Application of vinegar trogopterus dung water extract in preparation of product for protecting heart and / or resisting platelet aggregation thrombosis

Abstract

The invention discloses an application of a vinegar trogopterus dung water extract in preparation of a product for protecting heart and / or resisting platelet aggregation thrombosis. Research finds that the vinegar trogopterus dung water extract has a heart protection effect, and particularly, the heart rate, cardiac output and blood flow velocity are improved. The vinegar trogopterus dung water extract has the effect of resisting platelet aggregation thrombosis, and specifically, the dyeing intensity of heart red blood cells is increased. The vinegar trogopterus dung water extract is applied to heart protection and / or platelet aggregation thrombus resistance, and has high efficiency and safety.

Description

Technical Field

[0001] This application relates to the pharmaceutical or health care field, specifically to the use of vinegar-processed Trogopterus xanthipes aqueous extract in the preparation of products that protect the heart and / or prevent platelet aggregation thrombosis. Background Technology

[0002] Heart disease is a broad category of circulatory system diseases that affect the normal function of the heart, encompassing a variety of conditions such as heart failure, arrhythmia, coronary artery disease, pericardial disease, myocardial disease, and valvular heart disease. Globally, cardiovascular disease is one of the leading causes of death.

[0003] While various treatments exist for cardiovascular diseases, they generally suffer from high costs, low efficiency, and significant side effects. Drug therapy often leads to long-term dependence, interventional procedures are highly invasive, and surgical procedures carry high risks and slow recovery. Current cardiovascular medications can be categorized into Western and traditional Chinese medicine. Commonly used Western medications include antiplatelet drugs, nitrates, and statins. However, with long-term use, patients are prone to developing drug resistance, and some medications have potential liver and kidney damage. Therefore, there is an urgent need to develop a low-cost, highly effective drug with minimal side effects for treating cardiovascular diseases.

[0004] Water extracts of traditional Chinese medicine (TCM), with their natural components and multiple pharmacological effects, hold promise for filling gaps in existing treatment methods. By combining TCM theory with modern medicine, and through scientific extraction and purification, these extracts can act more precisely on the affected area. They leverage the holistic conditioning advantages of traditional Chinese medicine while improving the targeting and safety of treatment, thus opening new avenues for the treatment of cardiovascular diseases. Their preparation process is relatively simple, low-cost, and has fewer side effects. Summary of the Invention

[0005] Based on this, one embodiment of this application provides the application of vinegar-processed *Wulingzhi* water extract in the preparation of products that protect the heart and / or prevent platelet aggregation thrombosis, so as to realize the treatment of cardiovascular diseases with traditional Chinese medicine extracts.

[0006] In some embodiments, the use of the vinegar-processed aqueous extract of Trogopterus xanthipes is provided in the preparation of products that protect the heart and / or prevent platelet aggregation thrombosis.

[0007] In some implementations, protecting the heart refers to improving heart rate.

[0008] In some implementations, protecting the heart refers to improving cardiac output.

[0009] In some implementations, protecting the heart refers to improving blood flow velocity.

[0010] In some embodiments, the total terpene acid content in the aqueous extract of *Wu Ling Zhi* is greater than or equal to 300 mg / g.

[0019] , ,

[0020] , , and the total flavonoid content is greater than or equal to 89.2 mg·g -1 .

[0011] In some embodiments, in the aqueous extract of processed feces trogopterori, the total terpene acid content is 300 mg·g -1 ~650 mg·g -1 , and the total flavonoid content is 89.2 mg·g -1 ~311.4 mg·g -1 . <​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0021] Figure 1 The results of myocardial injury marker level detection in each group of rats are shown below. A is the comparison of serum CK levels, B is the dose-response curve of serum CK, C is the comparison of serum cNTi levels, D is the dose-response curve of serum CK, E is the comparison of serum LDH levels, and F is the dose-response curve of serum LDH. Group is the group and dose is the dosage.

[0022] Figure 2 The bar chart is for GO function analysis, where enrichment score, biological process, cellular component, and molecular function are represented.

[0023] Figure 3 Bubble chart for KEGG pathway enrichment analysis. Detailed Implementation

[0024] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0025] 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 description of the 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.

[0026] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0027] The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and "a combination of A and B."

[0028] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0029] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0030] In this application, the term "suitable" as used in "suitable combination", "suitable method", "any suitable method", etc., refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0031] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0032] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0033] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0034] In the present invention, in relation to "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0035] In this application, among the technical features described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions that include the listed features.

[0036] In this application, in relation to numerical intervals (i.e., numerical ranges), unless otherwise specified, the selectable numerical values are considered continuous within the above numerical intervals and include the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, when t is an integer selected from 1 to 10, it means that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when providing multiple range descriptions of features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0037] For the temperature parameters in this application, unless otherwise specified, both constant temperature treatment is allowed and fluctuations within a certain temperature range are allowed. It should be understood that the said constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed. <​​​​​​​​​

[0041] This application conducts an in-depth and systematic study on the cardioprotective mechanism of the aqueous extract of *Wulingzhi* (a type of plant extract), clarifying its specific targets and pathways of action. This will help promote its widespread application in the treatment of cardiovascular diseases and provide a reliable new treatment option for cardiovascular diseases.

[0042] In some embodiments, the use of the vinegar-processed aqueous extract of Trogopterus xanthipes is provided in the preparation of products that protect the heart and / or prevent platelet aggregation thrombosis.

[0043] In some implementations, protecting the heart means improving heart rate.

[0044] In some implementations, protecting the heart means improving cardiac output.

[0045] In some implementations, protecting the heart means improving blood flow velocity.

[0046] In some embodiments, the aqueous extract of vinegar-processed *Pteris vittata* contains fatty acids and their derivatives, aromatic carboxylic acids and phenolic acid derivatives, terpenes and their derivatives, steroids, alkaloid derivatives, and flavonoids and their derivatives.

[0047] In some embodiments, the total terpene acid content in the aqueous extract of *Wu Ling Zhi* (a type of plant extract) is greater than or equal to 300 mg / g. -1 and a total flavonoid content greater than or equal to 89.2 mg·g -1 .

[0048] In some embodiments, the total terpene acid content in the aqueous extract of *Wu Ling Zhi* (a type of plant extract) is 300 mg / g. -1 ~650 mg·g -1 The total flavonoid content was 89.2 mg / g. -1 ~311.4 mg·g -1 .

[0049] In some embodiments, the total terpene acid content in the vinegar-processed Trogopterus dung water extract is greater than or equal to 25 wt%, and the total flavonoid content is greater than or equal to 12 wt%.

[0050] It should be noted that the total terpene acid content was measured using a standard curve prepared with ursolic acid as the reference standard, and the total flavonoid content was measured using a standard curve prepared with rutin as the reference standard.

[0051] In a non-limiting sense, total terpenic acids include components such as Trogopterin D, deoxycholic acid, and cholic acid.

[0052] In a non-limiting sense, total flavonoids include components such as apigenin, paclitaxel flavonoids, and morin hydrate.

[0053] In some embodiments, the preparation method of the vinegar-processed Trogopterus xanthipes aqueous extract includes the following steps:

[0054] Mix the vinegar-processed five-spice powder with water, soak, decoct, and filter to obtain the filtrate.

[0055] The filtrate was concentrated and dried to prepare a water extract of vinegar-processed five-spice powder.

[0056] In some implementations, the products provided for protecting the heart and / or preventing platelet aggregation thrombosis include pharmaceuticals.

[0057] In some embodiments, the dosage form of the aforementioned drug includes one of oral solutions, tablets, pills, liquid suspensions, injections, granules, powders, and capsules.

[0058] In some implementations, the provided products for protecting the heart and / or preventing platelet aggregation thrombosis also contain pharmaceutically acceptable excipients.

[0059] In some embodiments, the aforementioned excipients include one or more of the following: diluents, wetting agents, binders, disintegrants, lubricants, color and flavor modifiers, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH adjusters, isotonic adjusters, and isotonic adjusters.

[0060] In some embodiments, the aforementioned diluent may be selected from, but is not limited to, starches, sugars, celluloses, and inorganic salts; one or a combination of multiple diluents may be selected.

[0061] In some embodiments, the aforementioned wetting agent may be selected from, but is not limited to, water and ethanol, and one or a combination of multiple agents may be selected.

[0062] In some embodiments, the aforementioned adhesive may be selected from, but is not limited to, starch paste, dextrin, sugar, cellulose derivatives, gelatin, povidone, and polyethylene glycol, and may be one or a combination of multiple.

[0063] In some embodiments, the aforementioned disintegrant is selected from dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, croscarmellose, surfactants, and effervescent disintegrants; one or a combination of multiple disintegrants may be selected.

[0064] In some embodiments, the aforementioned lubricant may be selected from, but is not limited to, talc, calcium stearate, magnesium stearate, magnesium dodecyl sulfate, micronized silica gel, and polyethylene glycol, and may be selected from one or a combination of multiple substances.

[0065] In some embodiments, the aforementioned color, flavor and aroma modifiers may be selected from, but are not limited to, pigments, fragrances, sweeteners, adhesives and odorants, and one or a combination of multiple can be selected.

[0066] In some embodiments, the aforementioned solvent may be selected from, but is not limited to, water, oil, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil, and ethyl acetate. One or a combination of multiple solvents may be selected.

[0067] In some embodiments, the aforementioned solubilizer may be selected from, but is not limited to, Tween compounds, maltose compounds, polyoxyethylene fatty alcohol ethers, soaps, sulfates, and sulfonates; one or a combination of multiple solubilizers may be selected.

[0068] In some embodiments, the aforementioned cosolvent may be selected from, but is not limited to, organic acids and their salts, amides and amines, inorganic salts, polyethylene glycol, povidone, and glycerol, and may be selected from one or a combination of multiple types.

[0069] In some embodiments, the aforementioned emulsifier may be selected from, but is not limited to, Span, Tween, Mize, benzyl esters, glycerol fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, tragacanth gum, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxides, silica, and bentonite. One or a combination of multiple emulsifiers may be selected.

[0070] In some embodiments, the aforementioned antioxidants may be selected from, but are not limited to, sulfites, metabisulfites, bisulfites, ascorbic acid, gallic acid and their esters, and one or a combination of multiple antioxidants may be selected.

[0071] In some embodiments, the aforementioned metal complexing agent may be selected from, but is not limited to, disodium ethylenediaminetetraacetate and polycarboxylic acid compounds; one or a combination of multiple compounds may be selected.

[0072] In some embodiments, the aforementioned inert gas may be selected from, but is not limited to, nitrogen and carbon dioxide, and one or a combination of multiple gases may be selected.

[0073] In some embodiments, the aforementioned preservatives are selected from parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, and volatile oils; one or a combination of multiple preservatives may be selected.

[0074] In some embodiments, the aforementioned local analgesic is selected from benzyl alcohol, chlorobutanol, lidocaine, and procaine, and one or a combination of multiple agents may be selected.

[0075] In some embodiments, the aforementioned pH adjuster may be selected from hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate, and citrate. One or a combination of multiple substances may be selected.

[0076] In some embodiments, the aforementioned isotonic and isotonic regulators are independently selected from glucose, sodium chloride, sodium citrate, sorbitol, and xylitol, and one or a combination of multiple can be selected.

[0077] In some embodiments of the present invention, a method for evaluating the efficacy of vinegar-processed Trogopterus xanthipes aqueous extract is provided, which is determined by the maximum detectable concentration (MTC): the maximum detectable concentration (MTC) for the cardioprotective efficacy of vinegar-processed Trogopterus xanthipes aqueous extract is 62.5 μg / mL, and the maximum detectable concentration (MTC) for the antiplatelet aggregation thrombosis efficacy is 1000 μg / mL.

[0078] In some embodiments of the present invention, a method for studying the efficacy and active substances of the aqueous extract of *Wulingzhi* (a type of plant extract) using network pharmacology is provided, identifying the active substances as flavonoids and terpene acids. The specific research method is as follows:

[0079] Using target prediction tools such as TCMSP, PubChem, and SwissTarget Prediction, we correlated the chemical components of drugs with potential biological targets to construct drug-target networks, revealing the interactions between drug molecules and targets in vivo. We also constructed disease-gene networks by integrating gene expression profiles and pathway information from disease databases to reveal the molecular mechanisms of disease development. Genes related to components in the aqueous extract of *Pteris vittata* (vinegar-processed *Pteris vittata*) were screened, and their potential roles in diseases were analyzed. Molecular docking software such as AutoDock was used to perform molecular docking between the active ingredients and key targets in *Phyllanthus urinaria* extract, predicting their binding affinity and possible mechanisms of action. Target functions were annotated using databases such as GO and KEGG to elucidate the pharmacological pathways of the active ingredients.

[0080] The following are specific embodiments. They are intended to provide a more detailed description of this application to help those skilled in the art and researchers better understand it. The technical conditions described do not constitute any limitation on this application. Any modifications made within the scope of the claims of this application are protected by the claims.

[0081] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods not specifying particular conditions in the examples were performed under conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0082] The instruments used in the examples are as follows: high-resolution mass spectrometer (UPLC-Q-ExactiveOrbitrap-MS, Thermo Fisher Scientific, USA), ultra-high performance liquid chromatograph (Waters H-Class, Waters Corporation, USA), 0.001 g balance (ME204E, Mettler Toledo), 0.1 g balance (XP26, Mettler Toledo), ultrapure water system (Milli-QDirect8 / 16 system, Merck), CNC ultrasonic cleaner (KQ5500DE, Kunshan Ultrasonic Instrument Co., Ltd.), mechanical split decoction pot (YMW, Chaozhou Gangdian Crafts Manufacturing Factory), rotary evaporator (Buchi R-100, Buchi GmbH, Switzerland), circulating water vacuum pump (SHZ-DШ, Gongyi Yuhua Instrument Co., Ltd.). Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Beilanbo Biotechnology Co., Ltd., China); High-speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany); Cardiac blood flow analyzer (ZebraBlood 3.4, ViewPoint Life Sciences, France); Fully automated rapid sample grinder (JXFSTPRP-24L, Shanghai Jingxin Laboratory Equipment Technology Department, China).

[0083] The reagents used in the examples are as follows: dimethyl sulfoxide (DMSO, batch number I2229063, Shanghai Aladdin Biochemical Technology Co., Ltd., China); arachidonic acid (batch number H2303046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); o-anisidine (batch number MKBX3619V, Sigma, USA); anhydrous sodium acetate (batch number E2317841, Shanghai Aladdin Biochemical Technology Co., Ltd., China); methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0084] Aspirin Enteric-coated Tablets (batch number BJ72163, Bayer Healthcare Co., Ltd.); Verapamil Hydrochloride (batch number A2007039, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Compound Danshen Dripping Pills (batch number 190203, Tasly Pharmaceutical Group Co., Ltd.). The processed Wulingzhi pieces were provided by Guangdong Yifang Pharmaceutical Co., Ltd. and identified by Professor Huang Haibo of Guangzhou University of Chinese Medicine. After being tested and meeting the pharmacopoeia standards, they were stored in the warehouse. Methanol and acetonitrile were of chromatographic purity, and other reagents were of analytical purity. The reference standards used were all purchased from institutions such as the National Institutes for Food and Drug Control of China, and their purity was greater than 98%. The grades of the reagents used all met the analytical requirements.

[0085] The experimental animals used in the examples were as follows: Zebrafish were all raised in fish-raising water at 28 °C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 450 - 550 μS / cm; pH was 6.5 - 8.5; hardness was 50 - 100 mg / L CaCO3). They were provided by the fish-raising center of our company. The license number for the use of experimental animals was: SYXK (Zhe) 2022 - 0004. The feeding management met the requirements of international AAALAC accreditation (accreditation number: 001458), and the IACUC ethical review number was: IACUC - 2024 - 8946 - 01.

[0086] Preparation of the water extract of processed Wulingzhi in Example 1

[0087] 1. Sample preparation

[0088] Take 100 g of the processed Wulingzhi pieces and place them in an electric ceramic pot. Decoct with water twice. For the first decoction, add 8 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500 W) and then keep it simmering gently over low heat (200 W) for 30 minutes. Filter the decoction through a 200-mesh sieve while it is still hot, and quickly cool the filtrate with cold water. For the second time, add 6 times the amount of water, bring to a boil over high heat (500 W) and then keep it simmering gently over low heat (200 W) for 25 minutes. Filter the decoction through a 200-mesh sieve while it is still hot, and quickly cool the filtrate with cold water. Combine the two decoctions. Transfer the decoction to a 5000 mL round-bottom flask and concentrate it under reduced pressure and low temperature using a rotary evaporator (temperature: 50 °C; vacuum degree: -0.10 MPa) to a 150 mL fluid extract; transfer it to a vacuum freeze dryer for freeze-drying, seal it, and determine the extract yield.

[0089] 2. Determination of the extract yield

[0090] Precisely weigh 5 g of the concentrated solution, place it in a pre-weighed evaporating dish, evaporate it to dryness in a water bath, dry it at 105 °C for 3 hours, cool it in a desiccator for 30 minutes, quickly weigh it, and calculate the extract yield according to the following formula.

[0091] Calculation formula: .

[0092] Ten batches of vinegar-processed Wulingzhi slices were prepared according to the preparation method under the "Sample Preparation" section, and the yield of the extract was determined. The results are expressed as mean ± standard deviation (SD), which is 14.54 ± 3.00%.

[0093] 3. Determination of total terpenic acid content

[0094] Preparation of reference solution: Take an appropriate amount of ursolic acid reference standard and prepare a reference solution containing approximately 0.3 mg of ursolic acid per 1 mL using 80% ethanol.

[0095] Preparation method of test solution: Take 0.2g of vinegar-processed quinoa extract, accurately weigh it, put it in a 100 mL Erlenmeyer flask, accurately add 50 mL of 80% ethanol, weigh it, sonicate (power 250W, frequency 50kHz) for 30 min, cool, filter, and take the filtrate to obtain the test solution.

[0096] Establishing the standard curve:

[0097] Accurately transfer 0.10, 0.20, 0.25, and 0.30 mL of the reference solution into 10 mL stoppered graduated test tubes, evaporate the solvent in a water bath, accurately add 0.2 mL of 5% vanillin-acetic acid solution and 0.8 mL of perchloric acid solution, shake well, heat in an 80 ℃ water bath for 15 min, cool under running water, accurately add 5 mL of acetic acid, shake well, and use 80% ethanol as a blank. Measure the absorbance at 545 nm wavelength using a UV spectrophotometer. Calculate the regression equation with ursolic acid mass (mg) on ​​the x-axis and absorbance on the y-axis.

[0098] Sample determination:

[0099] Take 2 mL of the test solution, and follow the standard curve establishment method to measure the absorbance. Substitute the absorbance into the regression equation to calculate the total terpene acid content. The results show that the total terpene acid content in the sample is 417.4 mg·g⁻¹. -1 Up to 610.1 mg·g -1 between.

[0100] 4. Determination of total flavonoid content

[0101] Preparation of reference solution: Accurately weigh an appropriate amount of rutin reference standard and dissolve it in methanol to prepare a rutin reference standard solution containing approximately 0.2 mg per mL.

[0102] Preparation method of test solution: Accurately weigh 0.2g of vinegar-processed quinoa extract, add 50mL of water, sonicate for 30min, filter, and collect the filtrate to obtain the test solution.

[0103] Establishing the standard curve:

[0104] Accurately pipette 1, 2, 3, 4, 5, and 6 mL of the reference solution into separate 25 mL volumetric flasks. Add water to a final volume of 6 mL, then add 1 mL of 5% sodium nitrite, shake well, and let stand for 6 min. Add 1 mL of 10% aluminum nitrate, shake well, and let stand for 6 min. Add 10 mL of 4% sodium hydroxide, shake well, and let stand for 15 min. Using the corresponding reagent as a blank, measure the absorbance at a wavelength of 595 nm. The concentration (X μg·mL) is used as the reference standard. -1 Plot a standard curve with absorbance (Y) on the x-axis and absorbance (Y) on the y-axis to obtain the regression equation.

[0105] Sample determination:

[0106] Take an appropriate amount of the test solution, operate according to the standard curve establishment method, measure the absorbance, and substitute it into the regression equation to calculate the total terpene acid content. The results show that the total flavonoid content in the sample is 89.2 mg / g. -1 Up to 311.4 mg·g -1 between.

[0107] Example 2 Component Analysis

[0108] 1. Sample Preparation

[0109] Take an appropriate amount of water extract, accurately weigh about 0.15 g, accurately add 25 ml of 90% methanol to determine the weight, reflux for 1 h, cool, remove and cool again, weigh, add the corresponding proportion of methanol to reduce the weight loss, shake well, and take the filtrate to obtain the test solution.

[0110] 2. Mass spectrometry detection

[0111] The determination was performed using a UPLC-Q-Exactive™ Orbitrap-MS system. A Waters ACQUITY UPLCHSS T3 column (2.1 mm × 100 mm, 1.8 μm) was used as the stationary phase, and a gradient elution was performed using acetonitrile (B) – 0.1% formic acid solution (A) as the mobile phase. The elution gradient was: 0–30 min, 5%–25% A; 30–50 min, 25%–40% A; 50–70 min, 40%–80% A. The flow rate was 0.3 mL / min. The column temperature was 30 °C, and the injection volume was 2 μL.

[0112] Heated electrospray ionization source (HESI), positive ion mode detection, sheath gas flow rate 35 L·min -1 Assisted airflow velocity 10 L·min -1The spray voltages were 3.8 kV and 3.2 kV, respectively; the capillary temperature was 350°C, and the auxiliary temperature was 350°C. The scanning mode was Full MS / dd-MS2, the scanning range was 100~1500 m / z, the Full MS resolution was 70000, the dd-MS2 resolution was 17500, and the collision energy was set to 20 eV.

[0113] 3. Ingredient Identification

[0114] Raw mass spectrometry data were imported into Compound Discoverer 3.3 software, and preprocessing was performed on the chromatographic peaks in the samples, including peak extraction and noise removal. The preprocessed mass spectrometry data was matched with the mzVault database and a self-built database. Compounds were assigned and identified using precise relative molecular masses and secondary mass spectrometry fragment ions from the databases. Simultaneously, online databases such as ChemSpider, Pubchem, and Scifinder, as well as relevant literature reports, were consulted to retrieve possible chemical structures and fragmentation fragments of the compounds. The identified components were compared and verified, and further verification was performed using standard comparisons to eliminate false positives. A total of 191 major chemical components were identified, mainly aromatic carboxylic acids and phenolic acid derivatives, terpenes and their derivatives (terpenic acids), flavonoids and their derivatives, etc. Among them, the flavonoid components mainly included: apigenin, cephalotaxine biflavonoids, and linalool hydrate; the terpenic acid components mainly included: trogopterin D, deoxycholic acid, and cholic acid, etc. The component identification results are shown in Table 1.

[0115] Table 1. Composition Identification Table

[0116]

[0117] Table 1 continues (1)

[0118]

[0119] Table 1 continues (2)

[0120]

[0121] Table 1 continues (3)

[0122]

[0123] Table 1 continues (4)

[0124]

[0125] Table 1 continues (5)

[0126]

[0127] Table 1 continues (6)

[0128]

[0129] Example 3: Dose-response relationship of cardioprotective effect

[0130] 1. Laboratory animals and grouping

[0131] SD rats (SPF grade, 200-240g, half male and half female, 48 in total) were acclimatized for 7 days.

[0132] Rats were randomly divided into 8 groups, with 6 rats in each group. The groups were: normal control group (NC), model control group (MC), positive control group (propranolol, PC), and various dose groups of vinegar-processed ginseng extract: 56.25 mg / kg (LL), 112.5 mg / kg (SL), 225 mg / kg (ML), 450 mg / kg (SH), and 900 mg / kg (HL) of vinegar-processed ginseng extract, respectively. The experiment was divided into two stages: (1) Establishment of heart failure model: The control group, positive control group (propranolol), and various dose groups of vinegar-processed ginseng extract were intraperitoneally injected with ISO (15.0 mg / kg / d) for 1 week (1 time / d, for 7 consecutive days) to establish a rat model of heart failure. The blank group was injected with physiological saline (10 ml / kg / d) as a control. (2) Drug administration stage: After modeling, the corresponding drug intervention was continued for three weeks. The positive control group was given propranolol (30 mg / kg / d); the different dose groups of the vinegar-processed *Wu Ling Zhi* aqueous extract were administered the corresponding dose of the extract by gavage; the blank control group and the model control group were given an equal volume of 0.1% sodium carboxymethyl cellulose solution. During the administration period, the general condition, activity level, and food and water intake of the rats were observed and recorded daily, and they were weighed weekly to adjust the dosage. After the last administration, the rats were fasted for 24 hours but allowed free access to water. They were then anesthetized, and serum was collected from the abdominal aorta. The heart was collected, and organ coefficients were calculated. The serum and urine were aliquoted and stored at -80°C.

[0133] 2. Observation Indicators

[0134] Serum samples were collected, and the levels of creatine kinase (CK), lactate dehydrogenase (LDH), and cardiac troponin I (cTnI) were measured using a fully automated biochemical analyzer to assess the degree of myocardial injury. Simultaneously, metabolic indicators such as blood glucose and blood lipids (TC, TG), as well as the expression levels of malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in myocardial tissue, were detected using ELISA to assess the degree of myocardial injury. All procedures were performed according to the manufacturer's instructions to ensure the reliability of the results.

[0135] Table 2 shows the changes in body weight of rats in each group. During the experiment, the weight of rats in the blank control group generally showed a stable growth trend, with a relatively constant weekly weight gain. The weight gain of rats in the model control group was significantly slower, and even decreased, during and after modeling. The weight gain trend of rats in each dose group of the vinegar-treated *Wulingzhi* extract was improved compared to the model group during the administration period, especially in the medium and high dose groups. The positive control group rats showed similar characteristics to the medium and high dose groups of vinegar-treated *Wulingzhi* extract, with steady weight gain and good overall condition.

[0136] The cardiac organ coefficients of each group of rats are shown in Table 3. Data on cardiac organ coefficients in each group of rats showed that the normal control group (NC) had a coefficient of 3.00±0.13 mg / g, representing the relative weight of the heart under normal physiological conditions; the model control group (MC) significantly increased to 5.00±0.36 mg / g, indicating the successful development of the ISO-induced heart failure model and the significant cardiac remodeling (myocardial hypertrophy) in the heart failure rats. The cardiac organ coefficients of each dose group (LL to HL) of the vinegar-processed Wulingzhi gradually decreased with increasing dose (4.70±0.22→3.30±0.13 mg / g), showing a significant dose-dependent improvement effect. The high-dose group (HL) showed the best effect, being closer to the normal level. The positive control group (PC, propranolol) had a coefficient of 3.50±0.13 mg / g, showing a clear improvement effect, while the high-dose vinegar-processed Wulingzhi group was more effective than the positive control group. In conclusion, vinegar-processed Wulingzhi can effectively improve cardiac remodeling in heart failure rats, and the effect is more significant at higher doses.

[0137] Table 2 Comparison of body weight changes in rats of different groups (n=6, mean±SD)

[0138]

[0139] Note: Compared with the NC group, P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001.

[0140] Table 3. Cardiac coefficients of rats in each group (n=6, mean±SD)

[0141]

[0142] Note: Compared with the NC group, P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001.

[0143] Figure 1Table 4 shows the results of myocardial injury marker level detection in each group of rats. Figure 1 In the table, A represents the comparison of serum CK levels; B represents the dose-response curve of serum CK; C represents the comparison of serum cNTi levels; D represents the dose-response curve of serum CK; E represents the comparison of serum LDH levels; and F represents the dose-response curve of serum LDH. The levels of myocardial injury markers (CK, LDH, cTnI) showed that, compared with the normal control group (NC), the serum CK, cNTi, and LDH levels in the model control group (MC) rats were significantly increased (P<0.0001), indicating that the myocardial injury model was successfully established. Low-dose vinegar-based gentian root extract (56.25 mg / g / d, 112.5 mg / g / d) did not significantly improve the above indicators. At the medium dose (225 mg / g / d), all three began to decrease significantly (P<0.05). The improvement effect was further enhanced in the second-highest (450) and high-dose (900) groups (P<0.0001), and the high-dose group was closer to the NC group level. Its improvement effect on CK and cNTi was better than that of the positive control group (PC). Dose-response relationship fitting analysis was performed on each indicator based on the dose-response relationship. The results showed that CK, LDH, and cTnI were all significantly negatively correlated with the administered dose (R0). 2 The value >0.95 indicates that the levels of myocardial injury markers continuously decreased with increasing dose, further validating the dose-dependent characteristics of vinegar-processed wulingzhi in repairing myocardial injury. Specifically, the dose-response curve for CK showed that the half-maximal inhibitory concentration (IC50) of vinegar-processed wulingzhi was 276.0 mg / kg / d, with a 95% confidence interval of 198.1–353.9 mg / kg / d; the dose-response curve for cNTi showed that the effective dose was 377.6 mg / kg / d, with a 95% confidence interval of 44.59–710.7 mg / kg / d; and the dose-response curve for LDH showed that the effective dose was 281.8 mg / kg / d, with a 95% confidence interval of 91.22–472.4 mg / kg / d.

[0144] Table 4 Comparison of myocardial injury marker levels in different groups of rats (n=6, mean±SD)

[0145]

[0146] Note: Compared with the NC group, P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001.

[0147] Example 4: Efficacy Testing

[0148] 1. Cardioprotective effects

[0149] (1) Determination of maximum detectable concentration (MTC)

[0150] Wild-type AB strain zebrafish, 2 days post-fertilization (2 dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The aqueous extract of *Wu Ling Zhi* prepared in Example 1 (concentration shown in Table 5) was administered in water. A normal control group and a model control group were also set up, with a volume of 3 mL per well. After 4 h of sample treatment, except for the normal control group, all other experimental groups were administered verapamil hydrochloride in water to establish a zebrafish heart failure model. After treatment at 28 ℃ for 1 h, the mean toxicity concentration (MTC) of the sample in the model zebrafish was measured. The results are shown in Table 5. Under the experimental conditions, the maximum detectable concentration (MTC) of the aqueous extract of *Wu Ling Zhi* for cardioprotective efficacy was 62.5 μg / mL.

[0151] Table 5 Results of the concentration exploratory experiment for cardioprotective efficacy of samples (n=30, mean ± SE)

[0152]

[0153] (2) Evaluation of cardioprotective efficacy

[0154] Two dpf wild-type AB strain zebrafish were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The aqueous extract of *Wulingzhi* prepared in Example 1 (concentration shown in Table 6) was administered in water, along with a positive control of 250 μg / mL of compound *Danshen* dripping pills. A normal control group and a model control group were also set up, with a volume of 3 mL per well. After 4 h of sample treatment, except for the normal control group, all other experimental groups were administered verapamil hydrochloride in water to establish a zebrafish heart failure model. After 1 h of treatment at 28 ℃, 10 zebrafish from each experimental group were randomly selected and placed under a cardiac blood flow analyzer for video recording. Heart rate, blood flow velocity, and cardiac output were analyzed and statistically analyzed. The cardioprotective efficacy of the samples was evaluated based on the statistical analysis results of these indicators. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance. As shown in Table 6, under the experimental conditions, the aqueous extract of vinegar-processed *Typhonium flagelliforme* has cardioprotective effects, specifically by improving heart rate, cardiac output, and blood flow velocity.

[0155] Table 6. Experimental results of cardioprotective efficacy evaluation of samples (n = 10, mean ± SE)

[0156]

[0157] Compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001

[0158] 2. Antiplatelet aggregation thrombotic effect

[0159] (1) Determination of maximum detectable concentration (MTC)

[0160] Zebrafish of the Albino strain with a 3-day-pass (dpf) melanin allele mutant were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The aqueous extract of *Wulingzhi* (prepared in Example 1) prepared in vinegar (concentration shown in Table 7) was administered to each well. A normal control group and a model control group were also included, with each well containing 3 mL. After treatment at 28℃ for 3 h, except for the normal control group, all other experimental groups were given arachidonic acid to establish a zebrafish platelet aggregation thrombosis model. After further treatment at 28℃ for 90 min, the MTC of the sample in the model zebrafish was measured. As shown in Table 7, under the experimental conditions, the antiplatelet aggregation thrombosis efficacy (MTC) of the aqueous extract of *Wulingzhi* was 1000 μg / mL.

[0161] Table 7 Results of the concentration exploratory experiment for antiplatelet aggregation thrombotic efficacy of samples (n = 30, mean ± SE)

[0162]

[0163] (2) Evaluation of antiplatelet aggregation thrombosis efficacy

[0164] Zebrafish of the Albino strain with a 3-day-pass (dpf) melanin allele mutant were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The aqueous extract of *Wulingzhi* prepared in Example 1 (concentration shown in Table 8) was administered in water, along with aspirin at a concentration of 60.0 μg / mL as a positive control. A normal control group and a model control group were also included, with a volume of 3 mL per well. After treatment at 28℃ for 3 h, except for the normal control group, all other experimental groups were given arachidonic acid in water to establish a zebrafish platelet aggregation thrombosis model. After further treatment at 28℃ for 90 min, o-anisidine staining was performed. After staining, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Data were collected using NIS-Elements D 3.20 advanced image processing software, and the staining intensity of zebrafish cardiac erythrocytes was analyzed. The statistical significance of this index was used to evaluate the antiplatelet aggregation thrombosis efficacy of the samples. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicated statistical significance. As shown in Table 8, under the experimental conditions, the aqueous extract of *Wulingzhi* (a type of medicinal herb) exhibited antiplatelet aggregation and thrombotic effects, specifically by increasing the staining intensity of cardiac erythrocytes.

[0165] Table 8. Results of the experiment evaluating the antiplatelet aggregation thrombotic efficacy of the samples (n = 10, mean ± SE)

[0166]

[0167] Compared with the model control group, **p < 0.01, ***p < 0.001

[0168] Example 5 Network Pharmacology Study

[0169] 1. Compound collection and target prediction

[0170] The main compounds in the aqueous extract of *Wu Ling Zhi* (a type of plant extract) were collected through online databases and literature searches, and their potential targets were predicted. Using tools such as SwissTargetPrediction and combined with literature validation, key targets related to cardioprotection and antithrombosis were screened, and a "compound-target" network was constructed to reveal its multi-component, multi-target mechanism of action. A total of 375 compounds were collected in this application. 156 compounds were selected for subsequent target prediction. Prediction using the SwissTargetPrediction database showed that these 156 compounds corresponded to a total of 809 potential targets.

[0171] 2. Establishment of the "Drug-Compound-Target-Disease" Network

[0172] A search of the Gene Cards (https: / / www.genecards.org / ) database (Cardiopathy) and targets related to qi stagnation and blood stasis revealed 102 key targets related to cardioprotection and antithrombosis. A "compound-target" network was constructed using Cytoscape 3.7.0 software, and visualization analysis revealed that the vinegar-processed Trogopterus dung water extract exerts cardioprotective and antithrombotic effects by acting synergistically on key targets through multiple components.

[0173] Drug-active ingredient-target network analysis revealed four main core components: protocatechuic acid, triterpenoid acid, paclitaxel flavonoids, and apigenin.

[0174] 3. Analysis of core components and core targets

[0175] The common target points of Wulingzhi and CAD were imported into the STRIGN database (https: / / cn.string-db.org / ) to construct a PPI network diagram. The node information of the target PPI network was downloaded and imported into Cytoscape 3.7.0 software. The cytoNCA plugin was used to score each node and select 10 key target points. These key target points include: ESR1, GSK3B, IL6, MTOR, BCL2, ALB, KRAS, RHOA, HSP90AA1, and CASP3.

[0176] Computer-aided design was used to verify molecular docking between active ingredients and targets. The results showed that the active ingredients in the aqueous extract of *Wu Ling Zhi* (a type of herb) exhibited high affinity for these key targets. The binding energies of these active ingredients to the core targets were all within the range of -6.7 to -8.9 kcal / mol (1 kcal = 4.186 kJ). The lower the binding energy, the better the docking effect, indicating that the binding between the active ingredient molecules and the core target receptor protein is more stable and the affinity is higher. This suggests that these flavonoids and terpenoids improve cardiovascular disease symptoms through multi-target and multi-pathway synergistic effects.

[0177] 4. GO Functionality Analysis

[0178] GO analysis can reveal gene function, and the results are as follows: Figure 2 As shown. GO enrichment analysis yielded 458 entries, including 313 biological processes (BP), 55 cellular components (CC), and 90 molecular functions (MF). BP mainly included phosphorylation, exogenous metabolic processes, positive regulation of gene expression, protein phosphorylation, peptidyl-threonine phosphorylation, estrogen metabolism, responses to exogenous stimuli, cellular responses to hydrogen peroxide, negative regulation of apoptosis, and smooth muscle contraction. MF mainly included protein serine kinase activity, nuclear receptor activity, same-protein binding, ATP binding, protein serine / threonine kinase activity, estrogen response element binding, steroid binding, monooxygenase activity, protein kinase activity, and tau protein binding. CC mainly included cytoplasm, cytoplasm, endoplasmic reticulum membrane, dendrites, organelles bound to the inner cell membrane, nucleus, outer mitochondrial membrane, membrane, nucleoplasm, and secretory granule lumen. These results indicate that the aqueous extract of vinegar-processed *Typhonium flagelliforme* (a type of plant extract) affects multiple key intracellular sites by regulating various biological processes and molecular functions, thereby achieving a comprehensive regulatory effect on cardioprotection.

[0179] 5. KEGG Functional Analysis

[0180] The KEGG signaling pathway is an important tool for reflecting intracellular signal transduction and metabolic pathways. Results are as follows: Figure 3As shown, KEGG enrichment analysis identified 94 relevant signaling pathways. These mainly include lipid and atherosclerosis, proteoglycans in cancer, viral carcinogenesis, measles, chemical carcinogenesis-receptor activation, cancer pathways, human cytomegalovirus infection, endocrine resistance, human immunodeficiency virus type 1 infection, EGFR tyrosine kinase inhibitor resistance, hepatitis B, prostate cancer, AGE-RAGE signaling pathway in diabetic complications, PI3K-Akt signaling pathway, p53 signaling pathway, alcoholic liver disease, chemokine signaling pathway, Alzheimer's disease, ErbB signaling pathway, and adhesion. These signaling pathways involve multiple aspects such as cell proliferation, apoptosis, and metabolism, further confirming the multi-target and multi-pathway mechanism of action of the vinegar-processed *Wulingzhi* extract in cardiovascular protection, providing a solid theoretical basis for its clinical application.

[0181] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0182] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Application of vinegar-processed Trogopterus xanthipes aqueous extract in the preparation of products for protecting the heart and / or preventing platelet aggregation thrombosis.

2. The application according to claim 1, characterized in that, Protecting the heart refers to improving heart rate.

3. The application according to claim 1, characterized in that, Protecting the heart refers to improving cardiac output.

4. The application according to claim 1, characterized in that, Protecting the heart refers to improving blood flow velocity.

5. The application according to any one of claims 1 to 4, characterized in that, The total terpene acid content in the aqueous extract of *Wulingzhi* (a type of plant extract) is greater than or equal to 300 mg / g. -1 and a total flavonoid content greater than or equal to 89.2 mg·g -1 ; Optionally, the total terpene acid content in the vinegar-processed *Wu Ling Zhi* aqueous extract is 300 mg / g. -1 ~650 mg·g -1 The total flavonoid content was 89.2 mg / g. -1 ~311.4 mg·g -1 .

6. The application according to any one of claims 1 to 4, characterized in that, The preparation method of the vinegar-processed Trogopterus xanthipes water extract includes the following steps: Mix the vinegar-processed five-spice powder with water, soak, decoct, and filter to obtain the filtrate. The filtrate was concentrated and dried to prepare the vinegar-based extract of *Wulingzhi*.

7. The application according to any one of claims 1 to 4, characterized in that, The products include pharmaceuticals.

8. The application according to claim 7, characterized in that, The dosage form of the drug includes one of the following: oral solution, tablet, pill, liquid suspension, injection, granule, powder, and capsule.

9. The application according to claim 7, characterized in that, It also includes pharmaceutically acceptable excipients.

10. The application according to claim 9, characterized in that, The excipients include one or more of the following: diluents, wetting agents, binders, disintegrants, lubricants, color and flavor modifiers, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH adjusters, isotonic adjusters, and isotropic adjusters.

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