserving their complete structure.

[0091] ③ Improve the purity of the target component: The decoction contains free small molecules, colloids and other interfering substances. Centrifugation can quickly separate nanoparticles from impurities in the supernatant and remove impurities.

[0092] ④ Adapt to the stability requirements of self-assembled nanoparticles: shorten processing time and reduce particle aggregation.

[0093] ⑤ Provide homogeneous samples for subsequent research: Verify the particle size distribution of nanoparticles obtained after centrifugation to ensure experimental repeatability.

[0094] In practice, we initially chose to use an ultrafiltration tube for extraction (model: Millipore ultrafiltration tube inner tube 15ml 10KD). When centrifuging at high speed (8000-10000rpm) with this tube, the filter membrane was prone to breakage (it is suspected that when the purity of the liquid in the bottle is not high enough, the filter membrane is prone to blockage at high speed, resulting in filter membrane breakage and leakage). In addition, the efficiency is low, and less than 15ml can be extracted at a time, so the overall cost performance is not high.

[0095] Based on this, we selected mild gradient centrifugation. Compared to traditional gradient centrifugation, which requires multiple adjustments to the rotation speed or changes to the gradient medium (such as sucrose or iodixanol), making the process cumbersome and prone to errors, the fixed-time-multi-rotation method only requires setting a single time variable, making it simpler to operate and particularly suitable for high-throughput screening or industrial production.

[0096] The differences between the fixed-time, multi-speed gradient centrifugation method and the traditional centrifugation method are as follows:

[0097] parameter Fixed-time multi-speed gradient centrifugation Traditional gradient centrifugation Operational complexity Low (single-time variable) High (requires gradient preparation, multi-step centrifugation) Media interference risk none (Sucrose / cesium chloride may affect particle size) Scope of application Wide coverage (covering particles from 50nm to 1μm) Narrow (gradient needs to be optimized for target particles) Equipment Requirements Standard centrifuge (≤10000rpm) Ultracentrifuge (>30,000 rpm) Time cost Short (30 minutes / rpm) Long (2 hours to 18 hours)

[0098] Example 2: Feasibility of the present invention's nano-phase extract nano-components of *Gynostemma pentaphyllum*

[0099] 1. Extraction

[0100] Extract using the steps described in the embodiments

[0101] 2. Verification

[0102] Dynamic light scattering (DLS) was performed using a particle size analyzer (laboratory instrument on the 25th floor of the Second Affiliated Hospital of Southwest Medical University). The results included particle size and PDI.

[0103]

[0104] The results showed that the particle size was around 120 nm, and the PDI was around 0.25, which is consistent with the nanoscale range for liver targeting. Furthermore, the PDI indicated good dispersibility and stability. Based on this, transmission electron microscopy (TEM) images were taken (see...). Figure 1 ).

[0105] Microscopic results showed that the extracted nanoparticles of *Gynostemma pentaphyllum* extract exhibited distinct nanoscale characteristics, with high density and uniform size per unit area. These experimental results demonstrate that the gradient centrifugation-dialysis method for extracting nanoparticles from *Gynostemma pentaphyllum* is a feasible approach.

[0106] 3. Characterization

[0107] 3.1 Experiment Content

[0108] In addition to the DLS and TEM detection methods mentioned above, the characterization of nano-phase *Gynostemma pentaphyllum* extract nanoparticles also includes: ultraviolet fluorescence analysis, fluorescence excitation and emission wavelength, X-ray diffraction (XRD), Fourier transform infrared (FTIR), thermogravimetric analysis (TGA), electrical conductivity, resistivity, surface tension, and simulation of artificial gastrointestinal fluid environment.

[0109] 3.2 Experimental results are shown in Figure 2 :

[0110] 3.3 Analysis of Experimental Results:

[0111] Figure 2 A: Tyndall effect manifestation. The intensity of scattered light is directly proportional to the concentration of nanoparticles; red light has strong refraction, indicating a high concentration of nanoparticles in the nano-phase extract of *Gynostemma pentaphyllum*.

[0112] Figure 2 BC: TEM results and particle size distribution statistics. TEM showed that the nanoparticles of the *Gynostemma pentaphyllum* extract had a high distribution density and uniform size. Statistical analysis of the nanoparticle size distribution presented by TEM using ImageJ software revealed a histogram distribution characteristic, with D = 125.66 ± 22.73 nm. The particle size was relatively concentrated, with the concentration point at 125.66 nm, which is consistent with the optimal nanoparticle size range for liver (100-150 nm).

[0113] Figure 2D: Ultraviolet fluorescence analysis. The 280nm shoulder peak confirms the presence of protein components. The peak shape is relatively symmetrical after 250nm, indicating good purity. The absorption is low in the 400-800nm ​​range, with low UV background, indicating good dispersibility.

[0114] Figure 2 E: Fluorescence excitation and emission wavelength results. The EX excitation wavelength is high and broad at 400-450 nm, indicating a conjugated structure of flavonoids. The EM emission wavelength is high and broad at 520-600 nm, representing an intramolecular charge transfer state and the energy release after core excitation.

[0115] Figure 2 F: Fluorescence emission spectrum. This shows the variation of fluorescence intensity with wavelength for samples of different concentrations. The results for 0.125 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml, and 5 mg / ml show that a plateau is reached at 2 mg / ml. This indicates that when preparing nano-formulations, setting the concentration between 1 and 2 mg / ml can obtain the best fluorescence characteristics with minimal variation and stability.

[0116] Figure 2 GH: Results of DLS particle size and potential measured over seven consecutive days. Particle size and potential were measured once each from day one to day seven. The nano-phase extract of *Gynostemma pentaphyllum* was concentrated at a size of about 140 nm, with a PDI of about 0.25 and a potential of -15 to -20, exhibiting moderate uniformity and stability.

[0117] Figure 2 I: XRD results. The main peak at 15° represents the amorphous polysaccharide network, while the micro-peak at 26.7° reflects the flavonoid nanocrystal nuclei. Combined with FTIR correlation at 1618 cm⁻¹, this indicates hydrophobic cores <5 nm microcrystals. The shoulder peak at 43° represents carbonization traces, matching the TGA residues, providing evidence of inorganic residues. Therefore, the amorphous polysaccharide network exhibits strong water solubility, easy modification of targeting groups, high drug loading capacity, sustained release, and a stable carbon skeleton.

[0118] Figure 2 J: TGA results. Decomposition temperature >100℃, stable at room temperature. Residual rate is relatively high ≈35%, organic matter weight loss ≈65%, indicating a high proportion of active ingredients. Overall, the temperature steadily decreases from 100℃ to 600℃ without obvious step-like decomposition. This is consistent with the typical characteristics of traditional Chinese medicine nanotechnology.

[0119] Figure 2 K: FTIR results. A broad peak and strong absorption at 3419.7 indicates OH stretching vibration (hydrogen bond association), suggesting enrichment of polysaccharides / polyphenols through water decoction. A sharp peak and moderate intensity at 1618.3 indicates aromatic ring C=C stretching or conjugated C=O, indicating dialysis concentration of the hydrophobic core. A sharp peak and strong absorption at 1070.1 indicates COC glycosidic bond vibration, reflecting a polysaccharide backbone-dominated structure.

[0120] Figure 2 LM: Conductivity and resistivity results. These reveal the pressure-responsive conductivity behavior of the nanoparticle system. The low-pressure sensitive region (<5MPa) reflects dominant particle rearrangement, with a sharp drop in resistivity, suitable for high-sensitivity sensing. The high-pressure saturation region (>5MPa) reflects the completion of structural densification, with conductivity tending to stabilize.

[0121] Figure 2 N: Surface tension results. The nanosuspension exhibits typical adsorption characteristics of interfacial active substances, and its low equilibrium IFT (0.0145 mN / m) and dynamic variation trend reveal the key role of nanoparticles in interfacial remodeling.

[0122] Figure 2 O: In a physiological saline environment, the particle size remained at 140–150 nm with almost no aggregation or degradation, making it suitable for intravenous injection or physiological environment applications. In an artificial intestinal fluid environment, the particle size decreased from 120 nm to 100 nm (a decrease of approximately 17%) within 0–6 hours, due to dehydration and shrinkage caused by intestinal fluid components (such as NaHCO3). After 6 hours, the particle size stabilized, indicating a dynamic equilibrium was reached. In an artificial gastric fluid environment, the particle size fluctuated slightly between 130–140 nm (fluctuation of approximately 7.7%), slightly lower than the physiological saline group, which is related to the low pH (approximately 1.2–3.0) and pepsin activity in the gastric fluid.

[0123] This invention successfully solves the problems of inaccurate separation of nanophases, low purity, and structural damage in existing technologies, providing new technical means for the modernization research of traditional Chinese medicine.

[0124] The following pharmacodynamic tests demonstrate the beneficial effects of this invention.

[0125] Experimental Example 1: Anticoagulant Effect Test of the Nano-Phase Extract of the Present Invention

[0126] 1. Experiment Content

[0127] 1.1 Application of Western Blot

[0128] Detecting p-Src and p-Syk levels can serve as markers for evaluating platelet activation. HUVECs cells were modeled with 100 μM ADP for 24 h, followed by administration of a nano-phase extract of *Gynostemma pentaphyllum* at 4 mg / ml for 24 h. Protein extraction was then performed using cold RIPA lysis buffer (Beyotime, CAT: P0013B), and protein quantification was performed using the bichomycin acid (BCA) method. Protein samples were separated using SDS-PAGE and then transferred to a PVDF membrane (Merck Millipore, CAT: IPVH00010). After incubation at 37°C for 2 hours in 5% bovine serum albumin (BSA), primary antibodies including GAPDH (1:1000), Src (1:1000), Syk (1:1000), p-Src (1:1000), and p-Syk (1:1000) were added overnight at 4°C. After washing, the cells were incubated with HRP-labeled secondary antibody at 37°C for 1 hour. The bands were visualized using ECL blot technology (Beyotime, CAT: P0018S).

[0129] 1.2 Four coagulation tests in rats

[0130] A male adult Wistar rat, weighing 350g, was obtained from the breeding area of ​​the experimental animal facility at the North Campus of Southwest Medical University. After routine anesthesia, the abdominal aorta was exposed, and blood was collected in 7.5ml blue-top anticoagulant tubes containing an anticoagulant-to-blood ratio of 1:9 (i.e., 1 part sodium citrate anticoagulant to 9 parts blood). After shaking and mixing, 1.5ml of the sample was transferred to 15 1.5ml EP tubes, divided into three groups of five tubes each. The first group was the normal control group (NC), with no drugs added; the second group was the ES enoxaparin sodium positive control group, with 10mg / ml enoxaparin sodium added and mixed well; the third group was the nano-phase *Gynostemma pentaphyllum* extract nano-drug group, with 40mg / ml added and mixed well. All samples were immediately sent to the Department of Laboratory Medicine at the Affiliated Hospital of Southwest Medical University for coagulation tests.

[0131] 2. Experimental results (see...) Figure 3 )

[0132] 3. Analysis of Experimental Results

[0133] Figure 3 AF: Results of anticoagulation indicators in Western Blot. Regardless of the presence of ADP, treatment with nano-phase *Gynostemma pentaphyllum* extract had no significant effect on the expression levels of total Syk and Src proteins in each group, indicating that nano-phase *Gynostemma pentaphyllum* extract significantly inhibited ADP-induced Syk and Src phosphorylation in a dose-dependent manner, but had no effect on total protein levels, suggesting that it works by blocking kinase activation (rather than reducing protein expression).

[0134] Figure 3 GL: Results of coagulation parameters in rats after blood collection and drug administration. The nano-phase *Gynostemma pentaphyllum* extract group prolonged clotting time and interfered with fibrin formation. Specifically, the nano-phase *Gynostemma pentaphyllum* extract showed the highest values ​​among all coagulation parameters, indicating that it delays the coagulation process by inhibiting extrinsic (PT↑) and intrinsic (APTT↑) coagulation pathways. Simultaneously, fibrinogen (K) and thrombin time (TT) increased, suggesting that the nano-phase *Gynostemma pentaphyllum* extract inhibits fibrinogen activation or fibrin cross-linking. The ES group's parameters were all between those of the NC and nano-phase *Gynostemma pentaphyllum* extract, showing partial anticoagulant effect, but weaker than that of the nano-phase *Gynostemma pentaphyllum* extract. The NC group, as the control group, had the lowest coagulation parameters, consistent with normal coagulation function.

[0135] Experimental Example 2: The nano-phase extract of *Gynostemma pentaphyllum* exhibits in vitro and in vivo anti-fibrotic effects.

[0136] 1. Experiment Content

[0137] 1.1 Animal Experiments

[0138] Forty-eight male C57 mice (weighing 20-22g, 6-8 weeks old) were obtained from Jicui Pharmaceutical Co., Ltd. and acclimatized for one week. The mice were adapted to 22±0.5℃ under a 12h light-dark cycle and had unrestricted access to regular diet and drinking water. The animal care and treatment protocol was approved by the Animal Research Committee of Southwest Medical University. The 48 mice were divided into 8 groups: a normal control group given a regular diet (ND), and the other 7 groups (n=6 per group) given a high-fat diet (HFD). All groups except the normal control group received intraperitoneal injections of 20% CCl4 at a dose of 20g / 100μl for 7 weeks. Animals fed with HFD were randomly divided into the following groups: untreated model group (Control), *Gynostemma pentaphyllum* suspension (HXX) drug administration group (100 mg / ml), *Gynostemma pentaphyllum* precipitate (CDX) drug administration group (100 mg / ml), *Gynostemma pentaphyllum* true solution (ZRY) drug administration group (100 mg / ml), *Gynostemma pentaphyllum* colloidal phase nano-JTX drug administration group (100 mg / ml), anhydrous ethanol extract drug administration group (Alcohol, 100 mg / ml), and positive control liver-soothing granules drug administration group (5.5 g / kg). All prepared extracts and drugs were administered orally once daily at a dose of 2 mL / 100 g body weight for 4 consecutive weeks.

[0139] The extraction methods for the suspension phase, precipitate phase, and true solution phase are as follows:

[0140] ① Suspension Phase: The aqueous decoction was separated by gradient centrifugation, with fractional centrifugation performed sequentially at speeds of 4000 rpm, 6000 rpm, 8000 rpm, and 10000 rpm. The supernatant was collected after each centrifugation and transferred to the next centrifugation stage for further centrifugation, ultimately obtaining a fractionated suspension phase. This method improves the enrichment efficiency of the target component by progressively separating particles of different sizes using varying centrifugal forces. (The suspension phase is the undialyzed crude extract.)

[0141] ② Precipitated phase: During the gradient centrifugation of the suspension phase, retain the precipitate after each centrifugation. After discarding the supernatant, add an appropriate amount of RO water (reverse osmosis pure water) to the precipitate, sonicate for 1-2 minutes to fully suspend the particles, collect the suspension and then freeze-dry it.

[0142] ③ True solution phase: Place the suspension in a dialysis bag and dialyze in deionized water for 1-2 days. Do not change the dialysate on the first day. On the second day, collect the dialysate outside the bag (i.e., the true solution phase), concentrate it by rotary evaporation, and then freeze-dry it for storage.

[0143] ④ Colloidal phase (nanophase): In the true solution extraction step, the liquid retained in the dialysis bag is the colloidal phase. The colloidal phase in the bag is obtained after 2 days and then lyophilized for later use.

[0144] 1.2 Hematoxylin-eosin (H&E) staining

[0145] After the experiment, tissues from the five internal organs were collected, fixed in 4% paraformaldehyde for 72 hours, then embedded in paraffin and cut into 4-micrometer-thick sections. The paraffin sections were dewaxed with xylene and then rehydrated using a gradient of ethanol (100%, 95%, 90%, and 85%). Cell nuclei were stained with hematoxylin for 10 minutes, followed by differentiation in acidified ethanol (0.5% hydrochloric acid in 70% ethanol) for 5 seconds. The cytoplasm was counterstained with eosin for 2 minutes. Afterward, the sections were dehydrated using an ethanol gradient, cleared in xylene for 5 minutes, and finally mounted with a neutral mounting medium for microscopic examination.

[0146] 1.3 Masson's Tricolor Staining Method

[0147] Aortic sinus tissue sections that had been dewaxed and rehydrated were first stained with hematoxylin to reveal cell nuclei, and then immersed in...

[0148] Myofibrils were labeled using a mixture of Ponceau S and acid fuchsin. Cell types were differentiated using 1% phosphomolybdic acid solution, while collagen fibers were stained with aniline blue. After dehydration, the sections were washed with xylene and mounted with neutral resin, ultimately revealing distinct color differences: collagen fibers appeared blue, myofibrils red, and cell nuclei bluish-black.

[0149] 1.4 Sirius Red Staining

[0150] Dewaxed tissue sections were incubated in 0.1% Sirius red staining solution (dissolved in saturated picric acid solution) for 1 hour. Excess dye was then removed by brief separation with 0.5% acetic acid, followed by thorough rinsing with running water. Cell nuclei were counterstained with hematoxylin. After dehydration and xylene clearing, sections were mounted with neutral resin.

[0151] 1.5 Immunofluorescence staining

[0152] Frozen sections of the five internal organs (4 μm thick) were first fixed with 4% glutaraldehyde, then permeabilized with 0.1% Triton X-100, and subsequently blocked with 5% goat serum at room temperature for 1 hour. Primary antibody was added and incubated overnight at 4°C. After three washes with PBS (including one extended wash of 10 minutes), the sections were incubated at 25°C with fluorescently labeled secondary antibody (1:500 dilution) for 1 hour. Cell nuclei were counterstained with DAPI (Beyotime, product number C1002) for 10 minutes, then placed in 20% glycerol, and finally observed using a confocal laser scanning microscope (Leica SP8).

[0153] 1.6 Western Blotting

[0154] Protein extraction was performed using ice-cold RIPA lysis buffer (Beyotime, product number P0013B), and protein quantification was performed using a colorimetric method (BCA method). After SDS-PAGE separation, protein samples were transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, product number IPVH0001O). The membrane was blocked with 5% bovine serum albumin (BSA) at 37°C for 2 hours, followed by overnight incubation with primary antibody (1:1000 dilution) at 4°C. After washing, the membrane was incubated with a horseradish peroxidase (HRP)-bound secondary antibody (1:5000) at 37°C for 1 hour. Protein bands were detected using enhanced chemiluminescence (ECL) blot substrate (Beyotime, product number P0018S).

[0155] 2. Experimental results (see...) Figures 4-12 )

[0156] 3. Analysis of Experimental Results

[0157] Figure 4Based on histopathological examination of the tissue sections, no significant structural abnormalities or pathological damage were observed in the heart, spleen, lungs, and kidneys in any of the treatment groups. Compared with the normal control group (NC), the organ tissue morphology of the alcohol treatment group (Alch) remained largely intact, with only slight loosening of cell arrangement in a few areas. The cell morphology and tissue structure of the drug intervention groups (CDX, ZRY, JTX, GS) were basically consistent with those of the control group (Control), with no lesion features such as inflammatory infiltration, fibrosis, or necrosis. The red and white pulp boundaries of the spleen were clear, the alveolar structure of the lung tissue was intact, the glomeruli and renal tubules of the kidneys were normal in morphology, and the myocardial fibers were tightly arranged. Combined with the H&E staining results of each organ, no significant toxic reactions were induced in any of the treatment groups, indicating good safety for the five organs under the experimental conditions.

[0158] Figure 5 Pathological staining results of the liver after drug administration

[0159] H&E staining

[0160] In the normal control group (NC), hepatocytes were neatly arranged, hepatic cords were clearly defined, and no abnormalities were observed in the central vein and portal area. The nuclei (blue) and cytoplasm (red) were uniformly stained. The model control group (Control) exhibited significant pathological changes, including hepatocyte steatosis (cytoplasmic vacuolation), inflammatory cell infiltration, and localized disordered hepatic cord structure. The experimental treatment groups (e.g., Alcohol, CDX, ZRY, JTX, GS, etc.) showed hepatocyte morphology close to that of the normal group, with reduced steatosis and inflammation, suggesting that these interventions have a hepatoprotective effect.

[0161] Masson staining

[0162] In the NC group, collagen fibers (blue) were minimal, distributed only around blood vessels. In the control group, collagen fibers were significantly increased and diffusely distributed, indicating progression of liver fibrosis, such as portal enlargement and fibrous septum formation. In the experimental treatment groups (e.g., JTX, CDX, GS), collagen deposition was reduced, and the fibrous septa were thinner.

[0163] Sirius Red Dye

[0164] In the NC group, only a small amount of type I collagen (orange-red) and type III collagen (green) were observed, mainly distributed around blood vessels. In the control group, type I collagen (bright red) proliferated significantly, forming large fibrous bundles, while the proportion of type III collagen decreased, consistent with late-stage fibrosis. In the experimental treatment groups (such as JTX, ZRY, etc.), type I collagen decreased, while type III collagen relatively increased, suggesting a trend of fibrosis reversal.

[0165] Figure 6 Changes in Fibronectin (FN) and α-SMA immunofluorescence in each group after drug administration

[0166] Analysis of the images showed that, compared with the NC group (normal control group), the expression of Fibronectin (green fluorescence) and α-SMA (red fluorescence) was significantly enhanced in the Control group (model group), manifested as a denser fluorescence signal network and more obvious fibrous structures, suggesting that the model group successfully induced extracellular matrix deposition and myofibroblast activation. In the drug intervention groups (CDX, ZRY, JTX, GS), the fluorescence intensity of Fibronectin and α-SMA was significantly weaker than in the Control group, with the JTX group showing the most significant decrease in fluorescence signal, approaching the level of the NC group, indicating that these interventions exerted their therapeutic effect by inhibiting the expression of fibrosis-related proteins. The fluorescence intensity of the Alcohol group was stronger than that of other drug-treated groups, suggesting that the alcohol extraction treatment had a slightly weaker effect in improving the fibrosis process. In summary, the Control group, as a disease model, exhibited typical fibrosis characteristics, while drug intervention, especially the JTX group, showed significant anti-fibrotic effects.

[0167] Figure 7 Changes in Collagen-I and Collagen-III immunofluorescence in each group after drug administration

[0168] Fluorescence staining results showed that the Control group (model group) exhibited significantly increased collagen deposition compared to the NC group (normal control group), with significantly enhanced green fluorescence signals for both Collagen-I and Collagen-III, indicating that the model group successfully induced fibrotic lesions. In the drug intervention groups, the fluorescence signal intensity of the JTX group was significantly lower than that of the Alcohol group and the GS positive drug group, especially in the expression of Collagen-III. Specifically, the green fluorescence distribution in the JTX group was sparser and the fluorescence intensity was weaker, approaching the level of the NC group, while the fluorescence signal in the Alcohol group was similar to that of the Control group, and although the GS group showed some improvement, the effect was not as good as that in the JTX group. This result indicates that JTX has superior efficacy in inhibiting collagen deposition and can more effectively reverse the fibrotic process, with better effects than the Alcohol group and the GS positive drug group.

[0169] Figure 8 Transcriptome results

[0170] In differential gene expression between MO and NC, the PI3K-AKT pathway plays a role in regulating coagulation-related processes. GO and KEGG enrichment analyses of the DE pathway indicate that the PI3K-AKT pathway is involved in regulating coagulation-related biological processes such as platelet activation (e.g., regulation of platelet activation) and angiogenesis (e.g., positive regulation of angiogenesis). Among the coagulation-related pathways screened in Figure FG, the MAPK signaling pathway (e.g., MAPK signaling pathway) and the regulation of inflammatory mediators (e.g., inflammatory mediator regulation of TRP channels) are also involved.

[0171] Cross-regulatory relationships exist within the PI3K-AKT pathway, suggesting that PI3K-AKT influences coagulation function synergistically with these pathways. Particularly in the comparison between nanophase extract of *Gynostemma pentaphyllum* and monoclonal antibodies (MO), the number of upregulated genes significantly decreased (from 1956 to 127), while coagulation-related pathways (such as hemostasis) remained significantly enriched, further indicating that the PI3K-AKT pathway plays a central role in coagulation regulation. These results are consistent with the known regulatory functions of the PI3K-AKT pathway in coagulation and inflammatory responses, providing clues for further investigation into the molecular mechanisms of this pathway under specific pathological conditions.

[0172] Figure 9 Changes in CD61 / CD31 and CD62 / CD31 immunofluorescence in each group after drug administration.

[0173] Compared to the NC group (normal control group), the Control group (model group) exhibited significant pathological changes. Regarding the expression of CD61 (platelet marker) and CD62 (P-selectin), the Control group showed significantly enhanced and densely distributed red fluorescence signals, while the green fluorescence signal of CD31 (endothelial cell marker) was weakened and its distribution was disordered. These characteristics indicate that the model group successfully induced vascular endothelial injury and excessive platelet activation. In the drug intervention groups, the JTX group showed a significant therapeutic advantage. Its CD61 and CD62 fluorescence intensities were significantly lower than those in the Alcohol group and the GS positive drug group, approaching the normal levels of the NC group. Simultaneously, the expression pattern and signal intensity of CD31 were significantly improved, and the morphology and distribution of endothelial cells were closer to normal. In contrast, the fluorescence signal characteristics of the Alcohol group were similar to those of the Control group, indicating that alcohol treatment failed to effectively improve vascular injury. Although the GS group showed some therapeutic effect, it was inferior to the JTX group in both inhibiting CD62 expression and restoring normal CD31 distribution. These results fully demonstrate that JTX is significantly more effective than the Alcohol group and the GS positive drug group in protecting vascular endothelial function and inhibiting platelet activation, achieving vascular protection through a more efficient molecular mechanism.

[0174] Figure 10 Results of coagulation tests (four tests)

[0175] A comprehensive analysis of six coagulation indicators revealed significant changes in coagulation function in the nano-phase *Gynostemma pentaphyllum* extract-JTX group. Regarding the PT index, reflecting the extrinsic coagulation pathway, the clotting time (approximately 25 seconds) in the nano-phase *Gynostemma pentaphyllum* extract-JTX group was significantly longer than that in the NC group (approximately 10 seconds) and the GS group (approximately 12 seconds). Simultaneously, its PTA value (approximately 70%) was significantly lower than that in the NC group (approximately 100%) and the GS group (approximately 120%), while its INR value (approximately 3.0) was significantly higher than the other two groups (NC approximately 1.0, GS approximately 1.2). These data indicate that the nano-phase *Gynostemma pentaphyllum* extract-JTX significantly inhibited the activity of the extrinsic coagulation pathway. Regarding the APTT index, an intrinsic coagulation pathway indicator, the nano-phase *Gynostemma pentaphyllum* extract-JTX group (approximately 50 seconds) also showed a longer clotting time than the NC group (approximately 20 seconds) and the GS group (approximately 30 seconds), suggesting that it also inhibits the intrinsic coagulation pathway. Notably, in terms of fibrinogen levels (FIB), the nano-phase *Gynostemma pentaphyllum* extract-JTX group (approximately 1.0 g / L) showed lower levels than the NC group (approximately 1.5 g / L) and the GS group (approximately 2.0 g / L). However, in terms of TT (thromboplastin time), it exhibited a shorter clotting time (approximately 10 seconds) compared to the NC group (approximately 15 seconds) and the GS group (approximately 20 seconds). This seemingly contradictory result suggests that the nano-phase *Gynostemma pentaphyllum* extract-JTX has multi-target characteristics in regulating the coagulation process, both reducing fibrinogen levels and accelerating fibrin formation through other mechanisms. Based on these changes in coagulation parameters, it can be inferred that the nano-phase *Gynostemma pentaphyllum* extract-JTX exerts its unique anticoagulant effect by simultaneously affecting multiple coagulation stages. This multi-pathway regulatory mode makes it significantly different from the GS group, providing important clues for further research into its mechanism of action.

[0176] Figure 11 Liver function (AST, ALT) results

[0177] The JTX group showed significantly better performance than the Alcohol group and the GS positive control group in terms of AST and ALT levels. Its AST (~100 U / L) and ALT (~50 U / L) levels were close to those of the normal control group (NC group). In contrast, the Alcohol group's AST (~900 U / L) and ALT (~500 U / L) levels remained significantly higher. While the GS group was better than the Alcohol group (AST ~200 U / L, ALT ~100 U / L), it still did not achieve the protective effect of JTX. This indicates that JTX has a greater advantage in repairing hepatocyte damage.

[0178] Figure 12 Western blotting in vitro validates that nano-phase extracts of *Gynostemma pentaphyllum* exert therapeutic effects through the PI3K-AKT-mTOR pathway.

[0179] Western blot results showed that the phosphorylation level of the PI3K-AKT-mTOR signaling pathway was significantly inhibited in a dose-dependent manner with increasing concentrations of nano-phase *Gynostemma pentaphyllum* extract (0-8 μg / mL). Under Ang II (3 μM) stimulation, the protein band intensities of p-mTOR (250 KD), p-PI3K (54 KD), and p-AKT (62 KD) gradually decreased with increasing concentrations of nano-phase *Gynostemma pentaphyllum* extract, with the phosphorylation level in the 8 μg / mL nano-phase *Gynostemma pentaphyllum* extract treatment group being close to that of the negative control (NC group). Notably, the total protein expression levels of mTOR, PI3K, and AKT remained relatively stable across all treatment groups, and the β-actin (42 KD) internal control also confirmed the uniformity of the sample loading. These data indicate that nano-phase *Gynostemma pentaphyllum* extract antagonizes the Ang II stimulation effect by specifically inhibiting the activation of the PI3K-AKT-mTOR signaling pathway, rather than affecting the total expression levels of related proteins. Especially at a concentration of 8 μg / mL, the nano-phase extract of Gynostemma pentaphyllum showed the most significant inhibitory effect on pathway phosphorylation, providing important clues for further research on its mechanism of action.

[0180] The above test results demonstrate that the JTX group of this invention is significantly superior to other groups, and there are significant differences.

[0181] Example 3: The nano-phase extract of *Gynostemma pentaphyllum* of this invention exerts an early anticoagulant effect on the liver by antagonizing AngII-AT1R signal transduction.

[0182] 1. Experiment Content

[0183] 1.1 Animal Experiments

[0184] Twenty-four male C57 mice (weighing 20-22 g, 6-8 weeks old) were obtained from Jicui Pharmaceutical Co., Ltd., and acclimatized for one week. The mice were adapted to a 12-hour light-dark cycle at 22±0.5℃, with unrestricted access to regular diet and drinking water. The animal care and treatment protocol was approved by the Animal Research Committee of Southwest Medical University. AT1R - / - The gene knockout identification results in mice were obtained from Jicui Pharmaceutical. Twenty-four mice were divided into four groups: a normal control group fed a standard diet (ND), and seven other groups (n=6 per group) fed a high-fat diet (HFD). All groups except the normal control group received intraperitoneal injections of 20% CCl4 at a dose of 20 g / 100 μl for seven weeks. The HFD-fed animals were randomly assigned to an untreated model group (MO) and an AT1R group. - / - Group, AT1R - / - + Nano-phase *Gynostemma pentaphyllum* extract nano-component administration group (nano-phase *Gynostemma pentaphyllum* extract dosage was 100 mg / ml). The prepared drug was administered orally once daily at a dose of 2 mL / 100 g body weight for 4 consecutive weeks.

[0185] 2. Experimental results (see) Figures 13-17 )

[0186] 3. Analysis of Experimental Results

[0187] Figure 13 Liver tissue pathological staining results

[0188] In the AT1R knockout group (AT1R - / - ) and AT1R knockout combined with nano-phase extract of Gynostemma pentaphyllum extract (AT1R) - / - In the nano-phase extract of *Gynostemma pentaphyllum*, the histomorphological appearance of the three staining results (HE, Masson, and Sirius Red) was highly similar. HE staining showed that the arrangement of hepatocytes and the degree of inflammatory infiltration were similar in both groups; Masson staining showed that the collagen fiber (blue) deposition pattern was basically the same in both groups; Sirius Red staining also showed a similar type I / III collagen ratio. This phenotypic convergence indicates that when AT1R was knocked out, the intervention of nano-phase extract of *Gynostemma pentaphyllum* failed to produce additional therapeutic effects. Combining the known mechanism of Ang II mediating fibrosis through AT1R, it can be inferred that the effect of nano-phase extract of *Gynostemma pentaphyllum* depends on the intact AT1R signaling pathway—its therapeutic effect is achieved by blocking the binding of Ang II to AT1R or inhibiting downstream signal transduction. When AT1R is absent, nano-phase extract of *Gynostemma pentaphyllum* loses its target and therefore cannot show a therapeutic effect different from the simple knockout group. This result, in turn, verifies the molecular mechanism by which nano-phase extract of *Gynostemma pentaphyllum* exerts its anti-fibrotic effect through the Ang II-AT1R axis.

[0189] Figure 14 Changes in Collagen-I and Collagen-III immunofluorescence in different groups after gene knockout and drug administration

[0190] In the Control group, the green fluorescence signals of collagen-I and collagen-III were significantly enhanced and densely distributed, indicating that the high-fat diet successfully induced fibrotic lesions. In the AT1R gene knockout group (AT1R...),... - / - In the study, the fluorescence intensity of both collagen proteins was significantly reduced, approaching the level of the normal control group (NC), indicating that AT1R deficiency itself has an anti-fibrotic effect. It is worth noting that AT1R... - / - + The fluorescence signal of the nano-phase extract of *Gynostemma pentaphyllum* was different from that of the AT1R knockout group (AT1R...). - / -The results were almost identical, with no significant difference between the two groups in the degree and distribution pattern of collagen deposition. This key result indicates that the intervention of nano-phase *Gynostemma pentaphyllum* extract failed to produce additional therapeutic effects after AT1R knockout, suggesting that the effect of nano-phase *Gynostemma pentaphyllum* extract is entirely dependent on the presence of AT1R. Combined with the known mechanism by which Ang II mediates fibrosis through AT1R, it can be confirmed that nano-phase *Gynostemma pentaphyllum* extract exerts its anti-fibrotic effect by acting on the Ang II-AT1R signaling axis, achieved by blocking the binding of Ang II to AT1R or inhibiting downstream signal transduction.

[0191] Figure 15 Changes in Fibronectin (FN) and α-SMA immunofluorescence in different groups after gene knockout and drug administration

[0192] In the control group, the fluorescence signals of both α-SMA and FN were significantly enhanced, the number of α-SMA-positive cells increased significantly and their distribution was dense, and the deposition of FN in the extracellular matrix also increased significantly, indicating that the high-fat diet successfully induced myofibroblast activation and extracellular matrix deposition. Notably, the fluorescence signal of the AT1R- / -+ nanophase extract group was significantly different from that of the AT1R knockout group (AT1R...). - / - ) are almost identical, while AT1R - / - The expression levels of α-SMA and FN in the nano-phase extract group were significantly reduced, approaching the levels in the NC group (normal control group). Based on the known mechanism by which Ang II promotes fibrosis through AT1R, it can be inferred that the nano-phase extract of *Gynostemma pentaphyllum* exerts its anti-fibrotic effect by acting on the Ang II-AT1R signaling pathway.

[0193] Figure 16 Changes in CD61 / CD31 and CD62 / CD31 immunofluorescence in different groups after gene knockout and drug administration

[0194] A comprehensive analysis of the immunofluorescence staining results of CD61 (platelet marker) and CD31 (endothelial cell marker) in the images confirmed the molecular mechanism by which the nano-phase extract of *Gynostemma pentaphyllum* exerts its angiogenic protective effect through the Ang II-AT1R axis. In the Control group (high-fat diet treatment), the red fluorescence signal of CD61 was significantly enhanced and showed a dense, punctate distribution, while the green fluorescence signal of CD31 exhibited a disordered, filamentous distribution pattern, indicating that the high-fat diet successfully induced platelet activation and endothelial cell damage. Notably, the AT1R... - / - Group (AT1R knockout) and AT1R - / -The CD61 fluorescence intensity in the nano-phase *Gynostemma pentaphyllum* extract group (AT1R knockout + nano-phase *Gynostemma pentaphyllum* extract treatment) was significantly lower than that in the control group, and the expression pattern and distribution of CD31 were closer to the normal morphology of the NC group (normal control group). Crucially, AT1R... - / - +CD61 and CD31 expression levels and AT1R in the nano-phase extract group of *Gynostemma pentaphyllum* - / - The results were almost identical between the two groups, with no significant differences in platelet activation and endothelial cell protection. This result fully demonstrates that the angiogenic protective effect of the nano-phase extract of *Gynostemma pentaphyllum* is entirely dependent on the presence of AT1R; when AT1R is knocked out, the nano-phase extract of *Gynostemma pentaphyllum* cannot produce additional therapeutic effects.

[0195] Figure 17 Western blotting in vitro validates that nano-phase extracts of *Gynostemma pentaphyllum* exert therapeutic effects through the PI3K-AKT-mTOR pathway.

[0196] A comprehensive analysis of Western blot results further validated the molecular mechanism by which the nano-phase *Gynostemma pentaphyllum* extract exerts its effects through the Ang II-AT1R axis. In the Ang II-only treatment group, the phosphorylation levels of p-mTOR, p-PI3K, and p-AKT were significantly increased, indicating that Ang II successfully activated the PI3K-AKT-mTOR signaling pathway. Notably, in the Ang II + nano-phase *Gynostemma pentaphyllum* extract group, the expression levels of these phosphorylated proteins were significantly reduced, approaching basal levels, consistent with previous findings, confirming that the nano-phase *Gynostemma pentaphyllum* extract can effectively inhibit Ang II-induced signaling pathway activation. A key finding emerged in the olmesartan treatment group: the phosphorylation level in the Ang II + olmesartan group was comparable to that in the Ang II + nano-phase *Gynostemma pentaphyllum* extract group, while the Ang II + nano-phase *Gynostemma pentaphyllum* extract + olmesartan group did not show a stronger inhibitory effect than either the nano-phase *Gynostemma pentaphyllum* extract or olmesartan alone. This result is highly significant, indicating that the nano-phase extract of *Gynostemma pentaphyllum* and olmesartan exert their effects through the same molecular target in inhibiting the Ang II signaling pathway. Given that olmesartan is a known AT1R-specific antagonist, this finding strongly supports the hypothesis that the nano-phase extract of *Gynostemma pentaphyllum* exerts its biological effects by acting on the Ang II-AT1R axis. Furthermore, the expression levels of total proteins (mTOR, PI3K, AKT) and the internal control protein (β-actin) remained stable across all groups, ruling out non-specific influences and further validating the reliability of the results.

Claims

1. A nano-phase extract of *Gynostemma pentaphyllum*, characterized in that: It is made by soaking and decocting the herb *Gynostemma pentaphyllum*, filtering, collecting the filtrate, centrifuging in stages to remove the precipitate, dialyzing the supernatant using a dialysis bag with a molecular weight cutoff of 3.5 kDa, and freeze-drying the dialysate to obtain the nano-phase extract of *Gynostemma pentaphyllum*.

2. The nano-phase extract of *Gynostemma pentaphyllum* according to claim 1, characterized in that: The particle size of the nano-phase extract of *Gynostemma pentaphyllum* is 50-200 nm.

3. The nano-phase extract of *Gynostemma pentaphyllum* according to claim 2, characterized in that: The particle size of the nano-phase extract of *Gynostemma pentaphyllum* is 100-150 nm, preferably 120 nm.

4. The nano-phase extract of *Gynostemma pentaphyllum* according to any one of claims 1-3, characterized in that: The nano-phase extract of *Gynostemma pentaphyllum* has a PDI of 0.25 and a potential between -15 and -20 mV.

5. A method for preparing a nano-phase extract of *Gynostemma pentaphyllum*, characterized in that: It includes the following steps: a. Soaking: Mix the herbs with purified water and soak at room temperature; b. Decoction: Heat and decoct the soaked medicinal materials; c. Filtration: After decoction, filter the liquid while it is still hot using a filter cloth or filter paper, collect the filtrate and discard the dregs; d. Centrifugation in stages: Place the filtered extract in a centrifuge and centrifuge to obtain the supernatant; e. Collection of supernatant: After each centrifugation, discard the precipitate and retain the supernatant; f. Dialysis bag selection: Use a dialysis bag with a molecular weight cutoff of 3.5kDa (MWCO 3.5kDa). Fill the dialysis bag with the supernatant and place the dialysis bag in deionized water for dialysis. g. Freeze-drying preservation: After dialysis, take the dialysate from the dialysis bag and freeze-dry it to obtain the nano-phase extract.

6. The method for preparing nano-phase extract of *Gynostemma pentaphyllum* according to claim 5, characterized in that: The centrifugation described in step d involves centrifuging at different speeds: 4000 rpm, 6000 rpm, 8000 rpm, and 10000 rpm, for a total of 30 minutes.

7. The method for preparing nano-phase extract of *Gynostemma pentaphyllum* according to claim 7, characterized in that: The dialysis time described in step f is 12h-48h, with the water changed every 3 hours.

8. Use of the preparation of the nano-phase extract of *Gynostemma pentaphyllum* according to any one of claims 1-4 in a medicament for treating fatty liver disease.

9. The use according to claim 8, characterized in that: The drug described is an anticoagulant that improves the anticoagulant effect in metabolic dysfunction-related steatohepatitis.

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