Plant extract with blood pressure lowering effect and preparation method and application thereof

Abstract

The application discloses a plant extraction compound for reducing blood pressure, a preparation method and application thereof. The compound is composed of high-purity silybin extract and a specific fusion polypeptide through molecular self-assembly. The sequence of the fusion polypeptide is shown in sequence SEQ ID NO:1, which innovatively integrates cell membrane penetration function, angiotensin II receptor 1 antagonism function and hydrophobic drug binding function. The core of the application is that through polypeptide-mediated active targeting, silybin is accurately delivered to the diseased blood vessel area, and the double synergistic mechanism of upstream ACE inhibition and downstream receptor blocking of the renin-angiotensin-aldosterone system is realized. In-vivo pharmacodynamic experiments prove that compared with single components, the compound can produce more significant and persistent antihypertensive effect, and can effectively reverse myocardial hypertrophy and fibrosis caused by hypertension, and show excellent synergistic therapeutic effect and target organ protection ability.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a plant extract with antihypertensive effects, its preparation method, and its application. Background Technology

[0002] Hypertension is a chronic cardiovascular disease characterized by persistently elevated systemic arterial pressure and is a significant risk factor for damage to target organs such as the heart, brain, and kidneys. Clinically, drug treatment for hypertension primarily focuses on regulating the renin-angiotensin-aldosterone system (RAAS), with angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) being commonly used first-line treatments. Although these drugs can effectively lower blood pressure and improve cardiovascular outcomes, they still present challenges such as a high incidence of adverse reactions, poor treatment response in some patients, and insufficient long-term adherence, resulting in an overall unsatisfactory control rate of hypertension.

[0003] In the exploration of safer and more multi-targeted treatment strategies, active ingredients derived from natural plants have attracted attention. Milk thistle (Silybum marianum) is a traditionally widely used medicinal plant, and its main active ingredient, silybin, possesses antioxidant and anti-inflammatory biological effects. Some studies suggest that silybin has a certain degree of inhibitory activity against angiotensin-converting enzyme (ACE). However, silybin still has significant limitations as a candidate for antihypertensive drugs, including poor water solubility, limited oral absorption, lack of tissue targeting in vivo distribution, and weak ACE inhibitory efficacy, making it difficult to achieve ideal antihypertensive effects on its own.

[0004] Meanwhile, peptide drugs, due to their high target specificity and pharmacological activity, are widely used in the field of new drug development. Previous studies have shown that some peptide fragments derived from angiotensin II (Ang II) structural modifications can act as antagonists of the angiotensin II type 1 receptor (AT1R). However, peptide drugs generally suffer from drawbacks such as insufficient in vivo stability, rapid degradation, short half-life, and limited tissue penetration, which restricts their application in the treatment of hypertension.

[0005] Existing technologies have also attempted to improve drug stability and delivery efficiency through compound formulations or the use of nanocarriers. For example, compound formulations of plant extracts and chemical antihypertensive drugs are mostly physically mixed, and their efficacy is usually only additive, making it difficult to achieve synergistic enhancement of the mechanism of action. While drug delivery systems such as liposomes and polymer nanoparticles can improve the pharmacokinetic properties of some active ingredients, they still face challenges such as biocompatibility, drug loading capacity, preparation complexity, and difficulties in large-scale production.

[0006] In summary, existing technologies face varying degrees of technical bottlenecks in areas such as the efficacy of natural active ingredients, the stability and targeting of peptide drugs, and the synergistic effects of compound formulations. Therefore, it remains necessary to provide a new technical solution to address these issues and further enhance the efficacy of related active substances in the treatment of hypertension. Summary of the Invention

[0007] To address the shortcomings of existing technologies, a complex with significant synergistic and targeted blood pressure-lowering effects is provided, along with its preparation method and applications.

[0008] To achieve the above objectives, this invention provides a complex composed of a silybin active extract and a novel fusion polypeptide. The silybin extract is prepared using an optimized extraction and purification process, ensuring a high silybin content. The sequence of the fusion polypeptide is shown in Sequence Identifier 1, and its molecular structure is rationally designed, containing three functionally defined modules from the N-terminus to the C-terminus: a cell-penetrating module composed of nine arginine residues to enhance cellular uptake efficiency; a receptor antagonistic module based on angiotensin II structural modification to specifically block angiotensin II receptor 1; and a hydrophobic module composed of eight leucine residues to bind to the silybin molecule via hydrophobic interactions, driving the spontaneous formation of the nanocomplex. The preparation of this complex can be achieved simply by mixing and incubating the two components under mild buffer conditions.

[0009] Compared with existing technologies, the plant extract complex provided by this invention exhibits several significant advantages. First, it achieves dual upstream and downstream blockade of key blood pressure regulation pathways through the ACE inhibition of silymarin and the AT1 receptor blocking effect of the fusion peptide, producing a powerful synergistic antihypertensive effect, with a significantly greater in vivo blood pressure reduction than any single component. Second, the fusion peptide acts as an intelligent delivery system, precisely targeting the silymarin it carries to the lesion site in the vascular system, thereby greatly increasing the local drug concentration at the target site. This not only enhances the ACE inhibition effect but also lays the foundation for reducing toxicity and increasing efficacy. Furthermore, the molecular self-assembly strategy employed in this invention is simple and mild, requiring no complex chemical coupling or external synthetic carriers, effectively preserving the bioactivity of each component. Most importantly, while efficiently lowering blood pressure, this complex also exhibits good cardiovascular protective function, reversing myocardial hypertrophy and interstitial fibrosis in hypertensive model animals, which has significant clinical implications for preventing serious complications such as heart failure. Attached Figure Description

[0010] Figure 1 Schematic diagram of HPLC detection results for fusion peptides.

[0011] Figure 2Standard curve for silymarin content determination. Detailed Implementation

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0013] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0014] Example 1: Design, preparation and testing of fusion peptides

[0015] 1. Design of the fusion peptide: In this embodiment, a novel fusion peptide was designed and synthesized, the amino acid sequence of which is shown in SEQ ID NO:1 (RRRRRRRRRGGGGGGSVYIHPILLLLLLLL, where S represents sarcosine (Sar)). The specific domain functions are analyzed below:

[0016] (1) Cell-penetrating peptide (Arg9): Arginine-rich sequences are recognized as structures that enhance cellular uptake and can promote the uptake and tissue enrichment of peptide structures through various energy-dependent or energy-independent endocytosis processes. Its introduction is expected to improve the local distribution of the complex in cardiovascular-related tissues and enhance the accessibility of the AT1R antagonist fragment.

[0017] (2) Flexible linker (Gly6): A flexible segment composed of six glycine residues is used to provide moderate spatial mobility, reduce direct spatial conflict between the transmembrane region and the receptor binding region, and improve conformational tunability during the formation of the complex structure.

[0018] (3) AT1R rational design antagonistic fragment (Sar 1 -Val 2 -Tyr 3 -Ile 4 -His 5 -Pro 6 -Ile 7 This sequence is based on a rational modification of the key binding structure of angiotensin II, with the introduction of sarcosine (Sar) at the N-terminus to reduce aminopeptidase degradation. This fragment is expected to have AT1R binding and antagonistic tendencies.

[0019] (4) Hydrophobic binding domain (Leu8): This segment consists of multiple leucine residues, forming a distinct hydrophobic region. It can non-covalently bind with hydrophobic plant bioactive molecules such as silymarin through hydrophobic interactions and van der Waals forces, thereby promoting the formation of a nanocomposite structure dominated by a hydrophobic core in an aqueous system. This design is expected to improve the drug loading capacity and colloidal stability of the complex.

[0020] 2. Preparation of the fusion peptide: The fusion peptide shown in SEQ ID NO:1 was sent to Shanghai Polymer Bioengineering Co., Ltd. for synthesis. The synthesis and HPLC purity were ≥98%. Figure 1 ).

[0021] 3. Self-assembly and characterization of the complex

[0022] (1) Preparation: Accurately weigh 100.0 mg of silymarin extract (prepared in Example 2) and 10.0 mg of the fusion peptide. Dissolve both in 10.0 mL of phosphate buffer (PBS, 10 mM, pH 7.4). Vortex the mixture vigorously for 1 minute, then transfer it to a 37°C constant temperature shaker and incubate with gentle shaking at 150 rpm for 2 hours.

[0023] (2) Filtration: After incubation, sterile filtration was performed using a 0.22 μm polyethersulfone (PES) microporous membrane to obtain a clear complex stock solution, which was stored at -80℃ for later use.

[0024] (3) Characterization (Dynamic Light Scattering, DLS): The average hydrated particle size (Z-Average) was measured to be 95.3 nm, the polydispersity index (PDI) was 0.14, and the Zeta potential was +17.5 mV. These results indicate that a PDI < 0.2 suggests that the composite nanoparticles have a uniform size distribution and good monodispersity. The positive Zeta potential originates from the positive charge carried by polyarginine in the fusion peptide, which contributes to the colloidal stability of the nanoparticles and may promote their interaction with the negatively charged cell membrane.

[0025] 4. Activity testing of fusion peptides

[0026] 4.1 Experimental objective: To verify the specific antagonistic activity of the fusion peptide against AT1R.

[0027] 4.2 Experimental Methods (Cell-Based Reporter Gene Detection Method)

[0028] (1) Cells and reagents: HEK293 cell line (HEK293-AT1R-NFAT-Luc, prepared in this study) stably transfected with human AT1R and NFAT response element-driven luciferase reporter genes was used. Ang II was used as the agonist in this experiment.

[0029] (2) Experimental procedure: The cells were placed in a container with 1×10⁶ cells per well. 4 Inoculate at a density of [number] cells / well in 96-well white culture plates and incubate at 37°C, 5% CO2 for 24 hours. Discard the old culture medium. Set up the following groups (n=4):

[0030] Blank control group: Contains only detection buffer.

[0031] Maximum effect group (100% activation): Add buffer containing 100 nM Ang II.

[0032] Antagonistic experimental group: First, add fusion peptides containing different concentrations (10 -10 M, 10 -9 M, 10 -8 M, 10⁻ 7 M, 10 - 6 Pre-incubate with buffer (M) for 30 minutes.

[0033] Negative control (0% Activation): No Ang II or peptides added.

[0034] After pre-incubation, add 100 nM Ang II to all wells except the blank group and negative control group. Incubate together for 5 hours. Discard the culture medium, add 50 μL of 1× Luciferase Assay Reagent to each well, incubate in the dark with shaking for 10 minutes, and then detect the chemiluminescence value (RLU) using a multi-mode microplate reader.

[0035] (3) Data processing: The inhibition rate of each concentration of peptide on the maximum luciferase activity induced by Ang II was calculated. The four-parameter dose-response curve was fitted by nonlinear regression, and the half-maximal inhibitory concentration (IC50) was calculated. 50 ).

[0036] 4.3 The results are shown in Table 1: the fusion peptide at 10 -10 -10 -6 Within the M concentration range, it exhibits typical concentration-dependent inhibition of AngII-induced AT1R activation, and luciferase activity gradually decreases with increasing concentration. At 10 -8 The inhibition rate reached 53.0% at time M, indicating that it had entered the significant antagonistic range. 10 -6 At a concentration of M, 92.1% inhibition was observed, close to the baseline level (93.2%) without Ang II stimulation, indicating that high-concentration fusion peptides can block the receptor activation effect of Ang II. The calculated IC50 value... 50 =8.5 ± 1.2 nM.

[0037] Table 1. Inhibition rate of different concentrations of fusion peptides on Ang II-induced luciferase activity

[0038]

[0039] Example 2: Preparation, purification and content determination of silymarin active extract

[0040] 1. Experimental objective: To extract, separate and purify an active extract with high silybin content from milk thistle seeds, providing a quality-controlled raw material for the subsequent construction of nanocomposites.

[0041] 2. Materials: Milk thistle seeds, ethanol (analytical grade, Sinopharm Group), distilled water, methanol (chromatographic grade), silybin reference standard (purity ≥98%).

[0042] 3. Experimental Procedure

[0043] 3.1 Raw material pretreatment: Weigh 500.0 g of milk thistle seeds and place them in a high-speed universal grinder. Grind them intermittently three times, 15 seconds each time. Pass the ground material through a 40-mesh sieve to obtain uniform seed powder, which is then set aside. The total mass of the powder is 485.6 g (yield 97.1%).

[0044] 3.2 Ethanol Reflux Extraction: Accurately weigh 300.0 g of the above seed powder and place it in a 5000 mL round-bottom flask. Add 4500 mL of 70% (v / v) ethanol solution at a material-to-liquid ratio of 1:15 (w / v). Connect the condenser and heat under reflux in a 60°C constant temperature water bath. Start timing from boiling and extract for 2 hours each time. After extraction, while still hot, use a Buchner funnel (lined with double layers of filter paper) for vacuum filtration to separate the extract from the residue. Place the residue back into the flask and perform a second and third extraction using the same method. Combine the filtrates from the three extractions; the total volume of the combined extract is 12850 mL.

[0045] 3.3 Concentration of Extract: The combined extracts were transferred to a rotary evaporator and concentrated under reduced pressure at a water bath temperature of 45°C and a vacuum degree of -0.09 MPa until the distillate had no alcohol odor, yielding a dark brown, viscous extract. The mass of the viscous extract was 118.4 g.

[0046] 3.4 Purification of Macroporous Adsorption Resin: Take 500 mL of AB-8 macroporous adsorption resin, soak it in 95% ethanol for 24 hours, and then pack it into a column using the ethanol wet packing method. Continue to wash with 2 column volumes (BV) of 95% ethanol at a flow rate of 2 BV / h until the eluent and water are no longer turbid at a ratio of 1:5. Then wash away the ethanol with 5 BV of distilled water at the same flow rate, and set aside. Resuspend 118.4 g of the viscous extract in about 600 mL of distilled water (about 5 times the volume), and stir to disperse it thoroughly to obtain a suspension. Slowly load the sample into the pretreated AB-8 resin column at a flow rate of 1.0 mL / min (about 0.2 BV / h). After loading, elute with 5 BV (2500 mL) of distilled water at a flow rate of 2 BV / h to remove water-soluble impurities. Collect the water wash, which is pale yellow, and HPLC analysis shows that it contains almost no silymarin. Next, elution was performed with 3 BV (1500 mL) of 10% ethanol at a flow rate of 2 BV / h to further remove some weakly polar impurities. This eluent was collected, and HPLC analysis showed extremely low silymarin content. Finally, elution was performed with 5 BV (2500 mL) of 70% ethanol at a flow rate of 2 BV / h. During this process, a distinct yellow band was observed washing down the column bed. This 70% ethanol eluent was accurately collected, with a total volume of 2480 mL.

[0047] 3.5 Obtaining the refined extract: The collected 70% ethanol eluent was concentrated to a small volume (approximately 150 mL) under reduced pressure at 45°C, and then transferred to a pre-weighed lyophilization bottle. It was pre-frozen in an ultra-low temperature freezer at -80°C for 12 hours, and then transferred to a freeze dryer and freeze-dried for 24 hours at a cold trap temperature of -50°C and a vacuum degree <10 Pa. The final product was a pale yellow, loose, spongy solid powder. The accurate weight of the lyophilized product was 25.8 g.

[0048] 3.6 Calculate the yield from raw materials to refined extract: (25.8 g / 300.0 g) × 100% = 8.6%.

[0049] 4. Determination of silymarin content

[0050] 4.1 Preparation of reference solution: Accurately weigh 5.12 mg of silymarin reference standard, place it in a 10 mL brown volumetric flask, dissolve it in methanol and dilute to the mark, shake well to prepare a reference stock solution with a concentration of 512 μg / mL.

[0051] 4.2 Preparation of test solution: Accurately weigh 10.15 mg of this product (silymarin extract) and place it in a 20 mL brown volumetric flask. Add approximately 15 mL of methanol and sonicate (300 W, 40 kHz) for 20 minutes to ensure complete dissolution. Remove the flask, allow it to cool to room temperature, and dilute to the mark with methanol. Shake well. Filter through a 0.45 μm microporous membrane and use the filtrate as the test solution.

[0052] 4.3 Standard Curve Construction: Accurately pipette an appropriate amount of the reference stock solution and serially dilute it with methanol to prepare a series of standard solutions with concentrations of 10.24, 25.6, 51.2, 102.4, and 204.8 μg / mL. Inject and determine the solutions under the chromatographic conditions described above. Perform linear regression of peak area (Y) against concentration (X, μg / mL). The standard curve is shown below. Figure 2 As shown.

[0053] 4.4 Determination: Accurately pipette 10 μL each of the reference solution (51.2 μg / mL) and the test solution into the liquid chromatograph and record the chromatograms. The results showed that the retention time of the silymarin peak in the reference solution (51.2 μg / mL) was approximately 16.8 minutes, and the peak shape was symmetrical. The chromatogram of the test solution showed a main peak at the same position as the reference solution with the same retention time, and its peak area was 426835 mAU*s. Based on the standard curve, its content was calculated to be 350 μg / mL.

[0054] 4.5 Calculate the percentage content of silymarin in the purified extract.

[0055] Content (%) = (C * V * D) / W * 100%; C: concentration of the test solution (350 μg / mL); V: volume to be made up (20 mL); D: dilution factor (1); W: sample weight (10.15 mg = 10150 μg).

[0056] Based on the formula above, the content (%) = (350 * 20 * 1) / 10150 * 100% = 69%.

[0057] Experimental Example 3: Study on the in vivo antihypertensive activity and mechanism

[0058] I. Laboratory Animals and Models

[0059] 1. Basic information about the animal

[0060] 1.1 Breed and Specifications: SPF-grade male spontaneously hypertensive rats (SHR) and homologous normotensive Wistar-Kyoto (WKY) rats, both 12 weeks old, weighing 220±20g (SHR) and 210±15g (WKY), were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0061] 1.2 Husbandry conditions: Four rats per cage were housed in an SPF-grade animal facility for one week for acclimatization. Feed consisted of SPF-grade complete pelleted feed for rats, available freely; water was sterile distilled water supplied via automatic waterers. Environmental parameters were strictly controlled: temperature 23±2°C, relative humidity 55±10%, 12h / 12h light / dark cycle (light hours 07:00-19:00), ventilation twice daily (30 minutes each time), and cage and environmental disinfection once weekly.

[0062] 2. Grouping and Dosing Regimen

[0063] 2.1 Grouping Method: SHR rats were randomly divided into 5 groups (n=8) using a random number table. Initial body weight and blood pressure were measured in each group after grouping to ensure no statistically significant differences between groups (P>0.05). A WKY normal control group (n=8) was also included. Grouping information is as follows:

[0064] WKY normal group: The same volume of normal saline was injected into the tail vein.

[0065] SHR model group: The same volume of physiological saline was injected into the tail vein.

[0066] Silymarin group: Silymarin extract (50 mg / kg, prepared in Example 2) was injected via tail vein.

[0067] Fusion polypeptide group: fusion polypeptide (5 mg / kg, prepared in Example 1) was injected via tail vein.

[0068] Complex group: tail vein injection complex (prepared in Example 1, wherein 50 mg / kg of silymarin extract + 5 mg / kg of fusion peptide).

[0069] Positive drug group: losartan (10 mg / kg, prepared as a 2 mg / mL suspension with normal saline) was administered by gavage.

[0070] 2.2 Administration Procedure: Before tail vein injection, rats were placed in a tail restraint device, and the tail tip was wiped with 75% alcohol to expose the tail vein. An injection was then administered using a 27G needle at a rate of 0.2 mL / s, with a total volume of 5 mL / kg. For losartan gavage, a 1 mL gavage syringe was used. Rats were fasted for 4 hours prior to gavage (with free access to water) to avoid interference with absorption from gastric contents. Administration was performed daily from 09:00 to 10:00 for 28 consecutive days.

[0071] II. Detection Indicators and Methods

[0072] 1. Non-invasive tail artery systolic blood pressure measurement: One day prior to measurement, rats underwent acclimatization training in a fixation box (10 minutes each time) to reduce stress. Before measurement, rats were preheated in a 37°C incubator for 15 minutes to dilate tail vessels, then placed in a soundproof, vibration-free measurement fixation box (temperature 23±1°C) for 5 minutes before measurement. Each rat was measured three times consecutively, with a 1-minute interval between each measurement. Outliers (deviation from the mean >10 mmHg) were removed, and the average of the remaining data was taken as the systolic blood pressure at that time point. Measurements were taken before drug administration (day 0) and on days 7, 14, 21, and 28 after drug administration.

[0073] 2. Serum ACE Activity Assay: 24 hours after the last administration, rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), fixed supine on the operating table, and the abdominal cavity was cut open to expose the abdominal aorta. 5 mL of blood was collected using a heparin-anticoagulated blood collection tube. After the blood was allowed to stand at room temperature for 30 min, it was centrifuged at 3000 rpm for 15 min at 4°C to separate the serum, which was then stored at -80°C for later analysis. The rat angiotensin-converting enzyme (ACE) ELISA kit was used, strictly following the manufacturer's instructions.

[0074] 3. Cardiac tissue sampling and pathological analysis

[0075] 3.1 Determination of cardiac weight index: After blood collection, the heart was quickly removed by thoracotomy and rinsed three times with pre-cooled physiological saline to remove blood vessels and connective tissue. The surface moisture was absorbed with filter paper, and the wet weight of the whole heart was weighed using a Mettler PL2002 electronic balance (accuracy 0.1 mg). The cardiac weight index (heart wet weight / rat body weight, mg / g) was calculated.

[0076] 3.2 Preparation of Pathological Sections: A tissue sample (approximately 0.5cm × 0.5cm × 0.3cm) from the apex of the left ventricle was taken and fixed in 4% paraformaldehyde fixative at 4°C for 24 hours. Gradual dehydration was performed: 70% ethanol for 2 hours → 80% ethanol for 2 hours → 90% ethanol for 2 hours → 95% ethanol for 1 hour → anhydrous ethanol twice (1 hour each time). Clearing was performed twice with xylene (30 minutes each time). Embedding was performed: paraffin infiltration at 60°C for 2 hours, followed by embedding in a paraffin block, and cooling to solidify at 4°C. Sectioning: 5μm thick sections were cut using a Leica RM2235 rotary microtome, mounted on poly-L-lysine-coated slides, and baked at 60°C for 2 hours.

[0077] 3.3 Staining and Observation:

[0078] HE staining: Dewaxing → Hematoxylin staining for 5 min → Hydrochloric acid differentiation for 30 s → Rinsing with tap water for 10 min → Eosin staining for 3 min → Gradient dehydration → Xylene clearing → Mounting with neutral resin. Observe under a 200x optical microscope. Eight slides are randomly selected from each group, and five non-overlapping fields are selected from each slide to record the morphology, arrangement, and nucleus size of cardiomyocytes.

[0079] Masson trichrome staining: dewaxing → hematoxylin staining for 5 min → hydrochloric acid differentiation → tap water rinsing → Ponceau S and Acid Fuchsin staining for 10 min → phosphomolybdic acid solution treatment for 5 min → aniline blue staining for 5 min → 1% glacial acetic acid differentiation for 30 s → gradient dehydration → xylene clearing → neutral resin mounting. Collagen volume fraction (collagen fiber area / total field of view × 100%) was calculated using ImageJ 1.8.0 image analysis software at 200x magnification. Eight slides were randomly selected from each group, and five non-overlapping fields of view were selected from each slide.

[0080] 4. Data Analysis Methods

[0081] Data analysis was performed using SPSS 26.0 statistical software. Quantitative data are expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons among multiple groups, and Tukey's post hoc test was used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0082] III. Experimental Results

[0083] 1. Changes in coccygeal artery systolic blood pressure are shown in Tables 2 and 3. Significant differences were observed in blood pressure changes before and after drug administration among the groups:

[0084] 1.1 WKY normal group: Blood pressure remained stable throughout the experimental period, with systolic blood pressure between 123-126 mmHg at each time point, without significant fluctuations (P>0.05).

[0085] 1.2 SHR model group: Blood pressure continued to rise with the duration of drug administration, from 195.3±6.8 mmHg on day 0 to 210.5±9.2 mmHg on day 28. Blood pressure at each time point was significantly higher than that in the WKY normal group (P<0.05), confirming that the hypertension model was successfully constructed and stable.

[0086] 1.3 Silymarin group: Blood pressure gradually decreased after administration, dropping to 182.6±6.9 mmHg on day 7 (a decrease of 11.5 mmHg from day 0) and to 171.2±7.8 mmHg on day 28 (a decrease of 22.9 mmHg from day 0). Blood pressure at each time point was significantly lower than that in the SHR model group (P<0.05).

[0087] 1.4 Fusion peptide group: The antihypertensive effect was better than that of silymarin group. Blood pressure dropped to 176.4±6.2 mmHg on day 7 (a decrease of 20.4 mmHg from day 0) and to 161.7±5.9 mmHg on day 28 (a decrease of 35.1 mmHg from day 0), which was significantly lower than that of SHR model group (P<0.05).

[0088] 1.5 Complex group: The antihypertensive effect was the most significant and the onset of action was rapid. Blood pressure dropped to 162.8±5.4 mmHg on day 7 (a decrease of 30.7 mmHg from day 0) and to 142.6±1.5 mmHg on day 28 (a decrease of 50.9 mmHg from day 0). Blood pressure at each time point was significantly lower than that of the silymarin group and the fusion peptide group (P<0.05), and there was no statistical difference from the positive control group (146.3±4.7 mmHg on day 28) (P>0.05).

[0089] Table 2. Results of tail artery systolic blood pressure tracking (mmHg)

[0090]

[0091] Table 3. Statistical analysis of changes in systolic blood pressure in the tail artery of rats in each group (mmHg, x±s, n=8)

[0092]

[0093] Note: *P<0.05 compared with the SHR model group at the same time point; #P<0.05 compared with the silymarin group and the fusion peptide group at the same time point.

[0094] 2. Serum ACE activity: The statistical results are shown in Table 4. Significant differences in ACE activity were observed among the groups. The serum ACE activity in the WKY normal group was 33.7±2.8 U / L, within the normal physiological range. The ACE activity in the SHR model group significantly increased to 70.2±6.1 U / L, an increase of 108.3% compared to the WKY normal group (P<0.05), consistent with the pathological characteristics of RAAS system activation in hypertensive rats. The ACE activity in the silymarin group decreased to 46.3±4.5 U / L, a decrease of 34.0% compared to the SHR model group (P<0.05), confirming that silymarin has a direct inhibitory effect on ACE activity. There was no statistically significant difference in ACE activity between the fusion peptide group (66.8±5.9 U / L) and the positive control group (67.5±5.2 U / L) and the SHR model group (P>0.05), consistent with their mechanism of action on the downstream of the RAAS (blocking angiotensin II receptors). The ACE activity of the complex group decreased to 39.1±3.6 U / L, which was 44.3% lower than that of the SHR model group and significantly lower than that of the silymarin group (P<0.05), but only slightly higher than that of the WKY normal group (P>0.05), suggesting that the fusion peptide can enhance the ACE-targeting inhibitory effect of silymarin.

[0095] Table 4. Serum ACE activity test results (U / L)

[0096]

[0097] Note: *P<0.05 compared with the SHR model group; #P<0.05 compared with the silymarin group.

[0098] 3. Cardiac weight index and myocardial fibrosis: Statistical results are shown in Table 5. Significant differences were found in cardiac pathological indicators among the groups.

[0099] 3.1 Cardiac weight index: The WKY normal group had an index of 2.79±0.13 mg / g, while the SHR model group showed an increase to 4.31±0.24 mg / g (P<0.05), indicating that hypertension led to significant myocardial hypertrophy. The silymarin group (3.84±0.20 mg / g) and the fusion peptide group (3.70±0.18 mg / g) were both lower than those in the SHR model group (P<0.05); the complex group decreased to 3.28±0.15 mg / g, significantly lower than the two single-component groups (P<0.05), and comparable to the positive control group (3.31±0.16 mg / g) (P>0.05).

[0100] 3.2 Collagen volume fraction: The WKY normal group had a volume fraction of 2.1±0.4%, while the SHR model group showed an increase to 8.9±1.2% (P<0.05), indicating severe myocardial interstitial fibrosis. All treatment groups showed improvement, with the complex group decreasing to 3.9±0.6%, significantly lower than the silymarin group (6.5±0.9%) and the fusion peptide group (5.8±0.8%) (P<0.05), but not significantly different from the positive control group (4.1±0.7%) (P>0.05).

[0101] 3.3 Pathological section observation: HE staining showed that cardiomyocytes in the SHR model group were disordered, enlarged, and had deeply stained nuclei; cardiomyocytes in the complex group were close to normal in morphology, neatly arranged, and had uniformly sized nuclei. Masson staining showed that a large amount of blue collagen fibers were deposited in the myocardial interstitium in the SHR model group; the area of ​​blue collagen fibers in the complex group was significantly reduced and evenly distributed.

[0102] Table 5. Statistical analysis of heart weight index and collagen volume fraction in each group of rats (x±s, n=8)

[0103]

[0104] Note: Compared with the SHR model group, *P<0.05; compared with the silymarin group and the fusion peptide group, #P<0.05.

[0105] IV. Experiment Summary

[0106] 1. Antihypertensive effect: This experiment confirmed that the complex has significant synergistic antihypertensive activity. After 28 days of continuous administration, the systolic blood pressure of SHR rats was reduced to 142.6±1.5 mmHg, with a reduction of 50.9 mmHg, which was significantly better than the single silymarin group (22.9 mmHg) and the fusion peptide group (35.1 mmHg). Moreover, the effect was comparable to that of losartan, a commonly used antihypertensive drug in clinical practice, indicating that silymarin and the fusion peptide have a synergistic effect in antihypertensive activity.

[0107] 2. Mechanism of action: The antihypertensive mechanism of the complex is related to the targeted inhibition of ACE activity. The fusion peptide can target and deliver silymarin to the periphery of vascular endothelial cells, increasing the local drug concentration and enhancing its inhibitory effect on ACE, thereby reducing serum ACE activity to near-normal levels and blocking the activation of the RAAS system, thus exerting an antihypertensive effect. However, the fusion peptide itself does not have direct ACE inhibitory activity, suggesting that it may assist in antihypertensive action through other pathways (such as improving vascular endothelial function).

[0108] 3. Target organ protection: The complex not only effectively lowers blood pressure, but also significantly reverses myocardial hypertrophy and interstitial fibrosis caused by hypertension, and reduces cardiac weight index and collagen volume fraction. Its effect is better than that of the single component group, suggesting that the complex has a significant advantage in protecting the target organ (heart) of hypertension, and provides potential value for the clinical treatment of hypertension complicated with myocardial injury.

[0109] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A fusion polypeptide, characterized in that, The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO:1, where S represents sarcosine.

2. A plant extract complex for lowering blood pressure, characterized in that, The complex is formed by the self-assembly of silymarin active extract and the fusion polypeptide of claim 1 through non-covalent interactions.

3. The complex according to claim 2, characterized in that, The silymarin active extract contains 69% silymarin by weight.

4. The complex according to claim 2, characterized in that, The mass ratio of the silymarin active extract to the fusion polypeptide is 10:

1.

5. The complex according to claim 2, characterized in that, The composite self-assembles into nanoparticles in an aqueous phase.

6. The complex according to claim 5, characterized in that, The nanoparticles have an average hydrated particle size of 95.3 nm, a polydispersity index of 0.14, and a zeta potential of +17.5 mV.

7. The application of the fusion polypeptide as described in claim 1 in the preparation of a blood pressure-lowering plant extract complex.

8. The use of the antihypertensive plant extract complex as described in claim 2 in the preparation of drugs for the prevention or treatment of hypertension.

9. The application according to claim 8, characterized in that, The drug is used to prevent or reduce myocardial hypertrophy caused by hypertension.

10. The application according to claim 8, characterized in that, The drug is used to prevent or reduce myocardial fibrosis caused by hypertension.

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