Food-derived product affinity screening method for lowering blood pressure, compositions and uses

Abstract

The application relates to the fields of biological analysis and biological medicine and functional food technology, and particularly relates to a food source product affinity screening method for reducing blood pressure, a composition and application, the screening method injects a sample of a food source product to be detected into an ACE / ET-1 composite affinity chromatographic column for chromatographic separation, and adopts ELSD to detect the peak time of the food source product to be detected; the food source product is divided into different binding classification according to the peak time difference between the food source product and a negative control group, and the food source product combination of different binding classification is selected to obtain a food source product composition for reducing blood pressure. The application also specifically discloses the screened composition or monomer compound for reducing blood pressure, PCR / WB and HE pathology prove that the composition can down-regulate ACE / ET-1 path-related indexes, and the monomer compounds of myricetin and myricitrin can also down-regulate ACE / ET-1 related mRNA and protein expression.

Description

Technical Field

[0001] This application relates to the fields of bioanalysis, biomedicine and functional food technology, specifically to an affinity screening method, composition and application of a food product for lowering blood pressure. Background Technology

[0002] Hypertension is a common cardiovascular metabolic disease characterized by a large patient population, long disease course, and a wide spectrum of complications. It is a major risk factor for events such as stroke, coronary heart disease, heart failure, and kidney damage. Clinical treatment is based on long-term, individualized intervention, and commonly used drugs include angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), calcium channel blockers, diuretics, and beta-blockers. Although the existing drug system is mature, there are still pain points in clinical application, such as efficacy differences, tolerability issues, and insufficient compliance. At the same time, food and medicine homology resources have potential in adjuvant antihypertensive treatment. How to efficiently and reliably identify the active ingredients that are truly related to antihypertensive targets in complex categories is a key link in research and development and industrial transformation.

[0003] From a mechanistic perspective, angiotensin-converting enzyme (ACE)-mediated peptide conversion and endothelin-1 (ET-1)-related targets jointly participate in the regulation of vascular tone and humoral homeostasis: the former affects vasoconstriction and aldosterone secretion through the Ang I → Ang II conversion, while the latter is a potent vasoconstrictive factor derived from the endothelium; both have a sustained impact on blood pressure. Traditional in vitro screening methods targeting this target often employ enzyme inhibition activity assays or cell model evaluations. However, for food-derived / medicinal food-derived samples, problems exist such as non-specific binding, interference from impurities like pigments and polysaccharides, large batch-to-batch variability, limited throughput, and insufficient reproducibility. This leads to the "separation and purification first, then validation" approach being costly, time-consuming, and having a high false positive rate. Affinity chromatography, using "target-ligand specific binding" as the criterion, can perform online identification and enrichment of complex samples under aqueous phase and mild conditions. It can reflect "whether there is a specific interaction with the target" through retention time / response differences, enabling a natural adaptability-guided separation and "screening before matching" research and development path. Compared to conventional methods, this approach helps to eliminate non-specific signals at an early stage and provides quantifiable and reproducible physicochemical indicators for subsequent formulation design and quality control.

[0004] However, existing affinity chromatography applications in the depressurization direction still face several technical bottlenecks: First, target selection is too singular, making it difficult to reflect the coupling effects of different pathways; second, insufficient stationary phase activity and column lifetime limit the stability and transferability of the methodology; third, the lack of grading thresholds linked to positive / negative controls makes it difficult to compare results across different batches and processes. Using only single-target screening often fails to account for multiple pathway mechanisms, easily leading to false positives due to non-specific adsorption, and the screening results are difficult to form reproducible formulation design rules and obtain consistent in vivo validation. Therefore, there is an urgent need to construct an affinity screening platform that can simultaneously cover multiple targets and achieve grading screening and formulation guidance through quantitative indicators. Summary of the Invention

[0005] To address the aforementioned technical challenges, this application proposes an integrated technical solution for target-guided screening and formulation design in complex food / food-medicine homology systems. This solution utilizes ACE and / or ET-1 as dual targets, constructs an ACE / ET-1 affinity chromatography column, and establishes HPLC-ELSD detection methods and rules for determining positive / negative controls. Based on this, various food-derived raw materials are subjected to affinity grading screening. Compositions for antihypertensive food-derived products are designed based on the grading results, and the correlation and mechanistic consistency between the screening and formulation are verified through animal efficacy and molecular biological indicators. This application provides a systematic solution based on the ACE / ET-1 dual-target affinity screening platform, encompassing target affinity, formulation design, efficacy verification, and mechanism confirmation. This solution is applicable to the development and quality control of antihypertensive food-derived products / health supplements / drugs. It provides a unified and operable technical pathway for the development, process optimization, and quality evaluation of antihypertensive compositions and monomeric active ingredients.

[0006] To achieve the above objectives, this application first provides an affinity screening method for food-derived products used to lower blood pressure, comprising the following steps: injecting a sample of the food-derived product to be tested into an ACE / ET-1 composite affinity chromatography column for chromatographic separation, and using ELSD to detect the peak time of the food-derived product to be tested; classifying the food-derived product into different binding affinity grades according to the difference in peak time between the food-derived product and the negative control group, and selecting combinations of food-derived products of different binding affinity grades to obtain a food-derived product composition for lowering blood pressure;

[0007] The stationary phase packed with the ACE / ET-1 composite affinity chromatography column is an ACE / ET-1@SiO2 composite stationary phase, and the carrier of the ACE / ET-1@SiO2 composite stationary phase is SiO2 particles. The SiO2 particles simultaneously immobilize two target molecules: angiotensin-converting enzyme ACE and endothelin ET-1.

[0008] The negative control group consisted of acetaminophen.

[0009] Preferably, the peak elution time difference between the food-derived product and the negative control group is Δt. R =t R样品 -t R阴性 , where t R样品 t represents the peak elution time of the food product. R阴性 The peak time is the time of elution in the negative control group, and the unit of the peak time is min.

[0010] Preferably, the different binding grade includes three types: strong binding, moderate binding, and general binding.

[0011] When ΔtR < 2, the food product is classified as a general combination.

[0012] When 2≤ΔtR<5, the food product is classified as a medium combination.

[0013] When ΔtR≥5, the food product is determined to be of a strongly bound type.

[0014] Preferably, the graded food products with strong binding include Eucommia ulmoides leaves, hawthorn, lotus seeds, corn silk, and bayberry;

[0015] The food products classified as medium-combined include kudzu root, bitter tea, celery seed, polygonatum, and fig.

[0016] The graded food products generally include cassia seed, astragalus, buckwheat, codonopsis, platycodon, pumpkin, and blueberry;

[0017] The mass percentages of the food-derived products with different binding affinity grades in the food-derived product composition for lowering blood pressure are as follows:

[0018] The quality of food-derived products classified as "strongly combined" in the single-ingredient category accounts for 10-15%.

[0019] The quality of single-flavor food products classified as moderately combined accounts for 3-5% of the total quality.

[0020] The quality of single-ingredient food products in the general combination category accounts for 0-3%.

[0021] Preferably, the chromatographic separation conditions are: column temperature 35℃, flow rate 0.15 mL / min, mobile phase 5 mM ammonium acetate, injection volume 25 μL, and pH of the mobile phase 7.2.

[0022] Preferably, the evaporation temperature for ELSD detection is 50-70℃, the drift tube temperature is 50-70℃, and the nitrogen flow rate is 1.0-2.0 L / min.

[0023] Preferably, the preparation method of the ACE / ET-1@SiO2 composite stationary phase is as follows: SiO2 particles are placed in PBS buffer, and solutions of two target molecules, angiotensin-converting enzyme (ACE) and endothelin (ET-1), are added respectively. After immobilization by stirring under low temperature conditions, the particles are centrifuged and repeatedly washed with PBS to remove unfixed target molecules to obtain the ACE / ET-1@SiO2 composite stationary phase.

[0024] As a preferred method, the preparation of the ACE / ET-1@SiO2 chromatographic column includes the following steps: Select a column core with a size of 50 mm × 4.6 mm. After washing the column core and column sleeve with ultrapure water, pack the column using a low-pressure wet packing method: Insert the column core into the column sleeve, place gaskets and caps at both ends, and slowly inject the PBS suspension of the ACE / ET-1@SiO2 composite stationary phase into the column core. Pressurize the column with a high-pressure pump at a low flow rate to allow the composite stationary phase to settle rapidly until the pressure at the column inlet no longer rises significantly. Open the top cap and add the PBS suspension of the composite stationary phase again. Repeat the above operation to fill the chromatographic column to obtain the ACE / ET-1@SiO2 chromatographic column. Store at 4°C for later use.

[0025] Preferably, the sample preparation method for the food product includes the following steps:

[0026] (1) Crush and sieve the food product, add petroleum ether and heat and reflux it. After reflux, cool, filter and collect the first filter residue.

[0027] (2) Add anhydrous ethanol to decolorize the first filter residue, heat and reflux again, cool, filter and collect the second filter residue after reflux.

[0028] (3) Add ultrapure water to the second filter residue, heat and reflux again, cool, filter and collect the supernatant after reflux, evaporate and concentrate the supernatant, add 95% ethanol to the concentrated supernatant to precipitate, centrifuge to collect the precipitate, wash the precipitate and vacuum dry to obtain the active ingredient of the food product.

[0029] (4) Prepare an aqueous solution of the active ingredients of the food product to obtain a sample of the food product.

[0030] Based on a general inventive concept, this application also provides a food-derived product composition for lowering blood pressure obtained by the above-described affinity screening method, including either composition one or composition two;

[0031] The composition of the first composition, by weight percentage, is as follows: Eucommia ulmoides leaves 15%, bayberry 15%, hawthorn 15%, corn silk 15%, lotus seeds 10%; kudzu root 5%, bitter tea 5%, celery seed 5%, polygonatum 5%, fig 3%; cassia seed 2%, astragalus 2%, tartary buckwheat 1%, platycodon root 1%, blueberry 1%;

[0032] The composition of the second composition, by mass percentage, is as follows: 10% Eucommia ulmoides leaves, 10% bayberry, 15% hawthorn, 15% corn silk, 15% lotus seeds; 5% kudzu root, 5% bitter tea, 5% celery seed, 5% Polygonatum sibiricum, 5% fig; 2% cassia seed, 2% Astragalus membranaceus, 1% tartary buckwheat, 2% Codonopsis pilosula, 1% Platycodon grandiflorus, 1% pumpkin, 1% blueberry.

[0033] Based on a general inventive concept, this application also provides an application of the above-mentioned affinity screening method in screening monomeric compounds with antihypertensive properties, wherein the monomeric compounds include one or more of myricetin, myricetin and arbutin.

[0034] This application selects angiotensin-converting enzyme (ACE) and endothelin ET-1 as parallel targets: ACE catalyzes the transition from Ang I to Ang II, thereby enhancing vasoconstriction and aldosterone secretion; ET-1 is a potent vasoconstrictor derived from the endothelium. The two work synergistically to regulate vascular tone and humoral homeostasis, forming a core pathway in the prevention and treatment of hypertension. Therefore, when components in the food product sample to be tested specifically bind to ACE / ET-1, a longer retention time will be obtained on affinity chromatography; using the retention time of the negative control as a benchmark, ΔtR = tR is defined. 样品 -tR 阴性 It is used as a unified quantitative indicator of binding strength. Under HPLC-ELSD conditions, with acetaminophen as a negative control and captopril and alpracitentan as positive controls, the information on whether and to what extent food-derived products "bind to the core antihypertensive target" can be converted into a measurable ΔtR signal. Based on this, ΔtR can not only determine "whether it binds" but also serve as a quantitative indicator of "binding strength" for early activity screening and formulation optimization.

[0035] It should be noted that this application uses ΔtR as a quantitative indicator of affinity binding strength for early screening and grading of food-derived products. ΔtR reflects the binding performance of the sample under ACE / ET-1 immobilization conditions and can effectively identify candidate active sources related to the target axis. However, this indicator does not have a strict linear correspondence with the final blood pressure-lowering effect in vivo. The blood pressure-lowering effect in vivo is also affected by factors such as the content of active ingredients in the raw materials, release and dissolution, absorption and metabolism, bioavailability, and multi-pathway physiological regulation. Therefore, the screening in this application does not simply pursue "the higher the proportion of strong binding types, the better," but follows the combination optimization approach of "clear main effect + synergistic effect + safety and sustainability": using raw materials with strong binding types as the main effect base to achieve targeted intervention on the ACE / ET-1 axis, while introducing raw materials with moderate and general binding types to supplement and amplify the multi-link effects related to blood pressure lowering (such as improving endothelial function, anti-oxidation / anti-inflammation, diuresis and sodium excretion, affecting absorption and metabolism, etc.), and improving the palatability and stability of the formulation, making the composition more suitable for long-term blood pressure lowering intervention scenarios. Based on this principle, this application provides recommended content ranges for three different types of raw materials—strong, medium, and general—in the composition to achieve the optimal overall balance of pressure reduction effect, stability, and safety.

[0036] The main benefits of this application are as follows:

[0037] Existing technologies for screening antihypertensive active substances and developing functional compositions often suffer from limitations such as single-target coverage, insufficient correlation between screening results and physiological mechanisms, or low screening efficiency and large batch-to-batch variability due to reliance on complex sample pretreatment processes. This application innovatively constructs an affinity screening system using ACE and ET-1 as dual targets. As core molecules regulating blood pressure homeostasis, ACE and ET-1 respectively mediate key pathways of vasoconstriction and fluid balance. This dual-target synergistic design not only better reflects the complexity of the pathological process of hypertension but also enhances the targeting of active ingredient screening at the mechanistic level. It effectively avoids overlooking multi-pathway synergistic active substances that may be missed in single-target screening, providing a more physiologically aligned technical direction for exploring the antihypertensive potential of food-derived raw materials.

[0038] Building upon this foundation, this application addresses the shortcomings of existing technologies, such as numerous interferences from impurities and a disconnect between activity screening and formulation design in complex foodborne systems. Utilizing affinity chromatography principles, it enables the screening of active ingredients under mild aqueous conditions, simplifying purification steps and reducing interference from inactive components like pigments and polysaccharides. Furthermore, by establishing positive / negative controls and clearly defined binding strength grading rules, the interaction between active ingredients and targets is transformed into quantifiable and verifiable chromatographic signals. Animal efficacy experiments, PCR / WB, and HE pathological verification of ACE / ET-1 index changes further complete the process from in vitro screening to in vivo mechanism validation. This solves the problem of difficulty in cross-referencing screening results from different raw materials and batches, significantly improving the stability and operability of the screening process.

[0039] This application further separates and purifies extracts of bayberry and blueberry to obtain several representative flavonoid or glycoside monomers. The structures of the obtained monomers are systematically identified using LC-MS. Finally, under the same ACE / ET-1 affinity chromatography conditions as the raw materials, the affinity behavior of the monomers obtained above is determined. The binding strength of the monomers with ACE and / or ET-1 is graded based on ΔtR to establish the correspondence between "raw material affinity grading and monomer affinity grading" and to provide a basis for subsequent formulation design and functional evaluation. Unlike existing technologies that only report the blood pressure-lowering trend of bayberry and blueberry at the overall extract level, this invention not only constructs an affinity screening platform covering both ACE / ET-1 targets and a raw material formulation grading principle based on ΔtR, but also conducts in-depth research at the monomer level. This not only effectively corroborates the results of raw material investigation but also reveals the effective components for lowering blood pressure. This multi-level consistency not only supports the screening and grading principle based on ACE / ET-1 affinity chromatography from a mechanistic perspective but also provides a referable technical paradigm for subsequent targeted monomer development and quality control in berry resources such as bayberry and blueberry. It realizes multi-level technical integration from raw materials to monomers, from target affinity to in vivo mechanisms, significantly enhancing the transformation potential and application value of this invention.

[0040] Hypertension involves the coupled regulation of ACE and ET-1-related vasomotor pathways, and is also accompanied by multiple effects such as oxidative stress, inflammatory response, and diuresis and sodium excretion. This application defines "strong binding type" as ingredients with a more significant affinity for the ACE / ET-1 target, providing a primary intervention targeting the ACE / ET-1 axis; while moderate / moderate binding types, although exhibiting moderate or moderate binding under affinity chromatography conditions, can contribute to the overall formulation in vivo through other mechanisms, such as: ① supplementing or amplifying the primary pathway effect: indirectly enhancing the antihypertensive effect of ACE / ET-1 axis inhibition by improving the endothelial NO pathway, antioxidation, anti-inflammation, or affecting vasomotor balance; ② some components can affect dissolution, absorption, or metabolic transformation, improving the bioavailability of the primary active ingredient; ③ reducing adverse reactions and improving palatability / stability, making the formulation more suitable for long-term functional food intervention scenarios. Therefore, this application distinguishes between three different types of combinations: strong, moderate, and general. Then, it optimizes the combination based on different types of products. By considering the main target effect, multi-stage synergy, and comprehensive performance, the composition can be designed to produce a product with outstanding blood pressure-lowering effect. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 The following are the scanning electron microscope (SEM) results of SiO2 and ACE / ET-1@SiO2 in Example 1, wherein... Figure 1 A is a scanning electron microscope image of SiO2. Figure 1 B is a scanning electron microscope image of ACE / ET-1@SiO2;

[0043] Figure 2 The results of ACE and ET-1 immunofluorescence of ACE / ET-1@SiO2 in Example 1 are shown below. Figure 2 A represents the immunofluorescence of SiO2. Figure 2 B represents the ACE immunofluorescence of ACE / ET-1@SiO2. Figure 2 C represents the ET-1 immunofluorescence of ACE / ET-1@SiO2;

[0044] Figure 3 This is the chromatogram of the negative / positive control in Example 2, where... Figure 3 A is the chromatogram of the acetaminophen negative control. Figure 3 B is the chromatogram of the captopril positive control. Figure 3 C is the chromatogram of the alpracitentan positive control;

[0045] Figure 4 The chromatograms of Eucommia ulmoides leaves, kudzu root, bitter tea, hawthorn, celery seed, and corn silk raw materials obtained in Example 3 by ACE / ET-1@SiO2 column chromatography are shown below.

[0046] Figure 5 The chromatograms of the raw materials cassia seed, astragalus, polygonatum, lotus seed, tartary buckwheat, and codonopsis obtained from Example 3 using an ACE / ET-1@SiO2 column are shown below.

[0047] Figure 6 The chromatograms of the raw materials of Platycodon grandiflorus, Myrica rubra, pumpkin, fig and blueberry obtained by ACE / ET-1@SiO2 column in Example 3 are shown below.

[0048] Figure 7 The image shows the LC-MS detection results of bayberry and its monomers in Example 4. Figure 7 A is the LC-MS analysis chromatogram of bayberry. Figure 7 B~ Figure 7 C represents the liquid chromatograms of myricetin and myricetin, respectively. Figure 7 D~ Figure 7 E represents the mass spectra of myricetin and myricetin, respectively;

[0049] Figure 8 The image shows the LC-MS detection results of blueberries and various monomers in Example 4. Figure 8 A is the LC-MS analysis chromatogram of blueberries. Figure 8 B is the liquid chromatogram of arbutin. Figure 8 C is the mass spectrum of arbutin;

[0050] Figure 9 This is the monomer affinity chromatogram from Example 4, where... Figure 9 A is the chromatogram of myricetin. Figure 9 B is the chromatogram of myricetin. Figure 9 C is the chromatogram of arbutin;

[0051] Figure 10 The results of the antihypertensive effect of each group in the animal trial in Example 6 are as follows: Figure 10 A is a graph showing the trend of systolic blood pressure changes in rats. Figure 10 B is a graph showing the trend of diastolic blood pressure in rats;

[0052] Figure 11 The results of the animal-level evaluation of the antihypertensive effects of each monomer in Example 6 are as follows: Figure 11 A is a graph showing the trend of systolic blood pressure changes in rats. Figure 11 B is a graph showing the trend of diastolic blood pressure in rats;

[0053] Figure 12The results of PCR detection of ACE / ET-1 mRNA expression under each formulation intervention in Example 6 are shown below. Figure 12 A represents the mRNA expression result of ACE. Figure 12 B represents the mRNA expression result of ET-1;

[0054] Figure 13 The results of PCR detection of ACE / ET-1 mRNA expression under the intervention of each monomer in Example 6 are shown below. Figure 13 A represents the mRNA expression result of ACE. Figure 13 B represents the mRNA expression result of ET-1;

[0055] Figure 14 The images show the Western blotting and quantitative results of ACE / ET-1 under different formulation interventions in Example 6. Figure 14 A is a Western blotting (WB) stripe plot. Figure 14 B represents the quantitative result of ACE. Figure 14 C represents the quantitative result of ET-1;

[0056] Figure 15 The images show the Western blotting and quantitative results of ACE / ET-1 under different monomeric interventions in Example 6. Figure 15 A is a Western blotting (WB) stripe plot. Figure 15 B represents the quantitative result of ACE. Figure 15 C represents the quantitative result of ET-1;

[0057] Figure 16 The results of HE examination of the hearts of rats in each group in Example 6 are shown. Detailed Implementation

[0058] The following embodiments are used to illustrate this application, but are not intended to limit the scope of this application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application are within the scope of this application.

[0059] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0060] Unless otherwise specified, the percentage sign "%" in this application refers to the mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0061] The weight units mentioned in this application may be well-known weight units in the art, such as μg, mg, g, kg, etc., or multiples thereof, such as 1 / 10, 1 / 100, 10 times, 100 times, etc.

[0062] Example 1: Preparation of ACE / ET-1@SiO2 chromatographic column, the specific steps are as follows:

[0063] Weigh 20 mg SiO2 into a 100 mL round-bottom flask, add 20 mL PBS (pH 7.4), and add 98 μL each of ACE and ET-1 solutions (5 U / 490 μL). Stir magnetically in an ice bath for 3 h. Centrifuge and wash 5 times with PBS to obtain the ACE / ET-1@SiO2 stationary phase. Select a 50 mm × 4.6 mm column core. Wash the column core and column sleeve with ultrapure water and pack the column using a low-pressure wet packing method: insert the column core into the column sleeve, place gaskets and caps at both ends, and slowly inject the PBS suspension of the stationary phase into the column core. Pressurize with a high-pressure pump at a low flow rate to allow the stationary phase to settle rapidly until the pressure at the column inlet no longer rises significantly. Open the top cap and add the PBS suspension of the stationary phase again. Repeat the above operation to fill the column to obtain the ACE / ET-1@SiO2 column. Store at 4℃ for later use.

[0064] The morphological characteristics of SiO2 and ACE / ET-1@SiO2 and the immunofluorescence detection results of ACE and ET-1 in ACE / ET-1@SiO2 are as follows: Figure 1 , Figure 2 As shown. Figure 1 A is a scanning electron microscope image of SiO2. Figure 1 B is a scanning electron microscope image of ACE / ET-1@SiO2, based on Figure 1 The results show that, compared to the smooth surface of SiO2, the ACE / ET-1@SiO2 stationary phase is uniformly coated with ACE and ET-1. Figure 2 A represents the immunofluorescence of SiO2. Figure 2 B represents the ACE immunofluorescence of ACE / ET-1@SiO2. Figure 2 C represents the ET-1 immunofluorescence of ACE / ET-1@SiO2. Figure 2 The immunofluorescence assay results further provide clear and intuitive evidence for the above conclusions, demonstrating that ACE was successfully bound to the ACE / ET-1@SiO2 surface. Figure 2 B (green fluorescence) and ET-1 ( Figure 2 (Red fluorescence in C).

[0065] Example 2 HPLC-ELSD conditions and system suitability

[0066] The ACE / ET-1@SiO2 column prepared in Example 1 was used to perform chromatographic analysis on the test samples. The HPLC-ELSD conditions were set as follows: column temperature 35°C; flow rate 0.15 mL / min; mobile phase 5 mM ammonium acetate (pH 7.2); injection 25 μL; ELSD evaporation temperature 63°C, drift tube temperature 60°C, and nitrogen 1.5 L / min. Positive controls captopril and alpracentemumtan were prepared at a concentration of 250 μg / mL, and the negative control acetaminophen was prepared at a concentration of 250 μg / mL. The chromatograms obtained after injection and detection are shown below. Figure 3 As shown, Figure 3 A is the chromatogram of the acetaminophen negative control. Figure 3 B is the chromatogram of the captopril positive control. Figure 3 C is the chromatogram of the alpracitentan positive control. According to... Figure 3 The results showed that the peak elution time (tR) for the positive control captopril was approximately 18.9 min, for aspirin it was approximately 18.4 min, and for the negative control acetaminophen it was approximately 9.8 min. The positive controls captopril and aspirin were used to verify that the two target molecules in the ACE / ET-1@SiO2 composite stationary phase retained their binding activity and to determine the suitability of the detection system. A non-specific retention baseline was obtained using acetaminophen as the negative control. When the positive control showed a significantly prolonged retention time compared to the negative control on the composite affinity column, the column activity and system operation were considered normal. If the positive control did not exhibit the expected retention behavior, the results of that batch of detections were not used as the basis for ΔtR grading.

[0067] Example 3 Screening and Grading of Food and Medicinal Herbs

[0068] (1) Sample preparation of food products

[0069] Seventeen kinds of food-derived raw materials (Eucommia ulmoides leaves, kudzu root, bitter tea, hawthorn, celery seed, corn silk, cassia seed, astragalus, polygonatum, lotus seed, buckwheat, codonopsis, platycodon, pumpkin, bayberry, fig, and blueberry) were taken, washed, and dried. The raw materials were then pulverized using a pulverizer and passed through an 80-mesh sieve. Ten times the amount of petroleum ether was added, and the mixture was heated to reflux at 75°C for 3 hours, followed by sonication for 30 minutes. After cooling, the mixture was filtered, and the first filter residue was collected. This process was repeated three times. The first filter residue was then decolorized with ten times the amount of anhydrous ethanol, heated to reflux at 80°C for 3 hours, followed by sonication for 30 minutes. After cooling, the mixture was filtered, and the second filter residue was collected. This process was repeated three times. Ten times the amount of ultrapure water was added to the second filter residue, and the mixture was heated to reflux at 100°C for 3 hours, followed by sonication for 30 minutes. After cooling, filter the sample and collect the residue. Repeat the above operation three times. Collect the supernatant by filtration, combine the supernatants, concentrate and enrich them using a rotary evaporator, precipitate with 5 times the volume of 95% ethanol overnight, centrifuge at 3500 rpm for 10 min, collect the precipitate, and wash the precipitate with ether, anhydrous ethanol, and acetone. After vacuum drying, obtain the active ingredients of the above-mentioned food products. Prepare 250 μg / mL aqueous solutions for each, which are the samples of the food products.

[0070] (2) Chromatographic separation and detection

[0071] Following the chromatographic method and conditions of Example 2, the chromatograms of each raw material are as follows: Figures 4-6 As shown, the peak elution time t of each food product sample was recorded. R样品 Then, using the retention time of the negative control in Example 2 as a benchmark, the peak time difference Δt between the food-derived product and the negative control group was calculated. R :

[0072] Δt R =t R样品 -t R阴性 , where t R样品 t represents the peak elution time of the food product. R阴性 The peak time is the elution time of the negative control group, and the unit of peak time is min. When two or more identifiable peaks appear on the ACE / ET-1 affinity chromatography column for the same foodborne product sample, it indicates that multiple components in the sample interact with the target to varying degrees. To standardize the grading criteria, the strongest binding determination principle is adopted, and the peak with the largest ΔtR in the sample is taken as the main determination peak for binding grading.

[0073] Based on the peak time difference Δt R To determine the binding strength between the samples and ACE and / or ET-1, the 17 food-derived products were classified into different binding strength grades, including strong binding, moderate binding, and moderate binding.

[0074] When ΔtR < 2, the food product is classified as a general combination.

[0075] When 2≤ΔtR<5, the food product is classified as a medium combination.

[0076] When ΔtR≥5, the food product is determined to be of a strongly bound type.

[0077] In this embodiment, the peak elution time of 17 different food products was detected, and the peak elution time difference Δt was calculated. R The results are shown in Table 1:

[0078] Table 1. t-values ​​of 17 raw materials on an ACE / ET-1 affinity column R样品 With Δt R statistics

[0079]

[0080] According to the results in Table 1, the food products classified as strongly combined are Eucommia ulmoides leaves, hawthorn, lotus seeds, corn silk, and bayberry; the food products classified as moderately combined are kudzu root, bitter tea, celery seeds, polygonatum, and fig; and the food products classified as generally combined are cassia seeds, astragalus, buckwheat, codonopsis, platycodon, pumpkin, and blueberry.

[0081] Example 4: Screening and grading of active monomers.

[0082] The raw materials of bayberry and blueberry from Example 3 were selected, and the raw materials were degreased, decolorized, and subjected to water extraction and ethanol precipitation according to the process of Example 3. The resulting precipitate was dried under vacuum and used as the crude extract for monomer separation. The crude extract of bayberry was separated using a C18 column under gradient elution conditions (mobile phase A: 0.1% formic acid water, mobile phase B: acetonitrile, 0–30 min, B 10%→40%). The eluted fractions corresponding to the time period of the strong retention peak (ΔtR≥5) in the ACE / ET-1 affinity fraction were collected, and compounds Y1 and Y2 were obtained after multiple purifications. Similarly, using the crude extract of blueberry as the starting sample, compound B1 was obtained under the same chromatographic conditions.

[0083] Compounds Y1, Y2, and B1 were dissolved in methanol and injected into an HPLC-LCMS system. Mass spectrometry was used for detection under the HPLC conditions of Example 2. The results are as follows: Figure 7 , Figure 8The results show that compound Y1 exhibits a quasi-molecular ion peak at m / z=463.31 in negative ion mass spectrometry mode, Y2 at m / z=317.24, and B1 at m / z=271.15, all corresponding to the quasi-molecular ion peak [MH]⁻ of each substance. The mass-to-charge ratio of each peak is consistent with the theoretical molecular weight of the corresponding substance (myricetin 464, myricetin 318, arbutin 272) after losing one proton. Combined with the HPLC retention time, it can be determined that Y1 is myricetin, Y2 is myricetin, and B1 is arbutin.

[0084] The ACE / ET-1@SiO2 column prepared in Example 1 and the HPLC-ELSD conditions established in Example 2 were used. The three monomers, myricetin, myricetin, and arbutin, were dissolved in ultrapure water to prepare 250 μg / mL concentration gradient solutions, with an injection volume of 25 μL. Under the stated conditions, the peak times of the three monomers on the ACE / ET-1@SiO2 column were as follows: Figure 9 As shown, the times are 16.1 min, 16.9 min, and 11.1 min, respectively.

[0085] Example 5: Formulation of a food-derived product composition for lowering blood pressure.

[0086] Based on the results of Example 3 above, the mass percentages of food-derived products with different binding affinity grades in the blood pressure-lowering food-derived product compositions are as follows:

[0087] The quality of food-derived products with strong flavor combinations accounted for 10-15% of the total.

[0088] The proportion of food-derived products with a medium-level combined flavor classification is 3-5%.

[0089] The quality of single-flavor graded food products is generally 0-3% of the combined food products.

[0090] Based on the above formulation principles, two representative formulations were set up for subsequent effect evaluation and verification of chromatographic screening results. The formulations are set as follows:

[0091] (1) Formula 1

[0092] Eucommia ulmoides leaves 15%, bayberry 15%, hawthorn 15%, corn silk 15%, lotus seeds 10%;

[0093] Kudzu root 5%, bitter tea 5%, celery seed 5%, polygonatum 5%, fig 3%;

[0094] Cassia seed 2%, Astragalus 2%, Tartary buckwheat 1%, Platycodon grandiflorus 1%, Blueberry 1%.

[0095] (2) Formula 2

[0096] Eucommia ulmoides leaves 10%, bayberry 10%, hawthorn 15%, corn silk 15%, lotus seeds 15%;

[0097] Kudzu root 5%, bitter tea 5%, celery seed 5%, polygonatum 5%, fig 5%;

[0098] Cassia seed 2%, Astragalus 2%, Tartary buckwheat 1%, Codonopsis pilosula 2%, Platycodon grandiflorus 1%, Pumpkin 1%, Blueberry 1%;

[0099] Comparative Example 1: Control Formulation of a Food-Derived Product Composition for Lowering Blood Pressure

[0100] To compare and verify with the two representative formulations in Example 5, formulation 3 was set up according to the formulation principle of "strong binding 5%; moderate binding 10-15%; general binding 0-3%", and formulation 4 was set up according to the formulation principle of "strong binding 5%; moderate binding 0-3%; general binding 10-15%". The specific formulations are as follows:

[0101] (1) Formula 3

[0102] Eucommia ulmoides leaves 5%, bayberry 5%, hawthorn 5%, corn silk 5%, lotus seeds 5%;

[0103] Kudzu root 15%, bitter tea 15%, celery seed 15%, polygonatum 10%, fig 10%;

[0104] Cassia seed 2%, Astragalus 2%, Tartary buckwheat 1%, Codonopsis pilosula 2%, Platycodon grandiflorus 1%, Pumpkin 1%, Blueberry 1%.

[0105] (2) Formula 4

[0106] Eucommia ulmoides leaves 5%, bayberry 5%, hawthorn 5%, corn silk 5%, lotus seeds 5%;

[0107] Kudzu root 2%, bitter tea 2%, celery seed 2%, polygonatum 2%, fig 2%;

[0108] Cassia seed 15%, Astragalus 10%, Tartary buckwheat 10%, Codonopsis pilosula 10%, Platycodon grandiflorus 10%, Pumpkin 10%.

[0109] Experimental Example 6: Animal-level efficacy study.

[0110] (1) Construction and grouping of rat hypertension model

[0111] In this experiment, a high-salt diet model with 8% NaCl in drinking water was used: male SD rats were fed a fixed amount of feed containing 8% NaCl starting from the 3rd week after weaning, and this continued for 6 weeks. The model was considered successful when the tail artery systolic blood pressure was ≥180 mmHg.

[0112] After the hypertension model was successfully established, the rats were randomly divided into the following groups (n=6) for subsequent experimental intervention and observation:

[0113] ① Blank control group: Rats were fed a regular diet to eliminate the interference of physiological changes caused by high-salt diet on the experimental results.

[0114] ② Model group: Rats continued to be fed a fixed amount of diet containing 8% NaCl without drug intervention, and the development of hypertension and changes in related physiological indicators were observed.

[0115] ③ Positive drug group: Rats were fed a high-salt diet of 8% NaCl and simultaneously administered captopril + alpracententan (30 mg / kg / day each) by gavage for 4 consecutive weeks;

[0116] ④-⑦ Experimental groups (divided into 4 groups according to formulations 1-4): Rats were fed a high-salt diet of 8% NaCl and were administered formulations 1-4 (200 mg / kg / day) developed in Example 5 and Comparative Example 1 by gavage for 4 consecutive weeks; the antihypertensive effect of the formulations in this high-salt hypertension model was evaluated.

[0117] Monomer groups (divided into 6 groups according to different monomers and dosages): Rats were fed a high-salt diet of 8% NaCl and were administered different doses of myricetin (low dose 100 mg / kg / day, high dose 200 mg / kg / day), myricetin (low dose 100 mg / kg / day, high dose 200 mg / kg / day), and arbutin (low dose 100 mg / kg / day, high dose 200 mg / kg / day) by gavage to investigate the antihypertensive effects of different monomers in a high-salt hypertension model.

[0118] (2) Study on the antihypertensive effect in rats

[0119] During the 4-week administration period, the systolic and diastolic blood pressure of rats in each group were measured regularly using a tail artery blood pressure monitor, and the data were recorded to observe changes in blood pressure. Results are as follows: Figure 10Blood pressure monitoring results in rats showed that the systolic and diastolic blood pressure in the blank control group remained at normal levels (systolic blood pressure ≈ 120 mmHg, diastolic blood pressure ≈ 80 mmHg) throughout the experimental period, without significant fluctuations. In the model group, after continuous feeding with a high-salt diet, both systolic and diastolic blood pressure showed a continuous upward trend, remaining at a high level for 4 weeks. In the positive drug group, after intervention with captopril + alpracententan, blood pressure showed a significant downward trend, with both systolic and diastolic blood pressure decreasing significantly after 4 weeks of intervention, demonstrating a clear antihypertensive effect. Among the experimental groups, formulations 1 and 2 showed the most significant antihypertensive effects: blood pressure began to decrease significantly after 1 week of intervention, and the antihypertensive effect continued to increase with the extension of intervention time. Their antihypertensive effects were close to those of the positive drug group and closest to the blood pressure level of the blank control group. In contrast, although the blood pressure of rats treated with formulations 3 and 4 decreased slightly compared to the model group, the blood pressure values ​​at each time point were significantly higher than those of formulations 1, 2, and the positive drug group, indicating a moderate antihypertensive effect. To further verify the correlation between affinity grading results and in vivo effects, representative monomers (myricetin, myricetin, and arbutin) screened, enriched, and structurally confirmed in Example 4 were compared and evaluated. The trends in blood pressure improvement in vivo were as follows: Figure 11 As shown, the improvement rates of myricetin and myricetin were higher than those of arbutin, and they exhibited a certain dose-dependent effect.

[0120] The above results indicate that formulations 1 and 2 of this invention possess significant in vivo antihypertensive activity, and their antihypertensive effect is consistent with the previous ACE / ET-1 binding screening hierarchy. This also verifies the rationality of the formulation design based on the ACE / ET-1@SiO2 chromatographic column provided in this application, which can screen out food-derived products with good affinity for the antihypertensive target and outstanding antihypertensive effect. Meanwhile, the comparative results of representative monomers further support this: under the screening system of this application, monomers with higher affinity (such as myricetin and myricetin) better reflect in vivo effects related to the ACE / ET-1 target axis; monomers with lower affinity (such as arbutin) also show a certain trend, but the magnitude is weaker.

[0121] (3) PCR detection of the relative mRNA content of ACE and ET-1 in the hearts of rats in each group

[0122] After euthanizing rats in each group, the hearts were quickly separated, and 100 mg were excised on an RNase-free operating table. The tissue was rinsed with physiological saline to remove blood, blotted dry with filter paper, and stored at -80 °C for later use. Each tissue sample was homogenized thoroughly with 1 mL of TRIzol reagent, and total RNA was extracted. Genomic DNA was removed by incubation with RNase-free DNase I at 37 °C for 30 min, and 1 µg of purified RNA was used to synthesize cDNA. The transcriptional levels of ACE and ET-1 (rat gene EDN1) were detected using SYBR Green real-time quantitative PCR (qPCR). The 20 µL reaction system consisted of: 10 µL of 2× qPCR Master Mix, 0.4 µL each of upstream / downstream primers (10 µM, final concentration approximately 0.2 µM), 2 µL of template cDNA (diluted 1:5), and nuclease-free water to make up the difference. Cycling conditions: 95 °C pre-denaturation for 30 s; followed by 40 cycles (95 °C 5 s, 60 °C 30 s), with end denaturation at 65-95 °C. °C melting curve analysis was used to confirm a single specific peak; negative controls included a template-free control (NTC) and a reverse transcription-free control (-RT). Data processing was performed using the C-value of ACE / ET-1 from the same sample. t Subtract internal reference C t ΔC t And the model group is used as the calibration group (ΔΔC) t =ΔC t样本 −ΔC t模型组 ), relative expression levels according to 2 −ΔΔCt The results were calculated and expressed as mean ± standard deviation. The final evaluation of the intervention's effect on ACE / ET-1 was based on comparisons of relative mRNA levels in each group. The results are as follows: Figure 12 and Figure 13 As shown.

[0123] Depend on Figures 12-13The results showed that the relative expression of ACE mRNA was low in the blank control group and significantly increased in the hypertension model group (P<0.01 compared with the blank control group), indicating that the ACE pathway was overactivated in the high-salt-induced hypertension model. The expression of ACE mRNA in the positive drug group and formulations 1, 2, and 3 was significantly inhibited (P<0.01 compared with the model group), and formulation 4 also showed a certain inhibitory effect (P<0.05 compared with the model group). Furthermore, the inhibitory effects of formulations 1, 2, and 3 were not significantly different from those in the positive drug group, indicating that the formulations in this invention can alleviate the overactivation of the renin-angiotensin system by downregulating the ACE gene transcription level. The relative expression results of ET-1 mRNA showed that the trends in each intervention group were consistent with the trends in the relative expression of ACE mRNA. Further comparison of the effects of formulations 1-4 within the experimental groups shows that: formulations 1 and 2 exhibit the best reduction effect, are closest to the blank control group, and are better than formulation 3. Although formulation 4 can downregulate ACE and ET-1 expression, its inhibitory effect is weaker than other formulations.

[0124] The relative expression of ACE / ET-1 mRNA after intervention with each monomer showed that the expression of ACE and ET-1 mRNA was significantly increased in the model group (P<0.01 compared with the blank control group); the positive control group significantly downregulated the expression of ACE and ET-1 mRNA (P<0.01 compared with the model group). Both myricetin and myricetin groups significantly decreased the expression of ACE and ET-1 mRNA (P<0.01), with a reduction similar to that in the positive control group; arbutin also downregulated the expression of related mRNAs, but the degree of inhibition was weaker than that in the myricetin and myricetin groups (P<0.05 compared with the model group).

[0125] The above results indicate that formulations 1 and 2 have a more prominent effect on simultaneously regulating the mRNA transcription levels of both ACE and ET-1 targets, superior to formulations 3 and 4. This further verifies the formulation advantages of formulations 1 and 2 designed based on ACE / ET-1 dual-target affinity screening in hypertension intervention. Furthermore, the monomer myricetin derived from the strongly binding raw material bayberry exhibits strong binding characteristics with myricetin on the ACE / ET-1 affinity chromatography column, demonstrating a significant in vivo antihypertensive effect and significantly downregulating the mRNA and protein expression of ACE and ET-1. In contrast, the arbutin monomer derived from the generally binding raw material blueberry only shows a general binding, with relatively weaker in vivo antihypertensive and ACE / ET-1 index improvement. This result is highly consistent with the antihypertensive effect of the raw material affinity grading in Example 3 and the formulation level in Example 6, proving that the grading principle based on ACE / ET-1 affinity chromatography in this application is not only applicable to raw material and formulation screening but can also predict the strength of the antihypertensive effect at the monomer level.

[0126] (4) Western blot was used to detect the protein expression levels of ACE and ET-1 in the heart tissue of rats in each group.

[0127] After sacrifice, heart tissue was rapidly separated from rats in each group (washed with PBS), blotted dry with filter paper, and stored at -80°C. 50 mg of tissue was placed in a pre-chilled homogenizing tube, and RIPA lysis buffer containing protease inhibitors and PMSF was added. The tube was homogenized on ice and allowed to lyse for 30 min. The supernatant was collected by centrifugation at 12000 g for 15 min at 4°C, and quantified using the BCA method. Protein was mixed with 50 µg of 5×SDS loading buffer per well, denatured at 95°C for 5 min, loaded onto 10% SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked in TBST with 5% skim milk powder at room temperature for 1 h. Primary antibodies (anti-ACE, 1:500), anti-ET-1, and internal control GAPDH) were incubated overnight at 4°C. After washing with TBST for 3×10 min, the membrane was incubated with HRP-labeled secondary antibody (1:3000, room temperature for 1 h). After washing again, the membrane was developed with ECL chemiluminescence reagent and acquired using a gel imaging system. Band grayscale analysis was performed using ImageJ software. The results are shown in Figure 1. Figure 14 , Figure 15 As shown.

[0128] Analysis of the expression levels of ACE and ET-1 proteins in the heart tissues of rats in each group showed that the protein expression trend was consistent with the relative mRNA content trend. The blank control group showed low abundance of both ACE and ET-1 proteins. The model group showed a significant increase in the gray value of ACE / ET-1 protein (P<0.01 compared to the blank control group), indicating that the ACE / ET-1-mediated vasoconstriction pathway was continuously activated under hypertension. Both the positive control group and the experimental group downregulated ACE / ET-1 protein expression, with formulations 1 and 2 showing the most significant inhibitory effects (P<0.01 compared to the model group), and close to the positive control group. Further comparison of the differences within the experimental groups revealed that formulations 3 and 4 showed weak inhibitory effects on ACE / ET-1 protein, slightly lower than the hypertension model group, while the relative expression levels of ACE / ET-1 protein in formulations 1 and 2 were significantly lower than those in formulations 3 and 4. The above results indicate that formulations 1 and 2 showed significantly better inhibitory effects on both ACE and ET-1 at the protein level than formulations 3 and 4, further supporting the scientific rationality of the formulation design based on dual-target affinity screening. The combination of active ingredients in formulations 1 and 2 more efficiently blocked the synthesis of key proteins in the vasoconstriction pathway, consistent with the superior antihypertensive effect observed in previous blood pressure monitoring, fully demonstrating the scientific validity and effectiveness of the formulation combination in this invention. The trends in ACE and ET-1 protein expression levels in rat heart tissue after monomeric intervention were consistent with the relative RNA expression. ACE and ET-1 protein expression was upregulated in the model group; the positive control group, myricetin group, and myricetin group all significantly reduced their protein levels, with no significant difference between the myricetin and myricetin groups and the positive control group; arbutin protein expression also decreased to some extent, but its inhibitory effect was less than that of the myricetin and myricetin groups.

[0129] (5) HE staining and pathological evaluation of rat myocardium in each group

[0130] After sacrifice, rats in each group were rapidly separated from their heart tissue (washed with PBS), fixed in 4% paraformaldehyde (pH 7.4) at 4°C for 24 h, then dehydrated using a routine gradient of ethanol, cleared with xylene, and embedded in paraffin. 4 μm sections were cut using a rotary microtome, dried at 60°C for 1 h, dewaxed with xylene for 2 × 10 min, and rehydrated with 100%, 95%, 85%, and 75% ethanol. Hematoxylin staining was performed for 3-5 min, followed by rinsing with tap water, rapid differentiation with 1% hydrochloric acid ethanol, or soaking in saturated lithium carbonate for 1-2 min, and rinsing with running water. Eosin staining was performed for 1-3 min, followed by gradient dehydration with 95% and 100% ethanol, clearing with xylene, and mounting with neutral resin. Microscopic imaging was then performed, and the results are shown below. Figure 16As shown in the results, the blank control group exhibited neat and dense myocardial fiber bundles with clear striations, normal cell morphology and nuclear staining, and no obvious edema or inflammatory infiltration in the interstitium. The model group, however, showed significant swelling and degeneration of myocardial cells, disordered fiber arrangement with visible breakage, increased eosinophilic cytoplasm, interstitial edema / fibrosis, and multifocal inflammatory cell infiltration, indicating significant histological damage. The positive drug group significantly alleviated the above pathological conditions, with myocardial structure and cell morphology approaching normal. In each experimental group, after intervention with formulations 1 and 2, myocardial fibers basically recovered to a regular parallel arrangement, and cell swelling and interstitial lesions were significantly reduced or disappeared. Formulations 3 and 4 showed only mild improvement, still exhibiting focal disordered arrangement and residual inflammatory response. These histological changes were consistent with the results of ACE and ET-1 dual-target regulation and blood pressure monitoring, indicating that formulations 1 and 2 effectively protected myocardial tissue at the structural level by inhibiting key molecules in the vasoconstriction pathway and improving microcirculation and myocardial stress. Their cardioprotective effect was superior to that of formulations 3 and 4, consistent with the screening results of affinity chromatography.

[0131] In summary, this application successfully constructed an enzyme affinity chromatography screening platform with ACE / ET-1 dual targets as its core. Combined with HPLC-ELSD detection and quantitative grading rules, it achieved precise screening of active ingredients in complex food-derived product systems, effectively solving the technical bottlenecks of traditional screening methods such as single target and impurity interference. Based on the hierarchical screening results of 17 medicinal and edible raw materials using this platform, this application designed and obtained food-derived compositions (formulas 1 and 2) according to the principle of "strongly binding raw materials as the core, and medium- and general binding raw materials in synergistic formulation". Animal experiments confirmed that they have significant antihypertensive activity, can downregulate the expression of ACE / ET-1 pathway-related mRNA and protein, and improve myocardial tissue pathological damage. Moreover, the effect is significantly better than the control formulations (formulas 3 and 4) that deviate from the affinity grading principle, thus verifying the scientificity and effectiveness of formulation design guided by dual-target affinity chromatography grading.

[0132] Building upon this foundation, this invention further conducted in-depth research on blood pressure reduction using medicinal and edible raw materials such as bayberry: Through purification, separation, and structural identification of bayberry and blueberry extracts, key monomeric components such as myricetin, myricetin, and arbutin were obtained. Their affinity behavior with the target was systematically evaluated under the same ACE / ET-1 affinity chromatography platform, clarifying the comparison between "strongly binding raw materials - strongly binding monomers" and "generally binding raw materials - generally binding monomers". Combined with in vivo efficacy evaluation in a high-salt-induced hypertension rat model, it was confirmed for the first time that bayberry-derived monomers (such as myricetin and myricetin) have significant and dose-dependent antihypertensive effects and can more effectively downregulate ACE / ET-1 pathway mRNA and protein expression, while the generally binding monomer arbutin, although having certain antihypertensive activity, has a relatively weak effect. This systematic study at the monomer level fills the gap in existing technologies that only focus on raw material or formulation screening and lack in-depth elucidation of the key material basis and mechanism of action of food and medicine homologous raw materials. It further proves the predictive value of the dual-target affinity grading index of this application for the in vivo antihypertensive effect, and realizes a comprehensive and systematic demonstration from raw material screening, monomer confirmation, formulation optimization, mechanism and efficacy evaluation. It not only provides a standardized and quantifiable technical solution for the research and development of antihypertensive functional products, but also provides new ideas and methods for the precise development and quality control of food and medicine homologous resources such as bayberry.

[0133] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this application.

Claims

1. An affinity screening method for food-derived products used to lower blood pressure, characterized in that, Includes the following steps: The sample of the food product to be tested was injected into an ACE / ET-1 composite affinity chromatography column for chromatographic separation, and the peak time of the food product to be tested was detected by ELSD. According to the difference in peak time between the food product and the negative control group, the food product was divided into different binding grade, and the food products of different binding grades were selected to obtain a food product composition for lowering blood pressure. The stationary phase packed with the ACE / ET-1 composite affinity chromatography column is an ACE / ET-1@SiO2 composite stationary phase, and the carrier of the ACE / ET-1@SiO2 composite stationary phase is SiO2 particles. The SiO2 particles simultaneously immobilize two target molecules: angiotensin-converting enzyme ACE and endothelin ET-1. The negative control group consisted of acetaminophen.

2. The method according to claim 1, characterized in that, The difference in peak elution time between the food-derived product and the negative control group was Δt. R =t R样品 -t R阴性 , where t R样品 t represents the peak elution time of the food product. R阴性 The peak time is the time of elution in the negative control group, and the unit of the peak time is min.

3. The method according to claim 2, characterized in that, The different binding gradations include three types: strong binding, moderate binding, and general binding. When ΔtR < 2, the food product is classified as a general combination. When 2≤ΔtR<5, the food product is classified as a medium combination. When ΔtR≥5, the food product is determined to be of a strongly bound type.

4. The method according to claim 3, characterized in that, The food-derived products classified as strongly bound include Eucommia ulmoides leaves, hawthorn, lotus seeds, corn silk, and bayberry; the food-derived products classified as moderately bound include kudzu root, bitter tea, celery seed, polygonatum, and fig; and the food-derived products classified as generally bound include cassia seed, astragalus, tartary buckwheat, codonopsis, platycodon, pumpkin, and blueberry. The mass percentages of these food-derived products in the composition of the food-derived product for lowering blood pressure are as follows: The quality of food-derived products classified as "strongly combined" in the single-ingredient category accounts for 10-15%. The quality of single-flavor food products classified as moderately combined accounts for 3-5% of the total quality. The quality of single-ingredient food products in the general combination category accounts for 0-3%.

5. The method according to claim 1, characterized in that, The chromatographic separation conditions were as follows: column temperature 35℃, flow rate 0.15 mL / min, mobile phase 5 mM ammonium acetate, injection volume 25 μL, and pH of the mobile phase 7.

2.

6. The method according to claim 1, characterized in that, The ELSD detection was performed at an evaporation temperature of 50-70°C, a drift tube temperature of 50-70°C, and a nitrogen flow rate of 1.0-2.0 L / min.

7. The method according to claim 1, characterized in that, The preparation method of the ACE / ET-1@SiO2 composite stationary phase is as follows: SiO2 particles are placed in PBS buffer, and solutions of two target molecules, angiotensin-converting enzyme (ACE) and endothelin (ET-1), are added respectively. After immobilization by stirring at low temperature, the particles are centrifuged and repeatedly washed with PBS to remove unfixed target molecules to obtain the ACE / ET-1@SiO2 composite stationary phase.

8. The method according to claim 1, characterized in that, The sample preparation method for the food product includes the following steps: (1) Crush and sieve the food product, add petroleum ether and heat and reflux. After reflux, cool, filter and collect the first filter residue. (2) Add anhydrous ethanol to decolorize the first filter residue, heat and reflux again, cool, filter and collect the second filter residue after reflux. (3) Add ultrapure water to the second filter residue, heat and reflux again, cool, filter and collect the supernatant after reflux, evaporate and concentrate the supernatant, add 95% ethanol to the concentrated supernatant to precipitate, centrifuge to collect the precipitate, wash the precipitate and vacuum dry to obtain the active ingredient of the food product. (4) Prepare an aqueous solution of the active ingredients of the food product to obtain a sample of the food product.

9. A food-derived product composition for lowering blood pressure obtained by the method according to any one of claims 1 to 8, characterized in that, Includes either composition one or composition two; The composition of the first composition, by weight percentage, is as follows: Eucommia ulmoides leaves 15%, bayberry 15%, hawthorn 15%, corn silk 15%, lotus seeds 10%; kudzu root 5%, bitter tea 5%, celery seed 5%, polygonatum 5%, fig 3%; cassia seed 2%, astragalus 2%, tartary buckwheat 1%, platycodon root 1%, blueberry 1%; The composition of the second composition, by mass percentage, is as follows: 10% Eucommia ulmoides leaves, 10% bayberry, 15% hawthorn, 15% corn silk, 15% lotus seeds; 5% kudzu root, 5% bitter tea, 5% celery seed, 5% Polygonatum sibiricum, 5% fig; 2% cassia seed, 2% Astragalus membranaceus, 1% tartary buckwheat, 2% Codonopsis pilosula, 1% Platycodon grandiflorus, 1% pumpkin, 1% blueberry.

10. The application of the method according to any one of claims 1 to 8 in screening monomeric compounds with antihypertensive properties, characterized in that, The monomeric compounds include one or more of myricetin, myricetin and arbutin.

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